The Mechanism Of Metformin In Treating Lupus Nephritis By Activating AMPK And Regulating Th17/Treg Balance

Systemic lupus erythematosus(SLE)is an autoimmune disease characterized by the formation of multiple autoantibodies,deposition of a large number of immune complexes and inflammatory damage to multiple target organs.Lupus nephritis occurs in approximately two-thirds of SLE patients,the leading cause of death in lupus patients.The pathogenesis of Lupus nephritis(LN)is associated with renal causes such as podocyte damage and glomerular basement membrane destruction,as well as extrarenal factors such as immune system disorder.Although LN can be controlled by glucocorticoids and immunosuppressants in some extent,but about 20-70% of patients with LN are resistant to treatment,with a recurrence rate of about4%-20% every year.Recently,several experiments have demonstrated that key point of therapy of LN is to maintain the balance of certain cell subsets in the immune system,such as T auto-reactive cells(Th17)and T regulatory cells(Treg).Metformin,which is a traditional hypoglycemic drug,has good clinical efficacy and safety in Type 2 diabetes mellitus.By activating AMPK molecules to regulate autoimmunity,metformin has certain therapeutic effects on inflammation,malignant tumors,and aging-related diseases.This drug just has the potential to treat autoimmune diseases.In this study,metformin was used as an intervention to explore the mechanism of regulating Th17/Treg cell differentiation,AMPK activation and podocyte protection in spontaneous lupus nephritis(B6/lpr)and Pristane induced lupus nephritis(PIL)animal models.It provides new evidence for metformin as one adjunct treatment to lupus nephritis.Firstly,we observed T cell subsets of children who were diagnosed as systemic lupus erythematosus and confirmed that CD4 cell count and CD4/CD8 ratio were increased in children with lupus erythematosus at onset and effective treatment,indicating the stronger correlation between CD4 cells with the disease.Further text mining revealed that Th17 and Treg cells in this subpopulation were closely related to SLE,and Th17 cell differentiation was negatively correlated with AMPK activation.Meanwhile,we observed the different expressions of AMPK,m TOR and podocyte injury markers in renal glomerulus of adult lupus nephritis patients and found that AMPK/m TOR pathway was activated in lupus nephritis,and podocyte was significantly damaged.Based on this,we concluded that the AMPK activation may contribute to SLE.The AMPK molecule mainly participates in glucose metabolism and act as an important regulator of biological energy metabolism in cells.Therefore,we plan to further study the causal relationship between glucose metabolism disorder and systemic lupus erythematosus,to confirm whether activation of AMPK can prevent or alleviate lupus.We use Two sample Mendelian randomization analysis(MR Egger,Weighted median,Inverse variance weighted,Weighted mode)to contrast variety of glucose metabolic disorder state(Type 1 diabetes,Type 2 diabetes,Obesity,High density lipoprotein cholesterol,Triglycerides,and Fasting blood sugar)with the causal relationship of systemic lupus erythematosus.Methods of Heterogeneity analysis,horizontal pleiotropic analysis,single SNP analysis and Leave-one-out analysis were used to test the sensitivity of Mendelian randomization results.Two-sample Mendelian Randomization analysis in this study suggests that there is a positive causal effect relationship between Type 1 and Type 2 diabetes,Obesity,Triglycerides with the risk of SLE.High density lipoprotein cholesterol,fasting blood glucose have negative causal effect relationship with SLE.We concluded that the disorder of glucose metabolism may has a causal relationship with systemic lupus erythematosus.Based on with previous findings,we speculated that activation of AMPK,optimization of glucose metabolism and improvement of cellular energy metabolism may effectively prevent the occurrence of lupus and control the activity of lupus.Metformin has been known to be an important AMPK activator in type 2diabetes mellitus for many years.We intended to investigate whether metformin could be used in the treatment for systemic lupus erythematosus.Firstly,we use bioinformatics methods by selecting metformin drug related genes and with the same part of the systemic lupus erythematosus were analyzed,and the obtained correlation proteins are about 206 species,GO analysis and Kegg analysis confirmed that the glucose metabolism related pathways(such as insulin resistance)weight is higher,and except for the cell internal protein,most of the remaining high-weight proteins are downstream signal molecules of AMPK.Therefore,we speculate that metformin has molecular basis for the intervention of SLE.Meanwhile,based on the previous research results,we focus on the regulation of metformin on Th17 and Treg cells and the renal protection through the activation of AMPK.We observed B6/lpr lupus-prone model mice at the beginning of the study,and the dose of metformin was selected by drug