Use of a traditional Chinese medicine composition containing sophora flavescens in preparation of a medicine for treating metabolic-associated fatty liver disease combined with atherosclerosis

Abstract

The application discloses application of a traditional Chinese medicine composition containing Sophora flavescens in preparation of a medicine for treating metabolic-related fatty hepatitis combined with atherosclerosis, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition takes Sophora flavescens, gentiana and pig gall powder as raw materials, plays a synergistic effect through specific proportioning, can significantly improve liver lipid accumulation and inflammatory reaction, inhibits atherosclerotic plaque formation, and protects liver and aortic mitochondrial function, and can be used for preparing a medicine for treating metabolic-related fatty hepatitis or metabolic-related fatty hepatitis combined with atherosclerosis. Long-term toxicity test proves that the traditional Chinese medicine composition has good safety, and provides a safe and effective treatment option for patients with metabolic-related fatty hepatitis and atherosclerosis combined with metabolic-related fatty hepatitis.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to a new use of a traditional Chinese medicine composition composed of Sophora flavescens, Gentiana scabra, and pig bile powder in the preparation of a drug for treating metabolic dysfunction-associated steatohepatitis (MASH) or metabolic dysfunction-associated steatohepatitis complicated with atherosclerosis (AS). Background Art

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive form of metabolic associated fatty liver disease (MASLD), which is characterized by hepatic steatosis accompanied by hepatocyte injury and inflammation, and can further develop into liver fibrosis, cirrhosis, and even hepatocellular carcinoma. It is worth noting that patients with MASH often have an increased risk of cardiovascular diseases, and atherosclerosis (AS) is one of the most common complications. There is an interactive effect between the two in the pathological mechanism, forming a complex situation of "liver-heart comorbidity". At present, the co-management of MASH complicated with AS is extremely challenging, and there is still a lack of an ideal drug that can effectively intervene in liver and vascular lesions simultaneously in clinical practice. Existing therapeutic drugs such as Resmetirom (a thyroid hormone receptor β agonist) mainly target hepatic lipid metabolism, while statins mainly act on vascular blood lipids. The combination of the two may increase the risk of adverse reactions and cannot synergistically regulate common pathological links (such as systemic inflammation, mitochondrial dysfunction). Therefore, the development of a drug that can simultaneously improve hepatic lipid accumulation, hepatic inflammation, and inhibit the formation of atherosclerotic plaques has important clinical significance and application prospects.

[0003] Traditional Chinese medicine follows the principle of syndrome differentiation and treatment, and combines the characteristics of multiple components, multiple targets, and multiple pathways of traditional Chinese medicine. Traditional Chinese medicine has unique advantages in the treatment of this disease. Traditional Chinese medicine treats it from the liver. The location of MASH is in the liver. The liver is responsible for dredging and regulating the qi of the whole body. If the liver fails to dredge and regulate, the qi mechanism is disordered, and the output of qi, blood, body fluid, and essence of the whole body is abnormal, resulting in the endogenous generation of phlegm-dampness, which transforms into heat when stagnated. After a long course of disease, blood stasis forms. Pathological factors such as phlegm, dampness, blood stasis, and heat are intertwined with each other, leading to the endogenous generation of turbid toxin, and finally developing into this disease. Longdan Kushen Pills (Kushen Pills), which originated from "Emergency Prescriptions Kept Up One's Sleeve" written by Ge Hong in the Jin Dynasty, are simple in formula and powerful in effect, and have the function of clearing heat and resolving dampness, and are used to treat the syndrome of damp-heat accumulation and steaming in jaundice. There is no report on its use in MASH or MASH complicated with atherosclerosis at present. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a novel application of a traditional Chinese medicine composition containing Sophora flavescens in the preparation of a medicament for treating metabolic-associated steatohepatitis (MAH) or MAH complicated with atherosclerosis. This composition uses Sophora flavescens, Gentiana scabra, and pig bile powder as raw materials, and through a specific ratio, it exerts a synergistic effect, significantly improving hepatic lipid accumulation and inflammatory response, inhibiting atherosclerotic plaque formation, and protecting liver and aortic mitochondrial function, thus providing a safe and effective treatment option for MAH and patients with atherosclerosis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of a traditional Chinese medicine composition containing Sophora flavescens in the preparation of a medicament for treating metabolic-associated steatohepatitis or metabolic-associated steatohepatitis complicated with atherosclerosis, wherein the raw materials for preparing the traditional Chinese medicine composition are composed of Sophora flavescens, Gentiana scabra and pig bile powder.

[0006] Sophora flavescens is a legume plant (Sophora flavescens). Sophora flavescensAit. The dried root of *Sophora flavescens* is cold in nature and bitter in taste, and enters the heart, liver, stomach, large intestine, and bladder meridians. Its traditional effects include clearing heat and drying dampness, killing parasites, and promoting diuresis; it is commonly used for damp-heat diarrhea, jaundice, leukorrhea, and vulvar swelling and itching. Modern pharmacological studies have shown that *Sophora flavescens* mainly contains various alkaloids such as matrine, oxymatrine, and sophoridine, which have anti-inflammatory, antioxidant, anti-liver fibrosis, lipid-lowering, and anti-atherosclerotic effects. Matrine components can inhibit lipid accumulation in hepatocytes, reduce the release of inflammatory factors, improve vascular endothelial function, and inhibit smooth muscle cell proliferation and foam cell formation. In this invention, *Sophora flavescens* is used as one of the principal herbs, playing a core role in clearing damp-heat in the liver meridian, detoxifying, and lowering lipids.

