A drug for ADAMTS18 gene target and a product for treating obstructive renal fibrosis

Abstract

The application discloses a kind of drugs for ADAMTS18 gene target and product for treating obstructive renal fibrosis, it is related to the field of biotechnology.The application provides a kind of drugs for ADAMTS18 gene target, the drug includes the following raw and auxiliary materials according to weight fraction: ari red 1-3 parts, 5-10 parts of poria cocos, 15-25 parts of radix astragali, 0.1-2 parts of rhubarb, 1-5 parts of curcuma, 8-12 parts of fahansha, 1-6 parts of rhodiola, 5-8 parts of melon seed, 1-3 parts of bamboo, 1-6 parts of hundred two gold, 10-30 parts of adhesive, 5-20 parts of antioxidant, 10-25 parts of cosolvent.The drug prepared in the application has obvious improvement effect on obstructive renal fibrosis, and the drug plays an improving role on ADAMTS18 gene target, has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a drug targeting the ADAMTS18 gene and a product for treating obstructive renal fibrosis. Background Technology

[0002] Renal fibrosis refers to the pathogenesis of kidney failure caused by damage to intrinsic cells of the kidney due to circulatory disorders, inflammatory responses, trauma, etc., resulting in fibrosis of intrinsic cells, damage to nephrons, loss of function, and ultimately renal failure. The progression of renal fibrosis mainly involves the following steps: (1) activation of immune inflammatory response; (2) activation and abnormal proliferation of fibroblasts; (3) formation and accumulation of extracellular matrix; (4) final formation of scar tissue.

[0003] Renal fibrosis mainly manifests as glomerular sclerosis and interstitial fibrosis. The process of glomerular sclerosis is quite similar to that of interstitial fibrosis. Both glomerular sclerosis and interstitial fibrosis are caused by an imbalance between excessive synthesis and reduced degradation of the extracellular matrix, as well as a large number of inflammatory reactions and the proliferation of myofibroblasts. Glomerular sclerosis can lead to the destruction of renal tubular structure and result in renal fibrosis.

[0004] Unilateral ureteral obstruction (UUO) can induce renal fibrosis and cause kidney damage by altering renal hemodynamics and metabolism. The UUO model can rapidly develop glomerular sclerosis, tubular atrophy, and interstitial fibrosis, and is currently the most widely used and reliable model of renal fibrosis.

[0005] The ADAMTS (ADisintegri and Metalloproteinase with Thrombospondin motifs) family of metalloproteinases is a subfamily of disintegrin metalloproteinases containing type I platelet-binding protein motifs. Comprising 19 members, they play crucial roles in various physiological and pathological processes, including embryonic development, organogenesis, tumors, inflammation, and disease, by cleaving or modifying extracellular matrix components. Each member contains an N-terminal signal peptide, followed by a propeptide, a disintegrin-like region, a central type I platelet-like protein, a cysteine-rich region, a spacer region, and a variable C-terminal TSR repeat sequence. ADAMTS18 is one such member, and research indicates it plays an essential role in normal growth and development. Its deletion, mutation, and methylation can lead to malformations and diseases.

[0006] Chinese patent CN110393713A discloses the use of nicotinamide in the preparation of drugs for treating obstructive nephropathy and related renal fibrosis. Specifically, this patent discloses a drug for treating obstructive nephropathy and related renal fibrosis, comprising nicotinamide and its pharmaceutically acceptable salts. This invention reveals the beneficial efficacy of nicotinamide in treating renal fibrosis caused by obstructive nephropathy. Animal studies have shown that nicotinamide can effectively reduce renal fibrosis induced by unilateral ureteral ligation. Cellular studies have shown that nicotinamide can alleviate fibrotic changes in mouse renal tubular epithelial cells induced by transforming growth factor-β (TGF-β).

[0007] Currently, there is limited research on obstructive renal fibrosis, and developing a drug that can effectively treat obstructive renal fibrosis is a pressing technical problem that needs to be solved. Summary of the Invention

[0008] The purpose of this invention is to provide a drug targeting the ADAMTS18 gene and a product for treating obstructive renal fibrosis.

