Rabbit polyclonal anti-PI3K, rabbit polyclonal anti-phospho-(p-)p85 PI3K, rabbit polyclonal anti-Akt and rabbit polyclonal anti-p-Akt (ser473) were obtained from Cell Signaling Technology, Inc. Mouse monoclonal anti-HBx antibody was obtained from Chemicon International; Thermo Fisher Scientific, Inc. Rabbit polyclonal anti-Bcl-2 and rabbit polyclonal anti-Bax were obtained from Santa Cruz Biotechnology, Inc. Cell Counting Kit-8 (CCK-8) was obtained from Nanjing KGI Biological Technology Development Co., Ltd. Caspase-3 and -9 activity assay kit and LY294002 were obtained from Sigma-Aldrich; Merck KGaA. An Annexin V-Fluorescein Isothiocyanate (FITC) and propidium iodide (PI) double staining kit were obtained from Nanjing KeyGen Biotech Co., Ltd. Artificially cultured C. sinensis extract [trade name Corbrin capsule (Bailing Jiaonang)] was provided by Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd. and was dissolved in sterile distilled water. The final working concentration was 40 mg/l according to 0.5% (v/v) dilution to the medium (24,25).

A human renal proximal tubular epithelial cell line (HK-2) was obtained from the China Center for Type Culture Collections. HK-2 cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich; Merck KGaA) containing 4.5 mM glucose, 100 µg/ml streptomycin, 100 U/ml penicillin and 10% fetal bovine serum in a humidified atmosphere of 5% CO₂ at 37°C. Cells were detached using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid. Before treatment, 80-85% of confluent cells were cultured in serum-free media for 12 h to arrest and synchronize the cell cycle. This method can block the cell cycle in the G0/G1 phase and subsequent treatments and cell cycle change analysis based on this method are more convincing (26). In the present study, six repetitions were conducted in each group in every experiment. All experiments were repeated three times. In the C. sinensis treatment group, cells were incubated with C. sinensis (40 mg/l) for 24 h. In the LY294002 (PI3K inhibitor) treatment experiments, cells received 40 µmol/l LY294002 treatment for 60 min before C. sinensis induction (17).

HK-2 cells were transfected with pCMV-HBx or pCMV-tag2A (empty vector) with Lipofectamine™ LTX and PLUS™ transfection reagents (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At 24 h after transfection, the cells were diluted to 1:10 and cultured in growth medium containing G418 (600 µg/ml) for 3 weeks. Stable transfected clones were chosen and maintained in a medium containing 300 µg/ml of G418 for further studies.

Cell viability was assayed by a colorimetric procedure using CCK-8 (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. The absorbance at 450 nm was determined by a microplate reader. The cell suspension was inoculated into 96-well plates (100 µl/well) and incubated overnight. CCK-8 (10 µl) was added to each well and the cells were continuously cultured at 37°C for 2 h. The optical density value (OD) at 450 nm was determined by a microplate reader. The cellular survival rate was calculated according to the following formulas: Survival rate (%)=(OD value of the test group/OD value of the control group) ×100. Inhibition of proliferation rate (%)=(1−survival rate) ×100.

HK-2 cells were harvested and centrifuged at 250 × g at 4°C for 5 min. Cells were washed twice with PBS (pH 7.4) at 4°C, then re-suspended in 50 µl cell lysis buffer at 4°C. All steps were performed on ice. The protein concentration was measured using a micro bicin-choninic acid (BCA) kit (Thermo Fisher Scientific, Inc.). Each 50 µl cell extract (containing 100 µg protein) was combined with equal volumes of 2X reaction buffer in a microplate and 5 µl peptide substrates of caspase-3 and -9. After incubating overnight in the dark at 37°C, the samples were examined using a microplate reader at 405 nm. The activity of caspase-3 and -9 was calculated as the absorbance ratio of treated/control samples.

Total cell proteins were extracted with radioimmunoprecipitation assay lysate (Sigma-Aldrich; Merck KGaA) containing phenylmethane sulfonyl fluoride. Protein concentration was determined using the BCA kit according to the manufacturer's protocol. Immunoblotting was performed with 50 µg protein, which was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked with 5% fat-free milk or 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in TBST [Tris-buffered saline (TBS; pH 7.4) containing 0.21% Tween-20] for 2 h at room temperature. The primary antibodies: HBx (1:1,000; cat. no. MA1-081), t-PI3K (1:1,000; cat. no. 3011T), p-p85 PI3K (1:1,000; cat. no. 4228T), t-Akt (1:1,000; cat. no. 4691S), p-Akt (ser473) (1:1,000; cat. no. 4060S), Bcl-2 (1:500; cat. no. PRS3335), or Bax (1:500; cat. no. SAB4502546) were respectively added and incubated at 4°C overnight. Next, the secondary antibodies, anti-rabbit IgG (1:5,000; HRP-linked antibody; cat. no. 7074S; Cell Signaling Technology, Inc.) or anti-mouse IgG (1:5,000; HRP-linked antibody; cat. no. 7076S; Cell Signaling Technology, Inc.), were added and incubated at room temperature for 2 h. Bound proteins were visualized using electrochemiluminescence (ECL kit; Pierce; Thermo Fisher Scientific, Inc.) and detected using a DNR BioImaging system 3.2 (DNR Bio-Imaging Systems, Ltd.). β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) was used as an internal control.

