A composition for preventing, treating or reversing pulmonary fibrosis and use thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a composition for preventing, treating or reversing pulmonary fibrosis and application thereof.The composition comprises at least one of hippuric acid, glycine, malic acid and fumaric acid, preferably a combination of the four in a ratio of 0.3 mg / mL, 0.6 mg / mL, 0.6 mg / mL and 0.4 mg / mL.The application is verified by a cell experiment, and the composition can significantly down-regulate the mRNA expression of a pulmonary fibrosis marker ACTA2 and an inflammatory factor IL-6 through multi-component synergistic effect, the inhibition rate of ACTA2 of some combinations is more than 90%, and the composition can reverse the formed pulmonary fibrosis phenotype, and a Bliss independence model verifies that the composition has a strong synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for the prevention, relief, treatment, or reversal of pulmonary fibrosis. More specifically, this invention relates to a composition comprising at least one or at least two active ingredients selected from hippuric acid, glycine, malic acid, and fumaric acid, its preparation method, and its application in inhibiting fibroblast activation, downregulating α-smooth muscle actin (ACTA2) gene expression, and inhibiting the expression of the inflammatory cytokine interleukin-6 (IL-6). Background Technology

[0002] Pulmonary fibrosis is a fatal interstitial lung disease characterized by the proliferation of fibroblasts in lung tissue and the accumulation of large amounts of extracellular matrix accompanied by inflammatory damage. Currently approved treatments such as nintedanib and pirfenidone can only slow disease progression, not reverse or cure it, and they are expensive and have significant side effects (such as hepatotoxicity and gastrointestinal reactions). TGF-β1 (transforming growth factor-β1) is a core cytokine driving the progression of pulmonary fibrosis. It strongly induces the activation of lung fibroblasts into myofibroblasts and highly expresses ACTA2, leading to excessive extracellular matrix deposition. Therefore, developing novel treatments that can effectively inhibit the TGF-β1 signaling pathway and have a higher safety profile is urgently needed.

[0003] Existing treatments have single targets, limited efficacy, significant side effects, and high costs. While some small-molecule organic acids (such as malic acid and fumaric acid) or amino acids (such as glycine) found in nature or through chemical synthesis have been mentioned in some studies as having potential biological activity, they are usually weak when used alone and require very high concentrations to be effective. They do not have practical therapeutic value and have not been explored for the treatment of pulmonary fibrosis.

[0004] The pathogenesis of pulmonary fibrosis involves a complex network of multiple signaling pathways, making it difficult to achieve significant therapeutic effects by targeting a single point. Single-component mechanisms of action are limited and cannot synergistically regulate multiple key targets; therefore, high doses are often required to achieve the desired effect, thus increasing the risk of toxic side effects.

[0005] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] This invention aims to address the problems of insufficient efficacy, significant side effects, and high cost of existing anti-pulmonary fibrosis drugs. Currently, most treatments only slow disease progression; reversing fibrosis represents a superior therapeutic effect, which is itself a significant technological advancement. This invention provides a composition, preferably composed of natural or dietary-derived ingredients, which, through the synergistic effect of multiple components, effectively inhibits fibroblast activation and inflammatory responses at low doses, thus providing a new, safer, and more effective solution for the prevention and treatment of pulmonary fibrosis.

[0007] This invention provides a composition for the prevention, treatment, or reversal of pulmonary fibrosis, wherein the active ingredient of the composition comprises at least one of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), fumaric acid (MAC-4), and their respective derivatives, pharmaceutically acceptable salts, preferably at least two or more. Preferably, the malic acid is L-malic acid or DL-malic acid.

[0008] According to a preferred embodiment, the active ingredient is selected from one or more of the following: hippuric acid (MAC-1); Glycine (MAC-2); Malic acid (MAC-3); Fumaric acid (MAC-4); hippuric acid (MAC-1) and glycine (MAC-2); hippuric acid (MAC-1) and malic acid (MAC-3); hippuric acid (MAC-1) and fumaric acid (MAC-4); Glycine (MAC-2) and malic acid (MAC-3); Glycine (MAC-2) and fumaric acid (MAC-4); Malic acid (MAC-3) and fumaric acid (MAC-4); hippuric acid (MAC-1), glycine (MAC-2), and malic acid (MAC-3); hippuric acid (MAC-1), glycine (MAC-2), and fumaric acid (MAC-4); hippuric acid (MAC-1), malic acid (MAC-3), and fumaric acid (MAC-4); Glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4); hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4); Particularly preferred is that the composition consists of malic acid (MAC-3) and fumaric acid (MAC-4), or glycine (MAC-2), malic acid (MAC-3) and fumaric acid (MAC-4).

[0009] According to a preferred embodiment, the active ingredient is composed of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4).

[0010] According to a preferred embodiment, the concentrations of each active ingredient are: hippuric acid (MAC-1) 0.1-0.5 mg / mL, glycine (MAC-2) 0.4-1.0 mg / mL, malic acid (MAC-3) 0.4-0.8 mg / mL, and fumaric acid (MAC-4) 0.2-0.6 mg / mL.

[0011] According to a preferred embodiment, the concentrations of each active ingredient are: 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.6 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4).

[0012] According to a preferred embodiment, the composition is a pharmaceutical preparation, a health food, or a functional food.

[0013] The present invention also provides the use of the foregoing composition in the preparation of a medicament for inhibiting ACTA2 gene expression, preferably in the preparation of a medicament for preventing, delaying progression, reversing or treating pulmonary fibrosis.

[0014] The present invention also provides the use of the foregoing composition in the preparation of a substance for inhibiting IL-6 gene expression, preferably in the preparation of a substance for anti-inflammatory purposes.

[0015] The present invention also provides the use of the foregoing compositions in the preparation of medicaments for the prevention, and / or treatment, and / or reversal of pulmonary fibrosis, and / or liver fibrosis, and / or kidney fibrosis.

[0016] The present invention also provides the use of the foregoing composition in the preparation of any of the following products: (1) Health foods or functional foods for the prevention or treatment of pulmonary fibrosis; (2) Medical aesthetic and dermatological products, selected from products for treating skin scars / anti-scars, or products for treating acne pits / acne marks, or products for anti-aging / anti-wrinkle.

[0017] The present invention also provides the use of the aforementioned composition in the preparation of a medicament for reversing pulmonary fibrosis.

[0018] The present invention also provides the use of the foregoing composition in the preparation of any of the following products: (1) Health foods or functional foods that reverse pulmonary fibrosis; (2) Skin care products selected for use in treating surgical / burn scars, gels, creams, silicone patches, or acne marks and pits.

[0019] According to a preferred embodiment, the concentrations of each active ingredient in the composition are: 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3, and 0.4 mg / mL MAC-4.

[0020] According to a preferred embodiment, the concentrations of each active ingredient in the composition are: 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 1 mg / mL MAC-3, and 0.4 mg / mL MAC-4.

[0021] According to a preferred embodiment, the concentrations of each active ingredient in the composition are: 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.3 mg / mL MAC-3, and 0.8 mg / mL MAC-4.

[0022] The present invention also provides the use of any one or more of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4) in the preparation of substances for the prevention, relief, reversal, or treatment of pulmonary fibrosis, and / or in the preparation of substances for the prevention, relief, reversal, or treatment of pulmonary fibrosis, preferably pharmaceuticals, and / or in the preparation of substances that inhibit fibroblast activation, downregulate α-smooth muscle actin (ACTA2) gene expression, and inhibit the expression of the inflammatory factor interleukin-6 (IL-6).

[0023] The present invention also provides the use of the foregoing composition in the preparation of any of the following products: (1) Pharmaceutical preparations, including tablets, capsules, injections, suspensions, emulsions, ointments, gels, aerosols, sprays, inhalers, suppositories, and patches; (2) Health foods or functional foods, including oral liquids, granules, powders, tablets, capsules, meal replacement foods, beverages, and confectionery products; (3) Medical aesthetic and dermatological products, including anti-scar products, preferably gels, creams, silicone patches, medical dressings, acne scar and pit repair products, anti-aging / anti-wrinkle products, and cosmeceuticals; (4) Medical device related products, including implant anti-fibrotic coatings, especially suitable for artificial joints, pacemakers, artificial blood vessels, intrauterine devices, glaucoma drainage valves, injectable bio-hydrogels / tissue regeneration scaffolds; (5) Veterinary drug products, including oral preparations, injections, topical preparations, and feed additives for animal pulmonary fibrosis, liver fibrosis, and kidney fibrosis; (6) Research tools, including cell culture additive kits, especially in vitro culture aids for inhibiting fibroblast activation.

[0024] The present invention also provides the use of the foregoing composition in the preparation of anti-inflammatory and / or immunomodulatory products, preferably, the use includes at least one of the following: intervention for fibrotic diseases, treatment for inflammatory diseases, improvement for metabolic diseases, treatment for neurological diseases, adjuvant therapy for cancer, treatment for dermatological diseases, and treatment for orthopedic and dental diseases.

[0025] According to a preferred embodiment, the intervention for fibrotic diseases includes its use in the preparation of drugs or health foods for treating or delaying pulmonary fibrosis, liver fibrosis / cirrhosis, and kidney fibrosis.

[0026] According to a preferred embodiment, the inflammatory disease treatment includes the use in the preparation of medicaments for treating rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), atherosclerosis, sepsis, chronic obstructive pulmonary disease, systemic lupus erythematosus, and multiple sclerosis.

[0027] According to a preferred embodiment, the improvement of metabolic diseases includes its use in the preparation of medicaments or health foods for improving type 2 diabetes and insulin resistance, non-alcoholic fatty liver disease.

[0028] According to a preferred embodiment, the treatment of neurological diseases includes its use in the preparation of medicaments for treating Alzheimer's disease, Parkinson's disease, and multiple sclerosis.

[0029] According to a preferred embodiment, the adjuvant cancer therapy includes its use in the preparation of a medicament for tumor-associated macrophage reprogramming, for use in combination with chemotherapy / radiotherapy / immune checkpoint inhibitors to enhance efficacy and alleviate cancer cachexia.

[0030] According to a preferred embodiment, the skin disease treatment includes its use in the preparation of a medicament or medical dressing for treating psoriasis, atopic dermatitis, or promoting the healing of chronic, difficult-to-heal wounds.

[0031] According to a preferred embodiment, the orthopedic and dental disease treatment includes the use in the preparation of a medicament or topical formulation for treating osteoarthritis, periodontitis, and peri-implantitis.

[0032] According to a preferred embodiment, the application is a non-therapeutic application, including its use in the preparation of high-end skincare additives for soothing sensitive skin and repairing the skin barrier, and in hair growth products for improving inflammation-related hair loss.

[0033] A key aspect of this invention is that it provides specific concentration ratios of the aforementioned active ingredients, such as, but not limited to, 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3, and 0.4 mg / mL MAC-4. This combination has demonstrated excellent synergistic inhibitory and reversal effects in experiments.

[0034] Regarding the inhibition of pulmonary fibrosis, this application employs a prophylactic dosing regimen, administering drugs before constructing a fibrosis model in normal human lung fibroblasts (NHLF) induced by 5 ng / mL TGF-β1. The positive control drugs nintedanib (2 μM) and SB431542 (5 μM) significantly downregulated ACTA2 mRNA expression, confirming the stability and reliability of the model and the prophylactic dosing system. Based on this, this application investigated the antifibrotic activity of different samples. Results showed that MAC-3 (IC50 = 1.04 mg / mL) and MAC-4 (IC50 = 0.72 mg / mL) inhibited ACTA2 expression in a dose-dependent manner, with high concentrations (e.g., 9.6 mg / mL MAC-3, 1.2 mg / mL MAC-4) achieving the highest inhibition rate of 99.45%. MAC-1 and MAC-2, even at high concentrations, did not show significant ACTA2 downregulation when used alone. Further research revealed that multi-component combinations containing two or more active ingredients exhibited a strong synergistic inhibitory effect (Bliss Independence Value ≥ 0.15). For example, the combination of 0.3 mg / mL MAC-1 + 0.6 mg / mL MAC-2 + 0.6 mg / mL MAC-3 + 0.4 mg / mL MAC-4 achieved an ACTA2 inhibition rate of 98.69%, far exceeding the effect of a single component at the same concentration. Furthermore, it effectively inhibited fibroblast activation and ACTA2 gene expression even at low doses. These results indicate that this composition, through the synergistic effect of multiple components, can effectively block fibroblast activation before TGF-β1 stimulation, and has the potential to prevent pulmonary fibrosis.

