Therapeutic Effect And Mechanism Of Triptolide In MRL/lpr Lupus Mice

Bckground:Tripterygium has been used in the traditional Chinese medicine for hundreds of years.As a treasure of natural botanical medicine in China,it has gradually been recognized by the world.However,there is still a lack of specific immunological and molecular biological mechanisms for studying the disease of lupus mice in vivo.Triptolide is considered to be the main active ingredient of Tripterygium wilfordii,and is one of the most immunosuppressive monomers in Tripterygium wilfordii.Systemic lupus erythematosus(SLE)is a common chronic autoimmune disease involving multiple organs.Its etiology is complex and its pathogenesis is still unclear.It has become the focus of international research today.However,due to the unclear etiology and pathogenesis of the disease,the lack of specific treatment methods,the in-depth study of the pathogenesis of SLE,the development of new treatment strategies have important clinical significance.Objective: This study aimed to explore the mechanism of Chinese medicine monomer triptolide in the treatment of SLE.MRL/lpr mice were selected and the therapeutic effect of triptolide was observed systematically.The possible mechanism of triptolide in this model was preliminarily investigated,and the effect of triptolide was further verified in vitro.The molecular mechanism provides new ideas for the treatment strategy of SLE.Methods:Female MRL/lpr mice were randomly divided into three groups: low dose triptolide group(0.2 mg/kg/d),high dose triptolide group(0.3 mg/kg/d),blank carrier solution group as placebo and cyclophosphamide group as a positive control.Each group of mice was administered from 7 weeks to 21 weeks of age,and the therapeutic effects of each group on MRL/lpr mice were observed.The urine protein concentration of mice was quantitatively detected by BCA method,and the serum anti-ds DNA antibody level was detected by ELISA.Mouse kidney specimens were stained with H&E.Semi-quantitative assessment of glomerular involvement and renal small vessel involvement by renal pathologists blindly.The mouse spleen lymphocytes were isolated and the Treg cell subsets were detected by flow cytometry.Western Blot was used to detect Foxp3 protein in spleens.The mouse spleen cells were cultured with anti-CD3 and anti-CD28 stimulation in vitro,and 0n M.1n M and 10 n M triptolide were added respectively.The ratio of Treg cells in spleen cells was detected by flow cytometry.The mouse spleen lymphocytes were isolated in vivo experiments and RT-PCR was used to detect the expression level of miR-125a-5p.The mouse spleen cells were cultured with anti-CD3 and anti-CD28 stimulation in vitro,and 0n M,1n M and 10 n M triptolide were added respectively.The expression of miR-125a-5p was detected.MiR-125a-5p inhibitor and inhibitor control was transfected into spleen cell cultured with anti-CD3 and anti-CD28 stimulatio in vitr by liposome,and 10 n M triptolide was added.The expression level of Foxp3 and miR-125a-5p were detected in spleen cells.The ratio of Treg cells in spleen cells was detected by flow cytometry.Results: Firstly,Triptolide can significantly alleviate lupus-like disease in MRL/lpr mice.The level of proteinuria in mice treated with triptolide was significantly lower than that in the placebo group.Anti-ds DNA antibody levels in the triptolide group were significantly lower than that in the placebo group.Renal pathology showed that the lupus nephritis was milder than the placebo group by assessed glomerular injury and perivascular inflammation score differences blindly.There are statistical differences.We further found that triptolide increased the percentage of spleen lymphocyte Treg cells in MRL/lpr mice.The results of in vitro experiments showed that 1n M and 10 n M triptolide were added to the spleen cells of mice,and the ratio of Treg cells increased compared with the vehicle control group.Then,triptolide increased the expression of miR-125a-5p in vivo experiments and in the low dose group of triptolide(0.2 mg/kg/d),the percentage of Treg cells is directly proportional to the expression of miR-125a-5p.In vitro experiments showed that 1n M and 10 n M triptolide were added to the spleen cells of mice,and the ratio of miR-125a-5p were increased compared with the vehicle control group.Lastly,miR-125a-5p inhibitor and inhibitor control was transfected into spleen cell and 10 n M triptolide was added.Compared with the transfected control group,the transfection of miR-125a-5p inhibitor decreased Foxp3 m RNA and the proportion of Treg cells(P<0.05).Conclusion: This study investigated the therapeutic effect of triptolide on MRL/lpr lupus mice.Triptolide can significantly alleviate lupus-like disease in MRL/lpr mice and could induce Treg cells and miR-125a-5p in vivo and in vitro.Inhibition the effect of miR-125a-5p could counteract the effect of triptolide on inducing Treg cells.