Application of osteomodulatory protein in preparation of medicine for treating membranous nephropathy

Abstract

The invention discloses application of osteomodulatory protein in preparation of a medicine for treating membranous nephropathy, and belongs to the technical field of biology. According to the application, an in-vitro research model, namely a subsoluble C5b-9 podocyte injury model, of membranous nephropathy and an in-vivo research model, namely a passive Heymann nephritis rat model are respectively used for verifying that the knock-down osteomodulin plays a role in treating the membranous nephropathy, and the mechanism of the knock-down osteomodulin relates to inhibition of kidney and podocyte pyroptosis of the membranous nephropathy. The invention innovates treatment targets and medicines for membranous nephropathy, and is expected to generate good economic and social effects.

Description

Technical Field

[0001] The invention relates to application of osteomodulin in preparing medicine for treating membranous nephropathy, and belongs to the field of biotechnology. Background Art

[0002] Membranous nephropathy (MN) is a common primary glomerular disease and one of the common causes of nephrotic syndrome. The long-term prognosis of MN is poor, and about one-third of MN patients with persistent massive proteinuria will progress to end-stage renal disease. The clinical application of glucocorticoids and immunosuppressants has improved the prognosis of MN patients to a certain extent, but they still face many problems such as the occurrence of malignant tumors, infection, kidney and reproductive system toxicity, as well as serious adverse reactions, uncertain clinical efficacy, and high recurrence rate after drug withdrawal. Recently, the long-term effectiveness of B cell targeted drugs is also poor, and new therapeutic drugs for MN are urgently needed. The pathological characteristics of MN are the damage of renal podocytes caused by the activation of the complement system after the deposition of subepithelial immune complexes in the glomerulus. Therefore, clarifying the key genes in the process of podocyte damage in MN is the key to discovering therapeutic targets for MN and preparing new therapeutic drugs for MN. The sublytic C5b-9 podocyte injury model and the passive Heymann nephritis rat model are classic in vivo and in vitro research models of membranous nephropathy, providing powerful tools for exploring therapeutic targets and new drugs for membranous nephropathy.

[0003] Osteomodulin (OMD) is a small proteoglycan rich in leucine repeat sequences. It was originally found in mineralized tissues such as bones and teeth. It can participate in the mineralization process of tissues through interactions with the extracellular matrix, but later osteomodulin was found to be related to a wider range of biological processes such as cell proliferation and migration. The inventors found that osteomodulin was significantly upregulated during membranous nephropathy kidney and podocyte damage, and may participate in membranous nephropathy kidney damage by promoting cell pyroptosis, a programmed necrotic cell death mode, suggesting the prospect of targeted inhibition of osteomodulin in the preparation of drugs for the treatment of membranous nephropathy. However, the prior art has not disclosed or suggested the use of osteomodulin in the preparation of drugs for the treatment of membranous nephropathy. Summary of the invention

[0004] The purpose of the present invention is to provide an application of osteomodulin in the preparation of a drug for treating membranous nephropathy, specifically, to provide a therapeutic effect and mechanism of knocking down osteomodulin on membranous nephropathy;

[0005] Furthermore, the knockdown of osteomodulin refers to the knockdown of osteomodulin expression using small interfering RNA;

[0006] Furthermore, the membranous nephropathy includes an in vitro research model of membranous nephropathy, namely, a sublytic C5b-9 podocyte injury model, and an in vivo research model, namely, a passive Heymann nephritis rat model;

[0007] Furthermore, the therapeutic effects include: reduced podocyte lactate dehydrogenase release in the sublytic C5b-9 podocyte injury model, reduced number of propidium iodide staining-positive cells, up-regulated expression of podocyte slit diaphragm protein and marker proteins Nephrin, Podocin, and WT-1; reduced 24-hour urine protein, blood low-density lipoprotein, creatinine, and urea nitrogen in the passive Heymann nephritis rat model, increased serum albumin, reduced diffuse fusion of podocyte foot processes, reduced foot process width, normal expression and distribution of podocyte slit diaphragm proteins Nephrin and Podocin, and down-regulated expression of podocyte injury marker protein Desmin;

[0008] Furthermore, the therapeutic mechanism includes inhibiting pyroptosis of membranous nephropathy kidneys and podocytes, including: down-regulation of pyroptosis signaling pathway proteins NLRP3, ASC, Caspase-1, IL-18, and GSDMD in the sublytic C5b-9 podocyte injury model, and reduction of expression and cell membrane translocation of pyroptosis execution protein GSDMD (N); down-regulation of pyroptosis signaling pathway proteins and execution proteins NLRP3, ASC, Caspase-1, Caspase-1p20, IL-1β, IL-18, GSDMD, and GSDMD (N) in rat renal tissue in the passive Heymann nephritis rat model, and reduction of blood IL-1β and IL-18;

[0009] The present invention also provides a drug capable of treating and / or alleviating renal injury caused by membranous nephropathy, wherein the drug can inhibit the expression of osteomodulin gene or protein; the drug treats and / or alleviating renal injury caused by membranous nephropathy by inhibiting osteomodulin and its downstream molecules; the drug is a gene therapy; the gene therapy is small interfering RNA and its vector; the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.3-SEQ ID NO.4.

[0010] The present invention also provides a method for studying the function of genes related to membranous nephropathy renal injury, or screening gene therapy targets and drugs for treating and / or alleviating membranous nephropathy renal injury, or analyzing the association between gene intervention and drugs for treating and / or alleviating membranous nephropathy renal injury. The method comprises using a small interfering RNA containing a specific knockdown of osteomodulin gene or protein and a vector thereof to reduce the expression of the osteomodulin gene or protein; the vector is a small interfering RNA vector; the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.3-SEQ ID NO.4.