[0007] Gentian is the plant *Gentiana linearifolia*, belonging to the Gentianaceae family. Gentiana manshuricaKitag .), Gentian (Gentiana) scabraBunge Gentian root and rhizome, etc., are cold in nature and bitter in taste, and enter the liver and gallbladder meridians. Traditionally, they are used to clear heat and dry dampness, purge liver and gallbladder fire, and are mainly used to treat damp-heat jaundice, vulvar swelling and itching, leukorrhea, liver-fire headache, and red eyes. Modern pharmacology has confirmed that gentian mainly contains iridoid glycosides such as gentiopicrin and swertiamarin, which have hepatoprotective, choleretic, anti-inflammatory, anti-fatty liver, and glucose and lipid metabolism-regulating effects. Gentianopicrin can promote fatty acid oxidation by activating the AMPK signaling pathway, reduce liver triglyceride deposition, and inhibit the activation of hepatic macrophages and the expression of inflammatory factors. In this invention, gentian is the second principal ingredient, assisting sophora flavescens in clearing liver and gallbladder fire and damp-heat, enhancing the overall choleretic and lipid-reducing effects of the formula.

[0008] Pig bile powder is a fine powder made from dried and pulverized pig bile. It is cold in nature, bitter in taste, and enters the liver, gallbladder, lung, and large intestine meridians. Traditionally, it is used for clearing heat and detoxifying, promoting bile secretion and reducing jaundice, relieving cough and asthma, and is indicated for red and swollen eyes, jaundice, constipation, and asthma. Modern research shows that pig bile powder mainly contains bile acids such as cholic acid, deoxycholic acid, and chenodeoxycholic acid, which promote bile secretion, dissolve cholesterol, lower blood lipids, have anti-inflammatory effects, and regulate intestinal flora. Deoxycholic acid can activate farnesol X receptor (FXR) and G protein-coupled bile acid receptor 5 (TGR5), thereby improving insulin sensitivity and reducing hepatic steatosis and inflammation. In this invention, pig bile powder is used as an adjuvant to enhance the bile-promoting and heat-clearing effects of Sophora flavescens and Gentiana scabra, harmonize the medicinal properties, and guide the other drugs directly to the affected area.

[0009] Preferably, in the traditional Chinese medicine composition, the weight ratio of Sophora flavescens, Gentiana scabra and pig bile powder is (3-5): (3-5): (0.5-1.5), and more preferably 4: 4: 1.

[0010] Preferably, the drug is used to simultaneously improve hepatic lipid accumulation, liver inflammation, and inhibit the formation of atherosclerotic plaques.

[0011] Preferably, the improvement of hepatic lipid accumulation and inhibition of atherosclerotic plaque formation are achieved by regulating one or more of the fatty acid degradation pathway, pentose phosphate pathway, and glycerol metabolism pathway; and / or, the improvement of liver inflammation is achieved by inhibiting one or more of the Ras signaling pathway and PI3K-Akt signaling pathway.

[0012] Preferably, the traditional Chinese medicine composition works by improving liver and / or aortic mitochondrial function.

[0013] Preferably, the traditional Chinese medicine composition works by reducing the release of extracellular vesicles derived from vascular endothelium in the liver and / or extracellular vesicles derived from the liver in the aorta.

[0014] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0015] More preferably, the excipients include any one or a combination of at least two of the following: diluent, flavoring agent, binder, or filler. The combination of at least two is, for example, a combination of binder and diluent, a combination of binder and flavoring agent, a combination of binder and filler, etc. Other combinations are also possible and will not be described in detail here.

[0016] The dosage form of the drug is pills, capsules, tablets, granules, powders, or oral liquids, preferably pills.

[0017] Compared with the prior art, the present invention has the following significant advantages: First-of-its-kind application: This invention reveals for the first time a novel application of a traditional Chinese medicine composition consisting of Sophora flavescens, Gentiana scabra, and pig bile powder in the treatment of MASH and MASH combined with AS, filling a technological gap in the treatment of this major comorbidity. Furthermore, its efficacy and treatment duration are significantly superior to existing drugs: Compared to the only two existing MASH treatments globally [Resmetirom (52-week treatment cycle, efficacy approximately 20%) and Semaglutide (72-week treatment cycle, efficacy approximately 28%)], the traditional Chinese medicine composition of this invention has a treatment cycle of only 4 weeks, significantly shortening the treatment course, and has demonstrated an efficacy rate of approximately 80% in animal experiments.

[0018] Synergistic effect and comprehensive efficacy: Through rigorous comparative experiments, it has been demonstrated that the Chinese herbal composition (whole formula) of this invention is significantly better than that of single herbs such as Sophora flavescens or Gentiana scabra in improving key indicators such as improving liver lipid accumulation and reducing serum low-density lipoprotein cholesterol (LDL-C) and transaminase (ALT), which reflects the synergistic effect of compound formulation and can simultaneously produce therapeutic effects on liver and vascular lesions.

[0019] Novel and multidimensional mechanism: This invention not only confirms that the composition can systematically improve lipid metabolism and inflammatory markers, but also elucidates its mechanism of action from multiple levels for the first time. Improve cellular energy factories: enhance the activity of the respiratory chain complex and ATPase in the liver and aorta mitochondria, repair mitochondrial ultrastructure, enhance autophagy, and fundamentally improve energy metabolism disorders.

[0020] Multi-pathway synergistic regulation: Proteomics analysis revealed that the composition can regulate lipid metabolism pathways such as "fatty acid degradation" and inflammatory pathways such as "Ras signaling pathway" in the liver; and regulate "pentose phosphate pathway", "glycerol lipid metabolism", "PI3K-Akt signaling pathway" in the aorta, etc., to achieve multi-dimensional and networked regulation of energy metabolism, lipid remodeling, inflammatory response and vascular homeostasis.

