Synergistic improvement effect of rectum peptide combined with pentahydroxydiphenylketone on renal fibrosis

Abstract

The invention discloses a synergistic improvement effect of rectum peptide combined with penta-hydroxydiphenylketone on renal fibrosis, and belongs to the technical field of biological medicines. Research results show that the Proctolin and the Macurin reduce extracellular matrix synthesis by inhibiting mRNA expression of fibrosis-related genes (Col1a1, Acta2 and Col3a1) and Col3a1 protein deposition, so that the renal fibrosis is relieved, the Proctolin and the Macurin show a remarkable renal fibrosis inhibiting effect in a TGF-beta induced cell model and a UUO animal model, and the combined use effect of the Proctolin and the Macurin is more remarkable. In conclusion, Proctolin and Macurin can effectively improve renal fibrosis of mice, results of in-vitro cell experiments are consistent with results of in-vivo animal models, the effect of mixed medication is better, and an experimental basis is provided for development of anti-fibrosis compound medicines.

Description

Technical Field

[0001] The present invention relates to the synergistic improvement effect of rectal peptide combined with pentahydroxydiphenyl ketone on renal fibrosis, and belongs to the technical field of biomedicine. Background Art

[0002] Chronic kidney disease (CKD) is a progressive, multisystem disease affecting approximately 10% of the general population worldwide. It is one of the leading causes of renal failure worldwide, and treatment options are limited. New therapeutic strategies are urgently needed. Regardless of the underlying etiology, CKD is characterized by loss of renal function and fibrosis. Renal fibrosis is the final pathological pathway for all types of progressive renal disease. Renal fibrosis (RF) is the primary pathological feature of the progression of CKD to end-stage renal disease. Glomerulosclerosis and interstitial fibrosis are the core pathological features, manifesting as abnormal accumulation of extracellular matrix components such as collagen (Col) and fibronectin (FN), ultimately leading to renal structural destruction and functional loss. The unilateral ureteral ligation (UUO) model, a classic animal model of renal disease, is widely used in renal fibrosis research. UUO significantly impairs renal function by altering hemodynamics, affecting glomerular filtration and renal metabolism, and inducing renal fibrosis. The UUO model has played a key role in elucidating the mechanisms of renal fibrosis at the molecular, genomic, and cellular levels.

[0003] Currently, some drugs are being studied to reduce the damage caused by chronic kidney disease. For example, renin-angiotensin system (RAS) inhibitors reduce glomerular hypertension, reduce proteinuria, and inhibit renal interstitial fibrosis by inhibiting the production of angiotensin II or its receptors; glucocorticoids and immunosuppressants inhibit immune inflammatory responses, reduce kidney damage, and indirectly delay fibrosis; chronic kidney disease is intervened through multiple approaches such as anti-inflammatory, anti-fibrosis, and control of metabolic abnormalities. However, there is currently a lack of specific drugs that can directly reverse fibrosis. The focus of treatment is still to delay progression. Terminal patients need to rely on renal replacement therapy, which imposes a heavy medical burden. Summary of the Invention

[0004] To address the above technical problems, the present invention provides a method for treating renal fibrosis using a rectal peptide, proctolin, and macurin. These peptides significantly improved the degree of fibrosis in mice with renal fibrosis. Mechanistically, proctolin and macurin inhibited the expression of fibrosis-related genes and proteins by downregulating the transcription of fibrosis markers Col1a1, Acta2, and Col3a1 and reducing the extracellular deposition of fibrotic proteins. Furthermore, proctolin and macurin improved renal function and alleviated structural damage by restoring glomerular filtration and reducing the degree of renal fibrosis. In both TGF-β and UUO models, the combined use of proctolin and macurin significantly improved fibrosis gene expression (Col1a1 / Acta2 / Col3a1), protein deposition (Col3a1), BUN levels, and fibrosis percentage compared to either drug alone. This combination of proctolin and macurin resulted in synergistic inhibition across pathways, enhancing the anti-fibrotic effect.

[0005] To achieve the above objectives, this solution first provides a use of a rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin in the preparation of drugs for preventing and treating fibrosis.

[0006] Preferably, the rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin are used in the preparation of a drug for preventing and controlling renal fibrosis damage caused by chronic kidney disease.

[0007] Preferably, the invention comprises the use of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin in the preparation of a drug for preventing and controlling the increase of blood creatinine caused by renal fibrosis.

[0008] Preferably, the invention comprises the use of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin in the preparation of a drug for preventing and controlling the increase of urea nitrogen caused by renal fibrosis.

[0009] Preferably, the invention comprises the use of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin in the preparation of a drug for preventing and controlling inflammatory response caused by renal fibrosis.

[0010] Preferably, the invention comprises the use of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin in the preparation of a drug for preventing and controlling oxidative stress caused by renal fibrosis.