[0011] In one embodiment of the present invention, the method can treat and / or alleviate membranous nephropathy renal injury in in vitro cells, or in animal models, or in clinical patients.

[0012] The present invention also provides an application of an agent for knocking down osteomodulin in the preparation of a drug for treating and / or alleviating renal injury in membranous nephropathy, characterized in that the agent comprises a small interfering RNA and a carrier thereof, the nucleotide sequence of the small interfering RNA being as shown in SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA being as shown in SEQ ID NO.3-SEQID NO.4.

[0013] In one embodiment of the present invention, the agent is a small interfering RNA that specifically inhibits the expression of osteomodulin gene or protein, or a lipid nanoparticle carrier, N-acetylgalactosamine-coupled modification or viral vector containing the osteomodulin small interfering RNA, or an osteomodulin gene or protein activity inhibitor.

[0014] In one embodiment of the present invention, the dosage form of the drug includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills or solutions.

[0015] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0016] In one embodiment of the present invention, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

[0017] The present invention also provides an application of osteomodulin in preparing a drug for treating and / or alleviating membranous nephropathy renal injury.

[0018] In one embodiment of the present invention, the active ingredient of the drug is an agent that can specifically inhibit the expression or activity of the osteomodulin gene or protein.

[0019] In one embodiment of the present invention, the reagent is a small interfering RNA that specifically inhibits the expression of osteomodulin gene or protein, or a lipid nanoparticle carrier, N-acetylgalactosamine coupling modification or viral vector containing the osteomodulin small interfering RNA, or an osteomodulin gene or protein activity inhibitor, the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.3-SEQ IDNO.4.

[0020] In one embodiment of the present invention, the inhibitor of osteomodulin gene or protein expression or activity comprises a small molecule compound.

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

[0022] (a) Relieve proteinuria;

[0023] (b) Relieve hypoproteinemia;

[0024] (c) Relieve hyperlipidemia;

[0025] (d) relief of nephrotic syndrome;

[0026] (e) Alleviate impaired renal function;

[0027] (f) Alleviate renal immune complex deposition;

[0028] (g) Alleviate renal complement activation;

[0029] (h) Alleviate renal basement membrane thickening;

[0030] (i) Alleviate kidney intrinsic cell damage;

[0031] (j) Alleviate renal intrinsic cell death.

[0032] In one embodiment of the present invention, the dosage form of the drug includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills or solutions.

[0033] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0034] In one embodiment of the present invention, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

[0035] Beneficial Effects

[0036] (1) The present invention utilizes an in vitro research model of membranous nephropathy, namely the sublytic C5b-9 podocyte injury model, and an in vivo research model, namely the passive Heymann nephritis rat model, to confirm that knocking down osteomodulin can reduce renal tissue and podocyte damage in membranous nephropathy, thereby clarifying the role of osteomodulin in the preparation of drugs for the treatment of membranous nephropathy.

[0037] (2) The present invention utilizes an in vitro research model of membranous nephropathy, namely, a sublytic C5b-9 podocyte injury model, and an in vivo research model, namely, a passive Heymann nephritis rat model, to confirm the therapeutic effect of osteomodulin on membranous nephropathy and further reveals the mechanism behind the therapeutic effect, namely, inhibiting pyroptosis of membranous nephropathy kidneys and podocytes, thereby clarifying the mechanism behind the role of osteomodulin in the preparation of drugs for the treatment of membranous nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1: Knockdown of osteomodulin alleviates podocyte injury caused by sublytic C5b-9; wherein, A is the release of lactate dehydrogenase (LDH) of podocytes in each group; B is the result of propidium iodide (PI) / DAPI fluorescence staining of podocytes in each group; C is the propidium iodide (PI)-positive cell rate of podocytes in each group; D is the protein immunoblot of podocyte Nephrin, Podocin, WT-1 and internal reference GAPDH in each group; EG is the relative expression levels of podocyte Nephrin, Podocin and WT-1 in each group.

[0039] Figure 2 :Osteoporosis of podocytes induced by sublytic C5b-9 was alleviated by osteomodulin; A is the result of GSDMD(N) / ZO-1 / DAPI fluorescence staining of podocytes in each group; B is the protein immunoblot of OMD, p38MAPK, p-p38MAPK(Thr180 / Tyr182), NLRP3, ASC, Caspase-1, IL-18, GSDMD and internal reference GAPDH of podocytes in each group; CJ is the relative expression of OMD, p38MAPK, p-p38MAPK(Thr180 / Tyr182), NLRP3, ASC, Caspase-1, IL-18 and GSDMD of podocytes in each group;

[0040] NC: control group; sC5b-9: sublytic C5b-9 podocyte injury model group; si-OMD: osteomodulin small interfering RNA knockdown group; sC5b-9+si-OMD: sublytic C5b-9 podocyte injury + osteomodulin small interfering RNA knockdown group; Mean ± SD Data were described, and variance analysis and least significant difference t-test or Welch's approximate variance analysis and Dunnett's T3 test were used to infer the differences between multiple groups and compared pairwise. P<0.05 was considered statistically significant. ns: no statistically significant difference; *: P<0.05; **: P<0.01.

[0041] Figure 3 : Knockdown of osteopontin alleviates renal damage in rats with passive Heymann nephritis; A is the electron microscopic observation results of glomeruli in rats in each group; B is the measurement results of foot process width of podocytes in glomeruli in rats in each group; C is the immunofluorescence staining results of Nephrin and Podocin in renal tissues of rats in each group; D is the immunohistochemical staining results of Desmin in renal tissues of rats in each group; E is the relative expression level of Desmin in renal tissues of rats in each group.