[0035] To evaluate the potential of drugs to reverse pulmonary fibrosis, this application used 5 ng / mL TGF-β1 to induce NHLF cells to construct a fibrosis model. After significant upregulation of ACTA2 expression, drug intervention was then administered. The positive control drug SB431542 (5 μM) effectively downregulated ACTA2 expression, confirming the successful and stable construction of this "model first, treatment later" reversal model. Based on this, this application investigated the reversal effects of different samples on existing fibrosis. The results showed that low concentrations of single components (such as 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, and 0.6 mg / mL MAC-3) had no significant reversal effect, while high concentrations of MAC-3 (2.4 mg / mL, inhibition rate 74.76%) and MAC-4 (1.2 mg / mL, inhibition rate 90.67%) showed certain reversal potential. Further studies found that multi-component combinations could significantly reverse existing pulmonary fibrosis, and the synergistic effect was significant (BlissIndependence Value ≥ 0.15). Some combinations showed ACTA2 inhibition rates exceeding 90%, such as the 1 mg / mL MAC-3 + 0.4 mg / mL MAC-4 combination achieving an inhibition rate of 93.9%, and the 0.3 mg / mL MAC-1 + 0.6 mg / mL MAC-2 + 0.3 mg / mL MAC-3 + 0.8 mg / mL MAC-4 combination achieving an inhibition rate of 86.71%, fully demonstrating the excellent fibrosis reversal efficacy of the compositions. In summary, this study shows that even after administration following fibroblast activation, the target drug can still significantly inhibit ACTA2 expression, suggesting that it may reverse the established fibrotic phenotype by intervening in key signaling pathways such as TGF-β1 / Smad, demonstrating its potential for treating pulmonary fibrosis.

[0036] Regarding anti-inflammatory effects, this application successfully induced an inflammation model in RAW264.7 macrophages using 10 ng / mL LPS. The positive control drug dexamethasone (10 μM) significantly downregulated IL-6 mRNA expression, confirming the successful and stable establishment of the model. Based on this, this application investigated the inhibitory effects of different samples on the inflammatory response. The results showed that low concentrations of single components had weak anti-inflammatory effects; only high concentrations of single components (such as 2.4 mg / mL MAC-2, 1.2 mg / mL MAC-3, and 1.2 mg / mL MAC-4) could reduce IL-6 mRNA levels by more than 50%. Further research revealed that multi-component combinations could exert a synergistic anti-inflammatory effect (BlissIndependence Value ≥ 0.15), effectively inhibiting IL-6 gene expression. For example, the combination of 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 showed an inhibition rate of 39.12%, while the combination of 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3, and 0.4 mg / mL MAC-4 showed an inhibition rate of 56.83%. These results indicate that the composition possesses both anti-fibrotic and anti-inflammatory activities, demonstrating dual therapeutic potential.

[0037] All active ingredients involved in this application are of natural or dietary origin. They do not precipitate after cell treatment at the experimentally set concentrations, demonstrating high safety. This approach avoids the problems of existing anti-pulmonary fibrosis drugs, such as large side effects, high cost, and limited efficacy, and provides a safe and effective solution for the prevention, treatment, and reversal of pulmonary fibrosis and related inflammatory diseases.

[0038] The composition can be formulated into pharmaceutical preparations, health foods, or functional foods. This combination, particularly with its synergistic effects and reversal potential, has broad application potential across multiple fields, including: 1. Medical Aesthetics and Dermatology. Skin scars are essentially fibrosis. Anti-scar products (prevention and treatment): Surgical / burn scars: Developed in the form of gels, creams, silicone patches, etc., for the prevention and improvement of hypertrophic scars and keloids after surgery, trauma, or burns.

[0039] Acne scars and pits: Atrophic scars (acne pits) and red / brown marks (acne marks) left after acne inflammation subsides also involve abnormal proliferation and repair of fibrous tissue. This composition can target and inhibit overactivated fibroblasts, promote the regeneration of normal skin tissue, and achieve a "skin resurfacing" effect.

[0040] Anti-aging / anti-wrinkle products: Skin aging and wrinkle formation are partly caused by the degradation, disorder, and abnormal deposition of collagen fibers in the dermis (a mild, chronic fibrotic process). This composition may improve skin elasticity and reduce wrinkles by regulating the health of fibroblasts and promoting ECM (extracellular matrix) remodeling.

[0041] Preferably, the product form includes: medical dressings, high-end functional skincare products, and cosmeceuticals.

[0042] 2. Coatings for medical devices and implants Anti-fibrotic coating for implants: Many implants (such as artificial joints, pacemakers, artificial blood vessels, intrauterine devices, glaucoma drainage valves, etc.) experience a foreign body reaction after implantation, leading to the formation of fibrotic capsules around the implant. This can cause device malfunction, pain, or the need for secondary surgery. Applying the anti-fibrotic composition of this application as a coating to the implant surface allows for localized and sustained drug release, inhibiting the formation of fibrotic capsules and significantly extending the implant's lifespan and safety.

[0043] Injectable hydrogel / scaffold: Preferably, this application also relates to an injectable biomaterial containing an anti-pulmonary fibrosis composition. After injection at the site of tissue damage (such as after myocardial infarction or cartilage defects), this material can act as a scaffold for tissue regeneration and continuously release anti-fibrotic drugs, guiding functional tissue regeneration rather than the formation of non-functional scar tissue. This is crucial for the repair of precious tissues such as the heart and nerves.

[0044] 3. Veterinary drug field The compositions of this invention have wide applications in the field of veterinary medicine, suitable for livestock (pigs, cattle, sheep, horses, etc.), poultry (chickens, ducks, geese, etc.), pets (cats, dogs, rabbits, etc.), aquatic animals (fish, shrimp, crabs, etc.), and economic animals such as foxes and minks. They can provide intervention programs for fibrotic diseases caused by idiopathic etiologies and the progression of chronic diseases (such as chronic inflammation, infection, poisoning, etc.) in various animals. In the pet field, they are applicable to idiopathic pulmonary fibrosis in some dog breeds (such as West Highland White Terriers) and liver / kidney fibrosis in cats / dogs secondary to chronic liver and kidney diseases. In the livestock field, they can be used to control liver fibrosis in cattle and sheep (such as that caused by the progression of chronic hepatitis) and kidney fibrosis in pigs (such as late-stage manifestations of chronic kidney disease). In the poultry field, they can help improve pulmonary fibrosis secondary to respiratory diseases. In aquatic animals and economic animals, they can provide support for addressing problems such as growth retardation and functional decline caused by organ fibrosis (such as liver, kidney, and lung fibrosis). This invention relates to the use of the above-mentioned composition in the preparation of veterinary drug formulations (such as oral preparations, injections, topical preparations, and premixes) or functional products for animal husbandry / aquaculture targeting pulmonary fibrosis, liver fibrosis, and kidney fibrosis.

[0045] 4. Research Tools and In Vitro Diagnostics: Transforming this technology into tools to serve life science research, including novel cell culture additives: developing compositions into kits that can be added to cell culture media to specifically inhibit fibroblast activation in vitro. This is of significant value for stem cell research, organoid culture, and cancer research (inhibiting cancer-associated fibroblasts), helping scientists better maintain cellular function.

[0046] This invention also relates to the specific uses of the composition in "preparation of anti-scar drugs / cosmetics", "preparation of implant coatings", and "preparation of veterinary anti-fibrotic drugs". Preferably, this invention also relates to specific dosage forms for different uses, and more preferably, to "an anti-scar gel for dermal administration" and "a sustained-release formulation for implant coatings".

[0047] The composition provided by this invention has the following significant technical effects: Highly effective inhibition of ACTA2 expression: In a TGF-β1-induced NHLF fibrosis model, the specific composition of this invention significantly downregulated the mRNA level of the myofibroblast marker ACTA2, with an inhibitory effect exceeding 50%. For example, the four-component combination (0.3 mg / mL MAC-1 + 0.6 mg / mL MAC-2 + 0.6 mg / mL MAC-3 + 0.4 mg / mL MAC-4) achieved an inhibition rate of 98.69% against ACTA2, significantly superior to single components at the same concentration. Strong synergistic effect: Calculated using the Bliss independence model, the multi-component combination showed a strong synergistic effect in inhibiting ACTA2 and IL-6 expression (Bliss Independence Value ≥ 0.15), with a maximum of 0.85, indicating that the effect is not a simple additive effect, but a true synergistic enhancement. Potential to reverse fibrosis: In a reversal model of "modeling first, then administration", the composition (e.g., 1 mg / mL MAC-3 + 0.4 mg / mL MAC-4) showed an inhibition rate of 93.9% against ACTA2, demonstrating that it can reverse the established fibrotic phenotype. Dual inhibitory function: This composition can not only inhibit the core target of fibrosis, ACTA2, but also synergistically inhibit the mRNA expression of the key inflammatory factor IL-6 in a 10 ng / mL LPS-induced macrophage (RAW264.7) inflammation model, thus having both anti-fibrotic and anti-inflammatory effects. High safety: All ingredients are naturally occurring or dietary sources. No precipitation was observed in cell culture at the experimental concentration, indicating higher safety and potentially avoiding the serious toxic side effects of existing drugs.

[0048] This invention reveals the synergistic effect of a specific combination of hippuric acid, glycine, malic acid, and fumaric acid in inhibiting pulmonary fibrosis and related inflammation, rather than the weak effects of any single component. The specific concentration ratio is key to achieving this synergistic effect.

[0049] The anti-fibrotic / anti-inflammatory efficacy of the composition described in this invention is verified by the experimental data results shown in the accompanying drawings. The active ingredient ratio of the composition has been experimentally confirmed to have a significant synergistic effect (Bliss Independence Value ≥ 0.15). Attached Figure Description

[0050] Figure 1 This is a bar chart showing the effect of a single component on ACTA2 gene expression in TGF-β1-induced NHLF cells. It illustrates the effect of different concentrations of a single active component on the mRNA expression level of the myofibroblast marker ACTA2 in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The vertical axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH, and the horizontal axis represents the experimental groups: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (2 μM nintedanib, 5 μM SB431542), and single-component treatment groups with different concentrations of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4). Error bars represent the standard error (SEM) of the experimental data for each group. This figure clearly shows that MAC-3 and MAC-4 downregulate ACTA2 expression in a dose-dependent manner, while MAC-1 and MAC-2 did not show significant ACTA2 downregulation within the experimental concentration range, providing basic data on the activity of single components for subsequent multi-component combination screening.

[0051] Figure 2This figure presents the dose-response curves and IC50 analysis of single components on ACTA2 gene expression. The semi-logarithmic graph shows the trend of inhibition rates of four single active ingredients at different concentrations on TGF-β1-induced ACTA2 mRNA expression in normal human lung fibroblasts (NHLF). The vertical axis represents the ACTA2 expression inhibition rate (%), and the horizontal axis represents the compound concentration (mg / mL, logarithmic scale). Different colors and shapes of the markers represent hippuric acid (MAC-1, green circle), glycine (MAC-2, red triangle), malic acid (MAC-3, blue triangle), and fumaric acid (MAC-4, orange diamond), respectively. Error bars represent the standard error of each experimental group. This figure clearly shows the dose-dependent inhibitory differences of the four components. Malic acid (MAC-3) and fumaric acid (MAC-4) achieved high inhibition rates at lower concentrations, while hippuric acid (MAC-1) and glycine (MAC-2) showed lower inhibition rates within the experimental concentration range, providing crucial dosage references for subsequent concentration optimization of multi-component combinations.

