Study On The Effect And Mechanism Of Huanglianwendan Decoction On The PI3K/Akt Signaling Pathway Of 3T3-L1 Cell

Objective:To optimize Huanglianwendan decoction preparation methods and to extract the drug containing serum of rat.To establish insulin resistance 3T3-L1 adipocyte model,and to screen the best concentration and action time of Huanglianwendan decoction drug containing serum,then to observe its effect on PI3K/Akt signaling pathway,to explore the molecular mechanisms of Huanglianwendan decoction improved insulin resistance.Methods:Experiment 1:Used the comprehensive rating of hesperidin and total polysaccharide content as an index,the orthogonal test was carried out to optimize Huanglianwendan decoction preparation methods about the influence factors,determine the final water addition content,decocting time and decocting times.Experiment 2:30 male SD rats were randomly divided into Huanglianwendan decoction group and blank group,each group had 15 rats,Huanglianwendan decoction group intragastric gavage with Huanglianwendan decoction diluent(2.1g/mL),blank group intragastric gavage with the asme volume of saline,total 5 times in a row,according to the serum pharmacology method preparation of Huanglianwendan decoction drug containing serum and blank serum.Differentiated the 3T3-L1 cells in 96well plates,using dexamethasone establish insulin resistance 3T3-L1 adipocyte model,then were randomly divided into 5 groups(each group 3 wells)and were give the corresponding treatment intervention 12h:model group(8%blank serum),high dose group(8%medicated serum),middle dose group(4%medicated serum),low dose group(2%medicated serum),metformin group(1mm/L metformin solution),and choose the differentiation of mature 3T3-L1 cells as normal group(8%blank serum).Used CCK 8 method to determine the cell vitality,used glucose oxidase method determination of glucose consumption and used ELISA method to detect the contents of glucose oxidase.The same method was used to determine the results of each group intervention of 24h and 36h,screened the best concentration and ation time of Huanglianwendan decoction drug containing serum.Experiment 3:Differentiated the 3T3-L1 cells in the 90mm dishes,randomly divided into 4 groups,the normal group,model group,Huanglianwendan decoction group and metformin group,intervention methods corresponding with determined the best concentration and action time in Experiment 2.Using real time qPCR method to detect 3T3-L1 adipocyte PTP1B,IRS1,PI3K p85α;PTEN,Aktl and GLUT4 mRNA gene transcription level,using western blot method to detect 3T3-L1 adipocyte IRS1,p-IRS1(Tyr612),PI3K-p85α,p-PI3K-p85α(Tyr607),Akt1,p-Akt1(Ser473),PTP1B,PTEN,GLUT4 protein expression levels.Results:1.Huanglianwendan decoction main factors affecting the preparation order:decocting times(C)>water addition content(A)>decoction time(B),the analysis of variance showed that decocting times(C)of Huanglianwendan decoction had a significant effect,ultimately determine the best process for A3B1C3,namely with 14 times the amount of water,decocted for 3 times,each time decoted 1 hour.2.Cell morphological observation showed that the differentiation rate of 3T3-L1 adipocyte was greater than 80%,and the cells were filled with fat droplets,which showed a typical "ring shape",which was consistent with the results of oil red staining.3.CCK8 method to determine the cell vitality results showed that the cell vitality was indifference of three dose group when intervened with12h and 24h,high and low dose group cell vitality had a downward trend with middle dose group when intervened with 36h.4.The results of glucose consumption showed that:when intervened with 12h,the glucose consumption of the model group was significantly lower than the other 5 groups(P<0.01).When intervened with 24h,the glucose consumption of each group was higher than 12h,but there were no difference between low dose group and model group(P>0.05),and the glucose consumption in the remaining group was still higher than the model group(P<0.05).When intervened with 36h,there were no difference between high dose group/low dose group with model group(P>0.05),only middle dose group and metformin group elevated glucose consumption compared with model group(P<0.05).5.The results of glucose oxidase content showed that when intervened with 12h and 24h,the concentration of GOD in each group was no difference(P>0.05).When intervened with 36h,the concentration of GOD in the model group was lower than middle dose group(P<0.05).6.The results of real time qPCR and western blot showed that:there was no difference in mRNA expression level of IRS 1,PI3K-p85α and Aktl in each group,but the expression level of p-IRS 1(Tyr612),p-PI3K-p85α(Tyr607)and p-Akt1(Ser473)in the model group was lower than the normal group(P<0.05,P<0.01).The expression levels of p-IRS1Tyr612),p-PI3K-p85α(Tyr607)and p-Akt1(Ser473)were higher than those in the model group(P<0.05,P<0.01).Compared with the normal group,the mRNA and protein expressions of PTP1B and PTEN in the model group were up-regulated(P<0.05,P<0.01),and the mRNA and protein expression of GLUT4 were down-regulated(P<0.05).Compared with the model group,the mRNA and protein expression of PTP1B in the Huanglianwendan decoction group and metformin group were down-regulated(P<0.05),and the mRNA and protein expressions of GLUT4 were up-regulated(P<0.05,P<0.01),while PTEN was unchanged.Conclusions:1.The preparation process of the Huanglianwendan decoction:Chinese Traditional Chinese medicine yinpian is added 14 times of water,decocted 3 times,each time decocted for 1 hour.2.No cytotoxicity was found in the drug serum containing Huanglianwendan decoction,and the optimal concentration of IR-3T3-L1 cells was 4%of the culture system,and the best time was 36 hours.3.Huanglianwendan decoction drug containing serum can increase IR-3T3-L1 adipocyte IRS1(Tyr612),PI3K-p85α(Tyr607)and Akt1(Ser473)protein phosphorylation levels,lead to downstream GLUT4 gene expression levels,complete IRS 1/PI3K/Akt1/GLUT4 pathways of signal transduction,increased glucose uptake,improve IR.4.Huanglianwendan decoction drug containing serum can inhibit the gene expression of PTP1B in IR-3T3-L1 adipocyte and reduce dephosphorylation,that may be one of the mechanisms to maintain the phosphorylation level of IRS1(Tyr612)protein.5.PI3K/Akt signaling pathway may be one of the main targets for the regulation of IR-3T3-L1 adipocyte in the drug serum of Huanglianwendan decoction.