Protective Effect Of Curcumin On High Glucose Induced Transdifferentiation Of HK-2 Cell By Up-regulating SIRT1

[Backgrounds and purposes] Uncontrolled hyperglycemia in diabetic patients can lead to a variety of complications,such as Diabetic Nephropathy(DN).DN is still the leading cause of end-stage renal failure.The lack of effective interventions for DN in clinic has been recognized as a health problem.Curcumin belongs to the polyphenols with high medical value.Silent Information Regulator 1(SIRT1)is a histone deacetylase which widely presents in human cells.SIRT1 is also highly expressed in Human renal tubular epithelialccell cell(HK-2).Studies have found that curcumin could activate SIRT1 in the treatment of head and neck squamous carcinoma cell[1],up-regulation of which has a protective effect on bile duct ligation(BDL)induced liver fibrosis model[2].Nuclear factor-?B(NF-?B)is a well-recognized proinflammatory cytokine,and there are many modification ways such as acetylation,phosphorylation,methylation to regulate inflammatory reaction[3].Some researchers confirmed that SIRT1 could deacetylate the subunit p65 of NF-?B,thereby reduce the inflammatory response in downstream.Thus,we speculated whether curcumin could inhibit the acetylation of NF-?Bp65 induced by up-regulating the expression of SIRT1,and then inhibiting the transdifferentiation of high glucose-induced HK-2 cells.In this research,we studied the mechanism of high glucose-induced transdifferentiation on HK-2 cells and whether curcumin had a protective effect on it,so as to provide an experimental basis for the treatment of diabetic nephropathy by curcumin and provide new ideas and directions for the pharmacotherapy of diabetic nephropathy.[Materials and methods] The HK-2 cells in the logarithmic growth phase were selected as the research object.(1)Effects of high glucose on transdifferentiation of HK-2 cells: The experimental groups were divided into normal control group(5.5mmol/L),high glucose group(30mmol/L),mannitol group,high glucose plus SIRT1 inhibitor(EX527).Observation indicators:(1)Observation of cell morphology.(2)The expressions of E-cadherin,?-SMA and SIRT1 protein in each group were detected by Western Blot.(3)The expression of IL-1? in each group was detected by ELISA.(2)Effects of different concentrations of curcumin on transdifferentiation of HK-2 cells: The experimental groups were high glucose group,high glucose plus curcumin with low concentration group(5?mol/L),high glucose plus curcumin with medium concentration group(10?mol/L),High concentration of glucose plus curcumin(20?mol/L).Observation indicators:(1)Observation of cell morphology.(2)The expressions of E-cadherin,?-SMA,SIRT1 and acetylated NF-?Bp65 in each group were detected by Western Blot.(3)The expression of IL-1? in each group were detected by ELISA.(3)The effect of SIRT1 expression on HK-2 transdifferentiation: The experimental groups were high glucose group,high glucose plus curcumin group(20?mol/L),high glucose plus curcumin and SIRT1 inhibitor group.Observation indicators:(1)Western Blot was used to detect the expression of E-cadherin,?-SMA,SIRT1 and acetylated NF-?Bp65 in each group.(2)The expression of IL-1? in each group was detected by ELISA.[Results](1)The cells in the normal control group were paved like cobblestone.Compared with them,the cells in the high glucose group were long spindle-shaped.The expression of E-cadherin and SIRT1 decreased and the ?-SMA and IL-1? expression increased compared with the normal control group.The high glucose plus SIRT1 inhibitor group had more obvious long spindle-shaped cells,but with less expressed E-cadherin and SIRT1 protein but more expressed ?-SMA and IL-1? when compared with high glucose group.There were no significant differences in cell morphology,expression of E-cadherin,SIRT1,?-SMA and IL-1? between the mannitol group and the normal control group.It showed that high glucose can induce the epithelial-mesenchymal transition(EMT)of HK-2 cells.(2)Compared with the high glucose group,the long spindle-shaped morphology of HK-2 cells changed slightly in the high glucose plus curcumin group.With the increase of curcumin concentration,the expression of E-cadherin and SIRT1 protein increased and the expression of ?-SMA?acetylated NF-?Bp65 but IL-1? decreased.Therefore,Curcumin could reduce the EMT of HK-2 cells in high glucose environment.(3)Curcumin inhibited the injury of HK-2 cells induced by high glucose,the inhibition of which weakened when co-treated with SIRT1 inhibitor.This research indicated that curcumin could reduce EMT of HK-2 cells induced by the high-glucose environment via up-regulating the expression of SIRT1.[Conclusions](1)High glucose could induce EMT of HK-2 cells.(2)Curcumin could reduce EMT of HK-2 cells induced by high glucose via up-regulating SIRT1 expression.