[0052] Figure 3 This is a bar chart illustrating the overall effect of multi-component combinations on ACTA2 gene expression in TGF-β1-induced NHLF cells. It systematically demonstrates the regulatory effects of different binary, ternary, and quaternary active ingredient combinations on ACTA2 mRNA expression levels in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The vertical axis represents the ratio of ACTA2 relative mRNA levels to the internal reference gene GAPDH. The horizontal axis represents, in descending order, the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (10 μM dexamethasone, 5 μM SB431542), and the different active ingredient combination treatment groups numbered Group 3 to Group 34. Error bars represent the standard error of the experimental data for each group. This chart visually presents the significant advantage of multi-component combinations over single components in inhibiting ACTA2 expression, clearly distinguishing the differences in anti-fibrotic activity among different combinations, and providing a comprehensive experimental data basis for subsequent screening of the optimal combination with strong synergistic effects.

[0053] Figure 4This is a bar chart showing the effect of different concentrations of a single active ingredient on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells under single-dose administration in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model, specifically the effect of these single active ingredients on the mRNA expression level of the myofibroblast marker ACTA2. The vertical axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. The horizontal axis represents, in descending order, the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 μM SB431542), and the single-component treatment groups with different concentrations of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4). Error bars represent the standard error of the experimental data for each group. This figure further verifies the differences in antifibrotic activity among the four single components, clarifying that malic acid (MAC-3) and fumaric acid (MAC-4) can exhibit certain ACTA2 inhibitory effects at relatively low single doses, while hippuric acid (MAC-1) and glycine (MAC-2) have no significant inhibitory effects at their respective single doses. This provides a more accurate single-dose activity reference for subsequent concentration gradient design of multi-component combinations and screening of active ingredients.

[0054] Figure 5 This is a bar chart showing the synergistic effect of multiple component combinations on ACTA2 gene expression in TGF-β1-induced NHLF cells. It illustrates the regulatory effects of different concentrations of single components and various binary, ternary, and quaternary active component combinations on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The vertical axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. The horizontal axis represents, in descending order, the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 μM SB431542), and different concentrations of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), fumaric acid (MAC-4), single components, and various combination treatments. Error bars represent the standard error of the experimental data for each group. This figure visually demonstrates the significant enhancement effect of multi-component combinations on inhibiting ACTA2 expression compared to single components, clearly distinguishing the differences in synergistic effect strength among different combinations. It provides a core experimental data foundation for subsequent quantitative analysis of synergistic effects using the Bliss independence model and screening of synergistic compositions with optimal anti-fibrotic activity.

[0055] Figure 6This is a bar chart showing the effect of the binary combination of 0.3 mg / mL hippuric acid (MAC-1) and 0.6 mg / mL malic acid (MAC-3) on ACTA2 gene expression in TGF-β1-induced NHLF cells. The chart illustrates the regulatory effect of this binary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The x-axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), 0.3 mg / mL MAC-1 single-component treatment group, 0.6 mg / mL MAC-3 single-component treatment group, and 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-3 combined treatment group. The y-axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-1 alone had no significant inhibitory effect on ACTA2 expression, and treatment with 0.6 mg / mL MAC-3 alone showed only a weak inhibitory effect. However, the combined treatment of the two significantly reduced the ACTA2 expression level, indicating that the binary combination has certain synergistic anti-fibrotic activity.

[0056] Figure 7 This is a bar chart showing the effect of the binary combination of 0.6 mg / mL glycine (MAC-2) and 0.6 mg / mL malic acid (MAC-3) on ACTA2 gene expression in TGF-β1-induced NHLF cells. The bar chart illustrates the regulatory effect of this binary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The x-axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), 0.6 mg / mL MAC-2 single-component treatment group, 0.6 mg / mL MAC-3 single-component treatment group, and 0.6 mg / mL MAC-2 and 0.6 mg / mL MAC-3 combination treatment group. The y-axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.6 mg / mL MAC-2 alone had no significant inhibitory effect on ACTA2 expression, and treatment with 0.6 mg / mL MAC-3 alone showed only a moderate inhibitory effect. However, the combined treatment of the two significantly reduced the level of ACTA2 expression, and the inhibitory effect was far superior to that of the single component, indicating that the binary combination has significant synergistic anti-fibrotic activity.

[0057] Figure 8 This is a bar chart showing the effect of the binary combination of 0.6 mg / mL malic acid (MAC-3) and 0.4 mg / mL fumaric acid (MAC-4) on ACTA2 gene expression in TGF-β1-induced NHLF cells. The chart illustrates the regulatory effect of this binary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The x-axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), 0.6 mg / mL MAC-3 single-component treatment group, 0.4 mg / mL MAC-4 single-component treatment group, and the combined treatment group of 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4. The y-axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone only showed weak ACTA2 inhibition, while the combined treatment with both significantly reduced ACTA2 expression levels, almost approaching the level of the positive control group. This indicates that the binary combination has extremely strong synergistic anti-fibrotic activity and is one of the core binary combinations with excellent anti-fibrotic potential in this application.

[0058] Figure 9The figure shows the effect of the ternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), and 0.6 mg / mL malic acid (MAC-3) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. This figure is a bar chart, showing the regulatory effect of this ternary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The horizontal axis represents, in descending order, the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), the single-component treatment group of 0.3 mg / mL MAC-1, the single-component treatment group of 0.6 mg / mL MAC-2, the single-component treatment group of 0.6 mg / mL MAC-3, and the combined treatment group of 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, and 0.6 mg / mL MAC-3. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 alone had no significant inhibitory effect on ACTA2 expression, and treatment with 0.6 mg / mL MAC-3 alone showed only a moderate inhibitory effect. However, the combined treatment of the three components significantly reduced the ACTA2 expression level, and the inhibitory effect was far superior to that of each single component and the aforementioned binary combination, indicating that the ternary combination has significant synergistic anti-fibrotic activity.

[0059] Figure 10The figure shows the effect of the ternary combination of 0.6 mg / mL glycine (MAC-2), 0.6 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. This figure is a bar chart, showing the regulatory effect of this ternary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), the single-component treatment group of 0.6 mg / mL MAC-2, the single-component treatment group of 0.6 mg / mL MAC-3, the single-component treatment group of 0.4 mg / mL MAC-4, and the combined treatment group of 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.6 mg / mL MAC-2 alone had no significant inhibitory effect on ACTA2 expression, and treatment with 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone showed only weak inhibitory effects. However, the combined treatment of the three drugs significantly reduced the ACTA2 expression level, almost approaching the level of the positive control group, indicating that the ternary combination has extremely strong synergistic anti-fibrotic activity.

[0060] Figure 11The figure shows the effect of a quaternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.6 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on ACTA2 gene expression in TGF-β1-induced NHLF cells. This figure is a bar chart, showing the regulatory effect of this quaternary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), the 0.3 mg / mL MAC-1 single-component treatment group, the 0.6 mg / mL MAC-2 single-component treatment group, the 0.6 mg / mL MAC-3 single-component treatment group, the 0.4 mg / mL MAC-4 single-component treatment group, and the above quaternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 alone had no significant inhibitory effect on ACTA2 expression, and treatment with 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone showed only a weak inhibitory effect. However, the combined treatment of the four drugs significantly reduced the ACTA2 expression level, almost equivalent to the level of the positive control group, with an inhibition rate as high as 98.69%, indicating that the quaternary combination has extremely strong synergistic anti-fibrotic activity.

[0061] Figure 12The figure shows the effect of the ternary combination of 0.6 mg / mL glycine (MAC-2), 0.3 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. This figure is a bar chart, showing the regulatory effect of this ternary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), the single-component treatment group of 0.6 mg / mL MAC-2, the single-component treatment group of 0.3 mg / mL MAC-3, the single-component treatment group of 0.4 mg / mL MAC-4, and the combined treatment group of 0.6 mg / mL MAC-2, 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.6 mg / mL MAC-2 alone had no significant inhibitory effect on ACTA2 expression, and treatment with 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone showed only very weak inhibitory effects. However, the combined treatment of the three drugs significantly reduced the ACTA2 expression level, almost approaching the level of the positive control group, indicating that the ternary combination has extremely strong synergistic anti-fibrotic activity.

[0062] Figure 13This is a bar chart showing the effect of the binary combination of 0.3 mg / mL malic acid (MAC-3) and 0.4 mg / mL fumaric acid (MAC-4) on ACTA2 gene expression in TGF-β1-induced NHLF cells. The chart illustrates the regulatory effect of this binary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The x-axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), 0.3 mg / mL MAC-3 single-component treatment group, 0.4 mg / mL MAC-4 single-component treatment group, and the combined treatment group of 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4. The y-axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone had only a very weak inhibitory effect on ACTA2 expression, while the combined treatment with both significantly reduced the level of ACTA2 expression, indicating that this low-concentration binary combination still has significant synergistic anti-fibrotic activity.

[0063] Figure 14The figure shows the effect of a quaternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.3 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on ACTA2 gene expression in TGF-β1-induced NHLF cells. This figure is a bar chart, showing the regulatory effect of this low malic acid concentration quaternary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), the 0.3 mg / mL MAC-1 single-component treatment group, the 0.6 mg / mL MAC-2 single-component treatment group, the 0.3 mg / mL MAC-3 single-component treatment group, the 0.4 mg / mL MAC-4 single-component treatment group, and the above quaternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 alone had no significant inhibitory effect on ACTA2 expression, while treatment with 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone showed only very weak inhibitory effects. However, the combined treatment of the four ingredients significantly reduced the ACTA2 expression level, almost to the level of the positive control group, indicating that even with a reduction in the concentration of malic acid, this quaternary combination still has extremely strong synergistic anti-fibrotic activity.

[0064] Figure 15This is a bar chart showing the effect of the binary combination of 0.6 mg / mL glycine (MAC-2) and 0.4 mg / mL fumaric acid (MAC-4) on ACTA2 gene expression in TGF-β1-induced NHLF cells. The bar chart illustrates the regulatory effect of this binary combination on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The x-axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 μM SB431542 + 5 ng / mL TGF-β1), 0.6 mg / mL MAC-2 single-component treatment group, 0.4 mg / mL MAC-4 single-component treatment group, and 0.6 mg / mL MAC-2 and 0.4 mg / mL MAC-4 combined treatment group. The y-axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.6 mg / mL MAC-2 alone had no significant inhibitory effect on ACTA2 expression, and treatment with 0.4 mg / mL MAC-4 alone only showed a certain degree of inhibitory effect. However, the combined treatment of the two significantly reduced the ACTA2 expression level, indicating that the binary combination has significant synergistic anti-fibrotic activity.

[0065] Figure 16 This is a bar chart illustrating the overall effects of single and multi-component combinations on IL-6 gene expression in LPS-induced RAW264.7 macrophages. It systematically demonstrates the regulatory effects of different concentrations of single active ingredients and various binary, ternary, and quaternary combinations on the expression level of the key inflammatory factor IL-6 mRNA in a 10 ng / mL LPS-induced RAW264.7 macrophage inflammation model. The vertical axis represents the ratio of IL-6 relative mRNA level to the internal reference gene GAPDH. The horizontal axis represents, in descending order, the blank control group (Vehicle), the model group (10 ng / mL LPS), the positive control group (10 μM dexamethasone, 5 μM SB431542), and the single and combined treatment groups of different active ingredients, numbered Group 3 to Group 34. Error bars represent the standard error of the experimental data for each group. This figure visually demonstrates the significant enhancement effect of multi-component combinations in inhibiting IL-6 expression compared to single components, clearly distinguishing the differences in anti-inflammatory activity among different combinations. It provides a comprehensive experimental data foundation for subsequent quantitative analysis of anti-inflammatory synergistic effects using the Bliss independence model and for screening the optimal combination with both anti-fibrotic and anti-inflammatory activities.

