Effect of <italic style="font-style: italic">Longchai Formula</italic> （龙柴方） Drug-containing Serum Combined with Entecavir on P-glycoprotein and PI3K/AKT Signaling Pathway in HepG2.2.15 Hepatocellular Carcinoma Cells

ZHENG Xue; YAN Zhanpeng; LI Kun; XU Tingting; WANG Pei; LIN Lin; ZHU Fangshi

doi:10.13288/j.11-2166/r.2022.23.013

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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

更新时间：2022-11-28

- 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
- Journal of Traditional Chinese Medicine Vol. 63, Issue 23, Pages: 2272-2278(2022)
- 作者机构：
  
  1.南京中医药大学第三临床医学院，江苏省南京市红山路十字街100号，210028
  2.江苏省中医药研究院
  3.江苏省中西医结合医院
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2022.23.013
  CLC：
- Received：05 April 2022，
  
  Revised：2022-05-22，
  
  Published：02 December 2022
- 稿件说明：
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郑雪,严展鹏,李堃等.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[J].中医杂志,2022,63(23):2272-2278.

ZHENG Xue,YAN Zhanpeng,LI Kun,et al.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[J].Journal of Traditional Chinese Medicine,2022,63(23):2272-2278.
郑雪,严展鹏,李堃等.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[J].中医杂志,2022,63(23):2272-2278. DOI： 10.13288/j.11-2166/r.2022.23.013.

ZHENG Xue,YAN Zhanpeng,LI Kun,et al.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[J].Journal of Traditional Chinese Medicine,2022,63(23):2272-2278. DOI： 10.13288/j.11-2166/r.2022.23.013.

摘要

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（15 μmol/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（15 μmol/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（

＞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（

＜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（

＜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（

＜0.05或

＜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（

＜0.05或

＜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。

Abstract

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 （15 μmol/ml entecavir∶medium = 1∶9）） and entecavir plus low-， medium- and high-dose

Longchai Formula

groups （15 μmol/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 （

＞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 （

＜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 （

＜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 （

＜0.05 or

＜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 （

＜0.05 or

＜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.

关键词

Keywords

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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

DOI：10.13288/j.11-2166/r.2022.23.013

摘要

Abstract

关键词

Keywords

references