The Effects Of (5R)-5-Hydroxytriptolide On Lupus Nephritis

Systemic lupus erythematosus（SLE）is a systemic autoimmune diseases associated with various organ injury,and one of the most serious complications is glomerulonephritis.Approximately 40–70% of lupus patients develop renal dysfunction and nephritis,which finally lead to kidney failure.The development of lupus nephritis（LN）has been characterized by increased production of autoantibody and cytokines,deposition of immune complex,and infiltration of leukocyte.The specific inflammatory cell population known to initiate and amplify kidney injury in MRL/lpr mice are T cell and macrophage.Neutrophil has been reported to release extracellular traps（NETs）which are associated with lupus nephritis in humans.Several chemokines including IP-10,RANTES,MCP-1,etc,mediate inflammatory immune cells influx into kidney in the pathological process of LN.All of these chemokines and receptors represent a promising therapeutic target in LN.Triptolide,is a diterpenoid triepoxide isolated from Tripterygium wilfordii Hook f,a well-known Chinese medicinal herb used in the treatment of autoimmune and inflammatory diseases.Triptolide,as an efficient immunosuppressant,shows a good therapeutic effect on autoimmune diseases including RA,EAE,SLE,etc.Nevertheless,due to its strong toxicity,the application of triptolide is restricted in clinic.（5R）-5-hydroxytriptolide（LLDT-8）is a triptolide derivative,retaining strong immunosuppressive activities but markedly reduced toxicities compared to triptolide.The CC₅₀ value of LLDT-8 was 256.6±73.8 nM,and the CC50 value of triptolide was 2.1±0.3 nM.Here we examined the effect of LLDT-8 on lupus nephritis in murine model.Firstly,8-week-old MRL/lpr mice were administrated LLDT-8（0.125mg/kg/2 day）orally for 9 weeks.The results showed that compared with vehicle group,different clinical parameters were improved upon LLDT-8 treatment: prolonged life-span of mice,decreased proteinuria,downregulated blood urea nitrogen and serum creatinine,reduced glomerular IgG deposits and ameliorated histopathology.Then,we investigated the therapeutic of three dose of LLDT-8（0.125mg/kg,0.063mg/kg and 0.031mg/kg）on MRL/lpr mice.The results showed that all of three different doses of LLDT-8 could reduce proteinuria,downregulate blood urea nitrogen and serum creatinine and decrease glomerular IgG deposits.These data verified again the therapeutic effect of LLDT-8 on lupus nephritis in MRL/lpr mice.Then we explored the pharmacological mechanism of LLDT-8,and found that: LLDT-8 could reduce the levels of cytokines including IFN-γ,IL-17,IL-6,TNF-α in kidney,but LLDT-8 had no influence on levels of cytokines in serum.According to these results,we speculated that LLDT-8 might have influence on cells secreting these cytokines in local kidney.So we isolated the KMNCs from mice kidney,and detected the T cells proportion via FACS.The results showed that LLDT-8 a decrease in CD3⁺ T cells in kidney after LLDT-8 treatment.In contrast,the fraction of T cell and its subsets in spleen did not differ,suggesting that LLDT-8 might hinder the recruitment and/or maintenance of T cells in kidneys.Furthermore,we examined the expression of CXCR3 on T cells from spleens and kidneys,and we found LLDT-8 treated group didn’t show a reduction of CXCR3 expression in T cells.However,the expression of Mig,IP-10 and RANTES in the kidneys of LLDT-8 treated mice was impaired compared to vehicle-treated mice.Besides T cells,we also found a decrease in macrophage cells and neutrophils percentage in kidney after treating with LLDT-8.According to these data,we conclude LLDT-8 can sabotage immune cells infiltration from spleen to kidney during LN by suppressing chemokines expression in kidney.Then,we choose another classic lupus nephritis model,female NZB/W F1 mice,to evaluate the therapeutic effect of LLDT-8.Mice were administrated LLDT-8（0.031mg/kg）orally for 18 weeks.We found that LLDT-8 could significant reduce the ratio of proteinuria and urea creatinine;improve the survival rate.The results suggested LLDT-8 also have great therapeutic effect on NZB/W F1 mice.Moreover,we tested the immune cells proportion in kidneys of NZB/W F1 mice.Consistent with the results in MRL/lpr mice,the proportion of CD3⁺ T cells,CD4⁺ Th cells and CD11b⁺ myeloid cells were significantly reduced in the kidneys of mice treated with LLDT-8.Finally,to directly test whether LLDT-8 can suppress related chemokines expression in kidney cells,we stimulated human proximal tubule epithelial cell line（HK-2）and mouse mesangial cell line（SV40 MES 13）with IFN-γ and IL-17,and after treating with LLDT-8,we detected expression of T cell/ myelocyte attracting chemokines in kidney cells.LLDT-8 greatly repressed IP-10,Mig,MCP-1 and IL-6 expression in HK-2 cells and MES cells.Moreover,we observed a synergy between IFN-γ and IL-17 proinflammatory effects on kidney cells.In conclusion,this study demonstrated the therapeutic efficacy of LLDT-8 in MRL/lpr mice and NZB.W F1 mice,and provided evidence in support of expending clinical indications for LLDT-8 in lupus nephritis.Our data showed that LLDT-8 ameliorated lupus nephritis in MRL/lpr mice via suppressing chemokines expression and inhibiting immune cells infiltration.Therefore,these results suggested that LLDT-8 could be a promising compound for the development of new treatment for lupus disease.Meanwhile,it provides new evidence and clues for research about triptolide compounds in the field of autoimmune diseases.