[0066] Figure 17This is a single-dose screening chart of the effect of a single component on IL-6 gene expression in LPS-induced RAW264.7 macrophages. The chart is a color bar graph showing the effect of different concentrations of a single active component on the expression level of the key inflammatory cytokine IL-6 mRNA under single-dose administration in a 10 ng / mL lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. The vertical axis represents the ratio of IL-6 relative mRNA level to the internal reference gene GAPDH. The horizontal axis represents, in descending order, the blank control group (Vehicle), the model group (10 ng / mL LPS), the positive control group (10 μM dexamethasone), and the single-component treatment groups with different concentrations of hippuric acid (MAC-1, red), glycine (MAC-2, yellow), malic acid (MAC-3, green), and fumaric acid (MAC-4, blue). Different colors distinguish different active components. Error bars represent the standard error of the experimental data for each group. This figure clearly shows the differences in anti-inflammatory activity among the four single components, clarifying that only high concentrations of glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4) can exhibit certain IL-6 inhibitory effects, while low concentrations of single components and hippuric acid (MAC-1) have no significant inhibitory effect within the experimental concentration range. This provides a precise single-dose activity reference for the concentration gradient design of subsequent multi-component anti-inflammatory synergistic combinations and the screening of active ingredients.

[0067] Figure 18 This is a screening chart of the synergistic effects of multiple component combinations on IL-6 gene expression in LPS-induced RAW264.7 macrophages. The chart is a color bar graph showing the regulatory effects of different concentrations of single active ingredients and various binary, ternary, and quaternary combinations on the expression level of the key inflammatory factor IL-6 mRNA in a 10 ng / mL lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. The vertical axis represents the ratio of IL-6 relative mRNA level to the internal reference gene GAPDH. The horizontal axis represents, in order, the blank control group (Vehicle), the model group (10 ng / mL LPS), the positive control group (10 μM dexamethasone), and the treatment groups (purple) with different concentrations of single ingredients (MAC-1, red), glycine (MAC-2, yellow), malic acid (MAC-3, green), and fumaric acid (MAC-4, blue) and various binary, ternary, and quaternary combinations. Different colors distinguish different active ingredients and combination types. Error bars represent the standard error of the experimental data for each group. This figure visually demonstrates the significant enhancement effect of multi-component combinations in inhibiting IL-6 expression compared to single components, clearly distinguishing the differences in the intensity of anti-inflammatory synergistic effects among different combinations. It provides a core experimental data foundation for subsequent quantitative analysis of anti-inflammatory synergistic effects using the Bliss independence model and for screening the optimal combination with both anti-fibrotic and anti-inflammatory activities.

[0068] Figure 19 This is a bar chart showing the effect of the binary combination of 0.6 mg / mL malic acid (MAC-3) and 0.4 mg / mL fumaric acid (MAC-4) on IL-6 gene expression in LPS-induced RAW264.7 macrophages. The chart illustrates the regulatory effect of this binary combination on the expression level of the key inflammatory factor IL-6 mRNA in a 10 ng / mL lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. The x-axis represents, in descending order: blank control group (Vehicle), model group (10 ng / mL LPS), positive control group (10 ng / mL LPS + 10 μM dexamethasone), 0.6 mg / mL MAC-3 single-component treatment group, 0.4 mg / mL MAC-4 single-component treatment group, and 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 combined treatment group. The y-axis represents the ratio of IL-6 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone only showed weak IL-6 inhibitory effects, while the combined treatment with both significantly reduced IL-6 expression levels, indicating that the binary combination has significant anti-inflammatory synergistic activity.

[0069] Figure 20This is a bar chart showing the effect of a quaternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.6 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on IL-6 gene expression in LPS-induced RAW264.7 macrophages. The quaternary combination regulates the expression level of the key inflammatory factor IL-6 mRNA in a 10 ng / mL lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. The horizontal axis represents, in descending order, the blank control group (Vehicle), the model group (10 ng / mL LPS), the positive control group (10 ng / mL LPS + 10 μM dexamethasone), the single-component treatment groups of 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3, 0.4 mg / mL MAC-4, and the above quaternary combination treatment group. The vertical axis represents the ratio of IL-6 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3, and 0.4 mg / mL MAC-4 alone showed only weak or no significant IL-6 inhibitory effects, while the combined treatment of the four components significantly reduced IL-6 expression levels, indicating that this quaternary combination has significant anti-inflammatory synergistic activity.

[0070] Figure 21This is a bar chart showing the effect of a binary combination of 0.3 mg / mL malic acid (MAC-3) and 0.4 mg / mL fumaric acid (MAC-4) on IL-6 gene expression in LPS-induced RAW264.7 macrophages. The bar chart illustrates the regulatory effect of this low-concentration binary combination on the expression level of the key inflammatory factor IL-6 mRNA in a 10 ng / mL lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. The x-axis represents, in descending order: blank control group (Vehicle), model group (10 ng / mL LPS), positive control group (10 ng / mL LPS + 10 μM dexamethasone), 0.3 mg / mL MAC-3 single-component treatment group, 0.4 mg / mL MAC-4 single-component treatment group, and 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4 combination treatment group. The y-axis represents the ratio of IL-6 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone had no significant inhibitory effect on IL-6 expression, while the combined treatment with both significantly reduced IL-6 expression levels, indicating that this low-concentration binary combination still has significant anti-inflammatory synergistic activity.

[0071] Figure 22 This is a bar chart showing the effect of the binary combination of 0.3 mg / mL hippuric acid (MAC-1) and 0.4 mg / mL fumaric acid (MAC-4) on IL-6 gene expression in LPS-induced RAW264.7 macrophages. The chart illustrates the regulatory effect of this binary combination on the expression level of the key inflammatory factor IL-6 mRNA in a 10 ng / mL lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. The x-axis represents, in descending order, the blank control group (Vehicle), the model group (10 ng / mL LPS), the positive control group (10 ng / mL LPS + 10 μM dexamethasone), the 0.3 mg / mL MAC-1 single-component treatment group, the 0.4 mg / mL MAC-4 single-component treatment group, and the 0.3 mg / mL MAC-1 and 0.4 mg / mL MAC-4 combination treatment group. The y-axis represents the ratio of IL-6 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that treatment with 0.3 mg / mL MAC-1 and 0.4 mg / mL MAC-4 alone had no significant inhibitory effect on IL-6 expression, while the combined treatment with both significantly reduced IL-6 expression levels, indicating that this binary combination has certain anti-inflammatory synergistic activity.

[0072] Figure 23This is a bar chart comparing the effects of multi-component combinations and clinical positive control drugs on ACTA2 gene expression in TGF-β1-induced NHLF cells. The chart systematically displays the regulatory effects of different binary, ternary, and quaternary active ingredient combinations and commonly used clinical anti-fibrotic positive control drugs on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The vertical axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. The horizontal axis represents, in order, the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (10 μM dexamethasone, 5 μM SB431542, 5 μM nintedanib), and the different active ingredient combination treatment groups numbered Group3 to Group33. Error bars represent the standard error of the experimental data for each group. This figure visually demonstrates that some multi-component combinations are superior to or approach the clinical positive control in inhibiting ACTA2 expression, clearly distinguishing the differences in antifibrotic activity between different combinations and the positive control drug. This provides a direct head-to-head comparative experimental data basis for subsequent screening of novel low-toxicity antifibrotic compositions with clinical application potential.

[0073] Figure 24This is an extended single-dose screening plot of the effects of single components on ACTA2 gene expression in TGF-β1-induced NHLF cells. This bar chart shows the effects of four single active components on the expression level of the myofibroblast marker ACTA2 mRNA in a wider concentration range in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mTGF-β1 + 5 μM SB431542), and the single-component treatment groups with different concentrations of hippuric acid (MAC-1, 0.3 mg / mL, 0.6 mg / mL, 2.4 mg / mL), glycine (MAC-2, 0.6 mg / mL, 2.4 mg / mL), malic acid (MAC-3, 0.3 mg / mL, 0.6 mg / mL, 1 mg / mL, 1.2 mg / mL), and fumaric acid (MAC-4, 0.4 mg / mL, 0.8 mg / mL, 1.2 mg / mL). The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that the ACTA2 expression level in the blank control group was extremely low, while the ACTA2 expression in the model group significantly increased after TGF-β1 induction. The positive control group SB431542 significantly inhibited ACTA2 expression. Across all concentration ranges tested, the ACTA2 expression levels in the single-component treatment groups (hippuric acid, glycine, malic acid, and fumaric acid) were close to or higher than those in the model group. Only 1.2 mg / mL fumaric acid (MAC-4) showed a weak inhibitory trend, but its effect was far weaker than the positive control drug. This figure systematically validates the antifibrotic activity of four single components at a wider concentration gradient, clarifying that even at high concentrations, single components are unlikely to effectively inhibit myofibroblast activation.

[0074] Figure 25This is a bar chart showing the expanded synergistic effect of multi-component combinations on ACTA2 gene expression in TGF-β1-induced NHLF cells. It illustrates the regulatory effects of single and multi-component combinations of various concentrations and combinations on the expression level of the myofibroblast marker ACTA2 mRNA in a 5 ng / mL TGF-β1-induced human normal lung fibroblast (NHLF) fibrosis model. The x-axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), single-component treatment groups with different concentrations of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4), and various binary, ternary, and quaternary active ingredient combinations. The y-axis represents the ratio of relative ACTA2 mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that the ACTA2 expression level in the blank control group was extremely low, while the ACTA2 expression in the model group increased significantly after TGF-β1 induction. The positive control group SB431542 significantly inhibited ACTA2 expression. The ACTA2 expression levels in all single-component treatment groups (including higher concentration gradients of MAC-1, MAC-2, MAC-3, and MAC-4) were close to those in the model group, with no significant inhibitory effect. This further systematically verifies the conclusion that single components are difficult to effectively inhibit myofibroblast activation even at high concentrations. Among the various multi-component combination treatment groups, several combinations showed significant ACTA2 inhibition effects. Among them, some core combinations (such as the quaternary combination of 0.3 mg / mL MAC-1 + 0.6 mg / mL MAC-2 + 0.3 mg / mL MAC-3 + 0.4 mg / mL MAC-4, the ternary combination of 0.6 mg / mL MAC-2 + 0.3 mg / mL MAC-3 + 0.4 mg / mL MAC-4, and the binary combination of 0.3 mg / mL MAC-3 + 0.4 mg / mL MAC-4) had inhibition effects close to or even better than the positive control drug SB431542.

[0075] Figure 26This is a bar chart validating the effect of the binary combination of 0.6 mg / mL malic acid (MAC-3) and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The chart further validated the regulatory effect of this binary combination on the expression level of the myofibroblast marker ACTA2 mRNA in an expanded concentration screening system. The horizontal axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 ng / mL TGF-β1 + 5 μMSB431542), 0.6 mg / mL MAC-3 single-component treatment group, 0.4 mg / mL MAC-4 single-component treatment group, and the combined treatment group of 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4. The vertical axis represents the ratio of the relative ACTA2 mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that the ACTA2 expression level in the blank control group was extremely low, while in the model group, after TGF-β1 induction, ACTA2 expression significantly increased to approximately 12.8. The positive control group SB431542 significantly inhibited ACTA2 expression to approximately 2.5. Treatment with 0.6 mg / mL MAC-3 alone resulted in an ACTA2 expression level of approximately 9.8, showing only a weak inhibitory effect; treatment with 0.4 mg / mL MAC-4 alone resulted in an ACTA2 expression level of approximately 11.0, with an even weaker inhibitory effect. However, combined treatment with both significantly reduced ACTA2 expression to approximately 5.3, demonstrating a much stronger inhibitory effect than any single component, indicating that this binary combination possesses stable and significant synergistic anti-fibrotic activity.