[0011] Preferably, the drug is any pharmaceutically approved preparation.

[0012] Preferably, the drug is in the form of oral solution, tablet, capsule or granule.

[0013] Preferably, the effective dose of the rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin is 10 mg / kg / d.

[0014] The mechanism of the drug prepared by rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin in this scheme for treating renal fibrosis damage is as follows:

[0015] The rectal peptide proctolin and macurin can reduce the mRNA expression levels of genes such as Col1a1, Acta2, and Col3a1 in TGF-β-induced renal fibrosis cells. These genes are related to the synthesis of extracellular matrix components, and inhibiting their expression can reduce extracellular matrix deposition, which is a key pathological feature of renal fibrosis.

[0016] The present invention provides a use of a compound in the preparation of a medicament for preventing and / or treating diseases and / or conditions related to renal fibrosis, wherein the compound is a rectal peptide Proctolin and / or a pentahydroxydiphenyl ketone Macurin;

[0017] The structural formula of the pentahydroxydiphenyl ketone Macurin is as follows:

[0018]

[0019] The structural formula of the rectal peptide Proctolin is as follows:

[0020]

[0021] In one embodiment, the drug is used for at least one of (a) to (e):

[0022] (a) Prevent and control renal fibrosis caused by chronic kidney disease;

[0023] (b) prevent and control elevated serum creatinine levels caused by chronic kidney disease;

[0024] (c) Prevent and control elevated urea nitrogen levels caused by chronic kidney disease;

[0025] (d) prevent and control inflammatory responses caused by renal fibrosis;

[0026] (e) Prevent and control oxidative stress caused by renal fibrosis.

[0027] In one embodiment, the drug contains the rectal peptide Proctolin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof.

[0028] In one embodiment, the drug contains rectal peptide Proctolin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof;

[0029] In one embodiment, the drug contains the rectal peptide Proctolin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof, and contains pentahydroxydiphenyl ketone Macurin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof.

[0030] In one embodiment, the drug further comprises at least one pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.

[0031] In one embodiment, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0032] In one embodiment, the drug is in the form of a powder, tablet, lozenge, capsule, granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.

[0033] In one embodiment, the dosage form of the drug is:

[0034] (a) formulated for intravenous injection, subcutaneous injection, oral administration, inhalation, nasal administration, topical administration, ocular administration or otic administration;

[0035] or (b) in the form of a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops, or ear drops.

[0036] In one embodiment, the drug contains rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin as main ingredients; the drug contains rectal peptide Proctolin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof, and contains pentahydroxydiphenyl ketone Macurin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof; in the drug, rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin are added in a ratio of 1:1.

[0037] The present invention also provides a pharmaceutical composition for preventing and / or treating diseases and / or conditions related to renal fibrosis, wherein the composition contains proctolin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof, and macurin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof; the drug further contains at least one pharmaceutically acceptable carrier; in the drug, proctolin and macurin are added in a ratio of 1:1.

[0038] In one embodiment, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0039] In one embodiment, the drug is in the form of a powder, tablet, lozenge, capsule, granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.

[0040] In one embodiment, the dosage form of the drug is:

[0041] (a) formulated for intravenous injection, subcutaneous injection, oral administration, inhalation, nasal administration, topical administration, ocular administration or otic administration;

[0042] or (b) in the form of a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops, or ear drops.

[0043] Beneficial effects

[0044] (1) This study demonstrated that proctolin and macurin have a protective effect on renal fibrosis by inhibiting TGF-β-induced Acta2, Col1a1, and Col3a1 mRNA expression, which will help develop new therapeutic strategies for renal fibrosis (RF) caused by chronic kidney disease (CKD) and provide a basis for the development and application of proctolin and macurin in clinical drug treatment.

[0045] (2) This study demonstrated that the combination of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin is more effective, suggesting that the two may synergistically regulate fibrosis genes through different pathways and have the potential to become a new therapeutic drug for the treatment of CKD and RF.

[0046] (3) After intervention with rectal peptide proctolin, macurin and their mixture in the UUO mouse model, the renal structure and pathological changes were significantly improved, further emphasizing the therapeutic potential of rectal peptide proctolin and macurin for RF. Treatment with rectal peptide proctolin and macurin can significantly reduce the degree of renal fibrosis and significantly enhance renal function.

[0047] (4) Proctolin and macurin can also reduce the expression of Acta2, Col1a1, and Col3a1 mRNA in the kidneys of UUO mice. Therefore, in the case of renal fibrosis, the blocking effect of proctolin and macurin on fibrosis contributes to renal protection. Proctolin, macurin, and their mixture have an inhibitory effect on renal fibrosis in UUO model mice, improving the pathological state of renal tissue and reducing the degree of fibrosis.

