Application of JSH-23 in preparation of medicine for improving renal dysfunction and / or inhibiting renal fibrosis

Abstract

The invention provides application of JSH-23 in preparation of drugs for improving renal dysfunction and / or inhibiting renal fibrosis, and relates to the technical field of drugs for chronic kidney diseases. According to the invention, JSH-23JSH-23 with small molecular weight can significantly reduce serum creatinine (Scr) and urea nitrogen (BUN) levels and improve renal dysfunction; the expression of fibrosis markers such as alpha-SMA (alpha-smooth muscle actin), Col1a1 (alpha-1 type collagen), FN1 (fibronectin 1) and the like can be effectively down-regulated, so that the renal fibrosis progress is effectively blocked, and the renal fibrosis process is effectively relieved. A new candidate medicine is provided for long-term treatment of the chronic kidney disease, and the medicine has wide scientific research value and industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for chronic kidney disease, and particularly to the use of JSH-23 in the preparation of drugs for improving renal dysfunction and / or inhibiting renal fibrosis. Background Technology

[0002] Chronic kidney disease (CKD) is a major health problem posing a serious threat to public health worldwide. Renal fibrosis is a key pathological mechanism in the progression of CKD to end-stage renal disease (ESRD), characterized by excessive deposition of extracellular matrix in the renal tubules and interstitium, ultimately leading to irreversible and progressive decline in renal function. Currently, there are no effective means to reverse or slow the progression of renal fibrosis. Therefore, in-depth exploration of its pathogenesis and the search for new therapeutic targets have significant clinical value and practical implications.

[0003] Current treatments for chronic kidney disease and renal fibrosis primarily rely on angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), glucocorticoids, or immunosuppressants to slow disease progression. However, these alternatives still have the following significant shortcomings: 1. Limited therapeutic effect: Traditional drugs mainly delay the deterioration of kidney function by regulating blood pressure, reducing proteinuria, or suppressing immune responses, but they cannot fundamentally inhibit renal interstitial fibrosis and abnormal extracellular matrix deposition, resulting in unstable efficacy.

[0004] 2. Significant side effects: Long-term use of hormones and immunosuppressants can cause adverse reactions such as infection, osteoporosis, and hyperglycemia. ACEI / ARB drugs can easily lead to hyperkalemia, hypotension, and further decline in renal function.

[0005] 3. Poor drug tolerance and adherence: Existing medications require long-term oral administration or combination therapy, resulting in poor patient compliance, and some patients have tolerance issues with ACEI / ARB drugs.

[0006] 4. Cost and accessibility issues: Imported immunosuppressants or novel antifibrotic drugs (such as pirfenidone and bexarotine) are expensive, and inadequate medical insurance coverage in some areas limits their large-scale clinical application.

[0007] Although the aforementioned drugs or treatments can delay the deterioration of kidney function to some extent, they still have significant limitations in inhibiting renal fibrosis, improving tissue structure, and protecting kidney function.

[0008] JSH-23 is a small molecule NF-κB inhibitor with the following chemical structure: It can inhibit the transcriptional activity of NF-κB p65 subunit by blocking its entry into the nucleus, thereby reducing the expression of downstream inflammatory mediators. However, there are no reports on the application of JSH-23 in chronic kidney disease and renal fibrosis, nor has its mechanism of action been revealed in improving renal dysfunction by reducing serum creatinine (Scr) and blood urea nitrogen (BUN) levels, or in inhibiting renal interstitial fibrosis by regulating fibrosis markers such as α-SMA, Col1a1, and FN1. Summary of the Invention

[0009] In view of this, the present invention proposes the application of JSH-23 in the preparation of drugs for improving renal dysfunction and / or inhibiting renal fibrosis. This invention is the first to discover that JSH-23 can exert a significant renal protective effect by improving renal dysfunction, inhibiting renal tubular damage, and reducing interstitial fibrosis, demonstrating its novel application and innovation in the prevention and treatment of chronic kidney disease.

