Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway

Figure 1 Components of Xihuang Pills. The fingerprint of the Xihuang pill was determined using high-resolution mass spectrometry. A: The 12 compounds were labelled according to chromatographic retention times, and their molecular structures were analysed; B: The chemical formulae of 12 compounds. The chemical structures of 12 compounds and chemical formulae obtained from chemsrc (https://m.chemsrc.com/mip/), according to the compound numbering scheme in Table 1.

Figure 2 Xihuang pills extract inhibits the growth and migration of SMMC7721 cells. Cells were treated with different concentrations of the Xihuang pills (XHP) extract (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h, and 48 h. Cell viability was measured using Cell Counting Kit-8. A: A representative graph showing the effects of treatment with various concentrations of the XHP extract for 12 h; B: A representative graph showing that treatment with 0.625 mg/mL XHP extract induces a decrease in cell viability after 12 h; C: The migration of SMMC-7721 cells was measured using a cell scratch assay with or without the administration of 0.625 mg/mL XHP for 48 h. Data are presented as the means ± SD. ^aP < 0.05 and ^bP < 0.01 compared with the control group.

Figure 3 SMMC-7721 cell apoptosis is induced by Xihuang pills extract. SMMC-7721 cells were stained with annexin V/propidium iodide and fluorescein isothiocyanate after treatment with or without 0.625 mg/mL Xihuang pills (XHP) extract for 12 h. A and B: Flow cytometry was used to detect the number of apoptotic cells (A) and the cell cycle distribution (B); C: Cleaved caspase-3 and cleaved caspase-9 protein expression levels were detected by Western blotting, and caspase-3 and caspase-9 mRNA expression levels were detected by reverse-transcription polymerase chain reaction (RT–qPCR); D: After treating SMMC-7721 cells with different concentrations of the XHP extract, cleaved caspase-3 and cleaved caspase-9 protein expression levels were determined using Western blotting, whereas caspase-3 and caspase-9 mRNA expression levels were determined using RT–qPCR. The experiment was repeated three times, and the data are presented as the means ± SD. ^aP < 0.05 and ^bP < 0.01 compared with the control group; ^cP < 0.05 and ^dP < 0.01 compared with the XHP 0.625 group; ^eP < 0.05 and ^fP < 0.01 compared with the XHP 1.25 group.

Figure 4 Inhibitory effects of Xihuang pills extract on the expression of components of the phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin signalling pathway both in vivo and in vitro. A: The protein expression levels and ratios of phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) and p-PI3K/p-Akt/p-mTOR in SMMC-7721 cells were detected using Western blotting; B: PI3K/Akt/mTOR mRNA expression levels in SMMC-7721 cells, as determined using reverse-transcription polymerase chain reaction (RT-qPCR); C: PI3K/Akt/mTOR mRNA expression levels in SMMC-7721 cells after treatment with different concentrations of Xihuang pills (XHP), as measured using RT–qPCR; D: Levels of the PI3K/Akt/mTOR and p-PI3K/p-Akt/p-mTOR proteins in SMMC-7721 cells treated with different concentrations of the XHP extract were detected using Western blotting. Relative protein and mRNA expression levels are shown in representative histograms. The experiment was repeated three times, and the data are presented as the means ± SD. ^aP < 0.05 and ^bP < 0.01 compared with the control group; ^cP < 0.05 and ^dP < 0.01 compared with the XHP 0.625 group; ^eP < 0.05 and ^fP < 0.01 compared with the XHP 1.25 group.

Figure 5 Xihuang pills treatment inhibits tumour growth in vivo. SMMC7721 cells were injected subcutaneously into the lower right side of 5-week-old BALB/c male nude mice. After a model of subcutaneous xenograft tumours was successfully established in nude mice, animals were randomly divided into two groups. Each group was then treated with either 0.2 mL of distilled water (control) or 78 mg/kg of body weight Xihuang pills. A: Representative images of subcutaneous xenograft tumours at the end of treatment; B: Subcutaneous tumour weights measured at the end of the treatment; C: Average subcutaneous tumour volume measured every 2 d; D: Average mouse body weight measured every 2 d. The experiment was repeated three times, and the data are presented as the means ± SD. The results were analysed using one-way analysis of variance, followed by the least significant difference test. ^aP < 0.05 and ^bP < 0.01 compared with the control group. XHP: Xihuang pills.