Effect Of Rapamycin On Different Cellular Inflammatory Signaling Pathways In High-fat Environment

Background and objective: With the incidence of obesity worldwide increased rapidly in the past two decades,the incidence of some obesity-related diseases such as type 2 diabetes is also rising rapidly,which brings a heavy medical burden to individuals,families and society.High-fat diet and lack of exercise are two important causes of obesity as well as related diseases.High-fat diets can not only cause obesity but also trigger insulin resistance;exercise improves insulin resistance induced by high-fat diets.Understanding the skeletal muscle insulin resistance mechanism and the physiological basis of exercise intervention is of great significance for finding pharmacological target sites for the prevention and treatment of obesity or obesity-related diseases.Studies have shown that m TORC1 sustained activation and chronic low-grade inflammation play an important role in obesity-induced insulin resistance,but the relationship between m TORC1 and inflammation is unclear.Exercise improves the skeletal muscle insulin resistance induced by high-fat diets,and the mechanism is unclear.This study intends to use different cells for related cytology experiments to observe the NF-?B activity and its related signaling proteins and inflammatory signals in different cells,and to elucidate the effects of high-fat diet on NF-?B signaling pathway regulation and inflammatory signals at the cellular level,and Effect of rapamycin on high fat-induced inflammatory factors.This study will help reveal the new mechanism of insulin resistance and the physiological basis of exercise intervention,and provide new ideas for the prevention and treatment of insulin resistance induced by obesity.Materials and methods:Firstly,rat skeletal muscle L6 cells,Primary fat cell,and liver BRL-3A cells were cultured in vitro,and then differentiated into mature cells,(1)then differentiated skeletal muscle cells,fat cells,and liver.The cells were incubated in a concentration of 0.5 m M and 0.2 m M in a palmitic acid incubation solution,and were taken at 0h,1h,3h,6h,12 h,24h for protein quantification and fluorescence PCR quantification.(2)The skeletal muscle cells,fat cells and liver cells incubated in the0.5 m M concentration of palmitic acid incubation solution were divided into two groups: high fat group,high fat + rapamycin group,and at 0h,1h,3h,6h.At 12 h,samples were taken for protein quantification and fluorescence PCR quantification.Results: After incubation with different concentrations of palmitic acid,P65 m RNA,IL6 m RNA and TNF-? m RNA were up-regulated in skeletal muscle cells,and Rel B m RNA was up-regulated only after incubation with 0.5 m M concentration of palmitic acid.Proteins P65,P-P65,Rel B,P-Rel B IL-6 and TNF-? were up-regulated.P65 m RNA,Rel B m RNA,IL6 m RNA and TNF-? m RNA were up-regulated in adipocytes,and P-P65,P-Rel B,IL6 and TNF-? were up-regulated.P65 m RNA and Rel B m RNA were expressed in hepatocytes.IL6 m RNA and TNF-? m RNA were up-regulated,and proteins P-P65 and TNF-? were up-regulated.When up-regulated,we can observe that 0.5m M concentration of palmitic acid can be used earlier than incubation with0.2m M concentration of palmitic acid.The ground reaches the peak level.(P<0.05)(2)P65 m RNA,Rel B m RNA,IL6 m RNA and TNF-? m RNA were up-regulated in skeletal muscle cells,and the expression of protein S6 and P-S6 was decreased in skeletal muscle cells after rametin-treated 0.5 m M palmitate-incubated cells.P-P65,Rel B,P-Rel B,IL-6 and TNF-? were all increased(P<0.05).P65 m RNA,Rel B m RNA,IL6 m RNA and TNF-? m RNA were decreased in adipocytes,and the expression of protein S6 and P-S6 was decreased.P-P65,Rel B,P-Rel B,IL-6 and TNF-? were all decreased(P<0.05);there was no significant change in gene index in hepatocytes(P>0.05),and the expression of protein S6 and P-S6 was decreased.Increased expression of P-P65,IL-6 and TNF-?(P<0.05)Conclusion: 1.Palmitic acid promotes the activation of NF-?B pathway in skeletal muscle cells,adipocytes and hepatocytes,causing an inflammatory response.At the same time,the activation of NF-?B signaling pathway and the up-regulation of inflammatory factors IL6 and TNF-?,which is related to the incubation time and dose dependence of palmitic acid..2.The effection of rapamycin in different tissue cells is different,and the inflammatory reaction caused by high fat is further deteriorated in skeletal muscle,which is certain in the liver.The secretion of inflammatory factors caused by high fat is inhibited in fat cells.To a lesser extent,the inflammatory response is exacerbated and may trigger insulin resistance.