| Objective:The mechanism of treatment of glucose lipid metabolism disorder in insulin resistance HepG2 cells by the drug-containing serum of Dangua-fang was discussed from insulin receptor signal transduction pathway,which is the basic research of diabetes mellitus and the treatment service of TCM.Method:1.The IR model was established by using low-sugar DMEM culture containing 0.25mmol/l soft acid to induce HepG2 cell 24 h,the glucose residue in cell culture medium was detected by glucose oxidase method,and the insulin sensitivity index was calculated to verify the success of the model.2.The experiment was divided into 5 groups: the blank ccontrol group,the high-fat group,Dangua-fang 1 group,the high-sugar and high-fat group,Dangua-fang 2 group,according to the grouping situation each group was given the corresponding induction of liquid intervention 24 h.The glucose residue in each group of cell culture medium was detected by glucose oxidase method,sugar consumption and insulin sensitivity index were calculated,glycogen kit was used to detect the glycogen content of each group of cells,he staining observed the morphological changes of each group,oil red o staining observed the lipid accumulation of each group of cells,TC and TG Kit detected the content of TC and TG,The expression of INSR,IRS-1,SH2B2,IGF-1,PRKAA2 and PTPN1 mRNA in each group of cells was detected by qPCR method,and the expression of blot,INSR and IRS-1 protein in cells was detected by Western SH2B2 method.The expression of leptin protein in each group of cells was detected by Elisa method.Results:1.Model establishment: After 24 h of intervention HepG2 cells with low sugar DMEM culture containing 0.25 mmol/L soft acid,the results of glucose oxidase determination showed that glucose consumption in the model group decreased significantly(P < 0.05)and the insulin sensitivity decreased(P < 0.05),it is suggested that the IR-HepG2 cell model was successfully established.2.Cell Vitality : The results of CCK8 method showed that there was no significant difference in cell activity between the blank control group,the high-fat group,Dangua-fang 1group,the high-sugar and high-fat group,Dangua-fang 2 group(P>0.05).3.Glucose consumption and insulin sensitivity index: Compared with the blank control group,the glucose consumption of the high-fat group cells decreased(P<0.05)and the insulin sensitivity index decreased(P < 0.05).Compared with the high-fat group,the glucose consumption of Dangua-fang 1 group cells increased(P<0.05)and nsulin sensitivity index increased accordingly(P < 0.05),the glucose consumption of the high-sugar and high-fat group cell sdecreased correspondingly(P <0.05)and the insulin sensitivity index decreased(P < 0.05).Compared with the high-sugar and high-fat group,the glucose consumption of Dangua-fang 2 group cells increased(P <0.05)and the insulin sensitivity index increased correspondingly(P<0.05).4.Glycogen content: Compared with the blank control group,The glycogen content of the high-fat group cells decreased(P < 0.05).Compared with the high-fat group,the glycogen content of Dangua-fang 1 group cells increased(P<0.05),the glycogen content of high sugar and high fat group cells decreased(P<0.05).Compared with the high-sugar and high-fat group,the glycogen content of Dangua-fang 2 group cells increased(P<0.05).5.TC Content: Compared with the blank control group,The TC content of the high-fat group cells increased(P < 0.05).Compared with the high-fat group,the TC content of Dangua-fang 1 group cells decreased(P < 0.05),the TC content of the high-sugar andhigh-fat group cells increased(P<0.05).Compared with the high-sugar and high-fat group,the TC content of Dangua-fang 2 group cells decreased(P<0.05).6.TG Content: Compared with the blank control group,The TG content of the high-fat group cells increased(P < 0.05).Compared with the high-fat group,the TG content of Dangua-fang 1 group cells decreased(P<0.05),the TG content of high-sugar and high-fat group cells increased(P<0.05).Compared with the high-sugar and high-fat group,the TG content of Dangua-fang 2 group cells decreased(P<0.05).7.Oil red o staining: Compared with the blank control group,the lipid droplets in the high-fat group increased.Compared with the high-fat group,the lipid droplets decreased in Dangua-fang 1 group cells,the lipid droplets in the high-sugar and high-fat group were increased and more dense.Compared with the high-sugar and high-fat group,the lipid droplets in the Dangua-fang 2 groups were decreased accordingly.8.HE staining: Compared with the blank control group,the high-fat group cells showed some degree of deformation and superficial cytoplasmic staining.Compared with the high-fat group,the cell morphology of Dangua-fang 1 groups was improved,the degree of cytoplasm staining was reduced;the deformation degree of high-sugar and high-fat group cells was increased,and the degree of cytoplasm staining was more obvious.Compared with the high-sugar and high-fat group,the cell morphology of Dangua-fang 2 groups was also improved,and the degree of cytoplasmic staining was reduced.9.QPCR: Compared with the blank control group,the expression of INSR,IRS-1,SH2B2,IGF-1 and PRKAA2 mRNA in the high-fat group decreased(P < 0.05),the expression of PTPN1 mRNA expression increased(P<0.05).Compared with the high-fat group,the the expression of INSR,IRS-1,SH2B2,IGF-1 mRNA in Dangua-fang 1 groups increased(P<0.05),the the expression of PRKAA2 mRNA increased(P>0.05),the the expression of PTPN1 mRNA decreased(P<0.05).Compared with the high-fat group,the expression of INSR,IRS-1,SH2B2,IGF-1 mRNA in the high-sugar and high-fat group decreased(P<0.05),the expression of PRKAA2 mRNA expression decreased(P>0.05),the expression of PTPN1 mRNA expression increased(P<0.05).Compared with the highsugar and high fat group,the the expression of INSR,IRS-1,SH2B2,IGF-1 mRNA in Dangua-fang 1 group increased(P<0.05),the expression of PRKAA2 mRNA increased(P<0.05),the expression of PTPN1 mRNA decreased(P<0.05).10.Western Blot: Compared with the blank control group,the expression of INSR,IRS-1 and SH2B2 protein decreased(P < 0.05)in the high-fat group.Compared with the high-fat group,the cells of Dangua-fang 1 group INSR,IRS-1,SH2B2 protein expression increased(P<0.05),the expression of INSR,IRS-1 and SH2B2 protein decreased(P<0.05)in the high-sugar-and high fat group.Compared with the high-sugar and high-fat group,the expression of INSR,IRS-1 and SH2B2 protein in Dangua-fang 2 group increased(P<0.05).11.ELISA: Compared with the blank control group,the expression of leptin protein in the high-fat group increased(P<0.05).Compared with the high-fat group,the expression of leptin protein in Dangua-fang 1 group decreased(P<0.05),the expression of leptin protein in the high-sugar and high-fat group increased(P<0.05).Compared with the high sugar and high fat group,the expression of leptin protein in Dangua-fang 2 group decreased(P<0.05).Conclusion:1.High sugar can increase the glucose and lipid metabolism disorder in HepG2 cells on the basis of high fat induction,and aggravate the degree of IR;2.The drug-containing serum of Dangua-fang can increase the glucose consumption and glycogen content in IR-HepG2 cells,improve cell insulin sensitivity,improve glucose metabolism disorders,reduce IR;3.The drug-containing serum of Dangua-fang can reduce the content of TC and TG in IR-HepG2 cells,reduce the accumulation of lipid ectopic in cells,and improve the disorder of lipid metabolism,repair cell morphological damage;4.The drug-containing serum of Dangua-fang improve glucose and lipid metabolism disorder and reduct IR mechanism may be related to the expression of related factors(INSR,IRS-1,SH2B2,IGF-1,PTPN1 and leptin protein)in insulin receptor signal transduction pathway. |
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