The Role Of Nutrition And Oxidative Stress In The Pathogenesis Of Nonalcoholic Fatty Liver Disease

Part 1Associations of nutrition,oxidative stress,aging markers with non-alcoholic fatty liver disease in population with different glucose metabolism statusBackgrounds:Although the detail mechanisms of non-alcoholic fatty liver disease(NAFLD)remain to be well clarified,nutrition intake and oxidative stress is involved in the pathogenesis of NAFLD.While dietary antioxidants intake may improve oxidative stress and alleviate NAFLD,previous studies have reported inconsistent results and epidemiological research on those associations based on Chinese population is limited.The therapeutic effect of vitamin E on non-alcoholic steatohepatitis may be different under different glucose metabolism status,therefore when studying the relationship between antioxidants intake and NAFLD,subgroup analysis based on different glucose metabolism status should be further explored.In addition,cellular aging markers leukocyte telomere legth(LTL)and mitochondrial DNA copy number(mtDNAcn)are closely associated with oxidative stress and also potential biomarkers of NAFLD.However,the association between aging markers and NAFLD was not consistent,and when studying the association between aging markers and NAFLD,the effect of potential confusing factors such as oxidative stress markers was seldom taken into account simultaneously by most researchers.Objectives:We aimed to explore the association between dietary antioxidants intake,oxidative stress and NAFLD,and to perform subgroup analysis according to whether the participants had diabetes,and further to compare the differences in that association;to explore the internal relationship between oxidative stress,aging markers and NAFLD to deepen our understanding of the mechanism of NAFLD and to find simple blood markers of NAFLD.Methods:A total of 307 participants with different glucose metabolism status(diabetes,n=66;non-diabetes,n=241)from a diabetes project in ChangPing district of Beijing were included in the cross-sectional analysis.NAFLD was diagnosed by abdominal ultrasound(NAFLD,n=103;Non-NAFLD,n=204).Dietary intake was assessed by a 24-h food recall.The mtDNAcn and LTL were detected using realtime PCR assay.Serum oxidative stress related markers superoxide dismutase(SOD),glutathione reductase(GR)and 8-oxo-2'-deoxyguanosine(8-oxo-dG)were measured by ELISA.Participants were divided into different groups according to whether they had diabetes and NAFLD.Logistic regression analysis was applied to explore the association of antioxidants intake,oxidative stress related markers,aging markers with NAFLD.Mediation model analysis was used to investigate if SOD was involved in the association between antioxidant vitamins intake and NAFLD.Results:1.Associations of nutrition and oxidative stress with non-alcoholic fatty liver disease in different glucose metabolism status(1)The whole population was divided into different groups according to whether they had NAFLD or diabetes.The consumption of dietary vitamin A(P=0.023)was lower while serum 8-oxo-dG(P=0.010)was higher in the NAFLD group compared to the non-NAFLD group.Total caloric,carbohydrate,?-tocopherol and vitamin C intake were higher in diabetic population compared to those without diabetes(P<0.05),while there were no significant differences in serum oxidative stress markers between groups.Spearman correlation analysis indicated that dietary vitamin A(r=0.128,P=0.025)and?-tocopherol intake(r=0.230,P<0.001)were positively correlated with SOD in whole population.Multiple logistic regression analysis founded that 8-oxo-dG(OR=1.607,95%CI:1.082-2.3 86,P=0.019)was positively while ?-tocopherol(OR=0.905,95%CI:0.819-1.000,P=0.050)was marginally associated with NAFLD after adjusting for conventional NAFLD risk factors and caloric intake in whole population.(2)In non-diabetic population,we found that ?-tocopherol and vitamin A intake and serum SOD were lower while 8-oxo-dG was higher in NAFLD group compared to those of the non-NAFLD group(P<0.05).Spearman correlation analysis indicated that dietary vitamin A(r=0.173,P=0.007)and ?-tocopherol(r=0.325,P<0.001)intake were positively correlated with SOD.Multiple logistic regression analysis found dietary vitamin A(OR=0.997,95%CI:0.994-0.999,P=0.007),?-tocopherol intake(OR=0.777,95%CI:0.658-0.919,P=0.003)and serum SOD(OR=0.963,95%CI:0.936-0.992,P=0.012)were inversely associated with NAFLD after adjusting for conventional NAFLD risk factors and caloric intake.Mediation analysis indicated that SOD significantly mediated the indirect effect of dietary vitamin A or ?-tocopherol intake on NAFLD.(3)In diabetic population,there were no statistical differences between NAFLD group and non-NAFLD group regarding dietary antioxidants intake and serum oxidative stress markers.Moreover,Spearman correlation analysis and logistic regression analysis indicated that there were no significant associations between antioxidants intake,oxidative stress markers and NAFLD.(4)No significant associations between vitamin C,?-/?-tocopherol,?-tocopherol,zinc,and selenium intake and NAFLD were observed in population with diabetes or without diabetes.2.Associations between oxidative stress,aging markers and non-alcoholic fatty liver disease(1)Participants with NAFLD had higher mtDNAcn compared to those without NAFLD(P-0.032),while there was no significant difference in LTL between groups(P=0.347).