Morphological variations in the nuclei of apoptotic cells were observed by staining with HO33342 (Sigma-Aldrich; Merck KGaA). The cell slides were taken out, washed with PBS for three times, fixed with 4% paraformaldehyde at 4°C for 20 min and washed with PBS again for three times. After HO33342 fluorochrome (5 mg/l) was added, the cell slides were incubated for 8 min at 37°C (protected from light) and rewashed with PBS three times. Observations and imaging were immediately conducted under a fluorescence microscope. A total of 3 sections were selected in each group and 20 fields of vision were randomly selected for each section. The number of apoptotic cells and the total cells in each random field were counted by two independent researchers. Then the statistical analysis was performed. Finally, the equation (apoptotic cells/total cells ×100%) was used to indicate the apoptotic rate.

The percentage of apoptotic cells was determined using an Annexin V-FITC Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd.), quantified by flow cytometry and analyzed with BD CellQuest™ Pro (version S7; BD Biosciences). Cells were stained at 4°C for 30 min according to the manufacturer's protocol and flow cytometry was conducted using a FACScan flow cytometer (Becton-Dickinson, and Company). Cells were divided into four quadrants: Left lower quadrant (Annexin V-FITC⁻ and PI⁻, representing normal live cells), left upper quadrant (Annexin V-FITC⁻ and PI⁺, representing cells with mechanical damages), right lower quadrant (Annexin V-FITC⁺ and PI⁻, representing early apoptotic cells), and right upper quadrant (Annexin V-FITC⁺ and PI⁺, representing late apoptotic cells). The results show the sums of early and late apoptosis.

All results are expressed as means ± standard deviations from three independent experiments, and data were analyzed with SPSS version 21.0 (IBM, Corp.). Results were compared using analysis of variance (ANOVA). When the ANOVA indicated a statistically significant difference, multiple comparisons were performed using Tukey's. P<0.05 was considered to indicate a statistically significant difference.

HK-2 cells stably expressing HBx were first established by transfection with pCMV-HBx. In the present study, the empty vector pCMV-tag2A was utilized as a negative control (Fig. 1). Western blotting confirmed no expression of HBx in control cells; on the other hand, a prominent protein band at 17 kDa, corresponding to the HBx protein, was detected in lysates from cells stably expressing HBx. Treatment with C. sinensis had no effect on the expression of HBx (Fig. 1A and B).

Cell apoptosis analyzed by flow cytometry indicated that C. sinensis treatment had no effect on the apoptosis of control cells (3.53±1.28 vs. 4.21±1.34%; Fig. 2A and C). By contrast, C. sinensis treatment significantly inhibited the apoptosis of cells in the HBx group (26.11±4.01 vs. 16.42±3.73%; P<0.05). In the control cells, the nuclei of the HK-2 cells showed clear outlines after HO33342 staining. In the HBx groups, nuclear fragmentations were observed and dissolutions and chromatin margination occurred in some nuclei. C. sinensis treatment significantly improved the damage described above (Fig. 2B and D).

Upregulated caspase-3 activity (17.72±2.93%) and caspase-9 activity (16.04±2.11%) were found in HBx-overexpressing cells. In addition, C. sinensis treatment decreased the activity of caspase-3 by 7.71±1.81% (P<0.05) and caspase-9 by 8.47±1.36% (P<0.05; Fig. 2E).

The effect of C. sinensis on proliferation was examined in HBx-overexpressing cells. The results showed that HBx over-expression inhibited cell proliferation by 25.27±2.14% and C. sinensis treatment significantly attenuated this inhibitory effect (inhibition of 15.02±2.96%, P<0.05; Fig. 3).

The phosphorylation level of proteins involving in the PI3K/Akt pathway was examined by western blotting analysis (Fig. 4A and B). The results show that HBx-overexpressing cells had significantly higher levels of the phosphorylated forms of these proteins (P<0.05 for all comparisons) and C. sinensis treatment significantly attenuated this effect.

Next, western blotting analysis was performed to examine the expression level of Bcl-2 and Bax (apoptosis regulators) in HK-2 cells (Fig. 4C and D). The results indicated that HBx-overexpressing cells had higher levels of Bax and lower levels of Bcl-2 (P<0.05 for both comparisons). However, C. sinensis treatment significantly attenuated these effects (P<0.05 for both comparisons).

The effect of the PI3K inhibitor LY294002 on HBx-induced activation of the PI3K/Akt pathway was further investigated (Fig. 5). The results showed that LY294002 treatment alone significantly attenuated the HBx-increased phosphorylated protein level in this pathway (P<0.05 for all comparisons). In addition, treatment with C. sinensis and LY294002 together led to further attenuation (Fig. 5A and B). LY294002 had similar effects on the expression of Bax and Bcl-2, and C. sinensis treatment also increased this effect (Fig. 5C and D).

Next, the role of the PI3K/Akt signaling pathway in regulating the apoptosis of HBx-overexpressing cells was examined. Consistent with above results, LY294002 attenuated HBx-induced apoptosis and inhibited proliferation, and C. sinensis treatment increased this effect (Fig. 6A and B). Similarly, treatment with LY294002 attenuated the HBx-induced changes in the activity of caspase-3 and -9, and C. sinensis treatment increased this effect (Fig. 6C). Taken together, the present data indicate that C. sinensis treatment attenuates HBx-induced apoptosis and inhibits proliferation by inhibiting the PI3K/Akt pathway in HK-2 cells.