[0010] The technical solution of this invention is implemented as follows: Application of JSH-23 in the preparation of drugs to improve renal dysfunction and / or inhibit renal fibrosis.

[0011] Furthermore, the JSH-23 is used in the preparation of drugs that downregulate the expression of α-SMA, Col1a1, and FN1.

[0012] Furthermore, the application of JSH-23 in the preparation of expression regulators that downregulate α-SMA, Col1a1, and FN1.

[0013] Furthermore, the use of JSH-23 in the preparation of drugs that lower serum creatinine and / or blood urea nitrogen levels.

[0014] Furthermore, the use of JSH-23 in the preparation of regulators that lower serum creatinine and / or blood urea nitrogen levels.

[0015] Furthermore, the drug uses JSH-23 as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0016] Furthermore, the excipient is one of solid, liquid, or semi-solid excipients.

[0017] Furthermore, the excipients include, but are not limited to, polyethylene glycol (PEG), sodium carboxymethyl starch, hydroxypropyl methylcellulose (HPMC), lactose, starch, glycerol, and other pharmaceutically acceptable carriers or solvents.

[0018] Furthermore, the dosage form includes one or more of tablets, capsules, injections, oral solutions, pellets, or sustained-release formulations.

[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention uses JSH-23 as a regulator, which can effectively reduce pathological damage to renal tissue by improving renal function disorders, inhibiting renal tubular damage and reducing interstitial fibrosis, thereby exerting a significant renal protective effect.

[0020] 2. Unlike conventional antifibrotic drugs (such as pirfenidone, ACEI / ARBs) which mainly work through the TGF-β or RAAS pathway, JSH-23 of this invention can significantly reduce serum creatinine (Scr) and blood urea nitrogen (BUN) levels, improving renal dysfunction; it can effectively downregulate α-SMA (α-smooth muscle actin) and Col1a1 (α-smooth muscle actin). α-1 type collagen The expression of fibrosis markers such as FN1 (fibronectin 1) can effectively block the progression of renal fibrosis and alleviate the process of renal fibrosis.

[0021] 3. JSH-23 can be formulated into a variety of pharmaceutically acceptable dosage forms, including tablets, capsules, injections, pellets, and oral solutions; the carrier can be a solid, liquid, or semi-solid excipient system to achieve stable release and good bioavailability.

[0022] 4. This invention effectively improves renal structure and function through a multi-target pathway, enhancing efficacy while significantly reducing toxic side effects and costs. JSH-23 possesses characteristics such as small molecular weight, well-defined metabolic properties, strong oral feasibility, a favorable pharmacokinetic safety window, and ease of formulation. It can be administered orally or by injection, demonstrating feasibility and industrialization potential as a treatment for chronic kidney disease. The novel applications disclosed in this invention provide new drug candidates for the long-term treatment of chronic kidney disease, extending to targeted delivery formulations, sustained-release formulations, and combination therapy systems.

[0023] 5. This invention can take advantage of the fact that JSH-23 is orally available, easily modified, and can be formulated into sustained-release or targeted delivery formulations, which can provide new treatment options for patients with chronic kidney disease who need to take medication for a long time. It can also be further expanded into drug combination therapy, oral formulation platform or functional medical products, and has broad scientific research value and industrial application prospects. Attached Figure Description

[0024] Figure 1Serum Scr and BUN levels in mice of each group; where **P<0.01, ***P<0.001, ****P<0.0001; Control group was blank group, UUO group was model group, JSH-23 5mg / kg was low-dose group, and JSH-23 10mg / kg was high-dose group; Scr represents creatinine, and BUN represents blood urea nitrogen; data are expressed as mean ± standard deviation.

[0025] Figure 2 The transcriptional expression levels of α-SMA, Col1a1, and fibrobectin (FN), indicators of fibrosis in mouse renal tissue, were detected by qPCR in each group. *P<0.05, **P<0.01, ****P<0.0001; Control group was the blank group, UUO group was the model group, JSH-23 5mg / kg was the low-dose group, and JSH-23 10mg / kg was the high-dose group; ACTA2 represents smooth muscle actin α2 gene, FN1 represents fibronectin 1, and Col1a1 represents... α-1 type collagen; data are expressed as mean ± standard deviation.

