Cell culture Hepatic stellate cell lines cirrhotic fat storing cell (CFSC) isolated from carbon tetrachloride (CCl4)- induced cirrhosis rats and acquired permanent natural properties were built and presented by professor Greenwel  of the United States. The cells frozen in liquid nitrogen were resuscitated and inoculated in Dulbecco’s modified Eagle’s medium (DMEM) (Boehringer Ingelheim Corporation, USA) containing 5% fetal bovine serum (FBS) (Hangzhou Sijiqing Biological Products Company, China) and 1% penicillin streptomycin mixed solution in humidified air at 37℃ with 5% CO2. When the cells were monolayer dense, they were digested by 0.25% trypsin and passaged.
Cell grouping and treatment The cells were synchronized for 24h, then divided into three groups: control group, TGF-β1 group, TGF-β1 + PI103 (Selleck Chemicals, USA) group. The control group was grown in complete medium for 48h, the TGF-β1 group was grown in complete medium with 5ng/mL TGF-β1 for 48h, and TGF-β1 + PI103 group was grown in complete medium with 5ng/mL TGF-β1 for 24h, then 4µmol/L PI103 was added for 24h. In addition, HSCs were treated in a dose-dependent manner by PI103 to observe the effect of PI103 on HSCs proliferation.
Cell proliferation assay HSCs proliferation of each group was determined by MTT (3–4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide). The cell suspension was seeded in 96-well plates at a density of 2×104 cells/mL and 200µl per well. Each group was comprised of five wells and an empty zero well, 20µL of 5g/L MTT (Sigma Company, USA), was added for 4h in each well and 200µL DMSO (Dimethyl sulfoxide) (Sigma Company, USA) was added, then cells were exposed to a miniature oscillator shock for 10min. The enzyme-labelled instrument was set to deliver a single wave excitation at 490nm to measure the cell absorbance value (A). The cell proliferation rate (PR) was calculated. PR = T/C×100%, where T is the A value of the treatment group and C is the A value of the control group.
Cell morphological changes assay Morphological changes of the cells were observed under transmission electron microscopy (HITACHI Company, Japan). The cells in the control group, TGF-β1 group and TGF-β1 + PI103 group were centrifuged and fixed. After dehydration, transparency, waxing, embedding, slicing and staining, the cellular internal structure was observed under transmission electron microscopy.
HSC apoptosis detection The cells in each group were digested, centrifuged and collected, and washed with cold PBS(phosphate buffer saline), 1xBinding buffer 195µL and 5µL Annexin V (MultiSciences Lianke Biotechnology Corporate Limited) were added into the cells, mixed gently for 30 min, centrifuged and precipitated, discarded the supernatant, then added in 1x Binding buffer 195µL and 5µL PI (Propidine iodide) (MultiSciences Lianke Biotechnology Corporate Limited), blow and mixed gently. The apoptosis of cells was examined with Flow cytometry (Becton, Dickinson Company).
Observation of Ca 2+ concentration The loaded cells were rinsed 2–3 times with PBS, and incubated at 37˚C, 5% CO2 for 20 min after adding 1mL DMEM (Boehringer Ingelheim Corporation), then was observed with a laser scanning confocal microscope (Olympus Corporation, Japan). The Fluo-3-Acetoxymethyl ester (Fluo-3AM) excitation wavelength was 488 nm and the emission wavelength was 530 nm, Ca2+ fluorescence absorbance was scanned. The average fluorescence intensity of the whole cell was calculated by the data from the laser scanning confocal microscope and the image processing software FV10-ASW1.7 Viewer. The relative value of fluorescence intensity was recorded in the experiment to observe the dynamic change of the Ca2+concentration. The fluorescence intensity changes when the concentration of Ca2+ changes in the HSCs loaded with Fluo-3AM. The change of Ca2+ concentration is indicated by fluorescence intensity, and the higher the fluorescence intensity, the higher the concentration of Ca2+. Therefore, the fluorescence intensity of each cell was measured to indicate the intracellular Ca2+ concentration. In each group, 6 HSCs were randomly selected, scanned for fluorescence, and the average intensity value was calculated.