[0048] (5) Proctolin exerts its anti-fibrotic effect mainly by inhibiting fibroblast activation and collagen synthesis pathways; macurin has significant efficacy in reducing oxidative stress and inhibiting the release of inflammatory factors. When the two are used in combination, the fibrosis gene inhibitory effect of proctolin and the antioxidant and anti-inflammatory effects of macurin are superimposed, forming a "multi-target" synergistic mechanism, thereby more comprehensively intervening in the fibrosis process. This synergistic mechanism not only breaks through the limitations of targeting a single signaling pathway, but also reduces the risk of toxic side effects that may be caused by high doses of a single drug.

[0049] (6) The safety and efficacy of the combined regimen have been repeatedly verified in multiple animal models, providing a solid experimental basis for future clinical translation. Since both rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin are known natural active substances or their derivatives, they have relatively clear pharmacokinetic characteristics and good tolerability, making subsequent clinical research more feasible. The combined drug strategy proposed in this invention can not only significantly improve the degree of fibrosis, but also prevent the progression of the disease in the early intervention stage, and has a strong advantage of both prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0051] Figure 1 This is a screening of safe concentrations of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin; A is to detect the effects of different concentrations of rectal peptide Proctolin (rectal peptide) on cell viability; B is to detect the effects of different concentrations of Macurin (2,3',4,4',6-pentahydroxydiphenyl ketone) on cell viability.

[0052] Figure 2 The effects of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin at a concentration of 10 μM on the expression of Acta2, Col1a1, and Col3a1 mRNA, respectively; A is a TGF-β-induced cell model experiment of renal tubular epithelial cells HK-2; B is an immunofluorescence image; C shows the quantitative analysis results of Col3a1 staining in different treatment groups.

[0053] Figure 3 The figures show the effects of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin on the unilateral ureteral obstruction (UUO) mouse model; A represents the effect on Serum BUN (mmol / L), and B represents the effect on Scr (μmol / L).

[0054] Figure 4 The figure shows the effects of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin on the animal model of unilateral ureteral obstruction (UUO); A is the kidney weight index of mice in different treatment groups, B is the effect on Acta2, Col1a1, and Col3a1 mRNA levels, C is the HE staining of histopathological sections, D is the Sirius red staining of histopathological sections, and E is the quantification of renal fibrosis. DETAILED DESCRIPTION

[0055] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0056] Technical terms:

[0057] Unless specifically defined herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art in the field of the present invention. The following definitions are provided to provide clarity regarding the terms used in the specification and claims to describe the present invention.

[0058] Renal fibrosis

[0059] Renal fibrosis and inflammation are predominant features of advanced renal disease. Tubulointerstitial fibrosis is a progressive process involving ongoing cellular damage, abnormal healing, activation of resident and infiltrating renal cells, cytokine release, inflammation, and phenotypic activation of renal cells to produce extracellular matrix. The extent of renal interstitial fibrosis in renal biopsy is an important indicator of CKD progression and prognosis. Renal fibrosis is characterized by scarring caused by the deposition of extracellular matrix (ECM). During the progression of chronic kidney disease, renal damage triggers the activation of inflammatory cells and fibroblasts, increasing the release of key ECM components such as collagen, fibronectin, and hyaluronic acid. Simultaneously, ECM-degrading enzymes are affected, inhibiting ECM degradation. As the disease progresses, excessive ECM deposition leads to changes in renal tissue and structure, resulting in pathological changes such as glomerulosclerosis and tubulointerstitial fibrosis. Although the ECM maintains renal tissue integrity, renal function is still reduced compared to pre-injury levels. However, there is currently no anti-fibrotic therapy for CKD (Huang R, Fu P, Ma L. Kidney fibrosis: from mechanisms to therapeutic medicines. Signal Transduction and Targeted Therapy, 2023, 8(1):129.)

[0060] UUO model

[0061] The rodent model of unilateral ureteral obstruction (UUO) surgery is a well-known model that accelerates the progression of human obstructive nephropathy and renal interstitial fibrosis. This model has been used in various studies to investigate therapies that delay renal interstitial fibrosis. This ureteral obstruction model can cause significant changes in renal hemodynamics and metabolism, leading to tubular damage, cell apoptosis and necrosis, and fibroblast proliferation. Myofibroblast transformation and excessive ECM deposition ultimately lead to renal fibrosis.

[0062] Unilateral ureteral obstruction (UUO) is an animal experimental model of kidney damage caused by obstruction of one ureter, leading to increased intrarenal pressure, decreased renal blood flow, and impaired renal tubular function. The UUO model typically involves ligating one ureter in an animal (usually a mouse or rat) to simulate renal obstruction. This prevents urine from draining normally on the obstructed side, causing changes in renal structure and function. The classic UUO model is divided into 7 days, 14 days and 21 days. This model can lead to reduced renal perfusion, glomerular sclerosis, tubulointerstitial fibrosis, induce renal tissue cell apoptosis and inflammatory response, ECM deposition in a short period of time, and thus promote the formation of renal fibrosis. It has become an important model for studying the mechanism of renal fibrosis and evaluating potential treatments to improve the effects of kidney disease (Wang Y, Deng X, Yang Z, et al. Global research trends in unilateral ureteral obstruction-induced renal fibrosis: Abibliometric and visualized study. Medicine, 2023, 102(32): e34713).