[0076] Figure 27This is a bar chart validating the effect of the ternary combination of 0.6 mg / mL glycine (MAC-2), 0.6 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The chart further validated the regulatory effect of this ternary combination on the expression level of the myofibroblast marker ACTA2 mRNA in an expanded concentration screening system. The horizontal axis represents, in descending order: blank control group (Vehicle), model group (5 ng / mL TGF-β1), positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), single-component treatment group (0.6 mg / mL MAC-2), single-component treatment group (0.6 mg / mL MAC-3), single-component treatment group (0.4 mg / mL MAC-4), and combined treatment groups (0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3, and 0.4 mg / mL MAC-4). The vertical axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. This figure clearly shows that the ACTA2 expression level in the blank control group was extremely low, the ACTA2 expression in the model group significantly increased to approximately 13.0 after TGF-β1 induction, and the positive control group (SB431542) significantly inhibited ACTA2 expression to approximately 2.7. Notably, treatment with 0.6 mg / mL MAC-2 alone not only failed to inhibit ACTA2 but also slightly increased ACTA2 expression levels to approximately 15.2; treatment with 0.6 mg / mL MAC-3 alone resulted in an ACTA2 expression level of approximately 9.8, showing only a weak inhibitory effect; and treatment with 0.4 mg / mL MAC-4 alone resulted in an ACTA2 expression level of approximately 11.2, with an even weaker inhibitory effect. However, the combined treatment of all three significantly reduced ACTA2 expression levels to approximately 7.5, demonstrating a much stronger inhibitory effect than any single component, indicating that this ternary combination possesses stable and significant synergistic anti-fibrotic activity.

[0077] Figure 28This is a bar chart validating the effect of a quaternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.6 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), each single-component treatment group, and the quaternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 had no inhibitory effect on ACTA2 expression, while 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 showed only a very weak inhibitory trend. However, the expression level of ACTA2 in the quaternary combination treatment group was significantly reduced, and the inhibitory effect was far superior to that of any single component, proving that the quaternary combination has stable and significant synergistic anti-fibrotic activity.

[0078] Figure 29 This is a bar chart validating the effect of a binary combination of 0.3 mg / mL hippuric acid (MAC-1) and 1 mg / mL malic acid (MAC-3) on ACTA2 gene expression in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), the 0.3 mg / mL MAC-1 single-component treatment group, the 1 mg / mL MAC-3 single-component treatment group, and the binary combination treatment group, respectively. The vertical axis represents the ratio of ACTA2 relative mRNA level to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. In the single-component treatment groups, 0.3 mg / mL MAC-1 had no inhibitory effect on the expression of ACTA2, and 1 mg / mL MAC-3 showed only a weak inhibitory trend. However, the expression level of ACTA2 in the combined treatment group was significantly reduced, and the inhibitory effect was far better than that of each single component, proving that the binary combination of hippuric acid and malic acid has significant synergistic anti-fibrotic activity.

[0079] Figure 30 This is a bar chart validating the effect of a binary combination of 1 mg / mL malic acid (MAC-3) and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), the 1 mg / mL MAC-3 single-component treatment group, the 0.4 mg / mL MAC-4 single-component treatment group, and the binary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. In the single-component treatment groups, 1 mg / mL MAC-3 and 0.4 mg / mL MAC-4 showed only a weak inhibitory trend on ACTA2 expression, while the ACTA2 expression level in the combined treatment group was significantly reduced, and the inhibitory effect was far better than that of each single component, proving that the binary combination of high concentration malic acid and fumaric acid has extremely strong synergistic anti-fibrotic activity.

[0080] Figure 31 This is a bar chart to validate the effect of a ternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), and 1 mg / mL malic acid (MAC-3) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), each single-component treatment group, and the ternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 had no inhibitory effect on ACTA2 expression, and 1 mg / mL MAC-3 showed only a weak inhibitory trend. However, the ACTA2 expression level in the three-component combined treatment group was significantly reduced, and the inhibitory effect was far superior to that of any single component, proving that the three-component combination has significant synergistic anti-fibrotic activity.

[0081] Figure 32 This is a bar chart to validate the effect of the ternary combination of 0.6 mg / mL glycine (MAC-2), 1 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on the expression of ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), each single component treatment group, and the ternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.6 mg / mL MAC-2 not only had no inhibitory effect, but also increased the expression level of ACTA2. 1 mg / mL MAC-3 and 0.4 mg / mL MAC-4 only showed a weak inhibitory trend. However, the ACTA2 expression level in the three-component combined treatment group was significantly reduced, and the inhibitory effect was far better than that of any single component, proving that the three-component combination has extremely strong synergistic anti-fibrotic activity.

[0082] Figure 33This is a bar chart validating the effect of a quaternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 1 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), each single-component treatment group, and the quaternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 had no inhibitory effect on ACTA2 expression, while 1 mg / mL MAC-3 and 0.4 mg / mL MAC-4 showed only a weak inhibitory trend. However, the ACTA2 expression level in the four-component combined treatment group was significantly reduced, and the inhibitory effect was far superior to that of any single component, proving that the quaternary combination has extremely strong synergistic anti-fibrotic activity.

[0083] Figure 34This is a bar chart to validate the effect of a quaternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.3 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), each single-component treatment group, and the quaternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 had no inhibitory effect on ACTA2 expression, while 0.3 mg / mL MAC-3 and 0.4 mg / mL MAC-4 showed only a very weak inhibitory trend. However, the ACTA2 expression level in the four-component combined treatment group was reduced, and the inhibitory effect was better than that of the single component, proving that the low-dose quaternary combination still has synergistic anti-fibrotic activity.

[0084] Figure 35 This is a bar chart validating the effect of a binary combination of 0.3 mg / mL malic acid (MAC-3) and 0.8 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), each single-component treatment group, and the binary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.3 mg / mL MAC-3 and 0.8 mg / mL MAC-4 showed only a weak inhibitory trend on ACTA2 expression, while the ACTA2 expression level in the combined treatment group was significantly reduced, and the inhibitory effect was far superior to that of any single component, proving that the binary combination has extremely strong synergistic anti-fibrotic activity.

[0085] Figure 36This is a bar chart to validate the effect of the ternary combination of 0.6 mg / mL glycine (MAC-2), 0.3 mg / mL malic acid (MAC-3), and 0.8 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μMSB431542), each single component treatment group, and the ternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.6 mg / mL MAC-2 not only had no inhibitory effect, but also increased the expression level of ACTA2. 0.3 mg / mL MAC-3 and 0.8 mg / mL MAC-4 only showed a weak inhibitory trend. However, the ACTA2 expression level in the three-component combined treatment group was significantly reduced, and the inhibitory effect was far better than that of any single component, proving that the three-component combination has extremely strong synergistic anti-fibrotic activity.

[0086] Figure 37This is a bar chart validating the effect of a quaternary combination of 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.3 mg / mL malic acid (MAC-3), and 0.8 mg / mL fumaric acid (MAC-4) on the expression of the ACTA2 gene in TGF-β1-induced NHLF cells. The horizontal axis represents the blank control group (Vehicle), the model group (5 ng / mL TGF-β1), the positive control group (5 ng / mL TGF-β1 + 5 μM SB431542), each single-component treatment group, and the quaternary combination treatment group, respectively. The vertical axis represents the ratio of the relative mRNA level of ACTA2 to the internal reference gene GAPDH. Error bars represent the standard error of the experimental data for each group. The experimental results showed that the expression level of ACTA2 in the blank control group was extremely low, while the expression of ACTA2 in the model group was significantly increased after induction with TGF-β1. The positive control group SB431542 could significantly inhibit the expression of ACTA2, indicating that the fibrosis model was successfully constructed. Among the single-component treatment groups, 0.3 mg / mL MAC-1 and 0.6 mg / mL MAC-2 had no inhibitory effect on ACTA2 expression, while 0.3 mg / mL MAC-3 and 0.8 mg / mL MAC-4 showed only a weak inhibitory trend. However, the ACTA2 expression level in the four-component combined treatment group was significantly reduced, and the inhibitory effect was far superior to that of any single component, proving that the quaternary combination of high fumaric acid ratio has extremely strong synergistic anti-fibrotic activity. Detailed Implementation

[0087] 1. Preparation of experimental materials 1.1 Compound Information (General for single-component analysis and combinatorial analysis, unless otherwise specified)

[0088] Among them, malic acid is L-malic acid.

[0089] 1.2 Cell Information

[0090] 2. Single-component analysis experimental protocol 2.1 Day 1: NHLF cell seeding Remove and discard the cell culture medium.

[0091] Briefly rinse the cell layer with preheated PBS, discard the PBS solution, and add 1 mL of preheated TrypLE solution (room temperature, 1-2 min) to the T75 culture flask.

[0092] Add 5 mL of preheated cell culture medium and gently blow to collect the cells.

[0093] Cells were counted using Vi-Cell.

[0094] Seed cells into 96-well plates according to the cell seeding plate plan, 100 μL per well.

[0095] Cells were incubated at 37°C and 5% CO2 for 24 hours.

[0096] 2.2 Day 2: Serum starvation treatment Preheat serum-free culture medium.

[0097] Discard the growth medium containing FBS, wash once with 100 μL of serum-free medium, and then add 100 μL of serum-free medium.

[0098] Incubate at 37°C and 5% CO2 for 24 hours.

[0099] 2.3 Day 3: Compound Treatment and Cytokine Stimulation The compound was added to the cell plate and incubated at 37°C and 5% CO2 for 1 hour.

[0100] Add 20 μL of 6×TGF-β1 (final concentration 5 ng / mL) and the working concentration of the test compound.

[0101] Incubate at 37℃ and 5% CO2 for 48 hours.

[0102] 96-well plate compound addition layout (partial key concentrations):

[0103] Note: The solvent control (Vehicle control) is 20% H2O + 0.1% DMSO.

[0104] 2.4 Day 5: RT-qPCR detection Cell lysis preparation: Add 35 μL of lysis buffer (Lysis Solution) for each reaction.

[0105] Preparation of Cells-to-CT lysates: a. Add 35 μL of room temperature lysis buffer or DNase / lysis buffer to the prepared cells and mix by pipetting and aspirating 5 times.

[0106] b. Incubate at room temperature for 5 minutes.

[0107] c. Add 2 μL of gDNA Remover to the PCR tube, then add 4 μL of cell lysis buffer, and pipette five times.

[0108] d. Incubate at room temperature for 5 minutes.

[0109] e. Place the lysate on ice and perform RT-PCR.

[0110] f. Set up the thermal cycler (or real-time PCR instrument) according to the table below, then load the samples and run the reaction.

[0111] RT reaction conditions:

[0112] 3. qPCR experiment: a. At room temperature, prepare the PCR mixture in a nuclease-free microcentrifuge tube according to the table below (add an extra 10%).

[0113] PCR mixture formulation (per 10 μL reaction volume):

[0114] b. Dispense the PCR mixture into the wells of the PCR tubes or real-time PCR plate at room temperature.

[0115] c. Tighten the cap or sealing plate and mix gently.

[0116] d. Briefly centrifuge to remove air bubbles and concentrate the liquid at the bottom of the tube or orifice.

[0117] e. Set up the real-time PCR instrument according to the table below, then load the samples and run the reaction.

[0118] qPCR reaction conditions:

[0119] f. Add 10 μL of reaction solution to each well of a 384-well plate, performing two replicates for each gene and each sample. Detect ACTA2 mRNA and GAPDH mRNA, only once.

[0120] 3. Analytical experimental scheme for the combination of four components 3.1 Day 1: NHLF cell seeding The procedure was the same as Day 1 of the single-component analysis protocol, with a cell seeding density of 20,000 cells / well, 100 μL per well, and incubation at 37°C and 5% CO2 for 24 h.

[0121] 3.2 Day 2: Serum starvation treatment The procedure is the same as Day 2 in the single-component analysis experimental protocol, incubating at 37°C and 5% CO2 for 24 hours.

[0122] 3.3 Day 3: Compound Treatment and Cytokine Stimulation Add the compound to a 96-well cell culture plate and incubate at 37°C and 5% CO2 for 1 h. Add 20 μL of 6×TGF-β1 (final concentration 5 ng / mL) and the working concentration of the test compound. Incubate at 37°C and 5% CO2 for 48 h.

[0123] 4.3 Subsequent RT-qPCR detection The procedure was the same as the RT-qPCR detection procedure on Day 5 of the single-component analysis experimental protocol, detecting IL-6 mRNA and GAPDH mRNA. The layout of the 384-well plate was as per the relevant experimental records. The amount of lysis buffer added was 40 μL per reaction. The RT reaction system and qPCR reaction system were the same as before.

