Effects Of Sanghuang Tongxie Formula On Glycolipid Metabolism And PI3K/Akt Signal Pathway In Drosophila With Insulin Resistance

Objectives By establishing drosophila insulin resistance（IR）diabetes model,water extract of Sanghuang Tongxie Formula（SHTXF）was selected to intervene,and the effects of this formula on glucose and lipid metabolism and PI3K/Akt signaling pathway were observed and the molecular mechanism was explored.Methods The Gal4/UAS expression system was used to specifically interfere with the function of insulin receptors in drosophila adipocytes and reduce the activity of insulin signal transduction pathway,resulting in insulin resistance.In the control group,the progeny of hybridization between female flies Cg-Gal4 and male flies w¹¹¹⁸ were collected.The progeny of hybridization between female flies Cg-Gal4 and male flies UAS-InR^K1409Awere collected from Model group,Metformin group and Sanghuang Tongxing group.Control group and Model group were fed with sucrose,corn meal and yeast standard medium,and Metformin group was fed with medium containing 0.00165 g/mL metformin.Sanghuang Tongxing group was fed with SHTXF aqueous extract medium with concentrations of 0.0125 g/mL,0.025 g/mL,0.05 g/mL and 0.1 g/mL,respectively.The samples of the third instar larval stage of each progenitor were collected,and the contents of circulating glucose,circulating trehalose,protein,triglyceride and glycogen were determined respectively to obtain the levels of various biochemical indexes,and the optimal intervention dose of Sanghuang Tongxie Formula was analyzed accordingly.The number,size and area of lipid droplets in adipocytes of each group were detected by Nile Red staining.Subsequently,the activity reporter gene tGPH of PI3K was introduced to monitor the in vivo activity of PI3K by observing the intracellular localization of tGPH in adipocytes.Western blot was used to detect the expression levels of Akt and p-Akt in drosophila adipose bodies.Finally,the gene fragment of Drosophila Forkhead box Protein O（dFoxO）fused with GFP was introduced to monitor the transcriptional activity of dFoxO protein by observing the intracellular localization of dFoxO-GFP.The level of 4EBP mRNA was determined by qRT-PCR.Results 1.Compared with the Control group,glucose and trehalose levels in the Model group were significantly increased by 67.5%and 40%,triglyceride and glycogen contents were decreased by 15.67%and 17.48%,and protein contents in each group had no significant change.2.Compared with the Model group,the glucose and trehalose of fruit fly larvae fed SHTXF water extract decreased significantly（p<0.05）,and the triglyceride content increased significantly（p<0.05）,and the group with the concentration of0.025g/mL had the best effect,and the glycogen content was lower than that of the Model group.3.Compared with the Control group,the activity of PI3K was decreased,the phosphorylation of Akt was decreased（p<0.05）,dFoxO was significantly located in the nucleus,and 4EBP mRNA was increased in the Model group（p<0.05）.Compared with Model group,PI3K activity and Akt phosphorylation level of 0.025 g/mL SHTXF were significantly increased（P<0.001）,dFoxO was distributed in cytoplasm,and 4EBP mRNA level was decreased（P<0.001）.Conclusions 1.SHTXF reduces circulating sugar levels,probably by converting sugars into TAG for nutrient storage rather than glycogen.2.SHTXF rescues the IR-induced lipid homeostasis disorder.3.SHTXF can enhance PI3K activity in adipocytes of the third instar larva,improve the phosphorylation level of Akt,inhibit the nuclear localization and transcription activity of dFoxO in cells and regulate the expression of 4EBP mRNA.Figure 15;Table 4;Reference 129...