| Objective: Acupuncture has a certain effect on weight loss,but individual differences exist.In this study,the effect of acupuncture on the autophagy signaling pathway mediated by m TOR/S6K1 in white adipose tissue of obese rats induced by high-fat diet was been observed.The experiment is aimed at investigating the autophagy signaling pathway mediated by m TOR/S6K1 of acupuncture for obesity,which will beneficial to acupuncture’s popularization in clinical practice and promote clinical effect.Methods: Ninety 3-week-olds male Wistar rats were randomly divided into normally chow diet group(chow group)and high fat diet group.After 10 weeks of feeding with general diet and high fat diet respectively,the experimental group defined as 20% body weight over normal control group.The rats were randomly divided into the diet-induced obesity(DIO)group,the electroacupuncture(EA)group and the sham electroacupuncture(SA)group,20 rats in the EA group and 10 rats in other groups.The electroacupuncture treatment was performed on “Zhongwan”,“Tianshu”,“Zusanli”,“Sanyinjiao” or sham acupoints(about 5 mm beside each acupoint,shallow needling).The rats were treated with electroacupuncture at 2 Hz/15 Hz,1 m A,1 time per day,30 mins per time,5 days per duration,rest 2 days between durations,4 courses of continuous treatment.The body weight,epididymal white adipose content were used to evaluate the effect of weight loss by acupuncture.Taking the weight loss≥5% as the effective judgment standard of electroacupuncture,the effects were divided into two levels of valid and invalid.Comparing adipocytes’ size were observed by HE staining techniques and the cell diameter and area,the m RNA and protein expression of m TOR、S6K1、LC3、Beclin-1、p62 in white adipose tissue were detected by RT-PCR,immunohistochemistry techniques.Results:1.Evaluation of the effect of weight loss Compared with the chow group,high-fat feeding for 10 weeks can significantly increase the body weight(P<0.01).Before treatment,the body weight of rats in the DIO group,EA group and sham acupuncture group were significantly higher than that of the chow group(P<0.01).There was no difference in the weight among the DIO group,the EA group and the SA group.After 4 weeks of electroacupuncture treatment,compared with the model group,the body weight and e WAT content of electroacupuncture group decreased significantly(P<0.01,P<0.05),while the body weight and epididymis fat content of sham electroacupuncture group decreased slightly,but the difference was not significant(P>0.05).2.Evaluation of individual differences in weight loss effect On the basis of clarifying the effect of weight loss,six rats whose weight loss was less than 5% were included in the ineffective group of electroacupuncture,and the rest were in the effective group.The weight and e WAT fat content of the electroacupuncture ineffective group showed a certain trend of reduction compared with the model group and the sham acupuncture group,but the difference was not significant(all P>0.05).Comparison between the effective and ineffective group,the difference of body weight and epididymal fat content was significant(P<0.01,P<0.05).3.Diameter and area of white adipocytes in epididymal tissue Compared with the chow group,the DIO group had increased the area and diameter of in e WAT cells significantly(P<0.01);compared with the DIO group and the SA group,the area and diameter of adipocytes in the EA valid group was decreased,significantly smaller than that in the DIO group(P<0.01,P<0.05),and not statistically different from that in the chow group;while the area and diameter of adipocytes in the electroacupuncture ineffective group did not change significantly(P>0.05).The difference of adipocyte area and diameter between the effective and ineffective group was significant(P<0.05).4.Detection of autophagy in epididymal white adipose tissue(1)the protein and m RNA expression of Beclin-1 m RNA and protein of Beclin-1 in the chow group had a certain amount of expression;as compared with the chow group,the DIO group has more significantly increased protein and m RNA of Beclin-1(all P<0.01);compared with the DIO group and the SA group,protein and m RNA of Beclin-1 in the EA valid group was decreased,significantly smaller than that in the DIO group(all P<0.05),and not statistically different from that in the chow group;the expression change in the electroacupuncture ineffective group was not significant(all P>0.05).The difference of protein and m RNA of Beclin-1 between the effective and ineffective group was significant(P<0.05).(2)the protein and m RNA expression of LC3 m RNA and protein of LC3 in the chow group had a certain amount of expression;as compared with the chow group,the DIO group has more significantly increased protein and m RNA of LC3(all P<0.01);compared with the DIO group and the SA group,protein and m RNA of LC3 in the EA valid group was decreased,significantly smaller than that in the DIO group(all P<0.05),and not statistically different from that in the chow group;the expression change in the electroacupuncture ineffective group was not significant(all P>0.05).The difference of protein and m RNA of LC3 between the effective and ineffective group was significant(all P<0.05).(3)the protein and m RNA expression of p62 As compared with the chow group,the DIO group has a more significantly decreased protein and m RNA expression level of the p62(all P<0.01);compared with the DIO group and the SA group,the protein and m RNA expression level of the p62 in the acupuncture valid group was significantly increased(all P<0.01),and lower than that in the chow group;the expression change in the acupuncture invalid group was not significant(all P>0.05).The difference of protein and m RNA of p62 between the effective and ineffective group was significant(all P<0.05).5.Detection of m TOR-S6K1 signaling pathway(1)the protein and m RNA expression of m TOR As compared with the chow group,the DIO group has a more significantly decreased protein and m RNA expression level of the m TOR(all P<0.01);compared with the DIO group and the SA group,the protein and m RNA expression level of the m TOR in the acupuncture valid group was significantly increased(all P<0.05),and lower than that in the chow group;the expression change in the acupuncture invalid group was not significant(all P>0.05).The difference of protein and m RNA of m TOR between the effective and ineffective group was significant(all P<0.05).(2)the protein and m RNA expression of S6K1 As compared with the chow group,the DIO group has a more significantly decreased protein and m RNA expression level of the S6K1(all P<0.05);compared with the DIO group,the protein and m RNA expression level of the S6K1 in the acupuncture valid group was significantly increased(all P<0.05),and lower than that in the chow group;the expression change in the acupuncture invalid group was not significant(all P>0.05).The difference of m RNA of S6K1 between the effective and ineffective group was significant(all P<0.05).Conclusion: 1.Electroacupuncture at “Zhongwan”,“Tianshu”,“Zusanli” and “Sanyinjiao” points can effectively reduces the body weight and epididymal white adipose tissue mass of DIO rats,and reduce the volume of epididymal adipocytes.2.There are individual differences exist in the effects of electroacupuncture on improving body weight,epididymal fat mass and adipocytes volume in obese rats,that is,effective and ineffective.3.There are differences in the expression levels of m TOR/S6K1 signaling pathway and Beclin-1,LC3 and p62 related molecules among individuals with different therapeutic effects of electroacupuncture on obese rats.The mechanism of individual differences in weight loss may be closely related to the degree of inhibition of m TOR / S6K1 mediated autophagy in the epididymal white adipose tissue. |
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