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] 5. Experimental Results and Analysis 5.1 Results of single-component screening and optimization (as shown in the table below)

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] Experiments showed that a good fibrosis model was established in NHLF cells induced by 5 ng / mL TGF-β1. The positive compounds 2 μM nintedanib and 5 μM SB431542 significantly downregulated ACTA2 mRNA expression under these test conditions as expected.

[0140] Under these test conditions, MAC-1 and MAC-2 did not show significant downregulation of ACTA2 mRNA; MAC-3 and MAC-4 showed significant dose-dependent downregulation of ACTA2 mRNA, with IC50 values ​​of 1.04 mg / mL and 0.72 mg / mL, respectively, and consistent data were observed in the combination study for 0.6 mg / mL MAC-3 and 0.6 mg / mL MAC-4.

[0141] Inhibition rates of each individual component on ACTA2 at different concentrations (partial data): MAC-1: Inhibition rate was 14.10%-25.66% at 1.2 mg / mL, 4.56%-18.44% at 0.6 mg / mL, -133.04%-15.22% at 0.3 mg / mL, 2.56%-15.61% at 0.15 mg / mL, 9.03%-15.58% at 0.075 mg / mL, and 7.34%-18.60% at 0.0375 mg / mL.

[0142] MAC-2: 21.36%-27.22% at 9.6 mg / mL, 4.76%-17.57% at 4.8 mg / mL, -17.79%-17.57% at 2.4 mg / mL, 8.04%-18.67% at 1.2 mg / mL, 13.37%-27.70% at 0.6 mg / mL, and -5.90%-26.30% at 0.3 mg / mL.

[0143] MAC-3: 98.58%-99.45% at 9.6 mg / mL, 98.02%-98.97% at 4.8 mg / mL, 98.02%-98.79% at 2.4 mg / mL, 90.68%-97.93% at 1.2 mg / mL, 10.28%-25.78% at 0.6 mg / mL, and 11.16%-27.62% at 0.3 mg / mL.

[0144] MAC-4: 98.98%-99.31% at 1.2 mg / mL, 1.14%-16.51% at 0.6 mg / mL, 1.16%-31.30% at 0.3 mg / mL, 9.44%-24.54% at 0.15 mg / mL, 13.84%-17.72% at 0.075 mg / mL, and -4.00%-20.09% at 0.0375 mg / mL.

[0145] Positive controls: 5 ng / mL TGF-β1 + 2 uM Nintedanib showed inhibition rates of 59.37%-71.29%; 5 ng / mL TGF-β1 + 5 uM SB431542 showed inhibition rates of 95.80%-97.66%; Vehicle showed inhibition rates of 96.98%-97.85%; and 5 ng / mL TGF-β1 showed inhibition rates of -19.03%-24.03%.

[0146] IC50 and maximum concentration of each compound:

[0147] 5.2 Screening and optimization results of the four-component system (ACTA2 detection) The basic information for this experiment mainly includes two core parts: compound treatment parameters and cell culture conditions. All key data and experimental details are as follows: This experiment involved six compounds. All compounds were tested using 96-well plates. The solvent control was consistently 20% water + 0.1% dimethyl sulfoxide. After 48 hours of treatment, no precipitation occurred in the culture medium for any of the compounds. The specific correspondences of the MAC series compounds are as follows: MAC-1 is hippuric acid, MAC-2 is glycine, MAC-3 is malic acid, and MAC-4 is fumaric acid. Detailed treatment parameters for each compound are as follows: Compound MAC-1 (serial number 1): dosage 9.71 mg, stock solution concentration 6 mg / mL, preparation volume 1618 μL, storage conditions: room temperature (RT); Compound MAC-2 (serial number 2): dosage 39.44 mg, stock solution concentration 100 mg / mL, preparation volume 394 μL, storage conditions: room temperature (RT); Compound MAC-3 (serial number 3): dosage 27.73 mg, stock solution concentration 100 mg / mL, preparation volume 277 μL, storage conditions: room temperature (RT); Compound MAC-4 (number 4): dosage 14.96 mg, stock solution concentration 6 mg / mL, preparation volume 2493 μL, storage conditions: room temperature (RT); Compound No. 5, dexamethasone: No specific dosage or preparation volume data (labeled as NA), stock solution concentration 50 mmol / L, storage conditions -20℃; Compound SB431542 (serial number 6): No specific dosage or preparation volume data (labeled as NA), stock solution concentration 20 mmol / L, storage conditions -20℃.

[0148] The detailed culture information of the cells used in this experiment is as follows: Basic cell information: The cell name of serial number 1 is NHLF, the passage number is P3, the cell type is human lung fibroblast, and the growth type is adherent. Culture medium preparation: The complete culture medium uses FBM basal medium as the base, with the addition of 0.1% insulin, fibroblast growth factor B (FGF-B), 0.1% GA-1000 antibiotic additive and 2% fetal bovine serum (FBS); Experimental treatment parameters: The cell treatment time was 48 hours, and the number of cells seeded per well was 20,000.

[0149] The experimental results are shown below:

[0150]

[0151]

[0152] The experimental results showed that 5 ng / mL TGF-β1 could effectively upregulate the expression of ACTA2 in NHLF cells, while 5 μMSB431542 could effectively downregulate the expression of ACTA2 induced by TGF-β1 in NHLF cells under the same test conditions.

[0153] MAC-3 at 1.2 mg / mL and MAC-4 at 1.2 mg / mL reduced ACTA2 mRNA levels by more than 50% in TGF-β1-induced NHLF cells, while lower concentrations of these compounds did not show a reduction of more than 50%; MAC-1 at 0.3 mg / mL and MAC-2 at 2.4 mg / mL, as well as lower concentrations of these two compounds, did not show an inhibitory effect.

[0154] Under these test conditions, the 10 combination groups showed a synergistic effect. The effects of 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4 alone were low, but the effects were significantly improved when combined with appropriate concentrations of MAC-1 and MAC-2 or in multiple combinations.

[0155] Analysis of the inhibition rate and synergistic effect of some combination groups on ACTA2: Group 7 (0.3 mg / ml MAC-1 and 0.6 mg / ml MAC-3): inhibition rate 58.31%, E_obs=0.58, E_exp=0.35, Bliss Independence Value=0.23, synergistic effect.

[0156] Group 8 (0.6 mg / ml MAC-2 and 0.6 mg / ml MAC-3): Inhibition rate 76.31%, E_obs=0.76, E_exp=0.27, Bliss Independence Value=0.49, Synergy.

[0157] Group 9 (0.6 mg / ml MAC-3 and 0.4 mg / ml MAC-4): Inhibition rate 96.62%, E_obs=0.97, E_exp=0.41, Bliss Independence Value=0.56, Synergy.

[0158] Group 10 (0.3 mg / ml MAC-1, 0.6 mg / ml MAC-2 and 0.6 mg / ml MAC-3): inhibition rate 94.65%, E_obs=0.95, E_exp=0.35, Bliss Independence Value=0.60, synergistic effect.

[0159] Group 11 (0.6 mg / ml MAC-2, 0.6 mg / ml MAC-3 and 0.4 mg / ml MAC-4): inhibition rate 96.28%, E_obs=0.96, E_exp=0.41, Bliss Independence Value=0.55, synergy.

[0160] Group 12 (0.3 mg / ml MAC-1, 0.6 mg / ml MAC-2, 0.6 mg / ml MAC-3 and 0.4 mg / ml MAC-4): inhibition rate 98.69%, E_obs=0.99, E_exp=0.48, Bliss Independence Value=0.51, synergy.

[0161] Group 16 (0.3 mg / ml MAC-3 and 0.4 mg / ml MAC-4): inhibition rate 77.47%, E_obs=0.77, E_exp=0.34, Bliss Independence Value=0.43, synergistic effect.

[0162] Group 18 (0.6 mg / ml MAC-2, 0.3 mg / ml MAC-3 and 0.4 mg / ml MAC-4): inhibition rate 97.39%, E_obs=0.97, E_exp=0.34, Bliss Independence Value=0.63, synergy.

[0163] Group 19 (0.3 mg / ml MAC-1, 0.6 mg / ml MAC-2, 0.3 mg / ml MAC-3 and 0.4 mg / ml MAC-4): inhibition rate 98.55%, E_obs=0.99, E_exp=0.41, Bliss Independence Value=0.57, synergy.

[0164] Group 20 (0.6 mg / ml MAC-2 and 0.4 mg / ml MAC-4): inhibition rate 58.46%, E_obs=0.58, E_exp=0.19, Bliss Independence Value=0.39, synergistic effect.

[0165] 5.3 Screening and optimization results of the four-component system (IL-6 detection) The experimental protocol for detecting inflammatory factors is as follows:

[0166]

[0167] Day 1: RAW264.7 cell seeding For 150 cm 2 Remove all culture medium except for 10 mL of medium from the culture flask. Use a cell scraper to scrape RAW264.7 cells from the culture medium. To prevent excessive cell aggregation, sieve the cells. Count the cells using Vi-Cell. Seed the cells into 96-well plates, 100 μL per well. Add 5 mL of preheated cell culture medium and gently aspirate the cells. Incubate the cells at 37°C in a 5% CO2 incubator for 24 hours.

[0168] Day 2: Compound treatment and LPS stimulation Add the compound to the cell culture plate. Incubate the plate at 37°C ± 2°C with 5% CO2 for 1 hour. Add 20 μl of 6X LPS (final concentration 10 ng / ml) and the working concentration of the test compound. Incubate the cells at 37°C with 5% CO2 for 24 hours.

[0169] Day 5: RT-qPCR testing Prepare cell lysate

[0170] Preparation of cell lysate a. Add 40 μL of room temperature lysis buffer or DNase / lysis buffer to the prepared cells and mix thoroughly by pipetting up and down 5 times.

[0171] b. Incubate at room temperature for 5 minutes.

[0172] c. Add 2 μL of DNA removal agent to the PCR tube, then add 4 μL of cell lysis buffer, and pipette up and down 5 times.

[0173] d. Incubate at room temperature for 5 minutes.

[0174] e. Place the lysis buffer on ice and perform RT-PCR.

[0175] Perform reverse transcription (RT) a. In a nuclease-free microcentrifuge tube on ice, prepare the required number of reactions according to the table below, adding 10% excess RT premix.

[0176]

[0177] b. Gently but thoroughly mix the RT Master Mix, then briefly centrifuge and place on ice.

[0178] c. Aliquot the RT Master Mix into nuclease-free PCR tubes or multi-well plates.

[0179] d. Add the sample lysis buffer to each RT Master Mix to make a final reaction volume of 20 μL.

[0180] e. Gently mix the reaction solution, then briefly centrifuge and collect the precipitate at the bottom of the reaction vessel.

[0181] f. Set up the thermal cycler (or real-time quantitative PCR instrument) according to the table below, then load the samples and run the reaction.

[0182]

[0183] Perform qPCR: a. At room temperature, prepare the PCR reaction solution in a nuclease-free microcentrifuge tube according to the table below, and add an additional 10% excess reagent.

[0184]

[0185] b. Dispense the PCR reaction solution into the wells of each PCR tube or real-time PCR plate.

[0186] c. Cover with the tube cap or plate cap and mix gently.

[0187] d. Briefly centrifuge to remove air bubbles and collect the contents to the bottom of the tube / hole.

[0188] e. Set up the real-time PCR instrument according to the table below, then load the samples and run the reaction.

[0189]

[0190] f. Add 10 μl to each well of a 384-well plate, with two replicates per gene / sample. Detect ACTA2 mRNA and GAPDH mRNA. Only one assay is required.

[0191]

[0192]

[0193] The experimental results are as follows:

[0194]

[0195] Experiments showed that 10 ng / mL LPS could effectively upregulate the expression of IL-6 mRNA in RAW264.7 cells, while 10 μM dexamethasone could effectively downregulate the expression of IL-6 mRNA in RAW264.7 cells under the same test conditions.