[0009] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0010] On one hand, the present invention provides a drug targeting the ADAMTS18 gene, wherein the drug comprises the following raw and excipient materials in parts by weight: 1-3 parts of Ali Red, 5-10 parts of Poria cocos, 15-25 parts of Astragalus membranaceus, 0.1-2 parts of processed rhubarb, 1-5 parts of turmeric, 8-12 parts of processed Pinellia ternata, 1-6 parts of Rhodiola rosea, 5-8 parts of Trichosanthes kirilowii seed, 1-3 parts of bamboo shavings, 1-6 parts of Lysimachia christinae, 10-30 parts of binder, 5-20 parts of antioxidant, and 10-25 parts of solubilizer;

[0011] Preferably, the drug comprises the following raw and auxiliary materials in parts by weight: 2-3 parts of Ali Red, 6-8 parts of Poria cocos, 15-20 parts of Astragalus membranaceus, 0.1-1 parts of processed rhubarb, 3-5 parts of turmeric, 9-11 parts of processed Pinellia ternata, 1-4 parts of Rhodiola rosea, 5-7 parts of Trichosanthes kirilowii seed, 1-2 parts of bamboo shavings, 2-5 parts of Lysimachia christinae, 18-28 parts of binder, 10-16 parts of antioxidant, and 14-20 parts of solubilizer.

[0012] More preferably, the drug comprises the following raw and auxiliary materials by weight: 2 parts of Ali Red, 7 parts of Poria cocos, 18 parts of Astragalus membranaceus, 0.5 parts of processed rhubarb, 4 parts of turmeric, 10 parts of processed Pinellia ternata, 2 parts of Rhodiola rosea, 6 parts of Trichosanthes kirilowii seed, 2 parts of bamboo shavings, 4 parts of Lysimachia christinae, 24 parts of binder, 10 parts of antioxidant, and 18 parts of solubilizer.

[0013] Specifically, the adhesive is composed of sodium carboxymethyl cellulose and polyvinylpyrrolidone, the antioxidant is composed of gallic acid, di-tert-butyl-p-cresol and vitamin E, and the cosolvent is composed of polyoxyethylene castor oil and dimethyl sulfoxide.

[0014] Furthermore, the mass ratio of sodium carboxymethyl cellulose to polyvinylpyrrolidone is (1-10):1;

[0015] Furthermore, the mass ratio of sodium carboxymethyl cellulose to polyvinylpyrrolidone is (2-6):1;

[0016] Furthermore, the mass ratio of sodium carboxymethyl cellulose to polyvinylpyrrolidone is 2:1.

[0017] Furthermore, the mass ratio of gallic acid, di-tert-butyl-p-cresol, and vitamin E is (0.2-2):(1-5):1;

[0018] Furthermore, the mass ratio of gallic acid, di-tert-butyl-p-cresol, and vitamin E is (0.5-2):(2-5):1;

[0019] Furthermore, the mass ratio of gallic acid, di-tert-butyl-p-cresol, and vitamin E is 0.5:3.5:1.

[0020] Furthermore, the mass ratio of the polyoxyethylene castor oil to dimethyl sulfoxide is (5-15):1;

[0021] Furthermore, the mass ratio of the polyoxyethylene castor oil to dimethyl sulfoxide is (6-9):1;

[0022] Furthermore, the mass ratio of the polyoxyethylene castor oil to dimethyl sulfoxide is 8:1.

[0023] Specifically, the dosage form of the drug, depending on the route of administration, includes, but is not limited to, gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0024] Furthermore, the gastrointestinal dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0025] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0026] Furthermore, the injectable dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0027] Furthermore, the respiratory drug delivery formulations include, but are not limited to, sprays, aerosols, and powder inhalers.

[0028] Furthermore, the skin delivery dosage forms include, but are not limited to, lotions, ointments, topical solutions, styrosine, pastes, and patches.

[0029] Furthermore, the mucosal drug delivery dosage forms include, but are not limited to, eye drops, nasal drops, ophthalmic ointments, sublingual tablets, and patches.

[0030] Furthermore, the cavity drug delivery dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pellets.

[0031] Furthermore, the dosage form of the drug is a soft capsule.

[0032] In another aspect, the present invention provides a method for preparing the above-mentioned drug, comprising the following steps:

[0033] (1) Take the following weight proportions of Alisma, Poria, Astragalus, Rhubarb, Curcuma, Pinellia, Rhodiola, Trichosanthes seed, Bamboo shavings, and Lysimachia christinae, add water and decoct. The number of decoctions is 1-3 times, each time for 1-5 hours. Filter and combine the extracts.

[0034] (2) Concentrate the combined extracts to a relative density of 1.2-1.5, cool, dry, and sterilize to obtain a dry powder of the traditional Chinese medicine composition;

[0035] (3) Add binder, antioxidant and solubilizer to the dry powder of traditional Chinese medicine composition and mix well to prepare medicine.

[0036] Specifically, the raw materials in step (1) include soaking, rinsing and crushing steps before frying.

[0037] Furthermore, the soaking time is 1-10 hours; even further, the soaking time is 5 hours.

[0038] Furthermore, the raw materials are pulverized and then passed through an 80-mesh sieve.