[0042] Figure 4: Knockdown of osteomodulin alleviates renal pyroptosis in rats with passive Heymann nephritis; A is the protein immunoblot of OMD, p38MAPK, p-p38MAPK(Thr180 / Tyr182), NLRP3, ASC, Caspase-1, Caspase-1p20, IL-1β, IL-18, GSDMD, GSDMD(N) and internal reference GAPDH in renal tissues of rats in various groups; B is the relative expression levels of OMD, p38MAPK, p-p38MAPK(Thr180 / Tyr182), NLRP3, ASC, Caspase-1, Caspase-1p20, IL-1β, IL-18, GSDMD, GSDMD(N) in renal tissues of rats in various groups; C is the level of IL-1β in serum of rats in various groups; D is the level of IL-18 in serum of rats in various groups;

[0043] NC: control group; si-OMD: osteomodulin small interfering RNA knockdown group; PHN: passive Heymann nephritis model group; PHN+si-OMD: passive Heymann nephritis + osteomodulin small interfering RNA knockdown group; n = 8 rats in each group; mean ± SD Data were described, and variance analysis and least significant difference t-test or Welch's approximate variance analysis and Dunnett's T3 test were used to infer the differences between multiple groups and compared pairwise. P<0.05 was considered statistically significant. ns: no statistically significant difference; *: P<0.05; **: P<0.01. DETAILED DESCRIPTION

[0044] The sublytic C5b-9 podocyte injury model involved in the following embodiments is a classic in vitro research model of membranous nephropathy, which can well simulate the pathological process of podocyte injury caused by complement activation in membranous nephropathy; the passive Heymann nephritis rat model involved in the following embodiments is a classic animal model of membranous nephropathy. The renal injury manifestations of this model are very close to human membranous nephropathy, and the model has a rapid onset, a stable course, and is easy to repeat. It is an ideal animal model for testing the effect of drug treatment.

[0045] The cells involved in the present invention are as follows:

[0046] The human immortalized glomerular podocyte cell line can be purchased from a commercial company. The human immortalized glomerular podocyte cell line of the present invention was donated by Professor Moin A. Saleem of the University of Bristol, UK. The specific culture method is as follows: the human immortalized glomerular podocyte cell line is placed in RPMI1640 culture medium containing 10% fetal bovine serum and 1% Insulin-Transferrin-Selenium (100×) at 33°C and 5% CO 2When the cells reached 60-70% confluence, they were transferred to 37°C and 5% CO 2 After 10-14 days of differentiation, the medium was replaced with serum-free culture medium for subsequent experiments.

[0047] The animals involved in the present invention are as follows:

[0048] Female Sprague-Dawley (SD) rats, weighing 150-180 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The rats were housed in an SPF environment at the Experimental Animal Center of Wuxi Medical College of Jiangnan University. All animals had free access to water and food and were allowed to adapt for 1 week before the formal experiment.

[0049] The main reagents and instruments involved in the present invention are as follows:

[0050] Reagents: Zymosan A (Merck, Darmstadt, Germany); Lipofectamine TM RNAiMAX (Thermo Fisher Scientific, USA); OMD small interfering RNA (Huzhou Hippo Biotechnology Co., Ltd.); lactate dehydrogenase (LDH) cytotoxicity detection kit, HRP-labeled goat anti-mouse / rabbit IgG, FITC-labeled goat anti-mouse / rabbit IgG, Cy3-labeled goat anti-rabbit IgG (Shanghai Biotech Co., Ltd.); propidium iodide (PI) solution, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (Beijing Solebold Technology Co., Ltd.); whole protein extraction kit (Jiangsu Keygen Biotechnology Co., Ltd.); nephrin, WT-1, NLRP3 antibodies; rat IL-1β ELISA kit, rat IL-18 ELISA kit (Wuhan Boster Biotechnology Co., Ltd.); nephrin, podocin, caspas e-1, IL-18, GSDMD, GSDMD(N), NLRP3, ASC antibodies (Jiangsu Qinke Biological Research Center Co., Ltd.); OMD antibody (Beijing Bioson Biotechnology Co., Ltd.); p38MAPK, p-p38MAPK (Thr180 / Tyr182), Caspase-1, IL-1β antibodies (Saixintong (Shanghai) Biological Reagent Co., Ltd.); ASC, ZO-1, GAPDH, Desmin, Podocin antibodies (Wuhan Sanying Biotechnology Co., Ltd.); GSDMD(N) antibody (Abcam (Shanghai) Trading Co., Ltd.), GAPDH antibody (Jiangsu Kangwei Century Biotechnology Co., Ltd.); Bradford protein concentration assay kit (Shanghai Biyuntian Biotechnology Co., Ltd.); DAB color development kit (Quanhui International Trading Co., Ltd.).

[0051] Instruments: Multifunctional microplate reader (MeiGu Molecular Instruments Co., Ltd.); laser scanning confocal microscope (Carl Zeiss Group); electrophoresis power supply / electrophoresis tank / transfer tank (Bio-Rad Biomedical Products (Shanghai) Co., Ltd.); fully automatic chemiluminescence image analysis system (Shanghai Tianneng Life Sciences Co., Ltd.); fully automatic biochemical analysis system (Beckman Coulter Co., Ltd.); transmission electron microscope (Hitachi High-Tech Group); upright optical microscope (Nikon Precision Machinery Co., Ltd.).

