The Mechanism Research Of Diosgenin Improving Diabetic Kidney Disease By Regulating SIRT6 Signaling Pathway

Objective: Based on studies presented in the literature on kidney injuries plus screening for the binding effects of the drug to Sirtuin 6(SIRT6),we aimed to discuss the mechanism of diosgenin improving DKD through SIRT6 signaling pathway.Methods: DKD model was established in spontaneous diabetic db/db mice.Animal experiment was in two parts.The first part includes four groups consisting of control(Con)group,model(Mod)group,low dose of diosgenin(DL)group and high dose of diosgenin(DH)group.The second part includes four groups consisting of control group,model group,DH+OSS＿128167(OSS,inhibitor of SIRT6)group,MDL800(agonist of SIRT6)group.MPC5 cell line was selected in cell experiment,which was mainly composed of six groups including Con group,palmitic acid(PA)group,PA+DL group,PA+DH group,PA+DH+OSS group,PA+MDL800 group.Some procedures such as transcriptomics,RTPCR and so on were used in the study to explore and verify the mechanism.Results: In the first part of animal experiment,an increase of mesangial matrix expansion,glomerular basement membrane(GBM)thickness,foot process(FP)width,urine albumin/creatinine(UACR),DESMIN,adipose Differentiation Related Protein(ADRP),and a decrease of NEPHRIN,PODOCIN,SIRT6 in Mod group were alleviated in DH group.In the second part of animal experiment,above results could bot improved in DH+OSS group comparing to Mod group,which could be improved in MDL800 group.The same results were also found in cell experiment.In addition,protein level and mRNA expression of pyruvate dehydrogenase kinase 4(PDK4)and Angiopoietin-like-4(ANGPTL4)were increased in PA group,which could be alleviated in DH group,MDL800 group.Conclusions: Diosgenin could protect against DKD through regulating SIRT6 signaling pathway.Part Ⅰ Effect of diosgenin on DKD in db/db miceObjective: To explore the protective effect of diosgenin on DKD.Methods: Db/db mice were used to establish the animal model of DKD,and db/m mice were selected as the control mice.All db/db mice were divided into Mod group,DL group and DH group according to random number table method.The control group was db/m mice.All mice were intervented at the age of 8 weeks.DL group and DH group were gavaged with diosgenin(30 mg/kg/d,90 mg/kg/d)mixed with sodium carboxymethylcellulose respectively.The normal group and model group were gavaged with 0.5% sodium carboxymethylcellulose solution in same volume.All groups were intervened for 4 weeks.At the fourth week of intervention,the metabolism cage was used to collect 24-hour urine,and the oral glucose tolerance test(OGTT)was implemented.Pathology,renal injury and glomerular injury markers were measured after the animals were killed.Results: Compared with Con group,the glomerular mesangial matrix fraction,basement membrane thickness,FP width and 24-hour urinary albumin creatinine ratio in Mod group were increased;compared with Mod group,the glomerular mesangial matrix fraction,basement membrane thickness,FP width and 24-hour urinary albumin creatinine ratio in DH group were decreased.Compared with Con group,NEPHRIN and PODOCIN in Mod group were decreased and DESMIN were increased;compared with Mod group,NEPHRIN and PODOCIN in DH group were increased and DESMIN were decreased.Compared with Con group,Mod group had abnormal glucose tolerance and body weight gain,but DL group and DH group had no significant improvement.Compared with Con group,SIRT6 in Mod group was decreased;compared with Mod group,SIRT6 in DH group was increased.Compared with Con group,glomerular lipid deposition,ADRP expression,Sterol RegUlatory Element Binding Protein-1c(SREBP-1C)and Microsomal Triglyceride Transfer Protein(MTTP)mRNA expression,and cysteine proteinase inhibitor C(CYSTATIN-C)in renal tubules were increased in Mod group;compared with Mod group,glomerular lipid deposition,ADRP,SREBP-1C and MTTP mRNA expression,and CYSTATIN-C in renal tubules were decreased in DH group.Conclusions: Diosgenin has an effect on protecting against DKD and podocyte injury,inhibiting lipid deposition,which is related with regulating SIRT6.Part Ⅱ The exploration on the mechanism of diosgenin against DKD through SIRT6 signaling pathwayObjective: Method of SIRT6 inhibition/activation was select to verify the mechanism of diosgenin targeting SIRT6 to improve DKD.Methods: Db/db mice were selected as the animal model of DKD,and db/m mice were selected as the control mice.All db/db mice were divided into Mod group,DH+OSS(20 