Regulation Mechanism Of Curcumin And Endurance Mediated Methylated Modification In Inflamm-Aging

Human aging leads to progressive inflammatory and epigenetics changes in our body.This chronic low-grade inflammatory aging phenomenon and abnormal epigenetic modifications,such as methylation,would cause damages to tissues and organs,which further facilitate aging and related diseases.The NF-?B/TNF-? pathway is the main mechanism that causes inflammaging.Both curcumin and exercise can alleviate inflamm-aging by inhibiting the NF-?B signaling pathway.However,the mechanism of how the epigenetic changes,such as DNA and histone methylation,contribute to aging remains to be studied.The objective of my PhD thesis study is to investigate the change of DNA methylation levels on the genes involved in the NF-?B signaling pathway by using mice models of inflamm-aging.Specifically,we'd like to investigate whether endurance exercise can improve inflamm-aging by regulating the epigenetic modification of DNA and histone methylation levels of those genes involved in the NF-?B signaling pathway in aging mice,and to explore if curcumin and endurance exercise can modulate histone methylation levels in the NF-?B signaling pathway through skeletal musclespecific histone enzyme SMYD2.Experiment 1:Methods 20 female BALB/C mice,which have been normally fed for 3 months,were used in this experiment at different ages,5 each after 2 months,9 months,12 months and 15 months.HE staining,Elisa,Western blot and MSP were carried out according to the required indexes.The image-pro plus 6.0 and curveexpert1.3 software were used to process and analyze the images,and the SPSS19.0 and GraphPad prism 8 statistical software were used to process and analyze experimental data.The results showed differences between groups according to normal distribution,single factor analysis using T test,double factor analysis using Two-way ANOVA variance analysis.Rank sum test and Kruskal-Wallis method were used to carry out pairwise analysis for the differences between groups that did not meet the normal distribution and homogeneity of variance.Results(1)Weight and viscera coefficient: Body weights of the 18-month-old mice were significantly lower than those of the 5-month-old and 12-month-old mice(p<0.001);Ovarian coefficient of 18-month-old mice decreased significantly compared with that of 5-month-old group(p<0.05).(2)Characteristics of histomorphology: Compared with 5-month-old mice,18-month-old mice had different degrees of inflammatory infiltration in their ovary,lung tissue and hippocampus tissue.(3)Changes in serum inflammatory factor concentrations: TNF-? concentrations were higher in 18-month-old mice than in 5-month-old mice(p<0.001)and 12-month-old mice(p<0.001).(4)Expression of NF-?B related proteins in skeletal muscle and myocardium: Skeletal muscle p50,TNF-?,COX2 levels were higher in 18-month-old mice than in 5-month-old(p<0.001),p65 levels were higher than those in the 5-month-old group(p<0.01),p50,p65 levels were higher than in the 12-month-old group(p<0.05);p50,COX2 level is higher than 15-month-old group(p<0.05);The level of myocardial p50,p65,TNF-? in 18-month-old mice was higher than that in 5 months(p<0.01),p50,p65,COX2 levels were higher than those in the 12-month-old group(p<0.05).(5)NF-?B p50 methylation: Reduced DNA methylation levels of skeletal muscle p50 genes in 18-month-old mice compared with 5-month-old mice(p<0.05).Conclusion(1)The NF-?B signal protein expression level of p50,p65,COX2,and TNF-? in skeletal muscle and myocardium was higher in aged mice,which means the presence of inflamm-aging in natural aging mice;(2)Altered methylation of the p50 promoter in aged mice suggests a potential epigenetic mechanisms in inflamm-aging.Experiment 2Methods Female BALB/C mice with different ages: 9-month-old mice(10),12-monthold mice(10),and 15-month-old mice(10)were fed and included in this experiment: 9-monthold mice with exercise group(E12),9-month-old control group(N12),12 month-old mice with exercise group(E15),12-month-old control group(N15),15 month-old mice with exercise group(E18),15 month-old control group(N18),each with 5 mice.The mice in the control group(N12,N15,N18)were fed normally and the mice in the exercise group(E12,E15,E18)were treated with exercise intervention(Running platform training;0% slope,3 times a week,60 min;speed of 10 m/min ? 