[0026] Figure 3 Sirius red and Masson staining were used to observe the degree of renal fibrosis in each group. The Control group was the blank group, the UUO group was the model group, JSH-23 5mg / kg was the low-dose group, and JSH-23 10mg / kg was the high-dose group. Detailed Implementation

[0027] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0030] The main experimental reagents and instruments involved in this invention are as follows: 1. The main reagents used in the experiment are shown in Table 1 below.

[0031] Table 1 Main reagents for the experiment

[0032] 2. The main instruments used in the experiment are shown in Table 2 below.

[0033] Table 2 Main Instruments for the Experiment

[0034] 3. The preparation methods for the main reagents are as follows: 3.1 Drug-related JSH-23 Dosage Solution: Weigh JSH-23 powder and dissolve it in 20% PEG-400 (the remainder being sterile water or 0.9% NaCl). If necessary, add ≤5% DMSO to assist dissolution. The solution should be protected from light and stored at 4 ℃ for a short period. Dilute with 0.9% NaCl to the working solution half an hour before administration (prepare and use immediately after preparation). Working solution concentration: 5 mg / kg for low dose; 10 mg / kg for high dose.

[0035] 3.2 General Liquids / Reagents for Surgery and Sampling (1) PBS stock solution (10X): Add 8g of sodium chloride, 0.2g of potassium chloride, 0.2g of dipotassium hydrogen phosphate and 2.08g of sodium dihydrogen phosphate to ultrapure water, mix and bring the volume to 100mL.

[0036] (2) PBS working solution (1X): Dilute 10XPBS storage solution with ultrapure water at a ratio of 1:9 to make 1XPBS working solution, adjust the pH to 7.2, place in a glass bottle, and sterilize at high temperature and high pressure for later use.

[0037] (3) 4% Paraformaldehyde: Add 4g of paraformaldehyde to 100mL of 1×PBS solution, along with a few drops of NaOH. Heat at 60℃ in a fume hood to dissolve, cool to room temperature, adjust the pH of the solution to 7.4 with HCl, and dispense into separate containers. Keep in a refrigerator at 20℃.

[0038] (4) 10% neutral formalin solution (pH 7.0): 4g of sodium dihydrogen phosphate and 6.5g of disodium hydrogen phosphate are added to 100mL of formaldehyde and 900mL of water.

[0039] 3.3 Masson staining, Sirius red staining and histology (1) Hematoxylin solution (3000mL): Add 6g of hematoxylin to 100mL of anhydrous ethanol and 150g of potassium aluminum sulfate to 2000mL of distilled water. After dissolving, add glycerol, sodium iodate and glacial acetic acid to mix and dissolve. After mixing, place in a light and ventilated place for 3 weeks until it turns dark red, then it can be used.

[0040] (2) Mason-alcohol solution: Add 0.5-1g of mason to distilled water and glacial acetic acid, filter with filter paper, dry and add 100mL of anhydrous ethanol.

[0041] (3) 0.2% glacial acetic acid aqueous solution: 0.2 mL of glacial acetic acid is added to 100 mL of distilled water.

[0042] (4) 0.5% glacial acetic acid aqueous solution: Add 0.5 mL of glacial acetic acid to distilled water and bring the volume to 100 mL.

[0043] (5) Preparation of 60% isopropanol: Add 100% isopropanol to 40mL of distilled water and store at 4℃ for later use.

[0044] (6) 1% phosphomolybdic acid aqueous solution: 1g of phosphomolybdic acid is added to 100mL of distilled water.

[0045] (7) 1% chlorophyll solution: Add 1g of chlorophyll to 100mL of distilled water.

[0046] (8) 0.1% Sirius Red-Picric Acid Solution: Weigh 1g of Sirius Red F3B (Direct Red 80), add it to 1000mL of saturated picric acid solution, stir thoroughly until completely dissolved, filter to remove insoluble matter, and store in a brown reagent bottle protected from light. This solution can be stored at room temperature for 1-2 months. When using, directly immerse the slides in this staining solution for staining.