mg/kg/d)group,MDL800(50 mg/kg)group according to random number table method.The Con group was db/m mice.All mice were intervened at the age of 8 weeks.Both the Con group and the Mod group were treated with 0.5% sodium carboxymethylcellulose solution in same volume.All groups were intervened for 4 weeks.At the fourth week of intervention,urine was collected in a metabolic cage for 24 hours.Pathology,renal injury and glomerular injury markers were measured after the animals were killed.Results: Compared with Con group,the glomerular mesangial matrix fraction,basement membrane thickness,FP width and 24-hour urinary albumin creatinine ratio in Mod group were increased;compared with Mod group,mesangial dilation fraction,basement membrane thickness,FP width and 24-hour urinary albumin creatinine ratio in MDL800 group were decreased,but there was no significant difference in DH+OSS group.Compared with Con group,glomerular NEPHRIN and PODOCIN in Mod group were decreased and DESMIN was increased;compared with Mod group,NEPHRIN and PODOCIN in MDL800 group were increased,DESMIN was decreased,and there was no significant difference in DH+OSS group.Compared with Con group,SIRT6 in Mod group was decreased;compared with Mod group,SIRT6 in MDL800 group was increased,and there was no significant difference in DH+OSS group.Compared with Con group,glomerular lipid deposition,ADRP expression,SREBP-1C and MTTP mRNA expression were increased in Mod group and inflammatory factors including Tumor necrosis factor-a(TNF α),Interleukin-6(IL6)and Interleukin-1 β(IL1 β)were not found different;compared with Mod group,glomerular lipid deposition,ADRP expression,SREBP-1C and MTTP mRNA expression in MDL800 group were decreased,and no significant difference was found in DH+OSS group.Compared with Con group,Mod group showed body weight gain was increased,but no significant difference was found in MDL800 group and DH+OSS group.Conclusions: The protective effect of diosgenin on DKD can be inhibited by OSS＿128167,MDL800 has the same protective effect as diosgenin on DKD.Diosgenin can protect against podocyte injury and DKD through SIRT6 signaling pathway.Part Ⅲ Exploration on the mechanism of diosgenin against podocyte injury through SIRT6 signaling pathwayObjective: To explore the protective mechanism of diosgenin on improving podocyte injury through SIRT6 signaling pathway.Methods: The MPC5 cell line was used as the research object,and the podocyte injury model was established by using PA.The experimental groups included Con group,PA group,PA+DL group,PA+DH group,PA+MDL800 group,PA+DH+OSS group.The concentration of PA is 100μM/L.The concentration of DL is 0.1μ M/L,DH concentration is 1μ M/L,the concentration of OSS＿128167 is 100 μM/L.The concentration of MDL800 is 10μM/L.Immunofluorescence and western blot were used to measure the expression of podocyte injury markers such as NEPHRIN and SIRT6.Transcriptomics and other methods were used to explore and verify whether diosgenin against podocyte injury is related to SIRT6 signaling pathway.Results: Compared with Con group,NEPHRIN and PODOCIN in PA group were decreased,DESMIN was increased and SIRT6 was decreased;compared with PA group,NEPHRIN and PODOCIN were increased,DESMIN was decreased and SIRT6 was increased in PA+DH group.Compared with PA group,there was no significant difference on NEPHRIN,PODOCIN,DESMIN and SIRT6 in PA+DH+OSS group;In PA+MDL800 group,NEPHRIN and PODOCIN were increased,DESMIN was decreased and SIRT6 was increased.Transcriptome analysis combined with protein-protein interaction(PPI)analysis showed that PDK4 and ANGPTL4 may be the downstream pathway of SIRT6.Compared with Con group,the expressions of PDK4 and ANGPTL4 protein and mRNA in PA group were increased;compared with PA group,the expressions of PDK4 and ANGPTL4 protein and mRNA in PA+DH group and PA+MDL800 group were decreased,but there was no significant difference in PA+DL group and PA+DH+OSS group.Animal experiments further verified the downstream pathways PDK4 and ANGPTL4 of SIRT6.Compared with Con group,the expressions of PDK4 and ANGPTL4 in glomerulus of Mod group were increased;compared with Mod group,the expressions of PDK4 and ANGPTL4 in glomerulus of DH group and MDL800 group were decreased,while there was no significant difference in DL group and DH+OSS group.In addition,molecular docking showed that diosgenin had a good affinity with SIRT6.Conclusions: Diosgenin can regulate SIRT6 to protect against podocyte injury,and the downstream pathway is related to PDK4 and ANGPTL4 proteins.