Protective Effect Of Sinomenine Hydrochloride On Mesangial Proliferative Glomerulonephritis In Rats

Background and Objective: Mesangial proliferative glomerulonephritis(Ms PGN)is a common pathological type of primary glomerular disease with glomerular mesangial cell proliferation and diffuse proliferation of the extracellular matrix.It is an immune-mediated inflammatory response disease.Hormones and immunosuppressive agents are often used clinically for treatment,but due to poor therapeutic effects and adverse reactions,their application has certain limitations.In recent years,China has vigorously developed traditional Chinese medicine,which has achieved good results in the treatment of various diseases.Sinomenii Caulis is a traditional Chinese herbal medicine,and its main active ingredient is sinomenine(SIN).As a water-soluble Hydrochloride of Sinomenine,Sinomenine Hydrochloride(SH)has pharmacological effects such as immunosuppressive,anti-inflammatory and inhibition of tumor cell proliferation.At present,it is clinically used as an adjuvant therapy for rheumatoid arthritis and a potential therapeutic drug for tumors.Ms PGN is an autoimmune disease with mesangial proliferation as the main pathological change.Therefore,we hypothesized whether SH could be used for the treatment of Ms PGN.This study aims to explore the protective effect of SH on Ms PGN rats and its mechanism by establishing an Ms PGN animal model,an anti-Thy-1 nephritis rat model,in order to provide theoretical basis for the clinical treatment of Ms PGN and the development and application of innovative drugs.Methods: Effect of SH on Ms PGN model rats:(1)Mice were intraperitoneally injected with OX-7 hybridoma cells and induced to produce ascites containing m Ab OX-7.The ascites produced were purified by Protein A affinity chromatography to produce anti-Thy-1 antibodies.(2)90 rats were randomly divided into control group,model group,SH low-dose group(21.6mg/kg),SH medium-dose treatment group(43.2mg/kg)and SH highdose treatment group(86.4mg/kg).Rats in the model group and the SH treatment group were injected with anti-Thy-1 antibody(2.5 mg/kg)through the tail vein to establish an anti-Thy-1 nephritis rat model.The rats in the control group were injected with the same volume of phosphate buffer.Rats in the SH treatment group were given continuous intragastric administration every day after modeling,and the control group and the model group were given the same volume of normal saline.Each group took samples on the 3rd,7th,and 14 th day after modeling;(3)PAS staining to observe the pathological changes of each group under light microscope;(4)Serum creatinine,blood urea nitrogen,urine albumin,and urine creatinine levels were measured in each group;(5)Immunohistochemical staining to observe the proliferation of mesangial cells,immune cells infiltration and p-STAT3 activation status;(6)Western blot was used to detect the protein expression changes of JAK2,p-JAK2,STAT3,p-STAT3 and TNF-α.Study on the mechanism of SH on the over proliferation of human renal mesangial cells(HRMC):(1)The cell proliferation model was established by PDGF-BB;(2)CCK-8 assay was used to determine the effective administration concentration of SH;(3)Cell proliferation and the expression of inflammatory factors were detected by Western blot.(4)The protein expressions of JAK2,p-JAK2,STAT3 and p-STAT3 were detected.Results: Anti-Thy-1 antibody was successfully prepared and a rat model of anti-Thy-1 nephritis was established.The results of PAS staining under light microscope showed that the mesangial cells in the model group dissolved on the third day of modeling.On day 7,the mesangial cell proliferation was significantly increased in the model group,and the proliferation of mesangial cells was improved in a dose-dependent manner in the medium and high dose treatment groups.On the 14 th day of model establishment,there was no statistical difference between the model group and the treatment group.Urine protein creatinine ratio results showed a dosedependent decrease in the medium-dose and high-dose treatment groups compared with the model group on day 7 after modeling.Immunohistochemical staining showed significant proliferation of mesangial cells,increased infiltration of macrophages,and increased phosphorylation of STAT3 on day 7,which were improved after medium and high doses of SH.CCK-8 results showed that 50ng/ml PDGF-BB stimulated HRMC for 48 h to induce cell proliferation,and the cell model was established successfully.The abnormal proliferation of HRMC was reduced by adding 50μM SH.In vivo and in vitro Western blot results showed that the protein expressions of p-JAK2 and p-STAT3 and the inflammation-related factor TNF-α were decreased after SH treatment.Conclusion: SH treatment can reduce the urinary protein creatinine ratio of anti-Thy-1 nephritis rats,reduce the pathological damage of kidney,and reduce the infiltration of macrophages.SH treatment can inhibit the abnormal proliferation of HRMC induced by PDGF-BB and reduce the expression of pro-inflammatory cytokine TNF-α.In this study,SH was used to treat Ms PGN model animals and its protective effect on Ms PGN rats was found,preliminarily confirming that SH may inhibit mesangial cell proliferation by regulating the JAK2/STAT3 signaling pathway and reduce local immune inflammatory response in the kidney.These results suggest that SH may be a potential therapeutic agent for Ms PGN,providing a theoretical basis for the application of SH in the treatment of renal diseases with mesangial cell proliferation as an important pathological manifestation.