[0047] 3.4 UUO Surgical and Nursing Reagents / Medications (1) Skin disinfection: 70% ethanol (anhydrous ethanol and sterile water 7:3); povidone-iodine (commercial preparation).

[0048] (2) Anesthesia: Inhalation of isoflurane (3–4% for induction, 1.5–2% for maintenance).

[0049] (3) Analgesia: meloxicam 1–2 mg / kg qd × 3 d.

[0050] (4) Postoperative fluid replacement: Preheat 0.9% NaCl 0.5–1 mL sc (5) Rinse / infuse: 1× PBS or 0.9% NaCl.

[0051] 3.5 Molecular biological mechanism verification experiments (1) DNA lysis buffer: Add 5 mL of 10% SDS, 10 mL of 0.5 M EDTA, 1 mL of 1 M Tris-HCl and 2 mL of 5 M NaCl to 82 mL of double-distilled water. (2) Preparation of DNase I stock solution: Dissolve DNase I dry powder (1500U) from the total RNA extraction kit in RNase-free double-distilled water (550μL), mix well, dispense into containers, and place in a container. Store in a refrigerator at 20℃ until needed.

[0052] Preparation of DNase I working solution: Place DNase I stock solution (the amount to be prepared depends on the number of samples) into a new RNase-free centrifuge tube and add RDD buffer.

[0053] 4. Statistical Analysis SPSS 22.0 statistical analysis software was used for data analysis. Quantitative data were expressed as mean ± standard deviation (Mean ± SD). The t-test was used for comparisons between two groups, and the one-test was used for comparisons among multiple groups. ANOVA was used to determine the statistical significance of a difference, with p < 0.05 considered statistically significant. All statistical results were plotted using GraphPad Prism 9.4.1 software.

[0054] Example 1 - Collection of Specimens 1. Preparation of JSH-23 drug solution sample JSH-23 drug solution: Weigh JSH-23 powder, prepare a stock solution with 20% PEG400 as solvent, and dilute with 0.9% physiological saline to the target drug solution concentration (5 mg / kg for low dose; 10 mg / kg for high dose) before gavage. Prepare and use immediately.

[0055] 2. Establishment and grouping of UUO mouse model Healthy male C57BL / 6J mice, weighing 20–25 g, were randomly divided into four groups after one week of acclimatization to establish a unilateral ureteral obstruction (UUO) model. The model was established as follows: under isoflurane anesthesia, an incision was made in the left flank of the mouse to expose the left kidney and ureter. Two ligations were made approximately 5 mm from the renal pelvis (with a ligation interval of approximately 2 mm), and the ureter was cut in the middle to completely block urine flow. In the sham-operated group, only the ureter was separated without ligation. After surgery, all mice underwent routine disinfection and were kept warm. The four groups of mice were as follows: (1) Control group: sham surgery, without ligation of the ureter; (2) UUO group: unilateral ureteral ligation; (3) UUO + JSH-23 low-dose group: unilateral ureteral ligation; (4) UUO + JSH-23 high-dose group: after unilateral ureteral ligation.

[0056] The low-dose UUO + JSH-23 group was administered JSH-23 solution at 5 mg / kg / day via gavage; the high-dose UUO + JSH-23 group was administered JSH-23 solution at 10 mg / kg / day via gavage. The control group and UUO group received the same volume of carrier solution. All groups were administered once daily (qd) for 10 consecutive days. 3. Blood sample collection and testing Mice were fully anesthetized by inhalation of isoflurane (3–4% for induction, 1.5–2% for maintenance). The scalp of the mouse's neck was grasped, and both sides of the neck were compressed until the mouse's eyes protruded and congestion of the retro-orbital venous plexus was visible. The eyeballs were quickly removed with forceps, and blood flowing from the orbit was collected in EP tubes to obtain blood samples. The mouse's head was then fixed, and the tail was grasped by the right hand near the mouse's body and pulled backward and upward, immediately causing cervical dislocation and death. Kidney tissue was immediately harvested. The remaining specimens were stored at -80°C. All test reagents were stored at 4°C.