RT-qPCR assay Total RNA was extracted from HSCs using RNA extraction kit (Invitrogen, Thermo Fisher Scientific, Inc., Rockford, IL, USA) following the manufacturer's instructions. The extracted RNA was reverse transcribed using M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Inc.). Subsequently, real-time fluorescence quantitative polymerase chain reaction PCR(RT-qPCR) was performed with SYBR-Green PCR kit (Thermo Fisher Scientific, Inc.) according to manufacturer's instructions. The following primers were used for qPCR: PI3K, forward 5’ TAG GCT CCA AAC CGT TCT TTA TG3’ and reverse 5’ GAT GAC GAG GAT TTG CTG ATG TA3’; Akt, forward 5’GCT GAG TAG GAG AAC TGG GGA AA 3’ and reverse 5’ TGA GAC CGA CAC CAG GTA TTT TG 3’. RT-qPCR was performed with 2µg total RNA and the target genes PI3K and Akt were simultaneously amplified using the above 50µL reaction system. Specific cycle parameters were as follows: RT: 41 ℃ 45 min; PCR: 95 ℃ pre-denaturation 10 min into the cycle, 95 ℃ denaturation 15sec, 60 ℃ annealing 1min, 72 ℃ extension 1.5min, 35 cycles after 72 ℃ extension 10min. Calculation of mRNA expression of target gene. In RT-qPCR, each sample was repeated three times, taking the mean value as Ct value. The Ct value is the cycle number of the fluorescence threshold in the heat cycle instrument. The fluorescence quantitative analysis was performed with the PCR amplification instrument and the △Ct value was calculated. Relative quantitative 2−ΔΔCt method was used to compare the difference of target gene mRNA expression.
Immunohistochemistry assay Immunofluorescence staining was performed using the streptomycin biotin-peroxidase immunohistochemistry kit (SP method) (Beijing Zhongshan Jinqiao company, China), the experimental procedure was performed according to the kit instructions, the primary antibody concentration (PI3K, Akt antibody-Beijing Biosynthesis Biotechnology, China; Type I, III collagen antibody-Wuhan Boster Biological Technology, China) was 1: 100, and PBS instead of primary antibodies was used as a negative control. The positive expression of protein was a diffuse brown yellow staining in the cytoplasm. The stained cells were observed under microscope. The optical density of images was detected by Image-Pro Plus software, the average optical density of each slice was randomly taken from 6 visual fields at a magnification, x200 as the expression of PI3K, Akt signal molecule and type I, III collagen.
Western blot assay After HSCs were treated with TGF-β1 and PI103, the total cell proteins were extracted with RIPA(Radio immunoprecipitation assay) lysis buffer (Wuhan Bobst Biotechnology Co., Ltd., China). After 30 min of complete lysis, the cells were centrifuged at 12000 r/min at 4℃ for 20 min. Protein concentration was determined using BCA Protein Detection Kit (Solarbio Science & Technology Co., Ltd., Beijing, China). Protein loading system was 20µg/10µl/well. Electrophoresis was performed on 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrohoresis) gel preparation kit (Zoman Biotechnology Co., Ltd., Beijing, China), and electrically transferred to 0.45 µm PVDF(Polyvinylidene fluoride) membrane. PVDF membrane was sealed with 5% skim milk for 2 h at room temperature, then incubated with primary antibody (anti-phosphorylated-Akt (p-Akt) antibody: 1:1500; #bsm-52130R; Bioss Biological Co. Ltd., Beijing, China; anti-cleaved-caspase 3 antibody: 1:1500; #AF7022; Affinity Biosciences. Co. Ltd., USA; anti-caspase 3 antibody: 1:1500; #AF6311; Affinity Biosciences. Co. Ltd., USA.) at 4℃ overnight, washed with TBST(Tris-buffered saline Tween) for 3 times, and incubated with horseradish peroxidase-conjugated anti-rabbit (1:5000; # ZB2307, Zsbio Commerce Store, Beijing, China) secondary antibody at room temperature for 1 h. Protein bands were detected using an ECL luminescent reagent kit (Pierce Biotechnology, Inc., USA) and Image J software (version 1.6.0; National Institutes of Health) analysis of protein gray values.
Statistical Analysis The obtained data were processed by SPSS 20.0 statistical analysis software. Results were described as means ± SD (x̄ ± s). The multiple groups were compared with a one-way analysis of variance (ANOVA), and the minimum difference significant method (LSD) was used for pairwise comparison. The difference in P < 0.05 was statistically significant.