| 【Purpose】To explore the intrinsic mechanism between cellular autophagy and the inflammatory state of obesity from a theoretical perspective,to elucidate the key pathogenesis of obesity,to explore the association between cellular autophagy and phlegm-dampness trapped in the spleen,and to explore the possible mechanisms regulating the role of cellular autophagy in the inflammatory state of phlegm-dampness evidence of obesity.The study also investigated the effects of Wendan decoction on the expression of inflammatory cytokines and autophagic activity markers in the perirenal adipose tissue of rats with high-fat diet-induced obesity and phlegm-dampness,and explored the intrinsic correlation between the intervention of Wendan decoction on the inflammatory state of obesity and phlegm-dampness and cellular autophagy,so as to elucidate the scientific connotation of the intervention of Wendan decoction on obesity and phlegm-dampness from the perspective of cellular autophagy.【Methods】1 Theoretical researchThrough the literature database platforms of China Knowledge Network（CNKI）,China Biomedicine（Sino Med）,Wanfang（Wanfang Database）and National Center for Biotechnology Information（NBCI）,we searched,organized and analyzed the Chinese and Western medicine related literature on the formation mechanism of the inflammatory state of obesity phlegm and dampness evidence from the perspective of cellular autophagy.First,to clarify the intrinsic association between cellular autophagy and the inflammatory state of obesity,based on modern medicine.Second,based on Chinese medical literature,explain that phlegm and dampness trapping the spleen is the key pathogenesis of obesity.Thirdly,comparing the effects of phlegm-dampness trapped in the spleen in Chinese medicine with modern medicine on the inflammatory state of obesity,it is clear that through the regulation of cellular autophagy is an important way to intervene in the inflammatory state of obesity phlegm-dampness evidence.2 Experiment research（1）High-fat diet modelling:One hundred male SD rats were randomly divided into a modeling group（referred to as the modeling group）of 70 rats and a normal control group of 30 rats.The modeling group was fed with a high-fat diet and the normal control group was fed with a basic diet for 6 weeks.（2）Wendan decoction intervention:After the successful establishment of the obese phlegm and dampness model,16 rats were selected in order of body mass,and randomly divided into 2 groups according to the principle of random allocation,namely the Wendan decoction intervention group（referred to as the drug group）and the model control group（referred to as the model group）,and the rats in the previous normal control group were set as the blank group,8 rats in each group were fed with basic chow.The drug group was gavaged at a dose of 15 g-kg-1,and both the model and blank groups were gavaged with an equal amount of distilled water,once/day,with free access to water,for a 6-week intervention period.（3）Sample sampling and index detection:After the completion of the Wendan decoction intervention,samples were taken from the rats,which were fixed on a rat splint,opened layer by layer,the abdominal aorta was isolated and blood（approximately 3 m L）was taken using a 5 m L EDTA-containing blood collection tube,placed at 4°C and left to extract the supernatant clear for testing lipid levels.Perirenal adipose tissue from three groups of rats was cut into rice-grain size and placed in 1.5 m L centrifuge tubes and stored at-20℃for subsequent extraction to detect the expression levels of the pro-inflammatory cytokines tumour necrosis factor-α（TNF-α）,interleukin-6（IL-6）,interleukin-1β（IL-β）,monocyte chemotactic protein-1（MCP-1）,and messenger ribonucleic acid（m RNA）and protein expression levels of B-cell lymphoma 2 binding protein-1（Beclin-1）,microtubule-associated protein light chain（LC3）,markers of autophagic activity.Perirenal adipose tissue was also taken from three groups of rats at the same site,cut into rice-grain size and preserved in glutaraldehyde fixative for electron microscopy,for subsequent transmission electron microscopy to observe changes in adipocyte ultrastructure and autophagic status.The obesity rate and Lee’s index（obesity index）were then calculated,and the serum total cholesterol（TG）,triglyceride（TC）,low-density lipoprotein cholesterol（LDL-C）and high-density