[0052] The siRNA nucleotide sequence involved in the present invention is as follows:

[0053] Osteoporin siRNA sequences for in vitro cells:

[0054] Sense strand (5'-3'): CCAAGAGCCAGAUGAUGAUUAdTdT (SEQ ID NO. 1);

[0055] Antisense strand (5'-3'): UAAUCAUCAUCUGGCUCUUGGdTdT (SEQ ID NO. 2).

[0056] Osteoporin siRNA sequences for animal models:

[0057] Sense strand (5'-3'): GGCACUUCUGAUUCAGUUAAAdTdT (SEQ ID NO. 3);

[0058] Antisense strand (5'-3'): UUUAACUGAAUCAGAAGUGCCdTdT (SEQ ID NO. 4).

[0059] The detection method involved in the present invention is as follows:

[0060] Lactate dehydrogenase (LDH) detection: Based on the INT colorimetric reaction catalyzed by diaphorase, the amount of LDH released from podocytes was detected by colorimetric method according to the instructions of the LDH cytotoxicity detection kit.

[0061] Propidium iodide (PI) staining: Podocytes were inoculated in a glass-bottomed culture dish, and PI 3.34 μg / ml and DAPI 15 μg / ml were added successively to stain the cells and nuclei. The images were observed and collected under a confocal fluorescence microscope, and the ratio of PI-stained positive cells to the total number of cells, i.e., the positive rate, was calculated.

[0062] Western blot: Total protein of cells and rat renal cortex was extracted using a total protein extraction kit. The protein concentration of each group of samples was detected by the BCA method and then balanced. The samples were added with loading buffer and denatured by boiling. After that, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. After blocking with 5% BSA, the following antibodies were incubated at 4°C overnight: Nephrin, Podocin, WT-1, OMD, p38MAPK, p-p38MAPK (Thr180 / Tyr182), NLRP3, ASC, Caspase-1, IL-1β, IL-18, GSDMD, GSDMD (N), GAPDH, diluted 1:500-3000; after incubation with HRP-labeled secondary antibody, the samples were developed by ECL chemiluminescence method, and images were collected in a fully automatic chemiluminescence image analysis system. The integrated optical density values ​​of the target protein and the internal reference band were calculated using Image-ProPlus 6.0 software, and the ratio between the two was calculated as the relative expression of the protein.

[0063] Immunofluorescence staining: Podocytes were inoculated on slides, fixed with 4% paraformaldehyde, blocked with 5% BSA, and incubated with 1:200 dilution of GSDMD (N) and ZO-1 antibodies at 4°C overnight. After incubation with FITC and Cy3 labeled secondary antibodies, DAPI 15μg / ml was used to stain the nuclei. Typical images were collected by confocal fluorescence microscopy. Frozen sections of rat kidney tissue were taken, blocked with 10% calf serum, and incubated with 1:100 dilution of Nephrin and 1:500 dilution of Podocin antibodies overnight. After incubation with FITC labeled secondary antibodies, typical images were collected by confocal fluorescence microscopy.

[0064] Urine protein detection: Bradford protein concentration assay kit was used to detect 24-hour urine protein (24h-UPro) in rats, and the steps were carried out according to the instructions of the Bradford protein concentration assay kit.

[0065] Blood biochemical test: The fully automatic biochemical analysis system was used to detect the levels of serum albumin (ALB), total cholesterol (T-CHOL), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), creatinine (SCr) and blood urea nitrogen (BUN) in rats.

[0066] Kidney pathology: cut 1mm 3 Fresh renal cortex of different sizes was fixed front and back, dehydrated, infiltrated, and embedded before and after ultrathin sections of 70 nm were cut and double-stained with uranyl acetate-lead citrate. The electron-dense material deposition under the glomerular epithelium and the fusion of podocyte foot processes were observed under a transmission electron microscope. The podocyte foot process width was calculated according to the formula (π / 4)×(Σbasement membrane length / Σnumber of foot processes on the basement membrane).

[0067] Immunohistochemical staining: Fresh renal cortex was taken, fixed, dehydrated, transparent, infiltrated, and embedded in a conventional manner. After 2.5 μm sections were sliced ​​with a paraffin slicer, they were dewaxed, rehydrated, and repaired with high pressure heat in Tris-EDTA (pH=8.0) repair solution for 10 minutes. After blocking with 10% calf serum, the Desmin antibody diluted 1:200 was incubated at 4°C overnight, and the HRP-labeled secondary antibody was incubated, DAB was used for color development, and hematoxylin was counterstained and observed under an upright optical microscope. Three fields of view containing glomeruli were randomly photographed for each section, and the integrated optical density of the glomerular staining positive area and the glomerular area were calculated using Image-Pro Plus6.0 software, and the ratio of the two was calculated. The average value was taken as the relative expression of Desmin in the sample.

[0068] Enzyme-linked immunosorbent assay: Rat IL-1β ELISA kit and rat IL-18 ELISA kit were used to detect the levels of rat serum IL-1β and IL-18, and the steps were carried out according to the instructions of the kit.

[0069] Statistical analysis: For cell experiments, all experiments were set up with one replicate well each time; for animal experiments, the sample size of each group was n = 8, and all experiments were repeated more than 3 times. SPSS 20.0 software was used to perform statistical description and statistical inference on the obtained data, and the mean ± standard deviation was used. Describe the data, combine analysis of variance with least significant difference t test (if the data variances are equal) or Welch's approximate analysis of variance with Dunnett's T3 test (if the data variances are unequal) to infer the differences between multiple groups and make pairwise comparisons. P < 0.05 was considered statistically significant.