[0063] fibrosis

[0064] When organs or tissues are stimulated by infection, trauma, chemicals, or other stimuli, an inflammatory response occurs. For example, in the lungs, following infection, TGF-β1 released by inflammatory cells stimulates alveolar epithelial and endothelial cells, prompting their transformation into fibroblasts. Fibroblasts proliferate rapidly and synthesize and secrete extracellular matrix components such as collagen. Normally, the synthesis and degradation of the extracellular matrix (including collagen and fibronectin) are in a dynamic equilibrium. However, during fibrosis, this balance is disrupted. For example, in liver fibrosis, activated hepatic stellate cells enhance their ability to synthesize collagen, while the activity of enzymes that degrade collagen (such as collagenase) is inhibited. As a result, collagen and other extracellular matrix components accumulate excessively in the liver, forming fibrosis. Fibrosis can disrupt the normal structure of organs and lead to loss of function. For example, in renal fibrosis, fibrous tissue proliferates around the glomeruli and tubules, impairing the kidney's filtration and reabsorption functions, ultimately leading to renal failure. Fibrosis is a complex pathological process involving multiple cellular and molecular mechanisms. Although there are currently some treatments that can slow the progression of fibrosis, completely reversing fibrosis remains one of the difficulties in medical research.

[0065] Case 1: A method for treating a disease or condition associated with complement activation in an individual in need thereof, the method comprising administering a therapeutically effective amount of a rectal peptide Proctolin and / or a pentahydroxydiphenyl ketone Macurin;

[0066] The disease is renal fibrosis.

[0067] Case 2: A pharmaceutical composition comprising the rectal peptide Proctolin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof, and pentahydroxydiphenyl ketone Macurin or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label or isomer thereof; the drug further comprises at least one pharmaceutically acceptable carrier.

[0068] In one embodiment of Case 2, an effective amount of the above-mentioned pharmaceutical composition is used in the preparation of a medicament for preventing and / or treating renal fibrosis diseases and / or conditions.

[0069] Case 3: Application of rectal peptide Proctolin and / or pentahydroxydiphenyl ketone Macurin in preparation of formulations.

[0070] The preparation is in the form of powder, tablet, lozenge, capsule, granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.

[0071] In one embodiment of Case 3, the preparation is:

[0072] (a) formulated for intravenous injection, subcutaneous injection, oral administration, inhalation, nasal administration, topical administration, ocular administration, or otic administration; or (b) in the form of a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops, or ear drops.

[0073] In one embodiment of Case 3, the preparation is used for the treatment of renal fibrosis.

[0074] Case 4: Use of rectal peptide Proctolin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating renal fibrosis;

[0075] Use of pentahydroxydiphenyl ketone Macurin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating renal fibrosis;

[0076] Use of rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin in combination in preparing a medicine for treating renal fibrosis.

[0077] Case 5: A pharmaceutical composition for preventing and / or treating diseases and / or conditions associated with renal fibrosis, comprising a rectal peptide (Proctolin) or a pharmaceutically acceptable salt, ester, solvate, prodrug, or isomer thereof, and a pentahydroxydiphenyl ketone (Macurin) or a pharmaceutically acceptable salt, ester, solvate, prodrug, or isomer thereof; the composition further comprises at least one pharmaceutically acceptable carrier;

[0078] Preferably, in the drug, rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin are added in a mass ratio of 1:1.

[0079] In one embodiment of Case 5, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine;

[0080] In one embodiment of Case 5, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

[0081] In one embodiment of Case 5, the dosage form of the drug is: powder, tablet, lozenge, capsule, granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository dosage form.

[0082] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0083] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0084] The pentahydroxydiphenyl ketone Macurin involved in the following examples was purchased from MCE Company, product number: HY-N3347; CAS No. 519-34-6;

[0085] The chemical formula structure is as follows:

[0086]

[0087] Proctolin (rectal peptide) was purchased from MCE, product number: HY-P0275; CAS No. 57966-42-4; sequence information: Arg-Tyr-Leu-Pro-Thr (RYLPT);

[0088] The chemical formula structure is as follows:

[0089]

[0090] Example 1: Screening of safe concentrations of Proctolin and Macurin

[0091] The specific steps are as follows:

[0092] 1. Preparation of solution

[0093] (1) Preparation of Proctolin Solution

[0094] Accurately weigh 0.648 mg of proctolin and dissolve it in 1 mL of sterile PBS. Mix thoroughly, label, and prepare a 1 mM stock solution in aliquots. Store at -20°C. Add 200 μL of the stock solution to 800 μL of DMEM medium for a final concentration of 200 μM; add 100 μL of the stock solution to 900 μL of DMEM medium for a final concentration of 100 μM; add 50 μL of the stock solution to 950 μL of DMEM medium for a final concentration of 50 μM; add 20 μL of the stock solution to 980 μL of DMEM medium for a final concentration of 20 μM; and add 5 μL of the stock solution to 995 μL of DMEM medium for a final concentration of 5 μM.