[0039] Furthermore, the decocting process in step (1) is repeated twice.

[0040] Furthermore, in step (1), the amount of water added is 6-12 times the weight of the raw materials.

[0041] Further, the decoction process described in step (1) is as follows: first, add 12 times the amount of water and decoct for 2.5 hours; second, add 8 times the amount of water and decoct for 1.5 hours; or;

[0042] The first time, add 8 times the amount of water and simmer for 2 hours. The second time, add 10 times the amount of water and simmer for 2 hours.

[0043] Specifically, the concentration temperature in step (2) is 40-70℃; further, the concentration temperature in step (2) is 60℃.

[0044] In another aspect, the present invention provides the use of the above-mentioned drug in the preparation of products for treating renal fibrosis.

[0045] Specifically, the renal fibrosis includes, but is not limited to, obstructive renal fibrosis, drug-induced renal fibrosis, and renal fibrosis caused by urinary tract infection.

[0046] Furthermore, the renal fibrosis described is obstructive renal fibrosis.

[0047] Specifically, the product in question is a pharmaceutical product.

[0048] Furthermore, the medicine may contain a pharmaceutically acceptable carrier.

[0049] Specifically, the drug targets the ADAMTS18 gene to improve obstructive renal fibrosis.

[0050] The beneficial effects of this invention are as follows:

[0051] This invention provides a drug targeting the ADAMTS18 gene. The drug prepared by this invention has a significant ameliorative effect on obstructive renal fibrosis, significantly increasing the kidney weight / body weight ratio in mice with obstructive renal fibrosis and reducing the expression level of the fibrotic molecule FN. Moreover, the drug has an ameliorative effect on the ADAMTS18 gene target. The raw materials of this drug are all traditional Chinese medicines, which have the advantages of low toxicity and side effects and significant efficacy. This invention can be developed into a new drug for the treatment of obstructive renal fibrosis and has good application prospects. Attached Figure Description

[0052] Figure 1 The image shows the Masson staining results for each group of mice. Detailed Implementation

[0053] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0054] Example 1

[0055] The medicine is prepared from the following raw materials in parts by weight: 2 parts of Ali Red, 7 parts of Poria cocos, 18 parts of Astragalus membranaceus, 0.5 parts of processed rhubarb, 4 parts of turmeric, 10 parts of processed Pinellia ternata, 2 parts of Rhodiola rosea, 6 parts of Trichosanthes kirilowii seed, 2 parts of bamboo shavings, and 4 parts of Hundred-Tael Gold.

[0056] The drug also includes excipients: 24 parts of binder, 10 parts of antioxidant, and 18 parts of solubilizer.

[0057] The adhesive is composed of sodium carboxymethyl cellulose and polyvinylpyrrolidone in a mass ratio of 2:1.

[0058] The antioxidant is composed of gallic acid, di-tert-butyl-p-cresol, and vitamin E, with a mass ratio of gallic acid, di-tert-butyl-p-cresol, and vitamin E of 0.5:3.5:1.

[0059] The co-solvent is composed of polyoxyethylene castor oil and dimethyl sulfoxide, with a mass ratio of polyoxyethylene castor oil to dimethyl sulfoxide of 8:1.

[0060] The preparation of the pharmaceutical composition includes the following steps:

[0061] (1) Take the following ingredients by weight: Alisma, Poria cocos, Astragalus membranaceus, Rheum palmatum, Curcuma longa, Pinellia ternata, Rhodiola rosea, Trichosanthes kirilowii seed, Bambusa textilis, and Lysimachia christinae. Soak them in water for 5 hours, rinse them 5 times, and let them dry.

[0062] (2) After crushing the raw materials obtained in step (1), pass them through an 80-mesh sieve and mix the raw materials together;

[0063] (3) Add water to the mixed raw materials and decoct. Add 12 times the amount of water and decoct for 2.5 hours. Filter to obtain the first extract. Add 8 times the amount of water and decoct for 1.5 hours. Filter to obtain the second extract. Combine the two extracts.

[0064] (4) The mixed extract was concentrated at 60°C to a relative density of 1.2-1.22, cooled, dried, pulverized into fine powder, and sterilized at high temperature to obtain the dry powder of the traditional Chinese medicine composition.

[0065] (5) Add binder, antioxidant and solubilizer to the dry powder of traditional Chinese medicine composition and mix well to prepare medicine.

[0066] Example 2

[0067] The medicine is prepared from the following raw materials in parts by weight: 2 parts of Ali Red, 6 parts of Poria, 15 parts of Astragalus, 0.1 parts of Prepared Rhubarb, 3 parts of Curcuma, 9 parts of Prepared Pinellia, 1 part of Rhodiola Rosea, 5 parts of Trichosanthes Seed, 1 part of Bambusa textilis, and 2 parts of Hundred-Taels Gold.