[0070] Example 1: Knockdown of osteomodulin alleviates podocyte injury caused by sublytic C5b-9 by inhibiting pyroptosis

[0071] The specific steps are as follows:

[0072] (1) Establishment of sublytic C5b-9 podocyte injury model

[0073] 1) Cell culture

[0074] The immortalized human glomerular podocyte cell line was kindly donated by Professor Moin A. Saleem of the University of Bristol, UK. The immortalized human glomerular podocyte cell line was placed in RPMI 1640 culture medium containing 10% fetal bovine serum and 1% Insulin-Transferrin-Selenium (100×) at 33°C and 5% CO 2 When the cells reached 60-70% confluence, they were transferred to 37°C and 5% CO 2 After 10-14 days of differentiation, the medium was replaced with serum-free culture medium for subsequent experiments.

[0075] 2) Establishment of sublytic C5b-9 podocyte injury model

[0076] Zymosan A boiled in physiological saline for 1 hour was added to fresh serum of healthy volunteers at 10 mg / ml and incubated at 37°C for 1 hour to activate complement to produce C5b-9. The Zymosan A-activated serum rich in C5b-9 was centrifuged at 4°C, 20,000g, for 10 min to remove Zymosan A, and after passing through a 0.2 μm filter, was added to the serum-free culture medium described in step 1) at 10% (v / v) and incubated at 37°C for 1 hour to establish a sublytic C5b-9 podocyte injury model.

[0077] Control serum: Zymosan A boiled in physiological saline for 1 hour was added at 10 mg / ml to fresh serum of healthy volunteers in which complement was inactivated at 56°C for 30 min, and incubated at 37°C for 1 hour. The serum was centrifuged at 4°C, 20,000 g, 10 min to remove Zymosan A, and after filtering through a 0.2 μm filter, it was added at 10% (v / v) to the serum-free culture medium described in step 1) and incubated at 37°C for 1 hour to establish a control.

[0078] (2) Osteoporin knockdown

[0079] Small interfering RNA liposome transfection method is used to knock down osteomodulin. The transfection reagent can be purchased commercially. In this example, the transfection reagent used is Lipofectamine TM RNAiMAX (Thermo Fisher Scientific, USA), the sequence of the sense strand (5'-3') of osteomodulin small interfering RNA is: CCAAGAGCCAGAUGAUGAUUAdTdT, and the sequence of the antisense strand (5'-3') is: UAAUCAUCAUCUGGCUCUUGGdTdT; small interfering RNA (pmol) and Lipofectamine TM RNAiMAX (μl) dosage ratio is 1:0.2-2.5, incubation time is 24-48 hours; In this embodiment, small interfering RNA (pmol) and Lipofectamine TM The dosage ratio of RNAiMAX (μl) was 1:0.3, and the incubation time was 24 hours. The specific knockdown method was as follows: 1) an equal volume of Opti-MEM TM Reduced Serum Medium dilutes small interfering RNA and Lipofectamine TM RNAiMAX; 2) Mix well and incubate at room temperature for 5 minutes; 3) Add the above mixture to the cells to be transfected, continue incubation for 24 hours and then perform subsequent detection.

[0080] (3) Grouping and processing

[0081] Divided into four groups:

[0082] 1) Control group: follow the method described in step (1), add control serum, and incubate at 37°C for 1 hour;

[0083] 2) Sublytic C5b-9 podocyte injury model group: according to the method described in step (1), add C5b-9-rich zymosan A activated serum and incubate at 37°C for 1 hour;

[0084] 3) Osteoporin small interfering RNA knockdown group: According to the method described in step (2), osteopontin small interfering RNA and Lipofectamine were added TM The RNAiMAX mixture was incubated at 37°C for 24 hours, and then control serum was added according to the method described in step (1) and incubated at 37°C for 1 hour;

[0085] 4) Sublytic C5b-9 podocyte injury + osteomodulin small interfering RNA knockdown group: osteomodulin small interfering RNA and Lipofectamine were added according to the method described in step (2). TM The RNAiMAX mixture was incubated at 37°C for 24 hours, and then, according to the method of step (1), zymosan A-activated serum rich in C5b-9 was added and incubated at 37°C for 1 hour.

[0086] (4) Detection indicators and statistical analysis

[0087] The release of lactate dehydrogenase (LDH) from podocytes was detected by colorimetric method based on INT color reaction catalyzed by diaphorase; propidium iodide (PI) 0.67-3.34 μg / ml and DAPI were used. 5-15 μg / ml were used to stain podocytes and cell nuclei. The images were collected by confocal fluorescence microscopy, and the ratio of PI-stained positive cells to the total number of cells, i.e., the positive rate, was calculated. Western blot was used to detect the expression of podocyte Nephrin, Podocin, WT-1, OMD, p38MAPK, p-p38MAPK (Thr180 / Tyr182), NLRP3, ASC, Caspase-1, IL-18, and GSDMD proteins, and the ratio of the integrated optical density of the target protein to the integrated optical density of the internal reference GAPDH was used as the relative expression of the protein. Immunofluorescence staining was used to detect the expression of podocyte GSDMD (N) and its co-localization with the cell membrane marker protein ZO-1. All experiments were performed with one replicate well each time and repeated for more than 3 times. SPSS20.0 software was used to perform statistical description and statistical inference on the obtained data, and the mean ± standard deviation was used. Describe the data, combine analysis of variance with least significant difference t test (if the data variances are equal) or Welch's approximate analysis of variance with Dunnett's T3 test (if the data variances are unequal) to infer the differences between multiple groups and make pairwise comparisons. P < 0.05 was considered statistically significant.