(2)Univariate logistic regression analysis revealed that mtDNAcn was positively associated with NAFLD(P=0.033).However,following further adjustment for 8-oxodG,the association between mtDNAcn and NAFLD was not significant(P=0.055).With further adjustment for glucose tolerance status,antioxidants intake and other conventional NAFLD risk factors,this trend did not change(P>0.05),while 8-oxo-dG was independently associated with NAFLD(P<0.05),suggesting that oxidative stress may affect the association between mtDNAcn and NAFLD.Spearman correlation analysis further confirmed that leukocyte mtDNAcn was positively correlated with 8-oxo-dG(r=0.123,P=0.031)and negatively correlated with SOD(r=-0.141,P=0.013).Conclusions:1.Dietary ?-tocopherol intake was marginally associated with NAFLD in whole population,and dietary vitamin A and ?-tocopherol intake were negatively associated with NAFLD in non-diabetic population,and this relationship was partly mediated by SOD,while no significant associations between antioxidants intake and the risk of NAFLD were observed in diabetic population.Thus,the relationship between antioxidants intake and NAFLD may be different in population with different glucose metabolism status.However,the sample size was relatively small,and therefore large-scale longitudinal studies and related mechanism studies are needed to further confirm this view.2.In a Chinese population with different glucose metabolism status,serum 8-oxo-dG,rather than leukocyte mtDNAcn,was an independent risk factor for NAFLD.The relationship between leukocyte mtDNAcn and NAFLD could be affected by oxidative stress,and the higher mtDNAcn may be partly due to a compensatory mechanism in response to elevated oxidative stress.Therefore,oxidative stress may be an important driver of NAFLD.Part 2Study on the protective effect and related mechanism of antioxidants on HepG2 cells with steatosisBackgrounds:The mechanisms of occurcence and development of NAFLD are very complex,and many factors such as abnormal lipid metabolism,oxidative stress and apoptosis may be involved.We found that in non-diabetic population,dietary ?-tocopherol and vitamin A intake were negatively associated with NAFLD,and basic studies are needed to confirm these findings.In addition,the detailed protective mechanism of ?-tocopherol on NAFLD is still unclear,and previous studies have reported inconsistent results on the relationship between vitamin A and NAFLD,and therefore more in-depth research is needed to clarify.Objectives:We aimed to explore the effects of ?-tocopherol(?-T)and a bioactive metabolite of vitamin A[retinoic acid,(RA)]on lipid deposition,lipid synthesis,fatty acid ?-oxidation,oxidative stress and apoptosis in palmitic acid(PA)-induced HepG2 cells.Methods:1.HepG2 cells were treated with different concentrations of PA for 24 hours,and the appropriate concentration of PA was selected according to the cell viability detected by CCK-8 and the lipid deposition detected by oil red O staining.2.HepG2 cells were treated with ?-T or RA combined with PA,and the appropriate concentrations of ?-T and RA were selected according to the cell viability detected by CCK-8.3.The cells were divided into control group,PA group and PA combined with ?-T(RA)group.The lipid droplet deposition was detected by oil red O staining.The intracellular triglyceride(TG),malondialdehyde(MDA)and superoxide dismutase(SOD)of HepG2 cells were detected by kit.4.Western blot was used to assayed the protein expression of lipogenesis related enzymes(Srebp-1,ACC?),CPT1,SOD and apoptosis related factors(BCL-2,Bax,Cleaved Caspase-3)in different drug treated groups.Results:1.As the increasement of PA concentration,the cell viability was decreased while the lipid deposition was increased in HepG2 cell compared to that of control group.The cell viability decreased significantly and the lipid deposition increased greatly when HepG2 cells were treated by PA with the concentration greater than or equal to 0.4 mmol/L.Thus,0.4mmol/L PA was used for subsequent experiments.2.The results of CCK-8 test showed that ?-T or RA together with PA treatment could increase cell viability compared to that of PA treatment.The concentration of ?-T and RA were determined to be 100umol/L and 1umol/L according to the cell viability,respectively.3.?-T together with PA treatment markedly decreased intracellular lipid droplets accumulation,intracellular TG and MDA(P<0.05),and increased the level of SOD(P<0.05)compared to that of PA treatment;RA together with PA also reduced lipid deposition and intracellular TG significantly compared to that of PA treatment(P<0.05).4.Western blot showed that ?-T together with PA treatment significantly increased the protein expression of CPT1,SOD and BCL-2(P<0.05)but significantly decreased the protein expression of lipogenesis related enzymes(Srebp-1,ACC?)and apoptosis related factors(Bax,Cleaved Caspase-3)(P<0.05)compared to PA treatment.In terms of RA toghther with PA treatment,the protein expressions of CPT1 and SOD were significantly up-regulated while Bax was down-regulated compared to PA treatment(P<0.05).Conclusition:?-T and RA treatment can protect PA-induced HepG2 cell to some extent.?-T and RA are not just antioxidants with antioxidant effect,their protective effects may also be achieved by improving cell lipid metabolism and reducing cell apoptosis,but the specific signaling pathways still need to be further explored.