The Research On The Mechanism Of Artesunate Improve The Renal Injury In Bleomycin-induced Systemic Sclerosis Model

BackgroundSystemic scleroderma(SSc),also known as diffuse scleroderma or systemic sclerosis,is an autoimmune disease associated with connective tissue.SSc is a rare disease,with which women are the main target population.Systemic sclerosis is predominantly characterized by vascular lesions,inflammation,collagen deposition and fibrosis in the skin and internal organs.Although great progress has been made in elucidating the pathogenesis of SSc in recent years,exact molecular mechanism remains unclear and it remains a challenging disease.Artemisinin and its derivatives have achieved remarkable results in anti-malaria.More and more studies have reported that artemisinin and its derivatives have significant pharmacological effects in anti-tumor,immune regulation and anti-inflammatory.Artesunate,as one of the derivatives of artemisinin,has played a positive role in the treatment of immune system diseases.This study focused on the mechanism of artesunate in improving systemic sclerosis induced by bleomycin,especially in renal injury.ObjectiveSystemic sclerosis is a connective tissue-associated autoimmune disease.Its main features are vascular lesions,inflammation,collagen deposition and fibrosis in the skin and internal organs.In recent years,many studies have reported that artesunate has kinds of effects on anti-inflammatory,anti-fibrosis and immunosuppression.Artesunate plays a positive role in the treatment and improvement of immune diseases such as rheumatoid arthritis and systemic lupus erythematosus.We used artesunate on bleomycin-induced systemic sclerosis model to explore the mechanism of artesunate improving the systemic sclerosis renal damage.MethodsTwenty-four mice were randomly divided into 4 groups: control group,model group,artesunate low dose group and artesunate high dose group,with 6 mice in each group,respctively.The control group was subcutaneously injected with0.1m L PBS.The other groups were subcutaneously injected with 500μ g / m L bleomycin on the back,once every other day for 0.1 m L,forfour weeks.The artesunate dosage of low-dose(LA)group is 1mg/kg and the artesunate dosage of high-dose(HA)group is 5mg/kg.The above two groups of mice were injected intraperitoneally artesunate 200 ul per day,for four weeks.All mice were sacrificed 24 hours after the last injection.The skin at the back of the injection site and the kidneys will be excised for histopathology and PCR.Human embryonic kidney 293 T cell culture and stimulation,divided into6 groups,the control group,bleomycin model group(500ng / ml),bleomycin plus artesunate low dose group(2μ M),bleomycin plus artesunate high dose group(10μ M),bleomycin plus artesunate low-dose group plus NF-κ Binhibitor(20um),bleomycin plus artesunate high-dose group plus NF-κ B inhibitor(20um),respectively.Cells were collected after stimulation for PCR and Western Blot detection.Results1 skin pathologyCompared with the control group,the dermal layer in the model group was significantly thicker and disorderly arranged,the interstitial space was obviously narrower or disappeared,the fat layer was thin or even missing,infiltration of inflammatory cells in the deep dermis and subcutaneous tissue sometimes accompanied by follicular shedding.Artesunate group(low dose and high dose)compared with the model group,the skin dermis thickness is relatively thin,collagen fiber bundle is relatively thin,widened gap,vascular proliferation and expansion,infiltration of inflammatory cells,less runoff.2 kidney pathologyThe model group showed large area tubular cell swelling,inflammatory cell infiltration,a large number of cell shrinkage and necrosis.Artesunate low dose group glomerular,tubular cells still appear enlarged.Artesunate high-dose group glomerular and tubular cell swelling was not significantly different from the control group.3 Inflammation related indicators gene detectionThe results of cell experiments showed that high-dose dihydroartemisinin group had the best effect at 30 minutes,which could significantly reduce the levels of IFN-γ,IL-1β,IL-23 and INOS in model group(P <0.01).Compared with the model group,the IL-6 and MCP1 levels in kidney of artesunate high-dose group were significantly decreased(P <0.01,P <0.05).4 NF-κ B,P62 detectionThe results of cell experiments showed that the NF-κ B in bleomycin group was significantly higher than that in normal group,the high dose artesunate group was significantly lower than that in bleomycin group.ConclusionsArtesunate can reduce renal damage by inhibiting NF-κ B activity,decreasing P62,promoting autophagy,decreasing IFN-γ,IL-1β,IL-23,INOS,IL-6 and MCP-1.