[0021] Regulation of inter-organ communication: This composition was found for the first time to reduce abnormal pathological signaling between the liver and vascular endothelium via extracellular vesicles (EVs) in disease states, suggesting that it may alleviate disease progression by regulating inter-organ communication.

[0022] In the treatment of MASH combined with AS, it is particularly important to simultaneously reduce TC, TG, and LDL-C in blood, liver, and vascular endothelial cells. Currently, there are no drugs in clinical practice that can simultaneously downregulate these three core lipid indicators. This composition can achieve a synergistic reduction of serum, liver, and aortic TC, TG, and LDL-C through the above multi-mechanism synergistic action, providing an effective solution for the clinical treatment of MASH combined with AS.

[0023] Treating both the symptoms and the root cause, in line with the holistic treatment concept: The composition of this invention has both lipid-lowering and anti-inflammatory pharmacological effects, which can achieve both symptomatic and root cause treatment.

[0024] The drug exhibits outstanding safety and avoids the pain point of adverse clinical reactions: Lipid-lowering drugs are commonly used in clinical practice to treat MASH and AS, but adverse reactions remain a pain point in clinical application. Statins and fibrates are prone to causing liver toxicity, rhabdomyolysis and other problems. However, long-term toxicity tests have confirmed that the composition of this invention has not shown obvious toxic side effects within the therapeutic dose range (the effective dose in mice is 500 mg / kg / d, which is far below its median lethal dose of 32,000 mg / kg), and the drug has a greater advantage in safety. Attached Figure Description

[0025] Figure 1 The results of H&E staining (×200) of mouse liver tissue in Experiment Example 1; Figure 2 The results of Oil Red O staining (×200) of mouse liver tissue in Experiment Example 1; Figure 3 The results of serum TG, TC, and LDL-C levels in mice in Experiment Example 1; Figure 4 The results of ALT and AST level detection in the liver of mice in Experiment Example 1; Figure 5 The results of serum TNF-α, IL-1β, and IL-6 levels in mice in Experiment Example 1; Figure 6 The results of detecting TNF-α, IL-1β, and IL-6 levels in the liver of mice in Experiment Example 1; Figure 7 The results of HE staining, Oil Red O staining, and MASSON staining of mouse liver tissue in Experiment Example 2 are shown. Figure 8 The results of HE staining, Oil Red O staining, and MASSON staining of the aorta in Experiment Example 2 are shown. Figure 9 The results of lipid, inflammation, and mitochondrial-related markers in the serum, liver, and aortic tissue of mice in Experiment Example 2 are as follows: P<0.05, P<0.01, P<0.001 (vs. model group). Figure 10 The ultrastructure of mouse liver tissue and aortic mitochondria in Experiment Example 2; Figure 11 A bubble chart showing the enrichment of proteomics pathways in the liver (A) and aorta (B) of mice in Experiment Example 2; Figure 12 The secretion levels of endothelial endothelial EVs (A) and hepatic endothelial EVs (B) in the liver of mice in Experiment Example 2 were ( ). P<0.05, P<0.01, P<0.001 (vs. model group); Figure 13 The effect of the traditional Chinese medicine composition of the present invention on the body weight of mice in Experimental Example 3; Figure 14 The effect of the herbal composition of the present invention on the organ index of mice in Experiment Example 3 (ns represents no significant difference compared with the control group). Figure 15 The effect of the herbal composition of the present invention on plasma biochemistry in mice on day 30 in Experimental Example 3 (compared with the control group). P < 0.01; P < 0.0001; ns represents no significant difference). Figure 16 The results of HE staining of the heart, liver, spleen, lung, kidney and brain tissues of mice on day 30 are shown in Experimental Example 3. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0027] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0028] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0029] The following is information on the sources of some of the main raw materials and reagents used in the examples: Sophora flavescens, Gentiana scabra, and pig bile powder were purchased from Luosiwan Traditional Chinese Medicine Market in Kunming City. According to the 2025 edition of the Chinese Pharmacopoeia, all samples were qualified.

[0030] Resmetirom (Shanghai Bide Pharmaceutical Technology Co., Ltd., batch number: ESL687).

[0031] 8ApoE - / - Male mice and C57BL / 6J mice were purchased from Beijing Speywood Biotechnology Co., Ltd. (Certificate number for the quality of experimental animals: 110324241105415934; Production license number for experimental animals: SCXK (Beijing) 2024 - 0001). All experimental animals were housed in the Animal Experiment Center of Yunnan University of Traditional Chinese Medicine. The experimental conditions and methods were reviewed and approved by the Ethics Committee of the Animal Experiment Center of Yunnan University of Traditional Chinese Medicine, and the animal ethics review document number is: R - 062023151.

[0032] The remaining main reagents are shown in Table 1 below.

[0033] Table 1 Source information of main reagents

[0034] Preparation of the traditional Chinese medicine composition in Example 1 This example provides a representative preparation method of the traditional Chinese medicine composition of the present invention.

[0035] Weigh 400 g of Sophora flavescens, 400 g of Gentiana scabra, and 100 g of pig bile powder by weight. First, add the cut pieces of Sophora flavescens and Gentiana scabra to 10 times the amount (w / v) of pure water, decoct for 1 hour, filter, add the filter residue to 10 times the amount of pure water again, and extract in the same way for 1 hour. Combine the two filtrates, add pig bile powder and mix well, concentrate under reduced pressure with a rotary evaporator at 65 °C, freeze - dry to obtain a freeze - dried powder, grind and mix well to obtain the dry extract powder of the traditional Chinese medicine composition, and place it in a desiccator for later use.

