Effect Of Liuwei Dihuang Decoction On The PI3K / Akt Signaling Pathway In Liver Of Type 2 Diabetic Rats

Objective To establish a high performance liquid chromatography method for the separation of Liuwei dihuang decoction water extract-alcohol soluble parts, and use this method for the determination of four glycosides in it; To observed the effect of IRS2、PI3K、Akt expression in PI3K/Akt signaling pathway of the liver in rat model of T2DM insulin resistance after the administration of Liuwei dihuang decoction water extract-alcohol soluble parts, and to investigate the mechanism of intervention of T2DM by Liuwei dihuang decoction water extract-alcohol soluble parts at gene and protein levels, to clarify the material foundation of medicinal effectiveness and action mechanism of the intervention of T2DM insulin resistance for this compound.Methods To separate and determine the content of morroniside, sweroside, paeoniflorin and loganin in Liuwei dihuang decoction water extract-alcohol soluble parts by RP-HPLC. The HPLC method was used at Hypersile C18column (4.6mmx250mm,5μm), the mobile phase consisted of methanol-water(27:73), the detective wavelength was236nm, the flow rate was1.0ml/min, the column temperature was25℃.Used the method of feed high sugar, high fat diet for four weeks, intraperitoneal injection of small doses of streptozotocin(STZ) and fasting blood glucose(FBG)≥16.7mmol/L to establish the rat model of T2DM, all rats were divided averagely into normal group, model group, Rosiglitazone group and Liuwei dihuang decoction water extract-alcohol soluble parts group, respectively gavage drugs or distilled water30days, detect the level of FBG、FINS in each group; take the fresh liver tissue of each model rats, determine the expression of IRS2、PI3K(p110)、 Akt mRNA in each group rats liver tissue by RT-PCR, and determine the expression of of IRS2、 PI3K(p85)、Akt total protein and phosphorylated protein by Western blot.Result1、The calibration curves of morroniside, sweroside, paeoniflorin and loganin were respectively linear in the range of0.24ug～2.40μg (r=0.9996),0.24ug～2.40ug (r=0.9991),0.14ug～1.38ug (r=0.9991),0.24ug-2.40ug(r=0.9994). The recovery and relative standard deviation of them were100.22%,100.83%,102.40%,101.42% and1.80%,1.92%,1.44%,1.50%. The average content of morroniside, sweroside, paeoniflorin and loganin in Liuwei dihuang decoction water extract-alcohol soluble parts were calculate in terms of alcohol soluble solids1.03%,0.14%,0.26%,0.65%; to the material were0.21%,0.03%,0.06%,0.13%.2、The level of FBG、FINS in rats of Liuwei dihuang decoction water extract-alcohol soluble parts group were decreased obviously than the model group(P<0.05).3、The expression of IRS2、PI3Kp110mRNA in liver of Liuwei dihuang decoction water extract-alcohol soluble parts group were increased obviously than the model group(P<0.05).4、The expression of IRS2、PI3Kp85total protein and phosphorylated protein in liver tissue of Liuwei dihuang decoction water extract-alcohol soluble parts group were increased than the model group(P<0.05).Conclusion The HPLC method has the good stability and reproducibility to determinate the content of morroniside, sweroside, paeoniflorin and loganin, and could be used for quality control of glycosides in Liuwei dihuang decoction water extract-alcohol soluble parts.Liuwei dihuang decoction water extract-alcohol soluble parts could regulate blood sugar level and improve IR, its mechanism may through the up regulation the key gene IRS2、PI3K(p110,p85). Akt expression in PI3K/Akt signaling pathway of the liver tissue in rats of T2DM insulin resistance, promoting the recovery of hepatic insulin signaling pass, intervening the IR, and to improve the T2DM. It may be one of the medicinal effective components of Liuwei dihuang to improve IR and regulate the PI3K/Akt signaling pathway. Morroniside, sweroside, paeoniflorin and loganin in it may be the one of main effective components to intervene IR and regulate the PI3K/Akt signaling pathway.