[0088] (5) Observation results

[0089] 1) Sublytic C5b-9 causes podocyte damage and pyroptosis

[0090] The results showed that compared with the control group, the release of lactate dehydrogenase (LDH) in the sublytic C5b-9 podocyte injury model group increased ( Figure 1 A), the positive rate of propidium iodide (PI) staining increased ( Figure 1 BC), the expression of podocyte slit diaphragm protein and marker proteins Nephrin, Podocin, and WT-1 were significantly downregulated ( Figure 1 DG), the expression of pyroptosis execution protein GSDMD (N) and its co-localization with cell membrane marker protein ZO-1 were significantly increased ( Figure 2 A), the pyroptosis signaling pathway NLRP3-ASC-Caspase-1-IL-18 / GSDMD was significantly upregulated, and the differences were statistically significant (all P < 0.01) ( Figure 2 B, FJ). The expressions of OMD and p-p38MAPK (Thr180 / Tyr182) were significantly upregulated with podocyte pyroptosis, and the differences were statistically significant (P < 0.01) ( Figure 2 BC, E). The above results also indicate that the sublytic C5b-9 podocyte injury model of the present invention was successfully established.

[0091] 2) Knockdown of osteomodulin alleviates podocyte injury caused by sublytic C5b-9 by inhibiting pyroptosis

[0092] The results showed that compared with the sublytic C5b-9 podocyte injury model group, the lactate dehydrogenase (LDH) release and propidium iodide (PI) staining positive rate in the sublytic C5b-9 podocyte injury + osteomodulin small interfering RNA knockdown group were reduced ( Figure 1 AC), Nephrin, Podocin, and WT-1 expressions were all upregulated ( Figure 1 DG). The expression of GSDMD(N) and its co-localization with ZO-1 were both reduced ( Figure 2 A), p-p38MAPK (Thr180 / Tyr182) and pyroptosis signaling pathway NLRP3-ASC-Caspase-1-IL-18 / GSDMD were down-regulated, and the differences were statistically significant (all P<0.05) ( Figure 2BC EJ). Knockdown of osteomodulin alone did not significantly affect podocyte homeostasis, as shown by the similar release of lactate dehydrogenase (LDH) in the control group and the osteomodulin small interfering RNA knockdown group ( Figure 1 A), Propidium iodide (PI) staining positive rate ( Figure 1 BC), Nephrin, Podocin and WT-1 expression ( Figure 1 DG), low GSDMD (N) expression and co-localization with ZO-1 ( Figure 2 A), low p-p38MAPK (Thr180 / Tyr182) and pyroptosis signaling pathway NLRP3-ASC-Caspase-1-IL-18 / GSDMD expression levels ( Figure 2 BC, EJ) (all P>0.05), indicating the safety of osteopontin knockdown in the treatment of membranous nephropathy.

[0093] Example 2: Knockdown of osteomodulin alleviates kidney and podocyte damage in rats with passive Heymann nephritis by inhibiting pyroptosis

[0094] (1) Establishment of passive Heymann nephritis rat model

[0095] 1) Experimental animals

[0096] Female Sprague-Dawley (SD) rats, weighing 150-180 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The rats were housed in an SPF environment at the Experimental Animal Center of Wuxi Medical College of Jiangnan University. All animals had free access to water and food and were allowed to adapt for 1 week before the formal experiment.

[0097] 2) Establishment of passive Heymann nephritis rat model

[0098] Take fresh Sprague-Dawley (SD) rat kidneys, cut the renal cortex, cut it into a paste on ice, pass it through a 150-mesh stainless steel sieve, add an appropriate amount of cold saline to the renal tubular homogenate after passing through the sieve, centrifuge at 4°C, 400g / min for 10min, discard the precipitate, repeat 3 times until there is no precipitate, then ultracentrifuge at 4°C, 78,680g for 45min, discard the supernatant, wash the precipitate with deionized water 3 times, and obtain Fx1A antigen after lyophilization. Dissolve 10 mg of Fx1A antigen in 0.5 ml of normal saline and mix with an equal volume of Freund's complete adjuvant. After sufficient emulsification, inject intradermally at multiple points to immunize New Zealand rabbits. The above immunization needs to be performed more than 3 times. After the immunization is completed, the ear vein is cut and blood is collected. Fx1A antiserum is obtained by centrifugation at 3,000 rpm for 10 minutes. The antiserum is identified by immunofluorescence titer, and 1:2000 is qualified. The antiserum is inactivated in a water bath at 56°C for 30 minutes. SD rats are taken and intraperitoneally injected with 2 ml of Fx1A antiserum. One hour later, 1 ml of Fx1A antiserum is injected again to establish a passive Heymann nephritis rat model.

[0099] Control: Blood was collected from the ear vein of normal New Zealand rabbits and centrifuged at 3,000 rpm for 10 min. The serum was inactivated by water bath at 56°C for 30 min. SD rats were intraperitoneally injected with 2 ml of normal rabbit serum, and then injected with 1 ml of normal rabbit serum again 1 hour later to establish a control.

[0100] (2) Osteoporin knockdown

[0101] 5'Chol+2'OMe modified small interfering RNA was used to knock down osteomodulin. The sense strand (5'-3') sequence of the osteomodulin small interfering RNA in this example was: GGCACUUCUGAUUCAGUUAAAdTdT, and the antisense strand (5'-3') sequence was: UUUAACUGAAUCAGAAGUGCCdTdT. The osteomodulin small interfering RNA was dissolved in saline and injected into the tail vein. The dose was 0.25-1nmol·g -1 ·d -1 ; This embodiment is preferred: 1nmol·g -1 ·d -1 , injected until the day when the rats were euthanized and samples were collected.