[0068] The drug also includes excipients: 18 parts of adhesive, 12 parts of antioxidant, and 14 parts of solubilizer.

[0069] The adhesive is composed of sodium carboxymethyl cellulose and polyvinylpyrrolidone in a mass ratio of 3.5:1.

[0070] The antioxidant is composed of gallic acid, di-tert-butyl-p-cresol, and vitamin E, with a mass ratio of gallic acid, di-tert-butyl-p-cresol, and vitamin E of 1:2:1.

[0071] The co-solvent is composed of polyoxyethylene castor oil and dimethyl sulfoxide, with a mass ratio of polyoxyethylene castor oil to dimethyl sulfoxide of 6:1.

[0072] The preparation method of the pharmaceutical composition is the same as in Example 1.

[0073] Example 3

[0074] The drug is prepared from the following raw and auxiliary materials in parts by weight: 3 parts of Ali Red, 8 parts of Poria cocos, 20 parts of Astragalus membranaceus, 1 part of processed rhubarb, 5 parts of turmeric, 11 parts of processed Pinellia ternata, 4 parts of Rhodiola rosea, 7 parts of Trichosanthes kirilowii seed, 2 parts of bamboo shavings, and 5 parts of Hundred-Tael Gold.

[0075] The drug also includes excipients: 28 parts of binder, 16 parts of antioxidant, and 20 parts of solubilizer.

[0076] The adhesive is composed of sodium carboxymethyl cellulose and polyvinylpyrrolidone in a mass ratio of 6:1.

[0077] The antioxidant is composed of gallic acid, di-tert-butyl-p-cresol, and vitamin E, with a mass ratio of 2:5:1.

[0078] The co-solvent is composed of polyoxyethylene castor oil and dimethyl sulfoxide, with a mass ratio of polyoxyethylene castor oil to dimethyl sulfoxide of 9:1.

[0079] The preparation method of the pharmaceutical composition is the same as in Example 1.

[0080] Example 4

[0081] The raw materials and excipients of the drug in Example 4 are the same as those in Example 1.

[0082] The preparation of the pharmaceutical composition includes the following steps:

[0083] (1) Take the following ingredients by weight: Alisma, Poria cocos, Astragalus membranaceus, Rheum palmatum, Curcuma longa, Pinellia ternata, Rhodiola rosea, Trichosanthes kirilowii seed, Bambusa textilis, and Lysimachia christinae. Soak them in water for 5 hours, rinse them 5 times, and let them air dry.

[0084] (2) After crushing the raw materials obtained in step (1), pass them through an 80-mesh sieve and mix the raw materials together;

[0085] (3) Add water to the mixed raw materials and decoct. Add 8 times the amount of water and decoct for 2 hours. Filter to obtain the first extract. Add 10 times the amount of water and decoct for 2 hours. Filter to obtain the second extract. Combine the two extracts.

[0086] (4) The mixed extract was concentrated at 60°C to a relative density of 1.2-1.22, cooled, dried, pulverized into fine powder, and sterilized at high temperature to obtain the dry powder of the traditional Chinese medicine composition.

[0087] (5) Add binder, antioxidant and solubilizer to the dry powder of traditional Chinese medicine composition and mix well to prepare medicine.

[0088] Comparative Example 1

[0089] The difference from Example 1 lies in the raw materials in the pharmaceutical composition; the excipients are the same as in Example 1. The raw materials in Comparative Example 1 are:

[0090] Ali Red 2 parts, Astragalus 18 parts, Pinellia ternata 10 parts, Trichosanthes kirilowii 6 parts, and Hundred-Tael Gold 4 parts.

[0091] The preparation of the pharmaceutical composition includes the following steps:

[0092] (1) Take the following weight proportions of Alihong, Huangqi, Fabanxia, ​​Gualouren, and Bailiangjin, soak them in water for 5 hours, take them out and rinse them 5 times, and dry them for later use.

[0093] (2) After crushing the raw materials obtained in step (1), pass them through an 80-mesh sieve and mix the raw materials together;

[0094] (3) Add water to the mixed raw materials and decoct. Add 12 times the amount of water and decoct for 2.5 hours. Filter to obtain the first extract. Add 8 times the amount of water and decoct for 1.5 hours. Filter to obtain the second extract. Combine the two extracts.

[0095] (4) The mixed extract was concentrated at 60°C to a relative density of 1.2-1.22, cooled, dried, pulverized into fine powder, and sterilized at high temperature to obtain the dry powder of the traditional Chinese medicine composition.

