| Objective:This study intended to use diet-induced obesity rats model as the carrier.From the hypothalamus Tscl-mTOR signaling pathway,which is considered as key link of simple obesity onset.Based on the early study which confirmed the Tscl gene methylation increases in DIO rats,to observe the methylation modification effect of hypothalamus DNA methylation transferase to Tscl gene,regulating downstream mTORC1 and the key factors in simple obesity onset mechanisms,to explore the effect and mechanism of electro-acupuncture treatment of simple obesity with"ST25", "SP6","CV12/RN12"and"ST36"in the perspective of epigenetics,which will provide a solid scientific basis for acupuncture treatment of simple obestiy in clinical practice.Methods:Choose just weanling clean level,4 weeks(70?90g weight),healthy male SD rats,randomly divided into modules and ordinary feed group after eliminating the rats which are not conform to the weight. Using the modified homemade high fat feedstuff to induce the animal models of DIO rats.Building successful DIO rats were radomly divided into obesity group(or model group) and acupuncture group.Ordinary feed group choose to be control group after screening.Acupuncture treatment:choose "Tianshu(ST25)","Zhongwan(CV12/RN12)","Sanyinjiao(SP6)","Housanli(ST36)"poi nts for electrical acupuncture intervention with homemade holder fixed,using the dilatational wave,2/15Hz,30min/d,5d/course of treatment,2 days off,a total of four courses.Control group and obesity group:with the same method fixed but not acupuncure. All the animals were weighed and fasted 12h after treatment. The next day after fixation,choose the brain tissue,hypothalamus ARC of the rats,to detect Dnmts(Dnmt2, Dnmt3a, Dnmt3b)proteins expression by using immunohistochemical technique methods,detect mTORC1 proteins expression by using Western Blot protein imprinting method,detect mTORC1and appetite prptides(AgRP,NPY and PoMC) mRNA and Tsc1 methylation by using RT-PCR method,comparing the differences among groups.Results:1.Weight results:The body weight of DIO rats was increased significantly in obestiy group and acupuncture group compared with control group(P<0.01).There were no significant differences between obesity group and acupuncture group(P>0.05).After electropuncture treatment,body weight and body fat and BFR in obesity group and acupuncture group reduced observably(P<0.01).2.The hypothalamus Tsc1 Methylation results:(1)Tsc1 fragment 1 methylation: Compared with control group,the expression of Tsc1 methylation increased in obesity group(P<0.05).After electropuncture treatment,the expression of hypothalamus Tsc1 methylation level in acupuncture group was lower than that of obesity group(P<0.05). There were no significant differences between control group and acupuncture group(P>0.05).(2)Tsc1 fragment 2 methylation:Compared with control group,the expression of Tsc1 methylation increased in obesity group(P<0.05).After electropuncture treatment,the expression of hypothalamus Tsc1 methylation level in acupuncture group was lower than that of obesity group without significant differences(P>0.05). There were no significant differences between control group and acupuncture group(P>0.05).(3)Tsc1 fragment 3 methylation:Compared with control group,the expression of Tsc1 methylation increased significantly in obesity group(P<0.01). After electropuncture treatment,the expression of hypothalamus Tsc1 methylation level in acupuncture group was lower than that of obesity group(P<0.05).There were no significant differences between control group and acupuncture group(P>0.05).3.The hypothalamus related test results:(1)Dnmt2:Compared with control group,the average optical density of hypothalamus Dnmt2 increased significantly in obesity group(P<0.01).After electropuncture treatment,the average optical density of hypothalamus Dnmt2in acupuncture group was lower than that of obesity group (P<0.01).There were no significant differences between control group and acupuncture group(P>0.05).(2)Dnmt3a:Compared with control group,the average optical density of hypothalamus Dnmt3a increased significantly in obesity group (P<0.01).After electropuncture treatment,the average optical density of hypothalamus Dnmt3a in acupuncture group was lower than that of obesity group(P<0.05).There were no significant differences between control group and acupuncture group(P>0.05).(3)Dnmt3b:Compared with control group,the average optical density of hypothalamus Dnmt3b increased significantly in obesity group(P<0.01).After electropuncture treatment,the average optical density of hypothalamus Dnmt3bin acupuncture group was lower than that of obesity group(P<0.05).There were no significant differences between control group and acupuncture group(P>0.05).(4)mTORCl mRNA and protein:Compared with control group,the expression of mTORC1 mRNA and protein increased significantly in obesity group (P?0.01).After electropuncture treatment,the expression of hypothalamus mTORC1 mRNA and protein in acupuncture group was lower than that of obesity group (P=0.01,P<0.05).And the expression of hypothalamus mTORC1 mRNA and protein in acupuncture group was higher than that of control group(P<0.05, P<0.01).(5)AgRP mRNA:Compared with control group,the expression of AgRP mRNA increased significantly in obesity group(P<0.01).After electropuncture treatment,the expression of hypothalamus AgRP mRNA in acupuncture group was obviously lower than that of obesity group(P<0.01).The expression of hypothalamus AgRP mRNA in acupuncture group was slightly higher than that of control group without significant differences(P>0.05).(6)NPY mRNA:Compared with control group,the expression of NPY mRNA increased significantly in obesity group(P<0.01).After electropuncture treatment,the expression of hypothalamus NPY mRNA in acupuncture group was obviously lower than that of obesity group(P<0.01).The expression of hypothalamus NPY mRNA in acupuncture group was higher than that of control group (P<0.01).(7)PoMC mRNA:Compared with control group,the expression of PoMC mRNA reduced significantly in obesity group(P<0.01).After electropuncture treatment,the expression of hypothalamus PoMC mRNA in acupuncture group was higher than that of obesity group(P<0.05).The expression of hypothalamus PoMC mRNA in acupuncture group was lower than that of control group(P<0.05).Conclusions:1.Choose"Tianshu(ST25)","Zhongwan(CV12/RN12)","Sanyinjiao(SP6)","Hous anli(ST36)"points for electrical acupuncture can effectively reduce the weight,body fat content and body fat ratio of DIO rats.2.The hypothalamus DNA methyltransferases(Dnmt2,Dnmt3a,Dnmt3b) activity increases in simple obesity rats,it is perhaps that acupuncture makes Tscl demethylation through negative regulation of Dnmt2,Dnmt3a and Dnmt3b,and inhibits mTORC1 activity,to control appetite ralated factors(NPY,AgRP and PoMC,et al),thereby ameliorates the obese state. |
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