[0036] The above - mentioned dry extract powder can be directly used in subsequent pharmacodynamic experiments, or it can be mixed with pharmaceutically acceptable carriers or excipients according to needs and in accordance with conventional techniques in the art to prepare dosage forms such as pills, capsules, tablets, granules, powders, or oral liquids. For example, mix the dry extract powder obtained in Preparation Example 1 with an appropriate amount of refined honey, knead into strips and make pills to obtain pills.

[0037] Preparation of the Sophora flavescens extract in Comparative Example 1 Weigh an appropriate amount of Sophora flavescens, first decoct with 10 times the amount of water for 1 h, filter, decoct the filter residue with 10 times the amount of water for 1 h again, filter, combine the two extracts, concentrate under reduced pressure with a rotary evaporator at 65 °C, freeze - dry to obtain a freeze - dried powder, grind and mix well, and place it in a desiccator for later use.

[0038] Preparation of the Gentiana scabra extract in Comparative Example 2 Weigh out an appropriate amount of gentian, decoct it with 10 times the amount of water for 1 hour, filter it, decoct the residue with 10 times the amount of water for 1 hour, filter it, combine the two extracts, concentrate them under reduced pressure in a rotary evaporator at 65℃, freeze dry to obtain lyophilized powder, grind and mix it, and then place it in a desiccator for later use.

[0039] Therapeutic effect of Experiment 1 on MASH model mice 1. Experimental Methods Sixty-six healthy male C57BL / 6 SPF-grade mice were acclimatized for 7 days and randomly divided into 11 groups of 6 mice each. Starting from 13 weeks of high-fat diet feeding, the mice were administered medication once daily by gavage. The model group and the control group were given the solvent by gavage and the MASH model was established by intraperitoneal injection of 0.5 mL / kg of 25% CCL4 olive oil solution every Friday morning (administration was performed 4 hours after model establishment). The control group was injected intraperitoneally with sodium chloride injection (animal grouping and administration details are shown in Table 2).

[0040] Table 2 Animal grouping and administration

[0041] Note: N, no CCL4 injection; Y, CCL4 injection. 2. Sample Collection During the experiment, the mice's food intake and weekly body weight were recorded. At the end of week 16, after the first administration, the mice were fasted for 12 hours, anesthetized with 2% sodium pentobarbital, and blood was collected from the abdominal aorta. After standing at room temperature for 4 hours, the blood was centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant was collected, aliquoted, and stored at -80°C. Simultaneously, the liver, heart, kidneys, lungs, and spleen were harvested, weighed, and organ coefficients were calculated. Tissue from the same part of the liver (0.5 cm² of the mid-section of the largest transverse section of the hepatic lobe) was fixed in 10% formalin solution. Colonic feces and cecal contents were frozen. The remaining tissue was aliquoted into centrifuge tubes and frozen at -80°C for later use.

[0042] 3. Observation of liver tissue morphology and pathology (1) H&E staining Liver tissue was collected, fixed with 4% paraformaldehyde for 24 hours, dehydrated with graded ethanol, cleared with xylene to remove ethanol, embedded in paraffin, sectioned with an ultramicrotome, stained in hematoxylin-eosin solution, differentiated, blued, washed, dehydrated in 95% ethanol for 1 minute, stained in eosin solution for 15 seconds, dehydrated with graded ethanol, mounted with neutral resin, and observed under a microscope.

[0043] (2) Oil Red O staining Liver tissue was collected and fixed with 4% paraformaldehyde for 24 hours. The liver tissue was then removed, embedded in OCT embedding medium, and rapidly frozen in a cryostat. Sections were cut into 5-10 μm thick sections, mounted on glass slides, and air-dried at room temperature for 10-30 minutes. The sections were then stained with Oil Red O solution in the dark for 10-15 minutes, and immersed in 60% isopropanol for 10 seconds to remove non-specific staining until the background was clean and the lipid droplets were bright red. The sections were washed with pure water to terminate differentiation, counterstained the nuclei with hematoxylin solution for 2 minutes, washed with pure water, and then blued for 1 second. The sections were mounted with glycerol gelatin and observed under a microscope.

[0044] 4. Serum and liver lipid marker detection Remove serum samples, thaw on ice, and measure serum TC, TG, ALT, AST, HDL-C, and LDL-C levels according to the kit instructions. Remove liver samples, thaw on ice, accurately weigh 0.1g, add 0.9mL of pre-cooled physiological saline and a grinding bead, and grind using a tissue homogenizer at 4℃ and 60Hz for 4 times, 30s each time. Then centrifuge at 5000rpm for 10min at 4℃, collect the supernatant, and measure liver TC, TG, ALT, AST, HDL-C, and LDL-C levels according to the kit instructions.

[0045] 5. Serum and liver inflammatory marker detection Serum and liver samples were prepared (method as in 4), and the levels of TNF-α, IL-1β, and IL-6 in serum and liver were detected according to the kit instructions.

[0046] 6. Statistical processing Data were processed using GraphPad Prism 9.5.1, and all values ​​are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons between groups, and the Student / St test was used for individual comparisons. P<0.05, P<0.01, P<0.001 indicates a statistically significant difference.

[0047] 7. Experimental Results (1) Effects on liver morphology in MASH mice Figure 1 shows H&E-stained sections of mouse liver. In the normal group, hepatocytes were neatly arranged with abundant and uniform cytoplasm; in the model group, the boundaries of liver lobules were indistinct, hepatocytes were disordered, hepatocytes were enlarged, and obvious lipid droplets and vacuoles were observed within the hepatocytes. After administration of the composition of this invention and its single herbal ingredients, the hepatocyte outlines were clear, and the lipid droplet area was reduced. This indicates that the herbal composition of this invention and its single herbal ingredients have an improving effect on liver tissue morphology and lipid accumulation in MASH mice, and the herbal composition of this invention has a stronger effect on improving lipid accumulation than the single herbal ingredients.