[0096] (5) Add binder, antioxidant and solubilizer to the dry powder of traditional Chinese medicine composition and mix well to prepare medicine.

[0097] Comparative Example 2

[0098] The difference from Example 1 lies in the raw materials in the pharmaceutical composition; the excipients are the same as in Example 1, while the raw materials in Comparative Example 2 are:

[0099] Prepared rhubarb 0.5 parts, turmeric 4 parts, poria 7 parts, rhodiola rosea 2 parts, bamboo shavings 2 parts.

[0100] The preparation of the pharmaceutical composition includes the following steps:

[0101] (1) Take the following weight proportions of prepared rhubarb, turmeric, poria cocos, rhodiola rosea and bamboo shavings, soak them in water for 5 hours, take them out and rinse them 5 times, and dry them for later use.

[0102] (2) After crushing the raw materials obtained in step (1), pass them through an 80-mesh sieve and mix the raw materials together;

[0103] (3) Add water to the mixed raw materials and decoct. Add 12 times the amount of water and decoct for 2.5 hours. Filter to obtain the first extract. Add 8 times the amount of water and decoct for 1.5 hours. Filter to obtain the second extract. Combine the two extracts.

[0104] (4) The mixed extract was concentrated at 60°C to a relative density of 1.2-1.22, cooled, dried, pulverized into fine powder, and sterilized at high temperature to obtain the dry powder of the traditional Chinese medicine composition.

[0105] (5) Add binder, antioxidant and solubilizer to the dry powder of traditional Chinese medicine composition and mix well to prepare medicine.

[0106] Comparative Example 3

[0107] The only difference from Example 1 is that the raw materials of the drug are different. The raw materials of Comparative Example 3 are: 5 parts of Ali Red, 15 parts of Poria cocos, 30 parts of Astragalus membranaceus, 3 parts of processed rhubarb, 6 parts of turmeric, 14 parts of processed Pinellia ternata, 8 parts of Rhodiola rosea, 10 parts of Trichosanthes kirilowii seed, 4 parts of bamboo shavings, and 8 parts of Lysimachia christinae. The excipients are the same as those in Example 1, and the preparation method is the same as that in Example 1.

[0108] Comparative Example 4

[0109] Compared with Example 1, the only difference is the type of adhesive; everything else is the same.

[0110] The adhesive is composed of starch and hydroxypropyl methylcellulose in a mass ratio of 2:1.

[0111] Comparative Example 5

[0112] The antioxidant is composed of butylated hydroxyanisole, α-tocopherol and vitamin C, with a mass ratio of butylated hydroxyanisole, α-tocopherol and vitamin E of 0.5:3.5:1.

[0113] Comparative Example 6

[0114] The cosolvent is composed of Tween 80 and propylene glycol, with a mass ratio of Tween 80 to propylene glycol of 8:1.

[0115] Comparative Example 7

[0116] Compared with Example 1, the only difference is that the mass ratio of the adhesive is different; in Comparative Example 7, the mass ratio of sodium carboxymethyl cellulose and polyvinylpyrrolidone is 11:1.

[0117] Comparative Example 8

[0118] Compared with Example 1, the only difference is that the mass ratio of antioxidants is different. In Comparative Example 8, the mass ratio of gallic acid, di-tert-butyl-p-cresol, and vitamin E is 3:6:1.

[0119] Comparative Example 9

[0120] Compared with Example 1, the only difference is the mass ratio of the cosolvent. In Comparative Example 9, the mass ratio of polyoxyethylene castor oil to dimethyl sulfoxide is 17:1.

[0121] Example 1: Effect of the drug composition on obstructive renal fibrosis

[0122] 1 Experimental Methods

[0123] 1.1 Grouping of experimental animals

[0124] Ninety 8-week-old male C57BL / 6 mice (20-25g) were randomly divided into 15 groups of 6 mice each: sham-operated group, model group, Examples 1-4, and Comparative Examples 1-9. The experimental animals were raised in a 12h:12h environment to simulate day and night changes, at a temperature of 20±2℃ and a humidity of 50±5%. The experiment began after one week of acclimatization.

[0125] 1.2 Establishment of a mouse model of UUO-induced obstructive renal fibrosis

[0126] Mice were injected intraperitoneally with 3% pentobarbital (80 mg / kg) based on their body weight. Mice were then fixed to a board, and the hair on the left dorsal side of the mouse was removed and disinfected. A dotted line was drawn along the left thigh root towards the spine to identify the area containing the lower nephron. The skin in the lower middle part of the mouse's back was cut open to expose the left kidney and its lower nephron, revealing the left ureter. The left ureter was lifted and ligated using absorbable sutures. The ureter was then sharply severed between the two ligatures to prevent retrograde urinary tract infection. Postoperatively, each mouse's wound was rinsed daily with 10,000 units of penicillin. The mice were then sutured. All mice received a subcutaneous injection of 0.5 ml of physiological saline for rehydration postoperatively. In the sham-operated group, the left ureter was isolated but not ligated; the remaining procedures were the same as in the model group. The procedures for Examples 1-4 and Comparative Examples 1-9 were the same as in the model group.

