| Purpose:In recent years,obesity has become a global public health problem and has been recognized by the World Health Organization as the fifth largest risk factor affecting human health.Obesity can not only cause bad health outcomes at the physical level,but also cause serious adverse effects on mental health.It is increasingly worrying,and it is particularly important to take effective measures to interfere with obesity.Chinese medicine believes that qi deficiency and phlegm and dampness are common types of obesity.Instructor Wangqi self-made "Huatan Qushi Decoction"(ie "Yizhi Granules")and "Yiqi Jianyun Decoction"(ie"Fuyuan Granules")clinically adjusted for weight loss,but it has never been effective The role of exploration at the cell level.This subject aims to observe the effects of the two decoctions on cell viability,inflammatory factors,and cell autophagy through in vitro cell experiments,and explore the possible mechanisms by which the two decoctions play a role.Method:Establish a palmitic acid(100μmol/L)-induced RAW264.7 macrophage inflammation model to simulate metabolic diseases,use different concentrations of Huatan Qushi liquid and Yiqi Jianyun liquid to intervene in the cells,through the CCK-8 method Perform cell viability determination,detect inflammatory factors(MIF,TNF-α,MCP-1,IL-6,IL-10,YM-1,Arg-1,TGF-β1)in cell culture medium by Elisa method,by Western Blot method detects cellular autophagy proteins and key indicators of inflammation(CD36,Beclin1,LC3,TFEB).Autophagy antibody chips are used to detect 20 important proteins related to autophagy.(Grouping:DMEM group,DMSO group,PA group(that is,palmitic acid group),TS-50 group,TS-100 group,TS-150 group(TS group is Huatan Qushi Decoction group),QX-50 group,QX-100 group,QX-150 group(QX group is Yiqi Jianyun Decoction group),HF group(namely Hefang group),TT group(namely atorvastatin calcium group),YX group(namely Ganlu granule medicine Liquid group))Results:①The cell viability after 24 hours was not statistically significant except that the QX-10 group had a slight effect on the cells.After 48 hours,the cell viability was affected by the drug solution.The TS-75,TS-100,TS-200 groups of Huatan Qushi liquid and Yiqi Jianyun liquid in all concentrations have a significant effect on the vitality of the inflammation model of RAW264.7 cells(P<0.01).②Overall,the pro-inflammatory factors in the cell culture medium of the PA group were higher and the anti-inflammatory factors(MIF,TNF-α,MCP-1,IL-6)were lower(P<0.05).After intervention by the two Chinese herbal medicines,the pro-inflammatory factors were lower and the anti-inflammatory factors(IL-10,YM-1,Arg-1,TGF-β1)were higher(P<0.05).③Compared with the PA group,the cells in the TS-100 group had higher CD36,the cells in the three concentrations of QX had lower CD36(P<0.05),and the cells in the QX-50 and QX-100 groups had higher Beclin 1(P<0.05 or P<0.01).Except for the QX-100 group,the expression of TFEB in the nucleus of the TT group was lower than that of the other groups(P<0.05 or P<0.01).Although there is no statistical difference between the other four test groups except the TT group,from the numerical point of view,the PA group is lower and the TS-100 group is higher.④Compared with the DMEM group,the cell LC3-Ⅱ/Ⅰ of the 6 concentration groups of the two drug solutions were higher(P<0.05 or P<0.01),and the cell LC3-Ⅱ/Ⅰ of the TT group was lower than the drug solution group(P<0.01).After adding Baf A1,the LC3-Ⅱ cells in PA group had more accumulation than the DMEM group,but there was no statistical difference.The expression levels of LC3-Ⅱ in the TS-100 group,QX-100 group,HF group,and TT group were higher than those in the DMEM group(P<0.05 or P<0.01).⑥After detection by autophagy antibody chip,there were 15 differentially expressed proteins in PA group andDMEM group,which were all up-regulated.There were 17 differentially expressed proteins in PA group and TS-100 group,16 were down-regulated differences,and ATG10 was up-regulated differences.There were 16 differentially expressed proteins in PA group and QX-100 group,14 were down-regulated differences,and ATG10 and ATG12 were up-regulated differences.Conclusion:①According to the results of cell viability measurement,taking into account the consistency of the concentration of the two drug solutions in the cells and subsequent experiments,the concentration gradient of the two drug solutions in the cells was determined to be 50mg/L,100mg/L and 150mg/L.②The palmitic acid-induced RAW264.7 macrophages were mainly M1 type with pro-inflammatory effect,and the model was established successfully.The effect of Huatan Qushi Decoction,Yiqi Jianyun Decoction and combination of the two decoctions on the inflammatory factors is generally opposite to the PA group,indicating that the two decoctions have a certain anti-inflammatory effect.The effect of atorvastatin calcium on inflammatory factors is generally opposite to that in the PA group,and it is used as a positive control drug.③Huatan Qushi Decoction may promote the production of anti-inflammatory factors by increasing the expression of CD36 protein in cells,so that CD36 exerts its anti-inflammatory effect as a scavenger receptor,thereby maintaining the steady state of macrophages.Yiqi Jianyun Decoction may affect the functional expression of CD36 as an exogenous long-chain fatty acid receptor,reducing the damage of cells caused by palmitic acid-induced inflammation.Palmitic acid may have an effect of inhibiting autophagy.Both decoctions can promote autophagy,and the combined effect is better.Atorvastatin calcium can also promote autophagy,but the rate of autophagy may be higher than the two decoctions.④Huatan Qushi Decoction may play a greater role in the degradation phase of autophagy,thus antagonizing the inhibitory effect of SNCA on the degradation of autophagolysosome,while Yiqi Jianyun Decoction may play a more important role in the initial phase of autophagy.Both decoctions may improve cell inflammation by promoting autophagy,so as to achieve the purpose of weight loss. |