[0048] As shown in Figure 2, Oil Red O stained sections of mouse livers, the hepatocytes in the normal group showed normal morphology with almost no red lipid droplets in the cytoplasm; the hepatocytes in the model group showed irregular morphology with scattered bright red lipid droplets of varying sizes in the cytoplasm. After administration of the herbal composition and its single herb of this invention, the lipid droplets in the cytoplasm were reduced to varying degrees, and the pathological condition of the liver was improved. This indicates that the herbal composition and its single herb of this invention have an ameliorative effect on lipid accumulation in MASH mice, and the herbal composition of this invention has a stronger effect on improving lipid accumulation than the single herb.

[0049] (2) Effects on serum and liver lipid metabolism indicators in MASH mice As shown in Figure 3, a high-fat diet was used to feed the animals for 16 weeks, and carbon tetrachloride was administered for the last 4 weeks to establish a MASH model. In the last 4 weeks, the animals were also given a combination of traditional Chinese medicine and its single herbs. Compared with the normal group, the serum TC, LDL-C, and ALT levels in the model group mice were significantly increased (P<0.001, P<0.05, P<0.05), indicating that a high-fat diet accompanied by carbon tetrachloride induced abnormal lipid metabolism in MASH mice. Compared with the model group, after intervention with the traditional Chinese medicine composition of this invention, the levels of TG, LDL-C, and ALT were significantly decreased (P<0.01, P<0.05, P<0.05), while the levels of TC and AST showed a decreasing trend, and HDL-C showed an increasing trend. After intervention with the single herb Sophora flavescens, the TG level was significantly decreased (P<0.001), the HDL-C level was significantly increased (P<0.001), and the ALT level showed a decreasing trend. After intervention with the single herb Gentiana scabra, the TG level was significantly decreased (P<0.001), and the HDL-C level was significantly increased (P<0.01). After intervention with the single herb pig bile powder, the HDL-C level was significantly decreased. The levels of lipids increased significantly (P<0.001), but the AST level increased significantly (P<0.01). The results indicate that the traditional Chinese medicine composition and its single herb of the present invention can regulate abnormal serum lipid levels in MASH mice, and the efficacy of the traditional Chinese medicine composition of the present invention is superior to that of the single herb.

[0050] As shown in Figure 4, the animals were fed a high-fat diet for 16 weeks, and carbon tetrachloride was administered for the last 4 weeks to establish a MASH model. The animals were also given the herbal composition and individual herbs of this invention for the last 4 weeks. Compared with the normal group, the levels of TC, TG, and LDL-C in the liver of mice in the model group were significantly increased (P<0.001, P<0.001, P<0.01), indicating that a high-fat diet accompanied by carbon tetrachloride induced abnormal lipid metabolism in the liver of MASH mice. Compared with the model group, after intervention with the traditional Chinese medicine composition of this invention, the levels of LDL-C and ALT were significantly decreased (P<0.01, P<0.05), while the levels of TC, TG, and AST showed a decreasing trend. After intervention with the single herb Sophora flavescens, the level of LDL-C was significantly decreased (P<0.05). After intervention with the single herb Gentiana scabra, the level of LDL-C was significantly decreased (P<0.01), but the levels of ALT and AST were significantly increased (P<0.05). After intervention with the single herb pig bile powder, the level of LDL-C was significantly decreased (P<0.05), but ALT showed an increasing trend. The results showed that the traditional Chinese medicine composition of the present invention and its single herbal ingredients could regulate abnormal liver lipid levels in MASH mice, and the efficacy of the traditional Chinese medicine composition of the present invention was superior to that of the single herbal ingredients.

[0051] (3) Effects on serum and liver inflammatory factors in MASH mice As shown in Figure 5, the animals were fed a high-fat diet for 16 weeks, and carbon tetrachloride was administered for the last 4 weeks to establish a MASH model. The animals were also given the herbal composition and individual herbs of this invention for the last 4 weeks. Compared with the normal group, the serum levels of TNF-α, IL-1β, and IL-6 in the model group mice were significantly increased (P<0.01, P<0.001, P<0.001), indicating that a high-fat diet accompanied by carbon tetrachloride-induced serum inflammatory response in MASH mice. Compared with the model group, after intervention with the traditional Chinese medicine composition of this invention, the levels of TNF-α, IL-1β, and IL-6 were significantly decreased (P<0.05, P<0.001, P<0.001); after intervention with the single herb Sophora flavescens, the levels of TNF-α, IL-1β, and IL-6 were significantly decreased (P<0.05, P<0.001, P<0.001); after intervention with the single herb Gentiana scabra, the levels of TNF-α, IL-1β, and IL-6 were significantly decreased (P<0.01, P<0.001, P<0.001); after intervention with the single herb pig bile powder, the levels of TNF-α, IL-1β, and IL-6 were significantly decreased. The levels increased significantly (P<0.001). The results indicate that the herbal composition and its individual herbs of this invention can alleviate serum inflammatory responses in MASH mice.

[0052] As shown in Figure 6, a high-fat diet was used to feed the animals for 16 weeks. In the last 4 weeks, carbon tetrachloride was administered to establish a MASH model, and the animals were also given a combination of traditional Chinese medicine and its single herbs in the last 4 weeks. Compared with the normal group, the levels of TNF-α and IL-6 in the liver of mice in the model group were significantly increased (P<0.001), indicating that a high-fat diet accompanied by carbon tetrachloride-induced liver inflammation in MASH mice. Compared with the model group, the levels of TNF-α, IL-1β, and IL-6 were significantly decreased after intervention with the traditional Chinese medicine combination (P<0.05, P<0.05, P<0.001); the levels of IL-1β and IL-6 were significantly decreased after intervention with the single herb Sophora flavescens (P<0.001, P<0.01); the levels of TNF-α, IL-1β, and IL-6 were significantly decreased after intervention with the single herb Gentiana scabra (P<0.05, P<0.01, P<0.05); and the levels of TNF-α, IL-1β, and IL-6 were significantly increased after intervention with the single herb pig bile powder (P<0.05, P<0.01, P<0.01). The results showed that the traditional Chinese medicine composition and its single herb composition of the present invention could alleviate the inflammatory response in the liver of MASH mice.