[0102] (3) Grouping and processing

[0103] Divided into four groups:

[0104] 1) Control group: According to the method described in step (1), 2 ml of normal rabbit serum was injected intraperitoneally, and 1 ml of normal rabbit serum was injected again 1 hour later;

[0105] 2) Osteoporin siRNA knockdown group: According to the method described in step (1), 2 ml of normal rabbit serum was injected intraperitoneally, and 1 ml of normal rabbit serum was injected again 1 hour later. Then, according to the method described in step (2), 1 nmol·g osteopontin siRNA dissolved in normal saline was injected into the tail vein on the same day. -1 ·d -1 ;

[0106] 3) Passive Heymann nephritis model group: According to the method described in step (1), 2 ml of Fx1A antiserum was injected intraperitoneally, and 1 ml of Fx1A antiserum was injected again 1 hour later;

[0107] 4) Passive Heymann nephritis + osteomodulin small interfering RNA knockdown group: According to the method described in step (1), 2 ml of Fx1A antiserum was injected intraperitoneally, and 1 ml of Fx1A antiserum was injected again 1 hour later. Then, according to the method described in step (2), 1 nmol·g osteomodulin small interfering RNA dissolved in physiological saline was injected into the tail vein on the same day. -1 ·d -1 .

[0108] The experiment lasted for 15 days. All rats were placed in metabolic cages to collect urine for 24 hours, blood was collected from the medial canthal vein and serum was separated, and after euthanasia, the kidneys were perfused with cold PBS until pale and then renal tissue was obtained, and the renal cortex was cut for subsequent testing.

[0109] (4) Detection indicators and statistical analysis

[0110] Bradford method was used to detect the level of 24-hour urine protein (24h-UPro) in rats. Fully automatic biochemical analysis system was used to detect the levels of serum albumin (ALB), total cholesterol (T-CHOL), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), creatinine (SCr) and urea nitrogen (BUN) in rats. Uranium acetate-lead citrate double staining was used to stain the renal cortex, and the electron-dense material deposition under the glomerular epithelium and the fusion of podocyte foot processes were observed under transmission electron microscopy. The width of podocyte foot processes was calculated according to the formula (π / 4)×(Σbasement membrane length / Σnumber of foot processes on the basement membrane). Immunofluorescence staining was used to detect the expression and distribution of glomerular slit diaphragm proteins Nephrin and Podocin Condition; Immunohistochemistry was used to detect the expression of glomerular injury marker protein Desmin and the ratio of its integrated optical density to glomerular area was used as its relative expression; Western-blot was used to detect the expression of renal OMD, p38MAPK, p-p38MAPK (Thr180 / Tyr182), NLRP3, ASC, Caspase-1, Caspase-1p20, IL-1β, IL-18, GSDMD, GSDMD (N) proteins and the ratio of the integrated optical density of the target protein to the integrated optical density of the internal reference GAPDH was used as the relative expression of the protein; Enzyme-linked immunosorbent assay was used to detect the levels of IL-1β and IL-18 in rat serum. The sample size of each group of rats was n=8, and SPSS20.0 software was used for statistical description and statistical inference of the obtained data. Mean ± standard deviation Describe the data, combine analysis of variance with least significant difference t test (if the data variances are equal) or Welch's approximate analysis of variance with Dunnett's T3 test (if the data variances are unequal) to infer the differences between multiple groups and make pairwise comparisons. P < 0.05 was considered statistically significant.

[0111] (5) Observation results

[0112] 1) Kidney damage and pyroptosis in rats with passive Heymann nephritis

[0113] Passive Heymann nephritis rats showed typical membranous nephropathy features such as nephrotic syndrome, renal tissue and podocyte pathological damage. Compared with the control group, the 24-hour urine protein (24h-UPro), total cholesterol (T-CHOL), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), creatinine (SCr), and blood urea nitrogen (BUN) in the passive Heymann nephritis model group were increased, and serum albumin (ALB) was decreased. The differences were statistically significant (all P < 0.01) (Table 1). Electron-dense deposits were found under the glomerular epithelium of passive Heymann nephritis rats, and the podocyte foot processes were diffusely fused and the width was increased. The differences were statistically significant (P < 0.01) ( Figure 3 AB); Nephrin and Podocin expression in glomeruli of rats with passive Heymann nephritis was downregulated, unevenly distributed and discontinuously granular ( Figure 3 C), the expression of Desmin was upregulated, and the difference was statistically significant (P<0.01) ( Figure 3 DE); passive Heymann nephritis rats showed renal podocyte pyroptosis, which was manifested by upregulation of the pyroptosis signaling pathway NLRP3-ASC-Caspase-1-IL-1β / IL-18 / GSDMD compared with the control group, and the differences were statistically significant (all P<0.01)( Figure 4 AB); OMD and p-p38MAPK (Thr180 / Tyr182) in rats with passive Heymann nephritis were upregulated along with renal podocyte pyroptosis, and the differences were statistically significant (all P<0.01)( Figure 4 AB); serum pyroptosis signaling pathway proteins IL-1β and IL-18 were increased in rats with passive Heymann nephritis, and the differences were statistically significant (P < 0.01) ( Figure 4 CD); the above results also illustrate that the passive Heymann nephritis rat model of the present invention was successfully established.