[0095] (2) Preparation of pentahydroxydiphenyl ketone Macurin solution

[0096] Accurately weigh 0.262 mg of macurin and dissolve it in 1 mL of sterile PBS. Mix, label, and prepare 1 mM stock solutions in aliquots and store at -20°C. Add 200 μL of stock solution to 800 μL of DMEM medium for a final concentration of 200 μM; add 100 μL of stock solution to 900 μL of DMEM medium for a final concentration of 100 μM; add 50 μL of stock solution to 950 μL of DMEM medium for a final concentration of 50 μM; add 20 μL of stock solution to 980 μL of DMEM medium for a final concentration of 20 μM; and add 5 μL of stock solution to 995 μL of DMEM medium for a final concentration of 5 μM.

[0097] 2. Cell experiments

[0098] The specific steps are as follows:

[0099] (1) HK-2 cells were digested and resuspended in complete culture medium; inoculated into 96-well plates, 8,000 cells per well, with 6 replicate wells per group; culture medium was added to 100 μL per well; and incubated in an incubator for 12-24 hours to allow cells to adhere stably.

[0100] (2) Remove the old culture medium from the cells in step (1) and slowly add the proctolin solution prepared in step 1 containing different concentrations: 0 μM (DMEM medium), 5 μM, 20 μM, 50 μM, 100 μM, and 200 μM; the volume per well remains 100 μL; continue incubation for 24 hours. Add 10 μL of CCK-8 solution to each well; continue incubation for 1-2 hours (protect from light); measure the OD value (450 nm) of each well using a microplate reader; record the data.

[0101] (3) Remove the old culture medium from the cells in step (1) and slowly add the macurin solution prepared in step 1 containing different concentrations: 0 μM (DMEM medium), 5 μM, 20 μM, 50 μM, 100 μM, and 200 μM; the volume per well remains 100 μL; continue incubation for 24 hours. Add 10 μL of CCK-8 solution to each well; continue incubation for 1-2 hours (protect from light); measure the OD value (450 nm) of each well using a microplate reader; record the data.

[0102] The results are as follows Figure 1 As shown;

[0103] The results show that Figure 1 To screen the safe concentrations of Proctolin and Macurin, the effects of the two drugs on cell viability were observed at concentrations of 5-200 μM.

[0104] Taking the cell viability of the control group (0 μM) as the benchmark value 1, the cell viability at each concentration of 5-200 μM was close to or slightly higher than 1 (eg, approximately 1 at 5 μM and not significantly lower than 1 at 200 μM).

[0105] The results showed that Proctolin and Macurin had no significant toxicity to cells within the concentration range of 5-200 μM, and cell viability was not inhibited, suggesting that the drug is relatively safe.

[0106] Figure 1 The results showed that Proctolin and Macurin did not significantly inhibit cell viability at concentrations as high as 200 μM, eliminating the interference of drug toxicity on the results of subsequent fibrosis experiments and providing a premise for subsequent mechanism research. 10 μM was selected as the detection concentration in subsequent experiments (such as mRNA expression and protein staining).

[0107] Example 2: TGF-β-induced cell model experiment of renal tubular epithelial cells HK-2

[0108] 1. Establish a TGF-β-induced cell model experiment of renal tubular epithelial cells HK-2. The specific steps are as follows:

[0109] (1) HK-2 cells were digested with trypsin, counted, and inoculated into 6-well plates; approximately 2 × 10 5 Cells were plated on a plate. Complete medium containing 10% FBS was added to 2 mL per well. The cells were cultured in a CO2 incubator for 12-24 hours until the cells adhered and reached approximately 80% confluence. After the cells adhered, the medium was removed and the cells were starved with serum-free medium for 6-12 hours to synchronize the cell cycle.