[0057] Example 2 The blood samples collected in Example 1 were allowed to stand for 30 minutes, centrifuged at 3000 rpm for 20 minutes, and the supernatant serum was aliquoted. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured using a BS-240 fully automated biochemical analyzer. The results are as follows. Figure 1 As shown.

[0058] Depend on Figure 1 It can be seen that, compared with the UUO group mice, the use of JSH-23 drug in the low-dose and high-dose gavage groups of mice in this invention can significantly reduce the levels of Scr and BUN in mice, improve renal function disorder, and inhibit renal tubular damage. This indicates that JSH-23 drug can significantly improve renal function in UUO chronic kidney disease mice at both high and low doses (10 mg / kg and 5 mg / kg).

[0059] Example 3 - qPCR detection of target gene expression levels The cortical portions of the two hemi-kidney tissues obtained in Example 1 were placed in 1.5 mL dry EP tubes and stored at -80°C for molecular biological detection. The specific procedure is as follows: 3.1 Extraction of total RNA from tissues (1) Pre-cool the centrifuge to 4°C and place the prepared RNA lysis buffer on ice; use RNase-free tubes and pipette tips for all consumables; (2) Tissue samples: Kidney tissues from each group were taken out at -80℃ and frozen quickly in liquid nitrogen; after being pre-cooled in liquid nitrogen, the mortar and pestle were ground into powder. (3) Add lysis buffer to the ground tissue powder according to the reagent instructions, mix quickly by blowing and stirring, and let stand at room temperature for 5 minutes to fully lyse; (4) Add chloroform according to the reagent instructions, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes; (5) Centrifuge at 4℃ and 12000rpm for 15 minutes; transfer the upper aqueous phase to a new RNase-free centrifuge tube; (6) Add an equal volume of isopropanol and mix well. Let stand at room temperature for 10 minutes, then centrifuge at 4°C for 10 minutes to precipitate RNA. (7) Discard the supernatant, add 75% ethanol to wash the precipitate, centrifuge at 4°C for 5 minutes; discard the supernatant and air dry at room temperature for 3 minutes (avoid over-drying); (8) Add an appropriate amount of RNase-free water to dissolve the RNA, gently mix by pipetting, and place on ice for later use; (9) Cell samples: Discard the culture medium, wash once with PBS, add lysis buffer directly to lyse the cells, and after thorough pipetting, complete the subsequent extraction steps according to the instructions.

[0060] 3.2 Determination of tissue RNA concentration and purity (1) RNA concentration and purity were determined using NanoDrop or similar spectrophotometers (A260 / A280, A260 / A230). (2) Calculate the required volume based on the concentration and unify the input volume of each sample; if necessary, dilute with RNase-free water to the same concentration; (3) Optional: Take a portion of the sample for agarose gel electrophoresis or use a bioanalyzer to detect RNA integrity.

[0061] 3.3 Removal of DNA Contamination (1) Prepare the reaction system according to the instructions of the DNase I kit; (2) Incubate at 37℃ for 10–20 minutes; (3) Perform the termination reaction or purification according to the instructions to obtain gDNA-free RNA.

[0062] 3.4 Reverse transcription to synthesize cDNA (1) Prepare the system (RNA template, RT buffer, dNTPs, reverse transcriptase, primers Mix / oligo dT+ random primers, etc.) according to the instructions of the reverse transcription kit. (2) Set the reaction program according to the kit; (3) After the reaction is complete, store the cDNA at -20℃.

[0063] 3.5 Primer Design and Internal Reference Selection (1) Primers for the target gene and internal reference genes (ACTA2, FN1, Col1a1) were designed according to the specifications (exon crossing preferred), dissolved and prepared into working solution; among them, ACTA2 was used to represent the α-SMA index.