lipoprotein cholesterol（HDL-C）levels were measured using a fully automated biochemical analyzer.The expression levels of pro-inflammatory cytokines TNF-α,IL-6,IL-βand MCP-1 in rat perirenal adipose tissue were measured by enzyme-linked immunosorbent assay（ELISA）;the expression levels of autophagy genes Beclin-1 and LC3 m RNA in rat perirenal adipose tissue were measured by real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）.Protein immunoblotting（Western blot）was used to detect the expression of autophagy genes Beclin-1 and LC3 in perirenal adipose tissue;transmission electron microscopy was used to observe the changes in ultrastructure and autophagic status of perirenal adipocytes.（4）Data analysis and statistics:SPSS 21.00 software was used to conduct statistical analysis of the collected and collated relevant data.【Consequence】1 Theoretical research（1）Obesity is a chronic inflammatory state induced by abnormal adipose tissue metabolism.Cellular autophagy can regulate the development of abnormal adipose tissue metabolism and inflammation,and abnormal cellular autophagy can exacerbate the development of the chronic inflammatory state of obesity,so it is believed that cellular autophagy plays an important role in the inflammatory state of obese adipose tissue.（2）Obesity is mainly the result of the action of phlegm and dampness trapped in the spleen.Physiologically,cellular autophagy is closely associated with the spleen and stomach in Chinese medicine,and pathologically,abnormal autophagy is similar to the mechanism of phlegm and dampness trapped in the spleen.2 Experiment research（1）Results of high-fat diet modelling:The rat model of obesity with phlegm and dampness was successfully established under a high-fat diet.The obesity rate of the modeled rats was 23.36%（more than 20%）and the Lee’s index was also significantly higher,and the comparison of body weight and Lee’s index between the two groups was statistically significant（P<0.01 or P<0.05）.（2）Intervention results of Wendan decoction:Compared with the model group,the rats in the drug group showed a significant decrease in obesity,body weight and Lee’s index（P<0.01 or P<0.05）,as well as a significant decrease in blood TC,TG and LDL-C levels（P<0.01）and a significant increase in HDL-C levels（P<0.01）,while the expression levels of pro-inflammatory cytokines in perirenal adipose tissue,TNF-α,IL-6,IL-1βand MCP-1,were significantly reduced（P<0.01）.The expression levels of TNF-α,IL-6,IL-1βand MCP-1 were significantly decreased（P<0.01）,the m RNA and protein expression levels of autophagy genes Beclin-1 and LC3 in perirenal adipocytes were significantly decreased（P<0.01）,and autophagosomes were reduced.Compared with the blank group,the rats in the model group had significantly higher body weight（P<0.01）,significantly higher blood TC,TG and LDL-C levels（P<0.01）and significantly lower HDL-C levels（P<0.01）,and significantly higher expression of pro-inflammatory cytokines TNF-α,IL-6,IL-1βand MCP-1 in perirenal adipose tissue（P<0.01）,and perirenal m RNA and protein levels of the adipocyte autophagy genes Beclin-1 and LC3 were significantly higher（P<0.01）,and autophagosomes were more abundant.【Conclusion】1 Theoretical researchThe formation of obesity is associated with the chronic inflammatory state of adipose tissue induced by abnormal cellular autophagy,and the key pathogenesis is phlegm-dampness trapped in the spleen,Moreover,abnormal cellular autophagy and phlegm-dampness trapped in the spleen are mutually causal and affect each other.This provides an objective basis for exploring the treatment of Chinese medical conditions from the perspective of modern medicine,and provides research ideas for exploring the treatment of obesity phlegm-dampness evidence from the perspective of cellular autophagy.2 Experiment researchWendan decoction can effectively reduce the body weight of obese rats and can effectively correct the phlegm-damp pathological state of obese rats.The mechanism is related to the ability of Wendan decoction to regulate cellular autophagy and influence the metabolism and inflammatory state of adipose tissues,thus improving the phlegm-damp evidence state of obesity.This provides a scientific and experimental basis for the use of the classical formula Wendan decoction to intervene in obese phlegm-dampness evidence. |
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