[0110] (2) The treatment solution was replaced according to the group. Each drug was pre-added to the culture medium and mixed for 30 minutes before adding TGF-β. The volume was controlled at 2 mL per well. The cells were cultured for 48 hours. The specific groups are as follows:

[0111] Control group: Serum-free medium without TGF-β and drugs was added to the cells obtained in step (1);

[0112] TGF-β group: TGF-β (10 ng / mL) was added to the cells obtained in step (1);

[0113] TGF-β+Proctolin group: TGF-β (10 ng / mL) + Proctolin (final concentration 10 μM) were added to the cells obtained in step (1);

[0114] TGF-β+Macurin group: TGF-β (10 ng / mL) + Macurin (final concentration 10 μM) were added to the cells obtained in step (1);

[0115] TGF-β+Mixture group: TGF-β (10 ng / mL) + Proctolin (final concentration 5 μM) + Macurin (final concentration 5 μM) were added to the cells obtained in step (1);

[0116] 2. Experimental results

[0117] The results are as follows Figure 2 As shown;

[0118] Figure 2 The effects of Proctolin and Macurin at a concentration of 10 μM, respectively, on the mRNA expressions of Acta2, Col1a1, and Col3a1. Acta2, Col1a1, and Col3a1 are commonly used molecular markers of renal fibrosis.

[0119] The results show:

[0120] (1) Figure 2 Figure A shows that Acta2, Col1a1, and Col3a1 mRNA levels were significantly upregulated in the TGF-β group, indicating that the model successfully induced fibrosis gene expression. In the in vitro cell model, the expression of fibrosis genes in the proctolin / macurin single-agent treatment group was significantly decreased (by approximately 50% compared to the TGF-β group, P < 0.05), suggesting that single-agent treatment can inhibit TGF-β-induced gene activation. In the combined treatment group, expression levels were further reduced to near normal levels, demonstrating a synergistic inhibitory effect (P < 0.01 vs. TGF-β group).

[0121] Therefore, it was shown that both Proctolin and Macurin could significantly inhibit the expression of Acta2, Col1a1, and Col3a1 mRNA induced by TGF-β, and the mixed use had a better effect, suggesting that the two may synergistically regulate fibrosis genes through different pathways.

[0122] (2) Figure 2 Figure B shows immunofluorescence images showing the expression of Col3a1 (type III collagen α1 chain) in cells treated with different methods. In the control group, Col3a1 expression was low, with weak red fluorescence. In the transforming growth factor-β (TGF-β)-treated group, red fluorescence was significantly enhanced, indicating that TGF-β can induce a significant increase in Col3a1 expression in cells and promote the expression of renal fibrosis-related proteins. In the TGF-β and rectal peptide co-treatment group, red fluorescence intensity was weaker than in the TGF-β group, indicating that rectal peptide can inhibit TGF-β-induced Col3a1 expression. In the TGF-β and macurin co-treatment group, red fluorescence intensity was also lower than in the TGF-β group, indicating that macurin can inhibit TGF-β-induced Col3a1 expression. In the TGF-β, rectal peptide, and macurin mixture-treated group, red fluorescence intensity was further reduced, suggesting that the mixture may be more effective in inhibiting Col3a1 expression.

[0123] (3) Figure 2 Figure C shows the quantitative analysis results of Col3a1 staining in different treatment groups, reflecting the changes in Col3a1 expression levels relative to the control group. The Col3a1 expression level in the control group was set as the reference value, and the fold change was close to 1; the Col3a1 expression in the TGF-β group was significantly increased, and the fold change was significantly higher than that in the control group, indicating that TGF-β had a significant effect on inducing Col3a1 expression; the fold changes in Col3a1 expression in the TGF-β+Proctolin, TGF-β+Macurin, and TGF-β+Mixture groups were significantly lower than that in the TGF-β group, indicating that rectal peptide, macurin, and the mixture of the two can inhibit TGF-β-induced Col3a1 expression, among which the value in the mixture group was relatively lower, showing a better inhibitory effect.

[0124] Experimental Example 3: Establishment of UUO model mice and drug administration

[0125] 1. Animal experiments

[0126] All animal experiments in this study were approved by Jiangnan University. The UUO model was established under clean and standardized conditions. Male C57BL / 6J mice, 6 weeks old, were purchased from Sibeifu Biotechnology Co., Ltd. (Nanjing, China). The mice were housed in a humidity- and temperature-controlled environment with free access to standard chow and clean water on a 12-h light-dark cycle. After one week of acclimation, we evaluated the effects of proctolin and macurin on the UUO mouse model.

[0127] The experiment was divided into five groups: Control: normal control group (no surgery); UUO: unilateral ureteral obstruction model group (no medication); UUO+Proctolin: UUO model + Proctolin treatment group; UUO+Macurin: UUO model + Macurin treatment group; UUO+Mixture: UUO model + Proctolin + Macurin mixed treatment group.

