Regents and chemicals
TAA (purity ≥ 99.0%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 20 (R)-G-Rg3 (purity ≥ 98.0%, HPLC) was obtained and qualified as described by our previous study26. mTOR inhibitor Rapamycin (Ra) and PI3K inhibitor LY294002 were obtained from Med Chem Express Biotech Co. Ltd. (New Jersey, USA). Catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), H&E staining kit, and Masson staining kit were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kit for TGF-β1 was purchased from R&D systems (Minneapolis, MN, USA). Autophagy-related antibodies of LC3 a/b (12741 S), ATG3 (3415 S), ATG5 (12994 S), ATG7 (8558 S), ATG12 (4180 S), ATG16L (8089 S), Beclin-1 (3495), mTOR (2972 S), p-mTOR (2971 S), p-ULK1 (14202), Akt (9272 S), p-Akt (13038), PI3K (4292 S), p-PI3K (422S8), and anti-HRP were from Cell Signaling Technology (Massachusetts, USA). p62 (18420-1-AP), α-SMA (23660-1-AP), TGF-β1 (21898-1-AP), β-actin (60008-1-Ig), and GAPDH (60004-1-Ig) were from Proteintech (Chicago, USA). All other reagents and chemicals, unless indicated, were obtained from Beijing Chemical Factory (Beijing, China).
Male-specific pathogen-free (SPF) ICR mice (6–8 weeks old) were bought from the Chang YISI Experimental Animal Co. Ltd. (Changchun, China), and housed under temperature 23 ± 2 °C and 12 h light/dark cycle with ad libitum access to diet, and acclimatized for 1 week prior to the study. All experiment protocol in this study was strictly conducted according to the Guide for Laboratory Animal care and use Committee of Jilin Agricultural University.
For induction of subacute hepatic injury, mice were randomly assigned into four groups (n = 10, per group): Normal, TAA group, and two G-Rg3 treated groups (5 and 10 mg/kg). The subacute hepatic injury model was established in a dose of 150 mg/kg for TAA according to our preliminary experiments27. Intraperitoneal injection with TAA was for 2 weeks before administration orally G-Rg3 at doses of 5 or 10 mg/kg and totally for 4 weeks. The doses of G-Rg3 were appropriately adjusted according to clinical equivalent dose and our preliminary experiments26,28.
For induction of chronic hepatic fibrosis, mice were randomly assigned into four groups (n = 10, per group): Normal, TAA group, and two G-Rg3 treated groups (5 and 10 mg/kg). In reference to the subacute hepatic injury model, 100 mg/kg was used for the first time, and 50 mg/kg was used in later injections, twice a week, for 10 weeks. G-Rg3 was orally administered with doses of 5 or 10 mg/kg body weight in chronic hepatic fibrosis model for 4 weeks. After 12 h fasting, the mice were subjected to euthanasia. Blood sample was placed at 4 °C for 40 min till next procedures and liver tissues and other organs washed with cooling saline were partially collected in 4% formalin, and others kept at 80 °C for further biochemical analysis.
Liver function and oxidative stress indicators tests
The AST and ALT levels in serum were measured by commercial kits. Meanwhile, the enzyme activities of SOD and CAT, and content of GSH and MDA were tested to examine changes in oxidative stress levels in TAA-induced liver fibrosis model. Briefly, liver tissue was physically dissociated in 9 volumes of ice-cold 0.9% NaCl and centrifuged twice at 3000 rpm for 10 min at 4 °C, then collected supernatant of liver homogenate. Serum samples were obtained from centrifugation at 1000 × g for 10 min at 4 °C. Then, serum samples with substrates or buffer solution were incubated together for 50 min at 37 °C, followed with a color developing agent and measured at a wavelength of 510 nm. BCA kit was used in all involved protein quantification experiments (Beyotime Biotechnology, China). Any abnormal data of the sample (very few maximum and minimum) would be excluded from the group.
Liver histology examination
Briefly, the liver tissues were immersed in 10% buffered formalin over 24 h embedded in paraffin and cut into a 5-μm-thickness slice. Pathological sections were examined to assess the extent of fibrosis with H&E and Masson’s staining kits. Liver sections were observed with a light microscope (Leica, DM2500, Germany). Representative views of liver sections are performed. In subacute model: the survival mice were re-numbered sequentially, the number of 2nd, 4th, and 6th were chosen for H&E staining. In chronic model: mice were randomly divided and numbered sequentially, the number of 3rd, 6th, and 9th were chosen for pathological observation (including H&E staining, Massons’ staining and Immunofluorescence staining). Masson-stained images were randomly captured from 10 fields (magnification ×100) in three mice/group.
