| ObjectiveTo investigate the effects of Wendantang on glucolipid metabolism,peritoneal adipocyte ultrastructure and autophagy status changes,autophagy activity markers,inflammatory cytokines and mRNA and protein expression of key molecules ofthe phosphatidylinositol-3-kinase-protein kinase B-mammalian target protein of rapamycin(PI3K-AKT-mTOR)signaling pathway in rats with obesity and phlegm-dampness,and to explore the material basis of the inflammatory state of obesity and the molecular mechanism of Wendantang to interfere with the inflammatory state of obesity and phlegm-dampness.Methods(1)Animal modeling:126 rats were randomly divided into 2 groups(110 rats in the modeling group and 16 rats in the blank group),and the blank group was fed with basal diet while the modeling group was fed with high-fat diet for 8 weeks to create an animal model of obesity and phlegm.(2)Grouping and drug administration:After successful moulding,48 obese rats were selected according to their body mass,and randomly divided into model control group,Orlistat group,Rapamycin group,and low,medium and high dose groups of Wendantang,with 8 rats in each group;8 rats in the blank group were randomly selected as normal control group.The RAPA group,the ORLI group and the WDD low,medium and high dose groups were gavaged at 2 mg/kg,32.40 mg/kg,4.45 g/kg,8.90 g/kg and 17.80 g/kg respectively(in raw dose),while the MC and NC groups were given equal amounts of distilled water for gavage once daily for 6 weeks.(3)Sample collection and index testing:The rats were fasted without water for 12 h before anesthesia treatment,and samples were taken after anesthesia,and body mass,Lee’s index,fat-to-body ratio and obesity rate were measured and calculated.The levels of total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and fasting blood glucose(FBG)in rats were measured by fully automated biochemical analyzer according to the kit requirements;fasting insulin(FINS)was measured by enzyme-linked immunosorbent assay(ELISA)and Homeostasis model assessment of Insulin resistance(HOMA-IR)was calculated;observing changes in ultrastructure and autophagic status of peritoneal adipocytes by transmission electron microscopy;the expression levels of B-cell lymphoma 2-interacting protein-1(Beclinl),p62,Unc-51-like autophagy-activated kinase 1(ULK1),autophagy-associated gene 5(Atg5)and microtubule-associated protein 1 light chain 3(LC3)Ⅰ/Ⅱ were measured by a two-step immunohistochemical assay;the expression levels of pro-inflammatory cytokines of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1β and monocyte chemotactic protein-1(MCP-1)in peritoneal adipocytes was measured by ELISA;real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of PI3K-Akt-mTOR signaling pathway key genes of class I-PI3K,phosphatidylinositol-3-phosphate(PIP3),Akt,mTORC1,ULK1,tuberous sclerosis complex 1/2(TSC1/2)in peritoneal adipocytes;the protein expression of PI3K-Akt-mTOR signaling pathway key genes of classⅠ-PI3K,PIP3,phosphorylated Akt protein(p-Akt),Akt,phosphorylated mTOR protein 1(p-mTORC1),mTORCl,phosphorylated ULK1 protein(p-ULK1),ULK1,TSC1,TSC2 were detected by protein blotting(Western blot)in peritoneal adipocytes.(4)Statistical processing and analysis:SPSS22.00 was used for statistical analysis of the experimental data,and the data information was expressed in terms of .When obeying normal distribution,two independent samples t-test was used to compare the data of two groups,and one-way ANOVA was used to compare the data of multiple groups.When the variance was flush,the LSD method was used between groups,and when the variance was not flush,Tamhane’s T2 test was used between groups,with P<0.05 indicating that the difference was statistically significant.Result(1)Moulding result:high-fat diet feeding successfully replicated the obese rat model,and the rate of obesity in the MC group was>20%,and body mass and Lee’s index were significantly higher in the MC group compared with the NC group(P<0.01).and there were signs of phlegm and dampness,such as obesity,reduced mobility,lusterless and tight skin,reduced responsiveness to the outside world,reduced appetite,reduced water intake and loose stools.(2)Results of pharmacological intervention:Compared with the NC group,rats in the MC group showed obesity and phlegm-dampness Syndrome,and the levels of body mass,Lee’s index,lipid-body ratio,blood lipids,FBG,FINS and HOMA-IR,autophagic activity marker protein expression in peritoneal adipocytes,pro-inflammatory cytokines were all changed to different degrees(P<0.05,P<0.01),and the key molecules of signaling pathway classⅠ-PI3K,Akt mRNA expression and signaling pathway key molecules classⅠ-PI3K,PIP3,p-Akt,Akt,p-mTORC1,mTORCl,p-ULK1,TSC1,TSC2 protein expression were significantly decreased(P<0.01),but PIP3,mTORC1,ULK1,TSC1/2 mRNA expression and ULK1 protein expression were significantly increased(P<0.01).Compared with the MC group,the performance of obese phlegm and dampness Syndrome was significantly improved in all dosing groups,and the levels of