[0127] On the first day after surgery, the drugs prepared in Examples 1-4 and Comparative Examples 1-9 were administered by gavage at a dose of 2 mg / kg / day. The drugs were dissolved in 2 ml of physiological saline. The sham surgery group and the model group were administered the same dose of physiological saline by gavage for 14 consecutive days.

[0128] Three mice were harvested on the third day post-surgery. Blood was collected from the orbital plexus, and the left kidney was divided into two portions. One portion was fixed in 4% paraformaldehyde. On the 14th day post-surgery, the mice were weighed and blood was collected through the retro-orbital venous plexus. All mice were then sacrificed, and the left kidney was removed and divided into two portions. The kidneys were washed with PBS, one portion was rapidly frozen in liquid nitrogen and stored at -80°C for later use, and the other portion was fixed in 4% paraformaldehyde.

[0129] 1.3 Detection Indicators

[0130] 1.3.1 Kidney weight / body weight ratio

[0131] Kidney weight / body weight ratio = kidney weight / mouse body weight × 100%.

[0132] 1.3.2 Masson staining

[0133] (1) The left kidney tissue was fixed with 4% paraformaldehyde for 24 hours at room temperature;

[0134] (2) After successful fixation, the tissue was removed and placed in an embedding box. It was then rinsed with tap water until there was no strong formaldehyde odor.

[0135] (3) Perform dehydration, clearing, and paraffin embedding according to the following procedure:

[0136] Pure water 30 min - 75% ethanol I 30 min - 75% ethanol II 30 min - 75% ethanol III 30 min - 85% ethanol 10 min - 95% ethanol 10 min - anhydrous ethanol I 10 min - anhydrous ethanol II 10 min - xylene I 10 min - xylene II 10 min - paraffin I 30 min - paraffin II 60 min - embed in a paraffin embedding machine and set aside for later use.

[0137] (4) Paraffin sections:

[0138] The microtome thickness parameter was adjusted to 4μm. The paraffin-embedded kidney tissue was placed and fixed in place. The tissue was manually sectioned. The tissue section was flattened in 45℃ warm water. The tissue section was then carefully and completely attached to the glass slide. Finally, the glass slide was placed in a 60℃ slide oven for 1 hour. After removal, it was stored at room temperature.

[0139] (5) Take 4μm tissue sections and dewax them;

[0140] (6) Place in Bouin solution at 4°C overnight, then rinse with water for 10 min;

[0141] (7) Apply azurite blue for 3 minutes, then rinse with water for 2 minutes;

[0142] (8) Stain with hematoxylin for 2 min, then rinse with water for 5 min;

[0143] (9) Differentiate with acidic ethanol for 10 seconds, then rinse with water for 10 minutes;

[0144] (10) Sautéed Ponted Red for 8 minutes, then rinsed with water for 3 minutes;

[0145] (11) Phosphomolybdic acid solution for 10 min, discard the supernatant, add aniline blue for 5 min, weak acid solution for 2 min, mount the slide, observe under a microscope, use Masson staining to stained the slide, randomly select 5 different renal interstitial regions at 200x magnification, use Image-Pro Plus 6.0 software to measure the percentage of collagen fiber area to total area in the selected region, and take the average value to represent the degree of fibrosis of the slide.

[0146] 1.3.3 Detection of ADAMTS-18 gene expression level

[0147] Kidney tissue was collected, ground into powder, and RNA was extracted using an RNA extraction kit (purchased from Tiangen Biotech Co., Ltd.). The RNA was then reverse transcribed into cDNA. Using the cDNA as a template, RT-PCR (PCR kit purchased from Thermo Fisher Scientific, USA) was performed to detect the relative expression level of the ADAMTS-18 gene. Primers were designed by Sangon Biotech Co., Ltd. GAPDH was used as a reference gene to analyze the expression level of ADAMTS-18 gene mRNA.

[0148] The PCR reaction conditions are as follows:

[0149] 40 cycles × (95℃, 30s; 60℃, 34s; 70℃, 10s; 95℃, 15s).