[0196] MAC-2 at 2.4 mg / mL, MAC-3 at 1.2 mg / mL, and MAC-4 at 1.2 mg / mL reduced IL-6 mRNA levels by more than 50% in LPS-induced RAW264.7 cells, while lower concentrations of these compounds did not show a reduction of more than 50%.

[0197] The 0.6 mg / ml MAC-3 and 0.4 mg / ml MAC-4 groups, the 0.3 mg / ml MAC-1, 0.6 mg / ml MAC-2, the 0.6 mg / ml MAC-3 and 0.4 mg / ml MAC-4 groups, the 0.3 mg / ml MAC-3 and 0.4 mg / ml MAC-4 groups, and the 0.3 mg / ml MAC-1 and 0.4 mg / ml MAC-4 groups showed a synergistic effect of IL-6 reduction in LPS-induced RAW264.7 cells.

[0198] Analysis of the inhibition rate and synergistic effect of some combination groups on IL-6: Group 9 (0.6 mg / ml MAC-3 and 0.4 mg / ml MAC-4): inhibition rate 39.12%, E_obs=0.39, E_exp=0.06, Bliss Independence Value=0.33, synergy.

[0199] Group 12 (0.3 mg / ml MAC-1, 0.6 mg / ml MAC-2, 0.6 mg / ml MAC-3 and 0.4 mg / ml MAC-4): inhibition rate 56.83%, E_obs=0.57, E_exp=0.25, Bliss Independence Value=0.32, synergy.

[0200] Group 16 (0.3 mg / ml MAC-3 and 0.4 mg / ml MAC-4): Inhibition rate 23.74%, E_obs=0.24, E_exp=0.00, Bliss Independence Value=0.24, Synergy.

[0201] Group 21 (0.3 mg / ml MAC-1 and 0.4 mg / ml MAC-4): inhibition rate 25.35%, E_obs=0.25, E_exp=0.11, Bliss Independence Value=0.15, synergy.

[0202] 6. Data Calculation Methods Inhibition rate (%) calculation formula: (1 - AVERAGE(GROUP) / AVERAGE(5ng / ml TGF-β1)) × 100 The synergistic effect analysis uses the Bliss independence model, and the calculation formula is as follows: Observed inhibition rate (E_obs): E_obs=1-AVERAGE(GROUP) / AVERAGE(5ng / ml TGF-β1) Expected inhibition rate (E_exp): For two compounds: E_exp=1-(1-inhibition rate of compound 1)×(1-inhibition rate of compound 2) For three compounds: E_exp=1-(1-inhibition rate of compound 1)×(1-inhibition rate of compound 2)×(1-inhibition rate of compound 3) For four compounds: E_exp=1-(1-inhibition rate of compound 1)×(1-inhibition rate of compound 2)×(1-inhibition rate of compound 3)×(1-inhibition rate of compound 4) Bliss Independence Value=E_obs-E_exp Criteria for determining synergistic effects: Bliss Independence Value≥0.15: Synergy -0.15<Bliss Independence Value<0.15: Additive effect Bliss Independence Value≤-0.15: Antagonism 7. Preparation of the composition Hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3) and fumaric acid (MAC-4) are mixed according to a selected concentration ratio (e.g., 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3 and 0.4 mg / mL MAC-4), and pharmaceutically acceptable excipients can be added as required to prepare pharmaceutical preparations (such as tablets, capsules, injections, etc.), health foods or functional foods.

[0203] 8. Experimental protocol for reversing pulmonary fibrosis Preparation of experimental materials Compound information: same as the "analysis of four-component combination" experiment, namely MAC-1 (hippuric acid), MAC-2 (glycine), MAC-3 (malic acid), MAC-4 (fumaric acid), and positive control drugs dexamethasone (Dexamethasone, 50 mM, stored at -20°C), SB431542 (20 mM, stored at -20°C), nintedanib (Nintedanib, 10 mM, stored at -20°C). The Vehicle control is 20% H2O+0.1% DMSO, and no precipitation is formed after treatment with each compound.

[0204] Experimental steps Day 1: NHLF cell inoculation Remove and discard the cell culture medium. Briefly rinse the cell layer with pre-warmed PBS, aspirate the PBS solution, and add 1 mL of pre-warmed TrypLE solution (room temperature, 1-2 min) to the T75 culture flask. Add 5 mL of pre-warmed cell culture medium, gently pipette to collect the cells, and count the cells using Vi-Cell. Seed the cells into 96-well plates at 100 μL per well (20,000 cells / well) and incubate at 37°C and 5% CO2 for 24 h.

[0205] Day 2: Serum starvation treatment Preheat serum-free medium, discard the growth medium containing FBS, wash once with 100 μL of serum-free medium, and then add 100 μL of serum-free medium. Incubate at 37°C and 5% CO2 for 24 h.

[0206] Day 3: Cytokine Induction (Constructing a Fibrosis Model) Add 20 μL of 6×TGF-β1 (final concentration 5 ng / mL) to FBM medium containing 3% FBS and free of growth factors, and add the working concentration of the test compound. Incubate at 37°C and 5% CO2 for 48 h to construct a pulmonary fibrosis cell model.

[0207] Day 5: Compound Treatment (Reversing Fibrosis) Remove the culture medium from each well and add fresh FBM medium containing 0.5% FBS and free of growth factors. Add the compound to the cell culture plate according to the following 96-well plate grouping layout and incubate at 37°C and 5% CO2 for 48 hours.

[0208] Day 7: RT-qPCR detection Cell lysis preparation: Add 35 μL of lysis buffer (Lysis Solution) for each reaction.

[0209] Preparation of Cells-to-CT lysates: a. Add 35 μL of room temperature lysis buffer or DNase / lysis buffer to the prepared cells and mix by pipetting and aspirating 5 times.

[0210] b. Incubate at room temperature for 5 min, add 2 μL of gDNA Remover to the PCR tube, then add 4 μL of cell lysis buffer, and pipette 5 times.

[0211] c. Incubate at room temperature for 5 minutes, place the lysate on ice, and perform RT-PCR.

[0212] Reverse transcription (RT) reaction: a. Prepare RT Master Mix (add an extra 10%) in nuclease-free microcentrifuge tubes on ice according to the table below.

[0213]

[0214] b. Gently mix the RT Master Mix, centrifuge briefly, place on ice, and divide into nuclease-free PCR tubes or multi-well plates. Add sample lysis buffer to each tube / well to make the final reaction volume 20 μL. Gently mix and centrifuge briefly.

[0215] c. Set up the thermal cycler and run the reaction under the following conditions:

[0216] qPCR experiment: a. Prepare the PCR mixture at room temperature in nuclease-free microcentrifuge tubes according to the following table (add an extra 10%):

[0217] b. Divide the PCR mixture into the wells of each PCR tube or real-time PCR plate, tighten the cap or seal the plate, mix gently and centrifuge briefly to remove air bubbles.

[0218] c. Set up the real-time PCR instrument and run the reaction according to the following conditions:

[0219] d. Add 10 μL of reaction solution to each well of a 384-well plate. Perform two replicates for each gene (ACTA2 mRNA, GAPDH mRNA) and each sample, and detect only once.

[0220] Results and Analysis of Experiments on Reversing Pulmonary Fibrosis Experimental model verification The experimental results showed that under the test conditions, 5 ng / mL TGF-β1 could effectively upregulate the expression of ACTA2 in NHLF cells, successfully constructing a pulmonary fibrosis cell model; 5 μM SB431542 could effectively downregulate the expression of ACTA2 in TGF-β1-induced NHLF cells, verifying the effectiveness of the model.

[0221] Single-component and combination-component effects on reversing pulmonary fibrosis Single ingredient effects: 0.3 mg / mL MAC-1 (group 3) and 0.6 mg / mL MAC-2 (group 4) negatively inhibited ACTA2 expression (inhibition rates of -5.86% and -17.41%, respectively), with no reversible effect; 0.6 mg / mL MAC-3 (group 5), 0.4 mg / mL MAC-4 (group 6), 1 mg / mL MAC-3 (group 13), 0.8 mg / mL MAC-4 (group 25), 2.4 mg / mL MAC-3 (group 32), and 1.2 mg / mL MAC-4 (group 33) all have a certain inhibitory effect on ACTA2, with inhibition rates of 24.93%, 13.66%, 23.53%, 19.55%, 74.76%, and 90.67% respectively. Only high-concentration single components (such as 2.4 mg / mL MAC-3 and 1.2 mg / mL MAC-4) show good reversal potential.

[0222] Synergistic effect of combined components: multiple combination groups showed significant ACTA2 inhibition rate (up to 93.9%), and calculated by the Bliss independence model, it showed a synergistic effect (Bliss Independence Value ≥ 0.15), as detailed below:

[0223]

[0224] The composition of the present invention, at a specific concentration ratio, can significantly inhibit the expression of ACTA2 in the TGF-β1-induced NHLF cell reversal pulmonary fibrosis model, the inhibition rate of some combinations exceeds 90%, and there is a strong synergistic effect, which proves that the composition has potential pulmonary fibrosis reversal efficacy and further expands its application scenarios in the treatment of pulmonary fibrosis.

[0225] The data calculation method is the same as above: Inhibition rate (%) = (1 - AVERAGE(GROUP) / AVERAGE(5 ng / ml TGF-β1)) × 100; Bliss Independence Value = E_obs - E_exp; The judgment criterion is that a Bliss Independence Value ≥ 0.15 is a synergistic effect, -0.15 < Bliss Independence Value < 0.15 is an additive effect, and a Bliss Independence Value ≤ -0.15 is an antagonistic effect.

[0226] Example 1: Anti-scar gel (four-component combination) and preparation thereof This embodiment provides an anti-scar gel containing a four-component combination of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4). 0.03 g of MAC-1, 0.06 g of MAC-2, 0.06 g of MAC-3, and 0.04 g of MAC-4 are dissolved in an appropriate amount of purified water to prepare the active ingredient solution. Separately, 0.5 g of carbomer 940 is dispersed in 80 mL of purified water and allowed to swell overnight to obtain the gel matrix. The active ingredient solution is added to the gel matrix while stirring, followed by the addition of 5.0 g of glycerin and 0.1 g of methylparaben, and stirred until homogeneous. The pH is adjusted to 5.5-6.5 with triethanolamine, and purified water is added to a final volume of 100 g. Stirring continues until homogeneous to obtain the anti-scar gel. The resulting gel is uniform and fine, suitable for the prevention and treatment of surgical scars, burn scars, and acne pits and marks.

[0227] Example 2: Anti-fibrosis coating for implants (four-component combination) and its preparation This embodiment provides an implant anti-fibrotic coating containing a four-component combination of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4). First, drug-loaded PLGA microspheres were prepared: 3 mg MAC-1, 6 mg MAC-2, 6 mg MAC-3, 4 mg MAC-4, and 200 mg PLGA (50:50) were dissolved in 5 mL of dichloromethane as the oil phase. The oil phase was slowly added dropwise to 100 mL of a 1% (w / v) polyvinyl alcohol (PVA) aqueous solution under stirring, and emulsified at high speed to form an O / W emulsion. The mixture was stirred at room temperature for 4-6 hours to allow the dichloromethane to evaporate and solidify the microspheres. The microspheres were collected by centrifugation, washed three times with purified water, and freeze-dried to obtain drug-loaded PLGA microspheres. The obtained drug-loaded microspheres were dispersed in an appropriate amount of medical-grade silicone adhesive and coated onto the surface of implants (such as pacemakers, artificial blood vessels, and glaucoma drainage valves) by dip-coating or spraying. Vacuum drying yielded an implant with an anti-fibrotic coating. The resulting coating can slowly release active ingredients, effectively inhibiting the formation of fibrous capsules around the implant.