[0064] (2) Conduct preliminary experiments on primer specificity and amplification efficiency to ensure a single-peak melting curve and appropriate efficiency; (3) Maintain consistent primer batches and concentrations in the same experiment.

[0065] 3.6 qPCR reaction system preparation (1) Use the SYBR Green or TaqMan system and prepare the reaction system according to the kit instructions (example: 2×qPCRMix, forward and reverse primers, template cDNA, nuclease-free water); (2) Each sample was set up with 3 replicate wells; (3) Set up negative controls: template-free control (NTC); if necessary, set up a reverse transcription-free control (-RT) to exclude the influence of gDNA; (4) Add the reaction system to the qPCR plate / tube, seal the plate and centrifuge to remove air bubbles.

[0066] 3.7 qPCR amplification program (1) Place the reaction plate in a real-time quantitative PCR instrument; (2) Set amplification conditions according to reagent requirements (Example: 95℃ pre-denaturation; followed by 40 cycles: 95℃ denaturation, 60℃ annealing / extension; based on the results of reagent and primer optimization). (3) Melting curve analysis is required for the SYBR system to confirm amplification specificity.

[0067] 3.8 Development and Exposure Prepare the required high-sensitivity developer mixture in the dark, use it immediately after preparation, expose the image using the automatic exposure mode, and save the exposed image.

[0068] 3.9 Data Processing and Analysis (1) Record the Ct value of each hole, and take the average value of the technical repeats after removing abnormal holes; (2) Correction using internal reference gene: Calculate ΔCt = Ct(target gene) Ct (internal reference); (3) The relative expression level was calculated using the 2^-ΔΔCt method for intergroup comparison (the control group was set as the calibration sample); (4) Statistical analysis was performed using GraphPad Prism software. The results are expressed as mean ± standard deviation. ANOVA was selected according to the experimental design.

[0069] The results are as follows: Figure 2 As shown.

[0070] from Figure 2 As can be seen, compared with the UUO model group mice, the use of JSH-23 drug in the low-dose and high-dose gavage groups of mice in this invention can significantly inhibit the expression of fibrosis markers α-SMA, Col1a1 and fibrobectin (FN) in mice, thereby reducing extracellular matrix deposition in renal interstitial cells and alleviating the progression of renal fibrosis.

[0071] Example 4 - Hematoxylin in Kidney Tissue Morphological observation of Masson staining and Sirius red staining After collecting kidneys from mice in each group in Example 1, two kidneys were removed from each mouse using tissue scissors. The renal pedicle and capsule were thoroughly removed, and the kidneys were longitudinally cut open with a sharp blade. Half of the kidney tissue was fixed in 10% neutral formalin solution for 24–48 hours for Masson's staining and Sirius red staining. The specific procedure is as follows: 5.1 Preparation of kidney tissue paraffin blocks (1) Mouse kidney tissue fixed in 10% neutral formalin, incubated overnight at 4°C, and washed with PBS for 30 minutes × 3 times; (2) Dehydration: Take the fixed sample and place it in 50%, 70%, 85%, and 95% ethanol solutions for 60 minutes each, and then place it in 100% ethanol solution for 60 minutes twice; (3) Transparency: Dewaxing with xylene I and II for 30 minutes each; (4) Infiltration: at 62°C, place in 50% paraffin and 50% xylene for 90 minutes, then in paraffin I (50°C), paraffin II (60°C), and paraffin III (60°C) for 60 minutes each; (5) Embedding: Place in a paraffin embedding machine for embedding and use; (6) Sectioning: The paraffin-embedded block is sectioned and then placed on a drying machine to bake for 1 hour to prevent it from falling off, thus obtaining paraffin sections.

[0072] (7) The dried paraffin-embedded blocks from each group were divided into two sections, one for hematoxylin extraction of kidney tissue and the other for hematoxylin extraction. Masson staining and Sirius red staining tests.