[0150] The primer sequences are as follows:

[0151] Table 1

[0152]

[0153] 1.3.4 Immunohistochemistry

[0154] Add 1000 μL of lysis buffer to every 100 mg of kidney tissue. Quickly mince the tissue with ophthalmic scissors on ice, homogenize three times, and incubate on ice for 30 min. Centrifuge at 12000 g, 4°C for 20 min, and collect the supernatant to obtain the total tissue protein product. Dilute the protein sample 10-fold and determine the protein concentration using the BCA method. Equalize the protein concentration of each group with distilled water, add 2× loading buffer, mix well, seal with sealing film, boil in water for 10 min, and then incubate on ice for 5 min. Aliquot according to the protein concentration and the amount of protein required for each electrophoresis, and store at 80°C. After separating the protein sample by SDSPAGE electrophoresis (20-30 μg), transfer the protein to a PVDF membrane using the Western blotting method. Block the membrane with blocking buffer containing 5% skim milk powder at room temperature on a shaker for 2 h. The membrane was washed with TBST blocking buffer, then incubated with primary antibody at a dilution ratio of GAPDH (1:4000) and FN (1:1000) for 2 hours on a shaker at room temperature. The membrane was then washed three times with TBST buffer for 10 minutes each time. The membrane was then incubated with the corresponding horseradish peroxidase (HRP)-labeled secondary antibody for 1.5 hours at room temperature, followed by three washes with TBST buffer for 10 minutes each time. Then, 200 μL each of reagents A and B from the chemiluminescent substrate kit were mixed and quickly placed on a PVDF membrane. The membrane emitted light, and after development, the optical density was analyzed using an ImageQuant LAS4000 gel imaging system to calculate the average optical density.

[0155] 1.4 Statistical Methods

[0156] Statistical analysis was performed on all data using SPSS 26.0. Quantitative data are expressed as mean ± standard deviation. The comparison of data among multiple groups was performed using one-way ANOVA, and P < 0.05 indicated that the difference was statistically significant.

[0157] 2 Experimental Results

[0158] 2.1 Kidney weight / body weight ratio

[0159] Table 2 Kidney weight / body weight ratio of mice in each group

[0160] Group Kidney weight / body weight ratio (mg / g) Sham surgery group 4.8±0.3 Model group 14.6±1.1** Example 1 Group 5.1±0.5## Example 2 group 5.4±0.3## Example 3 Group 6.0±0.2## Example 4 group 5.7±0.6## Comparative Example 1 12.5±1.3 Comparative Example 2 14.1±1.1 Comparative Example 3 Groups 11.6±1.6 Comparative Example 4 Groups 13.1±1.9 Comparative Example 5 Groups 13.6±1.5 Comparative Example 6 Groups 12.6±1.7 Comparative Example 7 Groups 12.9±1.6 Comparative Example 8 Groups 13.2±2.1 Comparative Example 9 Groups 13.7±1.8

[0161] Note: Compared with the sham surgery group, **p<0.01; compared with the model group, ##p<0.01; only significant differences between the example group and the comparative group and the model group are indicated.

[0162] As can be seen from the table above, the kidney weight / body weight ratio of the model group mice was significantly higher than that of the sham-operated group. Compared with the model group, administration of the drugs prepared in Examples 1-4 could significantly increase the kidney weight / body weight ratio of the mice, while there was no significant increase in the comparative groups 1-9.

[0163] 2.2 Masson staining

[0164] Masson staining was used to assess fibrosis. The Masson staining results are as follows: Figure 1 As shown in the Masson staining results, the obstructive renal fibrosis model was successfully constructed. The drug prepared in Example 1 had a certain alleviating effect on obstructive renal fibrosis in mice. However, in Comparative Examples 1-9, by adjusting the raw materials or excipients of the drug, or by making the ratio of raw materials and excipients outside the scope of protection of this invention, the improvement effect on obstructive renal fibrosis was small, and there was no significant difference compared with the model group.

[0165] 2.3 ADAMTS-18 gene expression level

[0166] The relative expression levels of ADAMTS-18 mRNA in each group of mice are shown in the table below. It can be seen that, compared with the sham-operated group, the relative expression level of ADAMTS-18 mRNA in the model group was significantly downregulated. The drugs administered to groups 1-4 in Examples 1-4 were able to effectively alleviate the downregulation trend, and the differences were statistically significant. Although groups 1-9 in Comparative Examples 1-9 could upregulate the relative expression level of ADAMTS-18 mRNA to some extent, it was not statistically significant (p>0.05).

[0167] Table 3. Relative expression levels of ADAMTS-18 mRNA in mice of each group

[0168]

[0169] Note: Compared with the sham surgery group, **p<0.01; compared with the model group, ##p<0.01; only significant differences between the example group and the comparative group and the model group are indicated.