[0053] Experiment 2: Therapeutic effect on MASH combined with atherosclerosis mouse model 1. Animal experiments to evaluate drug efficacy High-fat diet induces ApoE - / - MASH combined with AS model replicated in mice at 16 weeks of age: ApoE at 6-8 weeks of age - / - Male mice and C57BL / 6J male mice were acclimatized and fed for one week. The C57BL / 6J male mice served as the normal control group (CON, saline). ApoE - / - Mice were randomly divided into three groups: a model group (MOD, saline), an atorvastatin calcium group (AT, positive control), a low-dose group of the traditional Chinese medicine composition prepared in Example 1 (LDKSW-L, 1 g / kg / d), and a high-dose group of the traditional Chinese medicine composition prepared in Example 1 (LDKSW-H, 4 g / kg / d), with six mice in each group. Except for the normal control group, which was fed a standard diet, all other groups were fed a high-fat, high-cholesterol diet for 16 consecutive weeks. Starting from week 13, the mice were administered the medicine once daily via gavage for 4 weeks. Daily food intake and weekly body weight were recorded. At the end of the experiment, mice were fasted for 12 hours before being administered the medicine via gavage. One hour later, the mice were anesthetized, blood was collected from the abdominal aorta, and liver and aortic tissues were collected and stored at -80°C.

[0054] Serum, liver, and aortic biochemical markers were measured according to the kit instructions. Lipid levels (total cholesterol TC, triglycerides TG, high-density lipoprotein cholesterol HDL-C, low-density lipoprotein cholesterol LDL-C), liver damage (aspartate aminotransferase AST, alanine aminotransferase ALT), and inflammatory factor levels (interleukin-1β IL-1β, interleukin-6 IL-6, tumor necrosis factor-α TNF-α) were measured in serum, liver, and aorta.

[0055] Morphological examination of liver and aorta tissues: Liver and aorta were taken, fixed, dehydrated, embedded in paraffin, and prepared into slides. After HE staining, Oil Red O staining and MASSON staining, the morphology, lipid droplet accumulation and degree of fibrosis of liver and aorta tissues were observed under an optical microscope.

[0056] 2. Study on the mechanism of the traditional Chinese medicine composition of the present invention in alleviating MASH combined with AS (1) Mitochondrial function detection Ultrastructure and autophagosomes of liver and aorta mitochondria: The liver and aorta were fixed, dehydrated, embedded, sectioned, stained, and observed under a transmission electron microscope.

[0057] Mitochondrial-related marker detection: Follow the kit instructions and use an ELISA reader to detect the levels of ATPase and respiratory chain complex I / II in the liver and aorta.

[0058] (2) Proteomics analysis of mouse liver and aorta Protein extraction: Liver and aortic tissues were weighed separately, and an appropriate amount of protein lysis buffer (8 M urea + 1% SDS, containing protease inhibitors) was added. The tissues were then homogenized using a high-speed cryogenic homogenizer (3 times, 180 s each time); followed by non-contact cryogenic sonication for 30 min; centrifugation (8℃, 14000 g, 15 min), and the supernatant was collected. Protein content was determined using the BCA method according to the kit instructions, and SDS-PAGE electrophoresis was performed.

[0059] Enzymatic hydrolysis: Take 100 μg of protein sample, add lysis buffer, add 100 mM TEAB; add 10 mM TCEP, react at 37℃ for 60 min; add 40 mM Iodoacetamide, react at room temperature in the dark for 40 min; add pre-cooled acetone to each tube (acetone:sample v:v=6:1), precipitate at -20℃ for 4 h; centrifuge (10000 g, 20 min), collect the precipitate; dissolve the sample thoroughly with 100 µL of 100 mM TEAB; add Trypsin at a mass ratio of 1:50 (enzyme:protein) and hydrolyze overnight at 37℃.

[0060] Peptide desalting and quantification: After trypsin digestion, the peptides were dried using a vacuum pump; the dehydrated peptides were reconstituted with 0.1% trifluoroacetic acid (TFA); the peptides were desalted using HLB and dried using a vacuum concentrator; the peptides were quantified using UV spectrophotometry using a NANO DROP ONE (Thermo Scientific).

[0061] DIA mass spectrometry detection: Equal volumes of peptides were dissolved in mass spectrometry loading buffer for DIA detection and analysis. Peptide separation was performed using a Vanquish Neo (Thermo) chromatograph. The analytical column was a uPAC High Throughptu column (75 μm × 5.5 cm, Thermo, USA). Mobile phase A was water (2% acetonitrile + 0.1% formic acid), and mobile phase B was water (80% acetonitrile + 0.1% formic acid). The chromatographic time was 8 min. The nano-level high-performance liquid chromatography (HPLC) separated samples were analyzed by Orbitrap Astral mass spectrometry (Thermo) in DIA mode, with positive ion detection, an ion source voltage of 1.5 kV, and a scan range of 100–1700 m / z.

