龙柴方含药血清联合恩替卡韦对HepG2.2.15肝癌细胞中P-糖蛋白和PI3K/AKT信号通路的影响

Effect of Longchai Formula(龙柴方) Drug-containing Serum Combined with Entecavir on P-glycoprotein and PI3K/AKT Signaling Pathway in HepG2.2.15 Hepatocellular Carcinoma Cells

目的观察龙柴方对恩替卡韦抗乙型肝炎病毒(HBV)效果的影响,并探讨可能作用机制。方法24只SD大鼠随机分为空白组和龙柴方低、中、高剂量组,每组6只。龙柴方低、中、高剂量组大鼠分别给予龙柴方药液9.84、19.68、39.36 g/(kg·d)灌胃,空白组给予和龙柴方中剂量组等量生理盐水灌胃,连续给药7天后制备含药血清。将HepG2.2.15肝癌细胞分为空白对照组(等量培养基)、空白血清组(100%空白血清∶培养基=1∶9)、恩替卡韦组(15μmol/ml恩替卡韦∶培养基=1∶9)及恩替卡韦+龙柴方低、中、高剂量血清组(15μmol/ml恩替卡韦∶100%龙柴方低、中、高剂量含药血清∶培养基=1∶1∶8),培养24 h。MTT法检测各组细胞存活率,荧光定量PCR法测定HBV DNA表达量;ELISA法测定乙型肝炎病毒表面抗原(HBsAg)、乙型肝炎病毒e抗原(HBeAg)表达量;Western blot法检测P-糖蛋白(P-gp)、蛋白激酶B(AKT)、磷脂酰肌醇3-激酶(PI3K)、同源性磷酸酶-张力蛋白(PTEN)蛋白表达。结果各组HepG2.2.15细胞存活率差异均无统计学意义(P>0.05)。与空白血清组比较,恩替卡韦+龙柴方中、低剂量血清组HBV DNA水平、HBeAg表达量明显降低(P<0.01)。与空白组对照比较,恩替卡韦+龙柴方中剂量血清组HBsAg表达量降低(P<0.05)。与空白血清组和恩替卡韦组比较,恩替卡韦+龙柴方低剂量血清组PI3K蛋白表达降低,恩替卡韦+龙柴方中剂量血清组PI3K、AKT、P-gp蛋白表达均降低,PTEN蛋白表达升高(P<0.05或P<0.01)。与恩替卡韦组+龙柴方高剂量血清组比较,恩替卡韦组+龙柴方中剂量血清组HBV DNA水平降低,PTEN蛋白表达升高(P<0.05或P<0.01)。结论龙柴方对恩替卡韦抗HBV产生增效作用,其机制可能与上调PETN蛋白,抑制PI3K/AKT信号通路,从而下调P-gp表达有关,且以中剂量效果最佳。

Objective To explore the effect of Longchai Formula(龙柴方)adjuvant to entecavir on anti-hepatitis B virus(HBV)function,and to explore the possible mechanism.Methods Twenty-four SD rats were randomly divided into blank group and low-,medium-and high-dose Longchai Formula groups,with six rats in each group.Rats in the low-,medium-and high-dose Longchai Formula groups were given 9.84,19.68,and 39.36 g/(kg·d)of Longchai Formula liquid by gavage,respectively,while those in the blank group were given the same amount of normal saline as the medium-dose Longchai Formula group by gavage,for continous seven days to prepare the medicated serum.HepG2.2.15 liver cancer cells were divided into blank control group(equivalent medium),blank serum group(100%blank serum∶medium=1∶9),entecavir group(15μmol/ml entecavir∶medium=1∶9))and entecavir plus low-,medium-and high-dose Longchai Formula groups(15μmol/ml entecavir∶100%low-,medium-and high-dose Longchai Formula-containing serum∶medium=1∶1∶8),cultured for 24 h.The survival rate of cells was detected by MTT method,and the expression of HBV DNA was detected by fluorescence quantitative PCR method.The expression levels of hepatitis B virus surface antigen(HBsAg)and hepatitis B virus e antigen(HBeAg)were determined by ELISA.Western blot was used to detect the protein expressions of P-glycoprotein(P-gp),protein kinase B(AKT),phosphatidylinositol 3-kinase(PI3K)and homologous phosphatase-tensin(PTEN).Results There was no significant difference in the survival rate of HepG2.2.15 cells among the groups(P>0.05).Compared to those in the blank serum group,the HBV DNA level and HBeAg expression level in the entecavir plus medium-and low-dose Longchai Formula-containing serum groups significantly decreased(P<0.01).Compared to that in the blank control group,the HBsAg expression level in the entecavir plus medium-dose Longchai Formula-containing serum group decreased(P<0.05).Compared to those in the blank serum group and the entecavir group,the PI3K protein expression in the entecavir plus low-dose Longchai Formula group was reduced,and the PI3K,AKT,P-gp protein expression in the entecavir plus medium-dose Longchai Formula group decreased,while the PTEN protein expression increased(P<0.05 or P<0.01).The serum HBV DNA level in the entecavir plus medium-dose Longchai Formula group was significantly lower than that in the entecavir plus high-dose Longchai Formula group,and the expression of PTEN protein increased(P<0.05 or P<0.01).Conclusion Longchai Formula has a synergistic effect on entecavir against HBV,whose mechanism may be related to the up-regulation of PETN protein,the inhibition of PI3K/AKT signaling pathway,and thus the down-regulation of P-gp expression,and the middle dose has the best effect.