Biological Basis Of AMPK-HIF1-PKM2 Signaling Pathway On Metabolic Syndrome In Rats With Phlegm Syndrome

Objective:From the corresponding point of view of prescription and syndrome,Erchen Decoction was used as an intervention method to observe the changes of macro behaviors,liver pathological structure and energy substrates,key enzymes of energy metabolism,AMPK-HIF1-PKM2 signaling pathway in rats with metabolic syndrome with phlegm syndrome.The biological basis of MS phlegm syndrome was revealed from the perspective of energy metabolism by prescription test,which provides new targets and new ways for the prevention and treatment of MS.Methods:1.Literature study: relevant literatures on the treatment of MS from phlegm was collected,and randomized controlled trials meeting the standard were screened.Review Manager 5.3software was used for statistical analysis of the included studies.2.Experimental research:six-week-old male Wistar rats were randomly divided into two groups according to body weight.The blank group(n=12)was fed with common feed,and the model group(n=38)was fed with high-sugar,high-salt and high-fat feed for a total of 12 weeks.After 12 weeks,the model rats were selected according to the model standard,and they were randomly divided into the model group and the Erchen Decoction group according to body weight.The blank group(n=11)and the model group(n=12)were intragastrically administered with normal saline,and the Erchen Decoction group(n=12)was intragastrically administered with Erchen Decoction 5.3g/(kd.d)for four weeks.During the modeling,the body weight,abdominal circumference,blood pressure,lactic acid,FBG,HDL,and TG of rats in each group were dynamically measured every four weeks.At the end of the 12 th and16th weekends,FINS,ALT,AST,TC,and LDL were measured,and Lee's index and HOMAIR were calculated.Hematoxylin and Eosin staining and transmission electron microscopy were used to observe the microstructure of liver tissue;Plasma and hepatic lactate,hepatic glycogen and hepatic TG were measured by spectrophotometry.The levels of ATP,CS,CPT1,HIF-1? and LDH in serum and liver tissue were measured by enzyme linked immunosorbent assay.The m RNA expressions of AMPK?2,PKM2,HIF-1?,CS,CPT1 A and LDHA in liver tissue were detected by Real-time PCR.The protein expressions of P-AMPK?,AMPK?2PKM2 and P-PKM2 in liver tissue were detected by immunohistochemistry.Results:1.Literature study: a total of 15 articles were included,including 1234 subjects,involving 56 drugs.According to the top four medication frequency are Pinellia,Pericarpium Citri Reticulatae,Poria.Meta-analysis results showed that:(1)the curative effect of treatment of phlegm method combined with conventional western medicine on MS syndrome was better than conventional western medicine.(2)The efficacy of treatment of phlegm method combined with conventional western medicine in waist circumference,blood glucose and blood lipid was significantly better than that of conventional western medicine.2.Experimental research:(1)the MS phlegm syndrome rat models were established.After Erchen Decoction intervention,the body weight,abdominal circumference,blood pressure,blood lipid and blood glucose of the model rats were improved to varying degrees.(2)The energy metabolism of rats with MS phlegm syndrome was disordered.Compared with the blank group,the contents of plasma lactic acid,serum ATP,LDH,CS and CPT1 were significantly increased(P<0.05),the contents of ATP,TG,lactic acid,CPT1 and HIF-1? in liver tissue were increased(P<0.05),the m RNA expressions of CPT1 A,HIF-1? and PKM2 in liver tissue were significantly increased(P<0.05),the m RNA expression in liver tissue of AMPK?2was significantly decreased(P<0.05),the protein expression of P-PKM2 in liver tissue was increased,and the protein expression of AMPK?2 and P-AMPK? in liver tissue was decreased(P<0.05).(3)Erchen Decoction can correct the energy metabolism disorder of MS phlegm syndrome.Compared with the model group,the contents of plasma lactic acid,serum ATP,and CPT1 in Erchen Decoction group were significantly decreased(P<0.05),the contents of ATP,TG,lactic acid,LDH,CPT1 and HIF-1? in liver tissue were decreased(P<0.05),the m RNA expressions of CPT1 A and PKM2 in liver tissue were significantly decreased(P<0.05),the m RNA expression of AMPK? 2 was significantly increased(P<0.05),the protein expression of P-PKM2 in liver tissue was decreased,and the protein expressions of P-AMPK? were increased(P<0.05).(4)The structure and function of liver tissue in MS phlegm syndrome model rats were significantly damaged,and the serum ALT and AST were significantly increased.HE staining showed that there were scattered holes in the nucleus of the liver,a large number of vacuoles in the cytoplasm,and inflammatory cells in the portal area.The results of transmissio electron microscopy showed that lipid droplets with different sizes were seen in the cytoplasm.Mitochondria could see the fusion and disappearance of cristae,and the structure was not clear.After treatment,the liver structure and function of the model rats were significantly improved.Conclusion:1.Erchen Decoction is the basic prescription of treating MS from phlegm,and it has good curative effect.2.In the early stage of MS,phlegm syndrome is in a state of excess energy.There are energy metabolism changes such as enhanced ?-oxidation of fatty acids,impaired glucose utilization and enhanced anaerobic glycolysis.3.Liver structure and function injury is one of the pathological mechanisms of MS phlegm syndrome.4.The expression and activity of AMPK?2 in liver of MS phlegm syndrome rats were up-regulated,the expression of HIF-1? and the phosphorylation level of PKM2 were upregulated,which led to the overexpression of key enzymes in energy metabolism,resulting in energy metabolism disorder of MS phlegm syndrome,suggesting that AMPK-HIF1-PKM2 signaling pathway in liver may be one of the pathological mechanisms of MS phlegm syndrome.5.Erchen Decoction can regulate the AMPK-HIF1-PKM2 pathway,which may inhibit the energy metabolism disorder caused by the overexpression of HIF-1 and PKM2 protein,affect the expression of key enzymes in energy metabolism,correct the dynamic imbalance between glucose oxidation and fatty acid oxidation,and promote the transformation of anaerobic glycolysis to aerobic oxidation,so as to prevent the occurrence and development of MS in the early stage.The key genes and proteins of AMPK-HIF1-PKM2 pathway may be the modern biological basis of MS phlegm syndrome.