9m/min ? 8m/min each time)for 3 months.Elisa,Immunohistochemical,Western blot and MSP were carried out according to the required indexes.Statistical analysis method is the same as experiment 1.Results(1)Changes in serum inflammatory factor concentrations: The serum TNF-? concentration of mice did not change significantly after exercise.(2)Immunohistochemistry of p65 skeletal muscle: There were more p65 positive cells in gastrocnemius tissue in E15 mice than that in N15,and fewer p65 positive cells were in gastrocnemius and flounder in E18 than that in N18.(3)Expression of NF-?B related proteins in skeletal muscle and myocardium: E12 skeletal muscle COX2 levels were lower in mice than that in N12(p<0.05);E15 mice had higher skeletal muscle p50 levels than that in N15(p<0.01),Myocardial p50 levels were lower than in that in N15(p<0.01);Skeletal muscle p50,COX2 levels were lower in E18 mice than that in N18(p<0.05),TNF-? levels were lower than that in N18(p<0.01),Myocardial p50 levels were lower(p<0.01)and p65 levels higher(p<0.01)than those in N18.(4)NF-?B p50 and TNF-? methylation: the promoter methylation level of p50 in skeletal muscle in E18 mice was higher than that in N18;The change of TNF-? methylation was not obvious.Conclusion p50 and TNF-? protein expression changes in skeletal muscle in mice with exercise intervention were most obvious,but only the change of methylation level on p50 occurred,indicating that methylation modification on p50 of endurance exercise may be a potential mechanism by which it improves inflamm-aging through NF-?B signaling in aged mice,and its regulation on TNF-? does not involve methylation modification.Experiment 3Methods Female BALB/C mice with different ages: 2-month-old mice(5),18-monthold mice(20)were fed and included in this experiment: Young control group(YN),Elderly control group(ON),Curcumin intervention in elderly group(OC),Exercise intervention in elderly group(OE),Curcumin combined with exercise intervention in elderly group(OCE),each for 5.OC,OCE mice were given 1-month exercise intervention(platform training,0% slope,3 times a week,60 min;speed of 8 m/min each time),and OC,OCE mice were given curcumin for 1 month(5 times a week,at 100 mg/kg doses).HE staining,Elisa,Immunofluorescence assay,Western blot and MSP were carried out according to the required indexes.Statistical analysis method is the same as experiment 1.Results(1)Histomorphology characteristics: Intraperitoneal administration of curcumin did not cause liver injury in aged mice,OCE mice had less inflammatory infiltration than the OC and OE groups.(2)Changes in serum inflammatory factor concentrations: OCE mice had lower serum TNF-? concentrations than ON mice(p<0.001),IL-1? concentrations higher than OE(p<0.01).(3)Co-expression SMYD2/p65 Immunofluorescence: Skeletal muscle enhancement in OCE mice compared to ON mice(p<0.001),OC mice(p<0.01),OE mice(p<0.05);Myocardium in OCE mice decreased compared with OC mice(p<0.01).(4)Expression of NF-?B related proteins in skeletal muscle and myocardium: OCE mice had higher skeletal muscle SMYD2,H3K36me2 levels than ON mice(p<0.01),p65?SMYD2?H3K36me2 levels were higher than in OC mice(p<0.05),Myocardial TNF-? levels were lower in OCE mice than in ON mice(p<0.05),SMYD2 levels were higher than in ON and OC mice(p<0.01).(5)NF-?B p50 and p65 methylation: The DNA methylation level of p50 in skeletal muscle in OCE mice was higher than that of ON and OC mice;The change of p65 methylation was not obvious.Conclusion(1)Curcumin combined with endurance exercise could regulate the methylation level of p50 in skeletal muscle and myocardial tissue in aged mice,which might be the mechanism of DNA methylation regulation in inflamm-aging;(2)Curcumin combined with endurance exercise inhibits the SMYD2-H3K36me2-RelA/p65-TNF-? pathway through SMYD2 mediated histone methylation,which might be also one of the epigenetic mechanisms to regulate inflamm-aging.