[0114] 2) Knockdown of osteomodulin alleviates renal injury in rats with passive Heymann nephritis by inhibiting pyroptosis

[0115] Knockdown of osteomodulin alleviated kidney and podocyte damage in rats with passive Heymann nephritis by inhibiting pyroptosis. Compared with the passive Heymann nephritis model group, the 24-hour urine protein (24h-UPro), blood low-density lipoprotein (LDL), creatinine (SCr), and blood urea nitrogen (BUN) in the passive Heymann nephritis + osteomodulin small interfering RNA knockdown group were reduced, and serum albumin (ALB) was increased, and the differences were statistically significant (all P < 0.05) (Table 1); the diffuse fusion and width of podocyte foot processes in the passive Heymann nephritis + osteomodulin small interfering RNA knockdown group were alleviated, the expression and distribution of Nephrin and Podocin tended to be normal, and the expression of Desmin was downregulated, and the differences were statistically significant (all P < 0.05) ( Figure 3 AE); in the passive Heymann nephritis + osteomodulin small interfering RNA knockdown group, renal p-p38MAPK (Thr180 / Tyr182) and pyroptosis signaling pathway NLRP3-ASC-Caspase-1-IL-1β / IL-18 / GSDMD were downregulated, and blood IL-1β / IL-18 was reduced, and the differences were statistically significant (all P<0.05)( Figure 4 AD). Knockdown of osteomodulin alone had no significant effect on the rat kidneys, as shown by similar 24-hour urine protein (24h-UPro), serum albumin (ALB), total cholesterol (T-CHOL), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), creatinine (SCr), and urea nitrogen (BUN) levels in the control group and the osteomodulin small interfering RNA knockdown group (Table 1); similar glomerular podocyte morphology, foot process width, nephrin, podocin expression and distribution, and desmin expression ( Figure 3 AE); similar low p-p38MAPK (Thr180 / Tyr182) and pyroptosis signaling pathway NLRP3-ASC-Caspase-1-IL-1β / IL-18 / GSDMD expression levels in the kidney and low IL-1β / IL-18 levels in the blood (all P>0.05) ( Figure 3 AE, Figure 4 AD); again demonstrated the safety of osteopontin knockdown in the treatment of membranous nephropathy.

[0116] Table 1: Urine protein and blood biochemical test results of rats in each group

[0117]

[0118] Table Notes: NC: control group; si-OMD: osteomodulin small interfering RNA knockdown group; PHN: passive Heymann nephritis model group; PHN+si-OMD: passive Heymann nephritis + osteomodulin small interfering RNA knockdown group; n=8 rats in each group; *: compared with the control group, P<0.05; **: compared with the control group, P<0.01; #: compared with the passive Heymann nephritis group, P<0.05; ##: compared with the passive Heymann nephritis group, P<0.01.

[0119] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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. A drug capable of treating and / or alleviating renal damage caused by membranous nephropathy, characterized in that: The drug can inhibit the expression of osteomodulin gene or protein; the drug treats and / or alleviates membranous nephropathy renal damage by inhibiting osteomodulin and its downstream molecules; the drug is a gene therapy; the gene therapy is small interfering RNA and its vector; the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.3-SEQ ID NO.

4.

2. A method for studying the function of genes associated with membranous nephropathy renal injury, or screening gene therapy targets and drugs for treating and / or alleviating membranous nephropathy renal injury, or analyzing the association between gene intervention and drugs for treating and / or alleviating membranous nephropathy renal injury, the method comprising using a small interfering RNA containing a specific knockdown of osteomodulin gene or protein and a vector thereof to reduce the expression of osteomodulin gene or protein; the vector is a small interfering RNA vector; the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.3-SEQ ID NO.4; Preferably, the method can treat and / or alleviate membranous nephropathy renal injury in in vitro cells, or in animal models, or in clinical patients.

3. Use of an agent for knocking down osteomodulin in the preparation of a drug for treating and / or alleviating renal injury in membranous nephropathy, characterized in that: The reagent comprises small interfering RNA and a carrier thereof; the nucleotide sequence of the small interfering RNA is shown as SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA is shown as SEQ ID NO.3-SEQ ID NO.4; Preferably, the agent is a small interfering RNA that specifically inhibits the expression of osteomodulin gene or protein, or a lipid nanoparticle carrier, N-acetylgalactosamine-coupled modification or viral vector containing the osteomodulin small interfering RNA, or an osteomodulin gene or protein activity inhibitor.

4. The drug according to any one of claims 1 to 3, characterized in that The dosage form of the drug includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills or solutions; Preferably, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners can be added to the composition.

5. Use of osteomodulin in the preparation of drugs for treating and / or alleviating renal damage in membranous nephropathy.

6. The use according to claim 5, characterized in that: The active ingredient of the drug is an agent that can specifically inhibit the expression or activity of osteomodulin gene or protein.

7. The use according to claim 5 or 6, characterized in that: The reagent is a small interfering RNA that specifically inhibits the expression of the osteomodulin gene or protein, or a lipid nanoparticle carrier, N-acetylgalactosamine coupling modification or viral vector containing the osteomodulin small interfering RNA, or an osteomodulin gene or protein activity inhibitor; the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1-SEQ ID NO.2 or the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.3-SEQ ID NO.

4.

8. The use according to any one of claims 5 to 7, characterized in that: The inhibitor of osteomodulin gene or protein expression or activity contains small molecule compounds.

9. The use according to any one of claims 5 to 8, characterized in that: The drug is used for at least one of (a) to (j): (a) Relieve proteinuria; (b) Relieve hypoproteinemia; (c) Relieve hyperlipidemia; (d) relief of nephrotic syndrome; (e) Alleviate impaired renal function; (f) Alleviate renal immune complex deposition; (g) Alleviate renal complement activation; (h) Alleviate renal basement membrane thickening; (i) Alleviate kidney intrinsic cell damage; (j) Alleviate renal intrinsic cell death.

10. The use according to any one of claims 5 to 9, characterized in that: The dosage form of the drug includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills or solutions; Preferably, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners can be added to the composition.

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