[0062] Data Analysis: Raw DIA data were imported into the Spectronaut™ 19 software system for database search and analysis. Parameter settings were as follows: peptide length 7-52; restriction enzyme site: trypsin / P; maximum missed cleavage site: 2; variable modifications: oxidation (M) and acetyl (Protein N-term); fixed modification: carbamidomethyl (C); Protein FDR ≤ 0.01; Peptide FDR ≤ 0.01; Peptide Confidence ≥ 99%; XIC width ≤ 75ppm. MaxLFQ was used for protein quantification. All data were uploaded to the Meiji Cloud platform for analysis. The t.test function in R was used to calculate the p-value and fold change of differences between groups. Proteins with a significance test p < 0.05 and a fold change greater than 1.2 were considered differentially expressed proteins. The GO database was used to perform GO annotation analysis and functional clustering analysis on all differentially expressed proteins from three aspects: biological processes, cellular components, and molecular functions. The KEGG pathway database was used to analyze the metabolic pathways involved in the differentially expressed proteins.

[0063] (3) The regulatory effect of the traditional Chinese medicine composition of the present invention on extracellular vesicles (EVs) Isolation of EVs from liver and aortic tissues: Enzymatic digestion followed by ultrafiltration was used. Liver and aortic tissues were weighed separately, placed in 6-well plates, and 1640 medium (0.1 g / mL) was added. The tissues were gently cut into uniform 2×2×2 mm pieces using a sterile scalpel. Collagenase D (20 L / 0.1 g) and DNasel (2 L / 0.1 g) were added, and the plates were incubated in a shaker at 37°C (200 rpm, 30 min). The supernatant was filtered through a 70 μm filter and centrifuged (4°C, 300 g, 10 min). The supernatant was then centrifuged (2000 g, 20 min, 4°C). The supernatant was then centrifuged (16,500 g, 45 min, 4°C). The precipitate was resuspended in PBS, centrifuged (100,000 g, 2.5 h, 4°C), and stored at -80°C.

[0064] Changes in the secretion of endothelial endothelial EVs and liver-derived EVs in vascular endothelium in mouse livers after drug administration: Total EVs from liver tissue were collected and anti-CD31 magnetic beads (vascular endothelial markers) were added; total EVs from aortic tissue were collected and anti-Albumin magnetic beads (hepatocyte markers) were added. Both were incubated at 4°C for 1 hour. EVs were separated using a magnetic rack and washed three times with MACS buffer. CD63-FITC antibody was added and incubated at 4°C in the dark for 30 min. After washing, surface markers in EVs were detected and counted using nanoscale flow cytometry.

[0065] 3. Experimental Results The MASH combined with AS model was replicated in ApoE- / - mice on a high-fat diet for 16 weeks. After continuous gavage administration of the herbal composition of this invention for 4 weeks, it was found that hepatocyte swelling, degeneration, necrosis, and inflammatory damage were reduced, liver fibrosis was alleviated, and lipid droplets in liver tissue cells were decreased. Figure 7 The aortic lipid deposition decreased, the fibrous cap formed by the migration of foam cells and smooth muscle decreased, and the area and severity of plaques in the lumen were significantly reduced. Figure 8 The herbal composition of this invention can significantly reduce the levels of TC, TG, and LDL-C in serum, liver tissue, and aortic tissue, and improve lipid metabolism disorders in MASH-associated AS mice. Furthermore, the herbal composition of this invention significantly downregulates the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in serum, liver tissue, and aortic tissue, and significantly upregulates the activity of mitochondrial respiratory chain complexes I and II and Na+. + / K + -ATPase, Ca² + / Mg² + -ATPase activity, inhibiting liver and vascular endothelial inflammation activation, and improving mitochondrial energy metabolism disorders ( Figure 9Transmission electron microscopy results of liver tissue and aorta showed that the traditional Chinese medicine composition of this invention can improve mitochondrial ultrastructural damage and increase the number of autolysosomes. Figure 10 (Red arrow).

[0066] KEGG proteomic analysis of liver tissue showed that ( Figure 11 A), the traditional Chinese medicine composition of this invention can significantly regulate lipid metabolism pathways such as fatty acid degradation, α-linolenic acid metabolism, and peroxisome metabolism, and simultaneously affect inflammatory and oxidative stress-related pathways such as the Ras signaling pathway and histidine metabolism. Aortic proteomics KEGG enrichment analysis showed ( Figure 11 (B) The herbal composition of this invention can exert a vascular protective effect through multi-pathway synergistic regulation: on the one hand, it significantly intervenes in the pentose phosphate pathway, glycerol lipid metabolism, lysosomes and PI3K-Akt signaling pathway to improve vascular lipid metabolism disorders and inflammatory microenvironment; on the other hand, it also regulates extracellular matrix-receptor interaction and focal adhesion pathway, affecting the expression of key molecules such as collagen and fibronectin and the migration of vascular smooth muscle cells, thereby stabilizing plaque structure and inhibiting vascular remodeling.

[0067] The levels of endothelial endothelial EVs and hepatic EVs secreted in the endothelium of the liver were measured. The results showed that both hepatic and vascular EVs were significantly increased in the model group. However, the herbal composition of this invention reduced the release of EVs from the liver and blood vessels, suggesting that it may alleviate MASH combined with AS by regulating EVs. Figure 12 ).

[0068] Test Example 3: Long-term toxicity test 1. Animal grouping and administration Thirty C57BL / 6J mice were randomly divided into three groups of ten each: a high-dose group (4000 mg·kg⁻¹), a low-dose group (1000 mg·kg⁻¹) (equivalent to 8 times and 2 times the daily oral dose for adults, respectively), and a solvent control group. The mice were administered the herbal composition prepared in Example 1 via gavage once daily at a volume of 0.1 ml·10 g⁻¹ for 30 consecutive days. The solvent control group received the same volume of physiological saline. After the last administration, the animals were fasted but allowed free access to water for 12 hours. Blood was collected using the ocular hemorrhage method, and the necessary tissues were subsequently collected through dissection.