[0228] Example 3: Anti-scar gel (three-component combination) and its preparation This embodiment provides an anti-scar gel containing a three-component combination of glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4). 0.6 g of MAC-2, 0.6 g of MAC-3, and 0.4 g of MAC-4 were dissolved in an appropriate amount of purified water to prepare the active ingredient solution. Separately, 0.5 g of carbomer 940 was dispersed in 80 mL of purified water and allowed to swell overnight to obtain the gel matrix. The active ingredient solution was added to the gel matrix while stirring, followed by the addition of 5.0 g of glycerol and 0.1 g of methylparaben, and stirred until homogeneous. The pH was adjusted to 5.5-6.5 with triethanolamine, and purified water was added to a final volume of 100 g. Stirring continued until homogeneous to obtain the anti-scar gel. This three-component combination has been experimentally proven to have a strong synergistic effect (ACTA2 inhibition rate of 96.28%, Bliss value of 0.55), maintaining excellent anti-fibrotic efficacy while simplifying the formulation.

[0229] Example 4: Anti-fibrosis coating for implants (three-component combination) and its preparation This embodiment provides an anti-fibrosis coating for implants containing a three-component combination of glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4). First, drug-loaded PLGA microspheres are prepared: 6 mg of MAC-2, 6 mg of MAC-3, 4 mg of MAC-4, and 200 mg of PLGA (50:50) are dissolved in 5 mL of dichloromethane as the oil phase. The oil phase is slowly added dropwise to 100 mL of a 1% (w / v) polyvinyl alcohol (PVA) aqueous solution under stirring, and emulsified at high speed to form an O / W emulsion. The mixture is stirred at room temperature for 4-6 hours to allow the dichloromethane to evaporate and solidify the microspheres. The microspheres are collected by centrifugation, washed three times with purified water, and freeze-dried to obtain drug-loaded PLGA microspheres. The obtained drug-loaded microspheres are dispersed in an appropriate amount of medical-grade silicone sealant and coated onto the implant surface by dip-coating or spraying. Vacuum drying yields the implant with an anti-fibrosis coating. This three-component combination also showed excellent synergistic effects in a model of reversed pulmonary fibrosis (e.g., the inhibition rate of the 1 mg / mL MAC-3 + 0.4 mg / mL MAC-4 combination reached 93.9%), making it suitable for the prevention of implant-associated fibrosis.

[0230] Example 5: Topical gel for treating psoriasis and its preparation This embodiment provides a topical gel for treating psoriasis. 0.3 g hippuric acid (MAC-1), 0.6 g glycine (MAC-2), 0.6 g malic acid (MAC-3), and 0.4 g fumaric acid (MAC-4) were dissolved in 10 mL of purified water. 5.0 g glycerin, 5.0 g propylene glycol, and 0.1 g methylparaben were added and stirred until completely dissolved to obtain the active ingredient solution. Separately, 0.5 g carbomer 940 was dispersed in 80 mL of purified water and allowed to swell overnight to obtain the gel matrix. The active ingredient solution was slowly added to the gel matrix while stirring. The pH was adjusted to 5.5-6.5 with triethanolamine, and purified water was added to a final volume of 100 g. Stirring continued until homogeneous. The gel was then dispensed to obtain the topical gel for treating psoriasis. The resulting gel is suitable for topical application to psoriatic lesions 1-2 times daily and can alleviate symptoms such as scaling, erythema, and thickened skin.

[0231] Example 6: Cream for treating atopic dermatitis and its preparation This embodiment provides a cream for treating atopic dermatitis. 0.6 g glycine (MAC-2), 0.6 g malic acid (MAC-3), and 0.4 g fumaric acid (MAC-4) are dissolved in an appropriate amount of purified water. 5.0 g glycerin, 1.0 g Tween-80, and 0.1 g ethylparaben are added, and the mixture is heated to 75-80°C to obtain the aqueous phase. Separately, 3.0 g glyceryl monostearate, 2.0 g stearic acid, 5.0 g liquid paraffin, and 2.0 g cetyl alcohol are heated to 75-80°C to obtain the oil phase. The aqueous phase is slowly added to the oil phase while stirring continuously, maintaining the temperature for 30 minutes. The mixture is then slowly cooled to room temperature, and stirring continues until condensed. Purified water is added to bring the volume to 100 g, yielding the cream for treating atopic dermatitis. The resulting cream is suitable for topical application to atopic dermatitis lesions twice daily, and can relieve symptoms such as itching, erythema, and dry skin.

[0232] Example 7: Spray for treating psoriasis and atopic dermatitis and its preparation This embodiment provides a topical spray for treating psoriasis and atopic dermatitis. 0.6 g g of glycine (MAC-2), 0.6 g of malic acid (MAC-3), and 0.4 g of fumaric acid (MAC-4) are dissolved in 65 mL of purified water. 30.0 g of ethanol and 5.0 g of propylene glycol are added, the mixture is stirred until homogeneous, filtered to remove bacteria, and then filled into a spray bottle to obtain the topical spray. The resulting spray is suitable for large-area lesions of psoriasis and atopic dermatitis. It is applied 2-3 times daily, is convenient to use, and is especially suitable for areas such as the scalp and extensor surfaces of the limbs.

[0233] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. A composition for the prevention, treatment, or reversal of pulmonary fibrosis, characterized in that, The active ingredients of the composition include at least one of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), fumaric acid (MAC-4), and their respective derivatives, pharmaceutically acceptable salts, preferably at least two or more.

2. The composition according to claim 1, characterized in that, The active ingredient is selected from one or more of the following: hippuric acid (MAC-1); Glycine (MAC-2); Malic acid (MAC-3); Fumaric acid (MAC-4); hippuric acid (MAC-1) and glycine (MAC-2); hippuric acid (MAC-1) and malic acid (MAC-3); hippuric acid (MAC-1) and fumaric acid (MAC-4); Glycine (MAC-2) and malic acid (MAC-3); Glycine (MAC-2) and fumaric acid (MAC-4); Malic acid (MAC-3) and fumaric acid (MAC-4); hippuric acid (MAC-1), glycine (MAC-2), and malic acid (MAC-3); hippuric acid (MAC-1), glycine (MAC-2), and fumaric acid (MAC-4); hippuric acid (MAC-1), malic acid (MAC-3), and fumaric acid (MAC-4); Glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4); hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4); Particularly preferred is that the composition consists of malic acid (MAC-3) and fumaric acid (MAC-4), or glycine (MAC-2), malic acid (MAC-3) and fumaric acid (MAC-4).

3. The composition according to claim 1, characterized in that, The active ingredients consist of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4); Preferably, the concentrations of each active ingredient are: hippuric acid (MAC-1) 0.1-0.5 mg / mL, glycine (MAC-2) 0.4-1.0 mg / mL, malic acid (MAC-3) 0.3-1 mg / mL, and fumaric acid (MAC-4) 0.2-0.6 mg / mL; or, Preferably, the concentrations of each active ingredient are: 0.3 mg / mL hippuric acid (MAC-1), 0.6 mg / mL glycine (MAC-2), 0.6 mg / mL malic acid (MAC-3), and 0.4 mg / mL fumaric acid (MAC-4).

4. The composition according to any one of claims 1 to 3, characterized in that, The composition is a pharmaceutical preparation, health food, or functional food.

5. Use of the composition according to any one of claims 1 to 3 in the preparation of the following substances: Its use in the preparation of drugs for inhibiting ACTA2 gene expression, preferably in the preparation of drugs for the prevention, slowing progression, reversal or treatment of pulmonary fibrosis; or Its application in the preparation of substances for inhibiting IL-6 gene expression, preferably in the preparation of substances for anti-inflammatory purposes; or Application in the preparation of drugs for the prevention, treatment or reversal of pulmonary fibrosis.

6. The use of the composition according to any one of claims 1 to 3 in the preparation of any of the following products: (1) Health foods or functional foods for the prevention or treatment of pulmonary fibrosis; (2) Medical aesthetic and dermatology products, including products for treating skin scars / anti-scars, products for treating acne pits and marks, and anti-aging / anti-wrinkle products; (3) Health foods or functional foods that reverse pulmonary fibrosis; (4) Skin care products, including gels, creams or silicone patches for treating surgical scars / burn scars or acne marks and pits.

7. The application according to claim 6, characterized in that, The concentrations of each active ingredient in the composition are: 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.6 mg / mL MAC-3, and 0.4 mg / mL MAC-4; or The concentrations of each active ingredient in the composition are: 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 1 mg / mL MAC-3, and 0.4 mg / mL MAC-4; or The concentrations of the active ingredients in the composition are: 0.3 mg / mL MAC-1, 0.6 mg / mL MAC-2, 0.3 mg / mL MAC-3, and 0.8 mg / mL MAC-4.

8. The use of any one or more of hippuric acid (MAC-1), glycine (MAC-2), malic acid (MAC-3), and fumaric acid (MAC-4) in the preparation of substances for the prevention, relief, reversal, or treatment of pulmonary fibrosis, and / or in the preparation of substances for the prevention, relief, reversal, or treatment of liver / kidney fibrosis, preferably pharmaceuticals, and / or in the preparation of substances that inhibit fibroblast activation, downregulate α-smooth muscle actin (ACTA2) gene expression, and inhibit the expression of the inflammatory factor interleukin-6 (IL-6).

9. The use of the composition according to any one of claims 1 to 3 in the preparation of any of the following products: (1) Pharmaceutical preparations, including tablets, capsules, injections, suspensions, emulsions, ointments, gels, aerosols, sprays, inhalers, suppositories, and patches; (2) Health foods or functional foods, including oral liquids, granules, powders, tablets, capsules, meal replacement foods, beverages, and confectionery products; (3) Medical aesthetic and dermatological products, including anti-scar products, preferably gels, creams, silicone patches, medical dressings, acne scar and pit repair products, anti-aging / anti-wrinkle products, and cosmeceuticals; (4) Medical device related products, including implant anti-fibrotic coatings, especially suitable for artificial joints, pacemakers, artificial blood vessels, intrauterine devices, glaucoma drainage valves, injectable bio-hydrogels / tissue regeneration scaffolds; (5) Veterinary drug products, including oral preparations, injections, topical preparations, and feed additives for animal pulmonary fibrosis, liver fibrosis, and kidney fibrosis; (6) Research tools, including cell culture additive kits, especially in vitro culture aids for inhibiting fibroblast activation.

10. The use of the composition according to any one of claims 1 to 3 in the preparation of anti-inflammatory and / or immunomodulatory products; Preferably, the application includes at least one of the following: intervention for fibrotic diseases, treatment for inflammatory diseases, improvement for metabolic diseases, treatment for neurological diseases, adjuvant therapy for cancer, treatment for skin diseases, and treatment for orthopedic and dental diseases; Preferably, the intervention for fibrotic diseases includes its use in the preparation of drugs or health foods for the treatment or delay of pulmonary fibrosis, liver fibrosis / cirrhosis, and renal fibrosis; Preferably, the treatment of the inflammatory disease includes its use in the preparation of medicaments for treating rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, sepsis, chronic obstructive pulmonary disease, systemic lupus erythematosus, and multiple sclerosis, wherein... Inflammatory bowel disease is preferably Crohn's disease or ulcerative colitis; Preferably, the improvement of metabolic diseases includes its use in the preparation of drugs or health foods for improving type 2 diabetes and insulin resistance, and non-alcoholic fatty liver disease; Preferably, the treatment of the nervous system disease includes its use in the preparation of drugs for treating Alzheimer's disease, Parkinson's disease, and multiple sclerosis; Preferably, the adjuvant cancer therapy includes the use of drugs for the preparation of tumor-associated macrophage reprogramming, which are used in combination with chemotherapy / radiotherapy / immune checkpoint inhibitors to enhance efficacy and alleviate cancer cachexia; Preferably, the skin disease treatment includes its application in the preparation of drugs or medical dressings for treating psoriasis, atopic dermatitis, and promoting the healing of chronic, difficult-to-heal wounds; Preferably, the treatment of orthopedic and dental diseases includes its use in the preparation of medicaments or topical formulations for treating osteoarthritis, periodontitis, and peri-implantitis. Preferably, the application is a non-therapeutic application, including the preparation of skin care product additives for soothing sensitive skin and repairing the skin barrier, and the application in hair growth products for improving inflammation-related hair loss.

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