[0170] 2.4 Immunohistochemistry

[0171] The experimental results are shown in Table 4 below. As can be seen from the table, compared with the sham surgery group, the expression level of the fibrotic molecule FN in the model group was significantly increased, while the expression of FN was significantly reduced after drug intervention in the groups of Examples 1-4.

[0172] Table 4

[0173] Group FN average optical density Sham surgery group 7.3±0.9 Model group 20.1±4.1*** Example 1 Group 9.6±1.3## Example 2 group 10.3±2.4# Example 3 Group 11.5±1.9# Example 4 group 10.1±2.8# Comparative Example 1 18.3±3.9 Comparative Example 2 17.6±4.6 Comparative Example 3 Groups 15.4±3.3 Comparative Example 4 Groups 16.9±4.6 Comparative Example 5 Groups 15.2±5.2 Comparative Example 6 Groups 16.1±2.9 Comparative Example 7 Groups 14.8±3.7 Comparative Example 8 Groups 15.6±4.4 Comparative Example 9 Groups 16.2±5.1

[0174] Note: Compared with the sham surgery group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01; the example group and the comparative example group only indicate the significant differences from the model group.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A drug for treating obstructive renal fibrosis, wherein the drug is made from the following raw and excipient materials in parts by weight: 1-3 parts of Alihong, 5-10 parts of Poria cocos, 15-25 parts of Astragalus membranaceus, 0.1-2 parts of processed rhubarb, 1-5 parts of turmeric, 8-12 parts of processed Pinellia ternata, 1-6 parts of Rhodiola rosea, 5-8 parts of Trichosanthes kirilowii seed, 1-3 parts of bamboo shavings, 1-6 parts of Lysimachia christinae, 10-30 parts of binder, 5-20 parts of antioxidant, and 10-25 parts of solubilizer.

2. The drug according to claim 1, characterized in that, The drug is made from the following raw and auxiliary materials in parts by weight: 2-3 parts of Ali Red, 6-8 parts of Poria cocos, 15-20 parts of Astragalus membranaceus, 0.1-1 parts of processed rhubarb, 3-5 parts of turmeric, 9-11 parts of processed Pinellia ternata, 1-4 parts of Rhodiola rosea, 5-7 parts of Trichosanthes kirilowii seed, 1-2 parts of bamboo shavings, 2-5 parts of Lysimachia christinae, 18-28 parts of binder, 10-16 parts of antioxidant, and 14-20 parts of solubilizer.

3. The drug according to claim 2, characterized in that, The drug is made from the following raw and auxiliary materials in parts by weight: 2 parts of Ali Red, 7 parts of Poria cocos, 18 parts of Astragalus membranaceus, 0.5 parts of processed rhubarb, 4 parts of turmeric, 10 parts of processed Pinellia ternata, 2 parts of Rhodiola rosea, 6 parts of Trichosanthes kirilowii seed, 2 parts of bamboo shavings, 4 parts of Lysimachia christinae, 24 parts of binder, 10 parts of antioxidant, and 18 parts of solubilizer.

4. The drug according to any one of claims 1-3, characterized in that, The adhesive is composed of sodium carboxymethyl cellulose and polyvinylpyrrolidone, the antioxidant is composed of gallic acid, di-tert-butyl-p-cresol and vitamin E, and the cosolvent is composed of polyoxyethylene castor oil and dimethyl sulfoxide.

5. The drug according to claim 4, characterized in that, The mass ratio of sodium carboxymethyl cellulose to polyvinylpyrrolidone is (1-10):

1.

6. The drug according to claim 4, characterized in that, The mass ratio of gallic acid, di-tert-butyl-p-cresol, and vitamin E is (0.2-2):(1-5):

1.

7. The drug according to claim 4, characterized in that, The mass ratio of the polyoxyethylene castor oil to dimethyl sulfoxide is (5-15):

1.

8. A method for preparing the drug according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Take the following weight proportions of Alihong, Poria cocos, Astragalus membranaceus, processed rhubarb, turmeric, processed Pinellia ternata, Rhodiola rosea, Trichosanthes kirilowii seed, bamboo shavings, and Hundred-tael gold, add water and decoct. The number of decoctions is 1-3 times, each time for 1-5 hours. Filter and combine the extracts. (2) Concentrate the combined extracts to a relative density of 1.2-1.5, cool, dry, and sterilize to obtain a dry powder of the traditional Chinese medicine composition; (3) Add binder, antioxidant and solubilizer to the dry powder of traditional Chinese medicine composition and mix well to prepare medicine.

9. The use of the medicament according to any one of claims 1-7 in the preparation of a product for treating obstructive renal fibrosis.

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