[0069] 2. Observation Indicators After drug administration, the general condition of the mice was observed, including their appearance, respiration, behavior, food and water intake, oral and nasal secretions, urine and feces, and mortality and changes in body weight were recorded. Blood was obtained from the experimental animals by ocular blood sampling, and serum was immediately separated for the detection of biochemical indicators such as liver function (ALT: alanine aminotransferase, AST: aspartate aminotransferase, AKP: alkaline phosphatase), protein metabolism (TP: total protein, ALB: albumin), kidney function (BUN: blood urea nitrogen, Crea: creatinine), blood lipids (TC: total cholesterol, TG: triglycerides), and blood glucose (GLU: glucose). Blood samples were stored at a suitable temperature after separation. After blood collection, the experimental animals were quickly dissected, and major organs, including the heart, liver, spleen, lungs, kidneys, and brain, were removed. Fat and excess tissue were completely removed, and the organs were weighed using an electronic balance (accurate to 0.1 mg). The organ coefficient is calculated as: organ mass (g) / body mass (g) × 100%. Collected organ tissues were immediately fixed in 4% paraformaldehyde solution for 24-48 hours. After fixation, the tissues underwent gradient dehydration, paraffin embedding, and sectioning. The sections were stained with hematoxylin and eosin (HE), and pathological changes in the organs were observed and photographed using an optical microscope for analysis.

[0070] 3. Experimental Results (1) General physiological indicators of mice Within 30 days of continuous administration, the mice showed good appearance, behavior, diet, and fecal condition, with no abnormalities, obvious toxic reactions, or mouse deaths. Results showed that the body weight of mice in each dosage group of the herbal composition at each time point was not statistically significant compared with that of the normal control group (P>0.05). Figure 13 .

[0071] (2) Organ index like Figure 14 As shown, compared with the control group, the morphology of the heart, liver, spleen, lungs, kidneys and brain of mice in the low (1000 mg·kg⁻¹) and high (4000 mg·kg⁻¹) dose groups of the traditional Chinese medicine composition of the present invention showed no significant changes. Statistical analysis of the organ indices of the experimental mice in each group revealed no statistically significant differences between or within different dose groups (P>0.05). (3) Blood biochemical index detection Ten blood biochemical indicators were measured, including serum biochemical indicators such as ALT, AST, AKP, GLU, TP, BUN, Cr, ALB, TC, and TG. Results are shown below. Figure 15Compared with the normal control group, the blood urea nitrogen (BUN) levels in both the low-dose group (1000 mg·kg⁻¹) and the high-dose group (4000 mg·kg⁻¹) of the herbal composition of this invention were significantly reduced (P<0.01), and the creatinine (Cr) level in the 4000 mg·kg⁻¹ group was significantly reduced (P<0.001). This significant decrease may suggest that the herbal composition of this invention has a certain regulatory effect on renal metabolism in mice, potentially improving renal function or reducing renal burden. However, while other individual serum biochemical indicators in each dose group of the herbal composition of this invention showed trends of increase and decrease, they remained within the normal physiological reference range overall. Statistical analysis showed that, except for BUN and Cr, there were no significant differences in other biochemical indicators between the dose groups and the normal control group, and all measured values ​​remained within the normal physiological reference range, with no abnormalities observed.

[0072] (4) Histopathological examination like Figure 16 As shown, compared with the normal control group, the mice in the low and high dose groups of the traditional Chinese medicine composition of the present invention showed no obvious pathological changes in the major organs and tissues such as the heart, liver, spleen, lungs, kidneys and brain.

[0073] The above results demonstrate that the traditional Chinese medicine composition of this invention exhibits good safety even when taken at doses far exceeding the effective level for extended periods, providing a solid foundation for the reliability of its clinical application.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a traditional Chinese medicine composition containing Sophora flavescens in the preparation of a medicament for treating metabolic-associated steatohepatitis or metabolic-associated steatohepatitis complicated with atherosclerosis, characterized in that, The raw materials for preparing the traditional Chinese medicine composition consist of Sophora flavescens, Gentiana scabra, and pig bile powder.

2. The application according to claim 1, characterized in that, The weight ratio of Sophora flavescens, Gentiana scabra and pig bile powder is (3-5): (3-5): (0.5-1.5).

3. The application according to claim 2, characterized in that, The weight ratio of Sophora flavescens, Gentiana scabra and pig bile powder is 4:4:

1.

4. The application according to claim 1, characterized in that, The drug is used to simultaneously improve hepatic lipid accumulation, liver inflammation, and inhibit the formation of atherosclerotic plaques.

5. The application according to claim 1, characterized in that, The improvement of hepatic lipid accumulation and inhibition of atherosclerotic plaque formation are achieved by regulating one or more of the fatty acid degradation pathway, pentose phosphate pathway, and glycerol metabolism pathway; and / or, the improvement of liver inflammation is achieved by inhibiting one or more of the Ras signaling pathway and PI3K-Akt signaling pathway.

6. The application according to claim 1, characterized in that, The pharmaceutical composition works by improving liver and / or aortic mitochondrial function.

7. The application according to claim 1, characterized in that, The pharmaceutical composition works by reducing the release of extracellular vesicles of vascular endothelial origin in the liver and / or extracellular vesicles of liver origin in the aorta.

8. The application according to any one of claims 1 to 7, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

9. The application according to any one of claims 1 to 7, characterized in that, The dosage form of the drug is pills, capsules, tablets, granules, powders, or oral liquids.

10. The application according to claim 9, characterized in that, The drug is in the form of pills.

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