[0128] The specific steps are as follows:

[0129] Control: Oral administration of normal saline from day 1 to day 15; sham operation (Sham) was performed on day 2;

[0130] UUO: From day 1 to day 15, normal saline was administered orally; UUO surgery was performed on day 2;

[0131] UUO+Proctolin: Proctolin (10 mg / kg / day) was administered orally from day 1 to day 15; UUO surgery was performed on day 2;

[0132] UUO+Macurin: Macurin (10 mg / kg / day) was administered orally from day 1 to day 15; UUO surgery was performed on day 2;

[0133] UUO+Mixture: From day 1 to day 15, oral administration of Mixture (5 mg / kg / day Proctolin and 5 mg / kg / day Macurin) was performed; UUO surgery was performed on day 2;

[0134] The sham operation was as follows: the control group mice underwent the same anesthesia and surgical incision as the UUO group; the left ureter was dissected and exposed; the ureter was not ligated, and only the tissue was gently separated; the ureter was gently returned to the abdominal cavity, and the muscle layer and skin were sutured; subsequent feeding was consistent with the UUO group, and samples were collected after 14 days.

[0135] The UUO surgery was performed as follows: anesthesia was induced with 2% isoflurane (1.0 L / min flow rate). The mouse was placed on a thermostatically controlled operating table and connected to a small animal ventilator. After adequate anesthesia, the left abdominal hair was trimmed, and a laparotomy was performed to expose the left kidney. The left ureter was ligated at two locations, upper and lower, with 6.0 non-absorbable sutures, ensuring that they did not overlap, and the ureter was severed between the two ligatures. After ureteral ligation, the incision was sutured with 6.0 lines.

[0136] After the experiment, anesthesia was performed using an appropriate dose of anesthetic (2% isoflurane). After confirmation of adequate anesthesia, blood was collected by cardiac puncture and the animals were subsequently euthanized by cervical dislocation.

[0137] Use a 1mL syringe to puncture the left ventricle or right atrium of the heart and collect 0.5-1mL of blood into a non-anticoagulant blood collection tube. Allow to rest for 30 minutes. Centrifuge at 3000 rpm for 10 minutes at 4°C. Pipette the supernatant (serum) into a new enzyme-free EP tube and immediately store at -80°C for testing of blood urea nitrogen (BUN) and creatinine. Carefully separate the left kidney, avoiding damage to the renal capsule. Cut the kidney in half longitudinally, typically for pathological staining; half is used for molecular biology analysis.

[0138] 2. Experimental results:

[0139] (1) Serum BUN, Scr (μmol / L) test results are as follows Figure 3 As shown:

[0140] a. Serum BUN (mmol / L), also known as serum urea nitrogen (mmol / L), is one of the indicators reflecting renal function. An increase in BUN usually indicates renal impairment.

[0141] The results showed that the serum urea nitrogen levels (mmol / L) of the Control, UUO, UUO+Proctolin, UUO+Macurin, and UUO+Mixture groups were 5.625±1.585, 15.275±4.375, 10.475±1.825, 11.485±2.835, and 5.985±1.665, respectively.

[0142] Serum urea nitrogen levels in the UUO group were significantly higher than those in the control group, indicating that unilateral ureteral obstruction impaired renal function in mice. Serum urea nitrogen levels in each drug-treated group (UUO+Proctolin, UUO+Macurin, and UUO+Mixture) were lower than those in the UUO group, indicating that rectal peptide, macurin, and their mixture can improve renal function and reduce serum urea nitrogen levels in UUO model mice to a certain extent.

[0143] b. Scr (μmol / L), that is, serum creatinine (micromoles / liter), is also an important indicator for evaluating renal function. An increase in Scr reflects a decrease in glomerular filtration function.

[0144] The results showed that the serum creatinine (μmol / L) levels of the Control, UUO, UUO+Proctolin, UUO+Macurin, and UUO+Mixture groups were 41.65±11.45, 152.8±18.3, 94.4±15.8, 94.35±18.85, and 48.6±13.5, respectively.

[0145] As can be seen, the serum creatinine level in the UUO group was significantly higher than that in the control group, reflecting impaired renal function in the model mice. The serum creatinine levels in each drug-treated group were lower than that in the UUO group, indicating that rectal peptide, macurin, and their mixture can reduce serum creatinine levels in UUO model mice and improve impaired renal function.

[0146] (2) To investigate the effects of rectal peptide, macurin and their mixture on renal fibrosis in a mouse model; the results are as follows Figure 4 shown.

[0147] Figure 4 A in the figure shows the kidney weight index of mice in different treatment groups. It can be seen from the figure that the UUO group was significantly higher than the control group, indicating that obstruction caused an increase in kidney weight; each drug treatment group was lower than the UUO group, indicating that rectal peptide, Macurin and the mixture can reduce the degree of increase in kidney weight, and the mixture group reduced the increase in kidney weight most significantly.

[0148] Figure 4 In Figure B, Acta2, Col1a1, and Col3a1 mRNA were significantly upregulated in the UUO group, reflecting the activation of renal fibrosis genes in vivo; the expression of fibrosis genes in the single-drug treatment group (Proctolin / Macurin) was significantly decreased (down about 50% compared with the UUO group, P<0.05), indicating that a single drug can inhibit fibrosis gene expression in vivo; the mixed treatment group dropped to near normal levels, showing a synergistic effect in vivo (P<0.01vs UUO group).