body mass,lipid body ratio,blood lipids(except for the WDD-L group),FBG(except for the WDD-L group),FINS(except for the RAPA group)and HOMA-IR(except for the WDD-L group),autophagic activity marker protein expression in peritoneal adipocytes,pro-inflammatory cytokines were all improved to different degrees(P<0.05,P<0.01),and the key molecules of signaling pathway,classⅠ-PI3K,Akt mRNA expression and signaling pathway key molecules classⅠ-PI3K,PIP3,p-Akt,Akt,p-mTORC1,mTORCl,p-ULK1,TSC1,TSC2 protein expression were increased to different degrees(P<0.05,P<0.01),but ULK1 protein expression was significantly decreased(P<0.01),and the expression of PIP3,mTORCl,ULK1,and TSC1/2 mRNA expression were decreased to different degrees in RAPA,ORLI and WDD-H groups(P<0.05,P<0.01),TSC1/2 mRNA expression was decreased significantly in WDD-L group(P<0.01),and PIP3,mTORCl,and ULK1 mRNA expression were decreased to different degrees in WDD-M group(P<0.05,P<0.01).Compared with the RAPA group,there were significant differences in the expression of autophagic activity marker protein,mRNA expression of key molecules of signaling pathway(except classⅠ-PI3K and Akt in WDD-H group)and protein expression of key molecules of signaling pathway(except classⅠ-PI3K in WDD medium and high dose groups)in all dose groups of Wendantang(P<0.01),and the levels of blood lipids,FBG and HOMA-IR,and inflammatory cytokines(except TNF-α)were different in the WDD-L group(P<0.05,P<0.01),and the levels of blood lipids(except LDL-C),FBG and HOMA-IR,and inflammatory cytokines(except IL-6,MCP-1)were different in the WDD-M group(P<0.05,P<0.01),and different levels of lipids(only TG,HDL-C)in the WDD-H group(P<0.05,P<0.01),but the rest were not statistically different in the WDD-H group.Compared with the ORLI group,autophagic activity marker protein(except p62 in WDD-H group)and the mRNA expression of key molecules of signaling pathway(except mTORCl in WDD low and medium dose groups and Akt and ULK1 in WDD-H group)of peritoneal adipocytes in all dose groups of Wendantang were significantly different(P<0.01),and there were different degrees of differences in lipid(except TG),FBG and HOMA-IR levels,inflammatory cytokine IL-6 levels and signaling pathway key molecule protein expression in the WDD-L group(P<0.05,P<0.01),lipid(LDL-C,HDL-C)levels and signaling pathway key molecules(except classⅠ-PI3K)protein expression differed to different degrees in WDD-M group(P<0.05,P<0.01),body mass,inflammatory cytokine TNF-α levels and signaling pathway key molecules only PIP3,p-Akt,mTORC1,p-ULK1,TSC1 protein expression differed in WDD-H group(P<0.05,P<0.01),and the rest were not statistically different,among which the expression of autophagy activity marker proteins(Beclin1,Atg5),inflammatory cytokines TNF-α levels and PIP3 and mTORC1 mRNA expression in the WDD-H group showed different degrees of decrease(P<0.05,P<0.01),while classⅠ-PI3K mRNA expression was significantly increased(P<0.01).Compared with the NC group,the MC group had very serious structural damage,with severe surface protrusions,significant destruction of mitochondrial cristae,fragmentation and separation,a large number of vacuoles in the cytoplasm,and a large number of autophagosomes;compared with the MC group,the low-dose group of Wendantang had no significant improvement in structural damage,and the mitochondrial cristae were significantly damaged.Compared with the MC group,the structural damage in the low-dose group did not improve significantly,with significant disruption of mitochondrial cristae,disintegration and separation,large number of vacuoles in the cytoplasm and large number of autophagosomes;the structural damage in the middle-dose group improved,with partial disruption of mitochondrial cristae,vacuoles in the cytoplasm and a decrease in the number of autophagosomes;the structural damage in the high-dose group improved significantly,with partial disruption of mitochondrial cristae,a small number of vacuoles in the cytoplasm and a significant decrease in the number of autophagosomes.ConclusionThere are significant alterations in the inflammatory state of obesity phlegm and dampness syndrome such as weight gain,disturbed glucolipid metabolism,elevated insulin levels,insulin resistance,enhanced autophagic activity,and elevated levels of inflammatory cytokine secretion,the formation of which may be associated with PI3K-Akt-mTOR signaling pathway-mediated adipocyte autophagy.Wendantang can improve the pathological manifestations of phlegm and dampness in obese rats,reduce body weight,regulate glucose and lipid metabolism,lower insulin levels,reduce insulin resistance,inhibit autophagy and reduce the secretion level of inflammatory cytokines,and its mechanism of action may be related to the regulation of PI3K-Akt-mTOR signaling pathway to regulate cellular autophagy and thus improve the chronic inflammatory state of the body,which provides an experimental basis for the intervention of Wendantang in obese phlegm and dampness. |
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