dose gradient(10mg/100*d).Subsequently,the model B6/lpr mice were given intervention of metformin.Hydroxychloroquine and rapamycin were selected as control drugs for immune regulation and anti-proliferation effect at the same time.Final results of different groups were observed about 7 weeks after different interventions were administered.We found that metformin can effectively regulate the differentiation of Th17 and Treg cells in peripheral blood and spleen,showing that down-regulate of Th17 cells,upregulate Treg cells and decrease Th17/Treg ratio.In addition,metformin can downregulate neutrophil,and some B cell subsets can be regulated by metformin to some extent,which is beneficial to relieve autoimmune reaction and inflammatory reactions.In kidney,proteinuria level of metformin treated group was lower than that of model group,and renal podocyte morphology remained,basement membrane thickened less than that of model group under light microscope and electron microscope.Mouse glomeruli were obtained by sieving to eliminate the influence of renal tubules and interstitial on the results as much as possible.The results showed that the expression of Podocin and Nephrin were increased after metformin treatment compared with the model group,showing the protective effect of podocyte.The expression of AMPK was also significantly upregulated,metformin play a role in activating AMPK.In addition,we also detected the expressions of IL-17 a and Foxp3 molecules in these glomeruli,and the results were similar to the changes in Th17 and Treg cells in peripheral blood and spleen,confirming that Th17 and Treg cells may also be involved in kidney damage in LN.We hypothesized that metformin may protect glomerular podocytes and reduce glomerular damage in lupus nephritis by activating AMPK.To ensure the integrity of the study,we also conducted similar experiments in Pristane-induced lupus mice,which supplement to the environment-induced systemic lupus erythematosus model.We observed that the mice developed ascites soon after the intraperitoneal injection of Pristane and observed after 8 weeks of different interventions.The mouse in the metformin group were slimmer in body shape,which was related to the reduction of abdominal fat by metformin,but it may also reduce ascites.After 14 weeks of intervention,we found that the intraperitoneal lymphogranuloma of metformin group was less than that of model group,suggesting that metformin has certain anti-inflammatory effect.In terms of immune cell regulation,the expression of Th17 and Treg cells in peripheral blood and spleen was similar to the result of mouse in the B6/lpr model group.In addition,metformin can also reduce proteinuria in Pristane induced lupus nephritis.Under light microscope,the proliferation level of glomerular morphologic matrix was lower than that of model group.Under electron microscope,the morphology of podocyte in metformin group was fair and the foot process was still preserved,which was better than that of model group.The glomerulus was obtained by sieving method,and it was found that the molecular expression levels of Podocin and Nephrin were improved compared with the model group,reflecting the partial protective effect of kidney.At the same time,we found that the expression of LKB1 and AMPK was up-regulated and the expression of m TOR was down-regulated in the metformin treatment group,suggesting that metformin activates AMPK and may be involved in and affect the activation of AMPK signaling pathway.The expressions of IL-17 a and Foxp3 in glomerulus were also detected similar to those in peripheral blood and spleen.Meanwhile,we also detected the level of IL-10 which is secreted by Treg cells in lupus and found that the trend was similar to Foxp3,which supports the possibility that metformin regulating Th17/Treg to improve renal loss in the course of systemic lupus erythematosus.Through this part of the study,we came to the following conclusions: Intervention of metformin in pristane-induced lupus model can effectively regulate Th17 and Treg cells in peripheral blood and spleen,maintain Th17/Treg balance,and participate in the differentiation and regulation of neutrophils and some B cell subsets.Activation of AMPK and its upstream and downstream molecules may play a protective role in podocytes,that will maintain the structure and function of glomerular basement membrane,reduce kidney injury,and relieve proteinuria.To sum up,immune imbalance may play an important role in the onset and recurrence of SLE.To maintain the stability of podocyte morphology and function is helpful to reduce the renal loss in lupus nephritis.Metformin in B6/lpr spontaneous lupus model mice and Pristane-induced lupus model mice may play a protective role in glomerular podocytes by activating AMPK molecule and participating in the regulation of Th17/Treg cell differentiation to improve certain clinical symptoms in systemic lupus erythematosus.These results may provide theoretical basis for metformin as adjunctive drug in the treatment of systemic lupus erythematosus and lupus nephritis.