SIRT-6 Alleviates Nonalcoholic Fatty Liver Disease By Up-regulating PPAR-? Pathway And Reversing Senescence Of Hepatocytes

Objective To investigate whether SIRT-6,a member of the Sirtuin family,can simultaneously regulate lipid metabolism and senescence of hepatocytes in nonalcoholic fatty liver disease,and to explore specific mechanisms.Methods ?.Experiments in vitro.Male C57BL/6J mice were randomly divided into five groups as follows,(1)4w CD group: mice were fed with standardized diet for 4 weeks,n=5.(2)22w CD group: mice were fed with standardized diet for 22 weeks,n=10.(3)22w HF group: mice were fed with high-fatty diet for 22 weeks,n=22.(4)22w HF+LV-Sirt6 group: mice were fed with high-fatty diet for 22 weeks and injected with SIRT-6 overexpressing lentivirus at last 2-week,n=4.(5)22w HF+LV-NC group: All conditions were same as 22 w HF+LV-Sirt6 group with the exception that mice were injected with negative control lentivirus,n=4.When feeding time finished,all mice were sacrificed and the liver tissues were analyzed by staining(HE and oil red O)or other histological tests.The m RNA expression of SIRT-6,PPAR-a,PPAR-g,SCD-1,IL-6,CCL-8,HGF,GLB-1,MCM-2 and P21 were measured by RT-PCR.The serum levels of AST and ALT were also detected.II.Experiments in vivo.AML-12 cells were cultivated in standardized medium or 300 m(44)PA for 24 h,transfected with LV-Sirt6,SIRT6 RNAi or not.Cells were analyzed by oil red O staining.The m RNA and protein expression of SIRT-6,PPAR-a,PPAR-g,SCD-1,ACADM,CPT1 a,IL-6,CCL-8,HGF,GLB-1,MCM-2,P21 and g-H2 AX were measured by RT-PCR,Western blot or immunofluorescence.III.The mechanism of SIRT-6 up-regulating PPAR-a.(1)AML-12 cells were cultivated in standardized medium or 300 m(44)PA for 24 h,with or without prominent SIRT-6 activity cyanidin(100 m(44))or inhibitor catechin(10 m(44)).The protein expression of PPAR-a were measured by Western blot.(2)AML-12 with different expression levels of SIRT-6 were cultivated in standardized medium or 300 m(44)PA for 24 h.The promotor activity of PPAR-a were detected by dual luciferase reporter kit.Results I.Experiments in vitro.(1)By HE and oil red O staining,we observed marked steatosis in 22 w HF group,which can be alleviated significantly in 22 w HF+LV-Sirt6 group.(2)From 4w CD,22 w CD to 22 w HF,continuous decreasing of m RNA expression were detected in SIRT-6 and MCM-2(P<0.05),on the contrary,continuous increasing of m RNA expression were presented in IL-6,CCL-8,HGF and P21.Compared with 22 w CD,the m RNA expression of PPAR-a and the couple PPAR-g and SCD-1 in 22 w HF was down-regulated and up-regulated(P<0.05),respectively.Compared with 22 w HF+LV NC,the m RNA of IL-6,CCL-8,HGF,GLB-1 and P21 in 22 w HF+LV Sirt6 were down-regulated,and PPAR-a m RNA was increased(P<0.05),but no significant changes were found in that of PPAR-g and SCD-1.(3)The AST/ALT levels of mice in 22 w HF were increased sharply(P<0.05),which were relieved in 22 w HF+LV Sirt6(P<0.05).?.Experiments in vivo.(1)AML-12 cells treated with PA presented marked steatosis,which can be alleviated significantly when SIRT-6 was overexpressed.(2)By RT-PCR,Western blot or immunofluorescence,we detected decreased expression of SIRT-6,PPAR-a(11)ACADM,CPT1 a,MCM-2 in PA treated AML-12,and increased expression were showed in PPAR-g,SCD-1,IL-6,CCL-8,HGF,GLB-1,P21 and g-H2 AX.Except of PPAR-g and SCD-1,the changes of all above items were reversed when SIRT-6 was overexpressed(P<0.05).Similarly,we found increased expression of GLB-1(P<0.05)and decreased expression of PPAR-a(11)ACADM,CPT1a(P<0.05)when SIRT-6 was silenced.III.The mechanism of SIRT-6 up-regulating PPAR-a.(1)By Western blot,we found there were no influence of cyanidin or catechin on PPAR-a expression in standardized medium.But when treated with PA,an enhanced or inhibited effect were detected induced by catechin and cyanidin,respectively(P<0.05).(2)SIRT-6 up-regulated activity of PPAR-a promotor whether AML-12 were treated by PA or not(P<0.05).Conclusion(1)SIRT-6 alleviates steatosis based on its predominant effect of up-regulating PPAR-? pathway and then promotes fatty acid ?-oxidation.(2)SIRT-6 stimulates activity of PPAR-? promotor to increase expression of PPAR-?(13)((18))SIRT-6 may be a crucial factor to regulates NAFLD-associated senescence of hepatocytes due to its prominent effects of lipid metabolism and reversing aging.