Cell-culture conditions and drug treatment
Activated phenotype HSC-T6 cells and L02 cells were purchased from ATCC with STR authentication. The rat HSC-T6 cell lines are used for potential therapeutic intervention of activated HSCs owing to it exhibits an activated phenotype and a fibroblast-like morphology after transfection with SV40 sequences29. HSC-T6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) of defined class and grown in 5% CO2 humidified atmosphere at 37 °C. Furthermore, HSC-T6 and L02 cells were seeded in 96-plates and 6-plates at a density of 3 × 105 cells/well and 5 × 104 cells/well, respectively. Before G-Rg3 treatment, cells were grown to approximately 50% confluence, then exposed to G-Rg3 at different concentrations of 0, 2, 4, 8, 16, and 20 µM for different periods of 0, 4, 8, 12, and 24 h. Then the corresponding experiments were carried out. The mTOR inhibitor Rapamycin (Ra) and PI3K inhibitor LY294002 were added 1 h in advance and incubated for 24 h, respectively. For the autophagy induction experiment, the groups are Normal, G-Rg3, Ra (100 nM), G-Rg3+ Ra (100 nM), Ra (200 nM), G-Rg3 + Ra (200 nM), respectively. For the PI3K signaling research experiment, the groups are as follows: Normal, G-Rg3, LPS, LY294002 (20 μM), LPS + G-Rg3, LY294002 + G-Rg3, LPS + LY294002 + G-Rg3. In our current research, all the results carried out in cells from triplicate experiments.
To investigate the fibrogenic situation in TAA-induced liver fibrosis, the concentrations of TGF-β1 both in serum and supernatant of liver homogenate were detected by the R&D system ELISA kit in strict accordance with the manufacturer’s protocols.
Western blot analysis
Equal amounts of protein obtained from cells or liver tissue samples were run in vertical electrophoresis unit, then transferred to the PVDF membrane (Millipore, USA), and blocked with 5% nonfat milk in TBS-T (TBS + 0.4% Tween-20) for 2 h. Subsequently, the PVDF membrane was incubated with corresponding primary antibodies at a dilution of 1:1000 except GAPDH and β-actin at 4 °C overnight, followed by three washes with TBS-T. HRP-conjugated secondary antibodies were, respectively, used to combine primary antibodies for 2 h, followed by three washes in TBS-T for 8 min each. Finally, blots were incubated with ECL (Proteintech, USA). The intensity of bands was quantified using software Image J.
Briefly, for liver tissues, 5-μm-thickness sections were reprocessed with xylene followed by gradient ethanol. Then, sections were incubated with primary antibody involving rabbit anti-mouse TGF-β1 (1:500) and α-SMA (1:500) in a humidified dark box at 4 °C overnight; they were followed by SABC-dylight448-labeled secondary antibody (Boster, China), incubated for 37 °C, 30 mins. The Nucleus in liver tissues was stained by 4, 6 diamidino-2-phenylindole (DAPI) staining for 5 min, and then examined by a light microscope (Leica, DM2500, Germany).
For cells, HSC-T6 cells were seeded in 6-plates at a density of 5 × 104 cells/well. Prior to G-Rg3 treatment, cells were grown to approximately 50% confluence, then exposed to G-Rg3 for indicated concentration and time, followed by permeabilization of 4% paraformaldehyde with 1% Triton-X100, then incubating with p62 and α-SMA. Other processing steps were similar to the above. Finally, fluorescence was captured by a fluorescence microscope (Leica, DMIL LED, Germany). Related cell trials including western blot and immunofluorescence staining were duplicated three times, respectively.
All data were presented with mean ± standard deviation (mean ± S.D). The difference between groups was analyzed by two-tailed Student’s t-test or one-way analysis of variance (ANOVA). For statistical tests, *p < 0.05, *p < 0.01, *p < 0.001 was set to measure the level of significance. GraphPad Prism software package 8.2 was used for figures.