[0073] 5.2 Hematoxylin in Kidney Tissue Masson staining (1) Dewax the paraffin slices in xylene in sequence, 5 minutes each time for 3 times, and then immerse them in 100% ethanol, 95% ethanol I, 95% ethanol II and 80% ethanol in sequence, immersing for 1 minute each time, and rinsing with tap water for 5 minutes. (2) Wash with PBS for 5 minutes × 3 times, add 100 μL of hematoxylin and stain for 10 minutes; (3) 75% hydrochloric acid ethanol decomposes in a few seconds; (4) Rinse with running tap water for about 10 minutes until the cell nuclei turn blue; (5) Rinse the tissue sections with double-distilled water for 15 minutes, then add 0.5% masson staining agent for about 1 minute; (6) Rinse with distilled water 1 2 seconds, gradient alcohol dehydration (80% ethanol for 5 seconds, 95% ethanol for 2 minutes, anhydrous ethanol for 2 minutes); (7) Rinse with tap water for about 10 minutes until the cell nuclei turn blue; (8) Xylene clears the film and seal it.

[0074] (9) Observe and record the experimental results under a microscope. The results are as follows: Figure 3 As shown.

[0075] 5.3 Sirius Red Staining (1) Take paraffin sections of mouse kidney tissue from each group, with a thickness of 5 μm, and bake the sections at 60 ℃ for 1 h.

[0076] (2) Dewax the slices twice in xylene for 5 minutes each time, then dehydrate them in 100% ethanol, 95% ethanol and 80% ethanol for 2 minutes each, and rinse them with distilled water for 2 minutes.

[0077] (3) Place the slices in a 0.1% Sirius red-saturated picric acid solution and stain at room temperature for 60 min to fully stain the collagen fibers.

[0078] (4) After staining, rinse the sections gently twice in 0.5% glacial acetic acid solution for 1 minute each time to remove non-specific dyes.

[0079] (5) After rapid dehydration by 1 min each of 95% ethanol and 100% ethanol, place it in xylene twice for 2 min each time to achieve transparency.

[0080] (6) Take out the slice, add neutral resin to seal the slice, and air dry at room temperature.

[0081] (7) Under a normal light microscope, collagen fibers appear red and cell nuclei appear pale blue. Under a polarized light microscope, birefringence of different types of collagen fibers can be observed, with type I collagen mainly appearing yellow-orange-red and type III collagen appearing green.

[0082] (8) The results are as follows Figure 3 As shown.

[0083] Depend on Figure 3 The results of Masson staining and Sirius red staining showed that JSH-23 can effectively alleviate the progression of renal fibrosis.

[0084] In conclusion, JSH-23 can treat chronic kidney disease by improving renal dysfunction, inhibiting renal damage, and alleviating renal fibrosis, thus showing promise as a drug for the treatment of chronic kidney disease.

[0085] 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. The use of JSH-23 in the preparation of drugs for improving renal function disorders and / or inhibiting renal fibrosis; the chemical structural formula of JSH-23 is: 。 2. The application according to claim 1, characterized in that, The application of JSH-23 in the preparation of drugs that downregulate the expression of α-SMA, Col1a1, and FN1.

3. The application according to claim 2, characterized in that, The application of JSH-23 in the preparation of expression regulators that downregulate α-SMA, Col1a1, and FN1.

4. The application according to claim 1, characterized in that, The application of JSH-23 in the preparation of drugs that reduce serum creatinine and / or blood urea nitrogen levels.

5. The application according to claim 4, characterized in that, The application of JSH-23 in the preparation of regulators that lower serum creatinine and / or blood urea nitrogen levels.

6. The application according to any one of claims 1-5, characterized in that, The drug uses JSH-23 as the active ingredient and is formulated into a dosage form with excipients.

7. The application according to claim 6, characterized in that, The excipient is one of solid, liquid or semi-solid excipients.

8. The application according to claim 7, characterized in that, The excipients are selected from one or more of polyethylene glycol, sodium carboxymethyl starch, hydroxypropyl methylcellulose, lactose, starch, and glycerin.

9. The application according to claim 6, characterized in that, The dosage form includes one or more of tablets, capsules, injections, oral solutions, pellets, or sustained-release formulations.

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