2022, Vol. 26 ›› Issue (31): 4988-4995

Mechanism underlying the effect of Fuhebeihua Recipe on the proliferation of CD133+HepG2 hepatoma stem cells and related autophagy proteins

Li Zulong1, Rong Zhen2, Sun Hua1, Jiang Ruiyuan1, Zhong Xiaoting1, Mo Chunmei2   

Abstract: BACKGROUND: Hepatoma stem cells are key cells for the continuous development and high heterogeneity of liver cancer. At present, there is no research on Chinese medicine compound that targets hepatoma stem cell-related autophagy pathways. Therefore, it is very necessary to study Chinese medicine compound prescription to inhibit hepatoma stem cell autophagy.  
OBJECTIVE: To investigate the effect and mechanism of Fuhebeihua Recipe on PI3K/AKT/mTOR pathway and related autophagy proteins in CD133+HepG2 hepatoma stem cells.
METHODS:   CD133+HepG2 cells were selected by immunomagnetic bead method. CD133+HepG2 cells were treated with different volume fractions of Fuhebeihua Recipe-containing serum of 2%, 4%, 8%, and 16%. The cell proliferation activity was detected by CCK-8 assay. The best volume fraction of Fuhebeihua Recipe-containing serum was selected. CD133+HepG2 cells were divided into blank control group, serum control group, rapamycin group, and Fuhebeihua Recipe group. Cells were separately cultured with DMEM/F12 containing serum of 16%, serum-free DMEM/F12, DMEM/F12 containing 0.05% rapamycin, and DMEM/F12 with Fuhebeihua Recipe-containing serum of 16%. The percentage of CD133+HepG2 hepatoma stem cells was detected by flow cytometry in each group. The mRNA expression levels of PI3K, AKT, mTOR, LC3-II, and beclin-1 were detected by RT-PCR. The protein expression levels of PI3K, p-AKT, AKT, p-mTOR, mTOR, and LC3-II were detected by western blot assay. The expression levels of LC3 and LC3-II were detected by immunofluorescence in each group.  
RESULTS AND CONCLUSION: (1) The Fuhebeihua Recipe-containing serum inhibited the proliferation of CD133+HepG2 cells, and the inhibitory effect was the most obvious when the volume fraction was 16% (P < 0.05). (2) Compared with serum control group and blank control group, the percentage of CD133+HepG2 cells reduced (P < 0.05); protein expression levels of PI3K, AKT, p-mTOR, mTOR, and LC3-II were significantly decreased (P < 0.05); mRNA expression levels of PI3K, AKT, mTOR, LC3-II, and Beclin-1 were significantly down-regulated (P < 0.05); the fluorescence signal intensities of LC3 and LC3-II were significantly down-regulated (P < 0.05) in the Fuhebeihua Recipe group and rapamycin group. (3) Compared with the rapamycin group, the protein expression levels of PI3K, p-Akt, AKT, p-mTOR, mTOR, and LC3-II in CD133+HepG2 cells were significantly down-regulated (P < 0.05); the mRNA expression levels of PI3K, AKT, mTOR, LC3-II, and Beclin-1 were significantly decreased (P < 0.05); the fluorescence signal intensities of LC3 and LC3-II were significantly decreased in the Fuhebeihua Recipe group (P < 0.05). (4) It is concluded that Fuhebeihua Recipe can inhibit autophagy and proliferation of hepatoma stem cells, and its mechanism is related to the inhibition of PI3K/AKT/mTOR signaling pathway.
Key words: hepatoma stem cells, Fuhebeihua Recipe, PI3K, AKT, mTOR, pathway, LC3, autophagy