| Objective: Obesity is a disorder of lipid metabolism,which has a negative impact on each system of body.Obesity has become a public health problem.The number of obese people in the world will reach 1.12 billion by 2030.Obesity reduces life expectancy by 5-10 years and is closely related to increased mortality.In addition to metabolic effects,obesity also affects the function of skeletal muscle,thus reducing the activity ability of obese patients.The decrease of activity ability may lead to the decrease of activity level and energy consumption,and then further aggravate the degree of obesity.A vicious circle is formed and affects the quality of life seriously.Obesity is an important factor leading to structural changes and dysfunction of skeletal muscle.There are changes in substance metabolism,endoplasmic reticulum stress and skeletal myositis during obesity,so it has a great impact on the metabolism of skeletal muscle nutrients metabolism,the composition of muscle fiber types,the number and function of mitochondria in muscle cells and the systolic function of skeletal muscle and so on.Rhein is a monomer of anthraquinone derivatives,which is extracted from the rhizome of rhubarb.Rhein has a wide range of pharmacological effects,so it has a significant clinical medicinal value.Rhein plays a vital role in the treatment of metabolic diseases such as obesity,diabetes and its chronic complications,nonalcoholic fatty liver and hyperuricemia through a variety of complex and staggered signal pathways.The purpose of this study is to explore whether Rhein can improve the impaired endurance exercise function caused by obesity,and to further explore its possible mechanism.Methods: 1.Animal experiment: Eighteen 4-week-old C57BL/6J male mice were fed adaptively for a week.These mice were randomly divided into three groups: normal control group(normal control,NC),high-fat group(HFD),high-fat+ Rhein group(HFD+RH),(6 mice in each group).Mice in NC group were fed with standard diet,HFD group and HFD+RH group were fed with high fat diet.The mice in NC group and HFD group were given sodium cellulose every day,while mice in HFD+RH group were given Rhein(120mg/ kg,dissolved in 1% cellulose sodium solution).All mice were trained to run the treadmill once a week.After 3 months of intervention,the following experiments were carried out: a)The exhaustive test of treadmill in mice was carried out,and the exhaustion distance and time were recorded.b)Fasted overnight and weighed.c)Killed the mice,fat and skeletal muscle were separated and weighed.d)The gastrocnemius muscle of mice was isolated and prepared sections for H&E and DAB staining.e)The gastrocnemius muscle of mice was isolated and the content of ATP was detected by ultraviolet spectrophotometry.f)The protein content of Sirt1 and the phosphorylation level of AMPK in gastrocnemius muscle were detected by Western Blot.g)The m RNA contents of AMPK?1,AMPK?2,Sirt1 and ATPase in gastrocnemius muscle were detected by quantitative real time-PCR.2.Cell experiment: C2C12 muscle cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibodies in a cell incubator(condition: 37 ? and 5%CO2).When the cell density grew to about 80%-90%,the DMEM medium containing 2% horse serum and 1% double antibody was replaced to induce differentiation.After 5-6 days,the C2C12 cells differentiated into obvious myotubes,and the differentiation solution was discarded.According to different treatment methods,C2C12 cells were divided into three groups,one: control group,namely RH=0umol/L group(the same volume of Na OH solution),two: RH=10umol/L group,three: RH=50umol/L group.After intervention for 24 hours,the culture medium was abandoned,protein lysate was added and cellular proteins were collected.Western Blot method was used to detect the protein content of Sirt1 and the level of AMPK phosphorylation in each group.Mouse skeletal muscle cell line C2C12 cells were purchased from the Zhongqiao Xinzhou Company.Results: 1.Comparison of body weight in each group: after 6 weeks of feeding,body weight of the HFD group was higher than that of the NC group by 20%,indicating that the mouse obesity model was successfully established.After 7 weeks of feeding,compared with the HFD group,body weight of the HFD+RH group was significantly lower(P<0.05),and the average daily food intake had no significant difference(P>0.05).2.Comparison of fat tissue in each group: compared with NC group,epididymal fat and subcutaneous fat tissue in HFD group increased significantly,while epididymal fat and subcutaneous fat decreased significantly in HFD+RH group compared with HFD group.There was no significant difference in brown fat tissue among three groups(P>0.05).3.Comparison of skeletal muscle index in each group: compared with NC group,the index of gastrocnemius and quadriceps femoris in HFD group was significantly lower(P<0.001),but there was no significant difference between HFD group and HFD+RH group(P>0.05).4.Comparison of exhaustive ability of mice in each group: compared with NC group,the exhaustive exercise distance and time of HFD group were significantly shorter,and the exhaustive exercise distance and time of HFD+RH group was significantly longer than that of HFD group(P<0.05).5.Comparison of cross-sectional area of gastrocnemius muscle fiber in each group: compared with NC group,the cross-sectional area of gastrocnemius muscle fiber in HFD group decreased(P<0.001),while that in HFD+RH group increased significantly compared with HFD group(P<0.001).6.Comparison of the content of ATP in gastrocnemius muscle of mice in each group: compared with NC group,the content of ATP in gastrocnemius muscle in HFD group was significantly lower(P<0.001),while the content of ATP in gastrocnemius muscle in HFD+RH group was significantly higher than that in HFD group(P<0.001).7.Comparison of protein contents of p AMPK and Sirt1 in gastrocnemius muscle of mice in each group: the protein content of p AMPK and Sirt1 in gastrocnemius muscle tissue in HFD+RH group was significantly higher than that in HFD group(P<0.05).8.Comparison of m RNA contents of AMPK?1?AMPK?2?Sirt1 and ATPase in gastrocnemius muscle of mice in each group: quantitative real time-PCR showed that m RNA content of AMPK?1?AMPK?2 and Sirt1 in gastrocnemius muscle of HFD+RH group was significantly higher than that of HFD group(P<0.05).There was no significant difference in m RNA content of ATPase(P>0.05).9.Comparison of Sirt1 DAB staining results in gastrocnemius muscle of mice in each group: the results of DAB staining showed that the expression of Sirt1 in gastrocnemius muscle of HFD+RH group was significantly higher than that of HFD group.10.Comparison of p AMPK and Sirt1 protein contents of C2C12 cells in each group: compared with RH=0umol/L group,there was no significant difference in p AMPK and Sirt1 protein contents in RH=10umol/L group(P>0.05),but p AMPK and Sirt1 protein contents in RH=50umol/L group was significantly higher than that in RH=0umol/L group(P<0.05).Conclusion: Rhein can significantly prolong the exhaustive exercise distance and time,increase the fatigue resistance and improve the exercise endurance of obese mice,and its mechanism may be related to the activation of AMPK-Sirt1 pathway in skeletal muscle tissue by Rhein. |
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