[0149] Therefore, it was shown that in the in vivo UUO model, both Proctolin and Macurin alone could effectively reduce the mRNA expression of Acta2, Col1a1, and Col3a1, and the combination of drugs further enhanced the inhibitory effect, verifying the in vivo validity of the in vitro experimental conclusions.

[0150] Figure 4Figure C shows a histopathological section stained with HE staining, showing the renal tissue morphology of mice in the different treatment groups. The control group showed normal tissue structure; the UUO group showed disordered structure and pathological changes. The UUO+Proctolin, UUO+Macurin, and UUO+Mixture groups showed less pathological changes compared to the UUO group, demonstrating that proctolin, macurin, and their mixture improve renal tissue morphology.

[0151] Figure 4 D in the figure is a Sirius red staining image of the tissue pathology section, which shows that the fibrosis components in the kidney tissue of the control group were less; the fibrosis components in the UUO group were significantly increased; the fibrosis components in the UUO+Proctolin, UUO+Macurin, and UUO+Mixture groups were less than those in the UUO group, which intuitively shows that drug treatment can reduce the degree of kidney tissue fibrosis.

[0152] Figure 4 Figure E is a quantitative diagram of renal fibrosis, showing that the percentage of renal fibrosis in the UUO group was significantly higher than that in the control group; the percentage of renal fibrosis in the UUO+Proctolin, UUO+Macurin, and UUO+Mixture groups was lower than that in the UUO group, quantitatively indicating that rectal peptide, macurin, and their mixture can reduce the degree of renal fibrosis in UUO model mice.

[0153] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of a compound in the preparation of a medicament for preventing and / or treating diseases and / or conditions related to renal fibrosis, characterized in that: The compound is rectal peptide Proctolin and / or pentahydroxydiphenyl ketone Macurin; The structural formula of the pentahydroxydiphenyl ketone Macurin is as follows: The structural formula of the rectal peptide Proctolin is as follows:

2. The use according to claim 1, characterized in that The drug is used for at least one of (a) to (e): (a) Prevent and control renal fibrosis caused by chronic kidney disease; (b) prevent and control elevated serum creatinine levels caused by chronic kidney disease; (c) Prevent and control elevated urea nitrogen levels caused by chronic kidney disease; (d) prevent and control inflammatory responses caused by renal fibrosis; (e) Prevent and control oxidative stress caused by renal fibrosis.

3. The use according to claim 1 or 2, characterized in that The drug contains rectal peptide Proctolin or its pharmaceutically acceptable salt compound, ester compound, solvate, prodrug or isomer thereof, and / or contains pentahydroxydiphenyl ketone Macurin or its pharmaceutically acceptable salt compound, ester compound, solvate, prodrug or isomer thereof; The medicine further comprises at least one pharmaceutically acceptable carrier and pharmaceutically acceptable excipient.

4. The use according to claims 1 to 3, characterized in that: The carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

5. The use according to claims 1 to 4, characterized in that: The dosage form of the drug is: powder, tablet, lozenge, capsule, granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.

6. The use according to claims 1 to 4, characterized in that: The drug is in the form of an intravenous injection, a subcutaneous injection, an oral preparation, an aerosol, a nasal preparation, a topical preparation, an ocular preparation or an otic preparation.

7. The use according to any one of claims 1 to 6, characterized in that: The drug contains rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin as main ingredients; Preferably, the drug contains the rectal peptide Proctolin or its pharmaceutically acceptable salt compound, ester compound, solvate, prodrug or isomer thereof, and contains pentahydroxydiphenyl ketone Macurin or its pharmaceutically acceptable salt compound, ester compound, solvate, prodrug or isomer thereof; Preferably, in the drug, the rectal peptide Proctolin and the pentahydroxydiphenyl ketone Macurin are added in a mass ratio of 1:

1.

8. A pharmaceutical composition for preventing and / or treating diseases and / or conditions related to renal fibrosis, characterized in that: The composition contains rectal peptide Proctolin or its pharmaceutically acceptable salt compound, ester compound, solvate, prodrug or isomer thereof, and contains pentahydroxydiphenyl ketone Macurin or its pharmaceutically acceptable salt compound, ester compound, solvate, prodrug or isomer thereof; the drug further contains at least one pharmaceutically acceptable carrier; Preferably, in the drug, rectal peptide Proctolin and pentahydroxydiphenyl ketone Macurin are added in a mass ratio of 1:

1.

9. The pharmaceutical composition according to claim 8, characterized in that The carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

10. The pharmaceutical composition according to claim 8 or 9, wherein the dosage form of the drug is: powder, tablet, lozenge, capsule, granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.

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