Xiaochaihutang is a traditional Chinese Medicine. Xiaochaihu granule was purchased from Baiyunshan Guanghua Pharmaceutical Co., Ltd. (approval number K13711, Guangzhou, China), which has been embodied in the Chinese Pharmacopeia Committee of China (2015). The Xiaochaihu granule composition were Bupleurum falcatum L. (1.5 g), Scutellaria baicalensis Georgi (0.56 g), Codonopsis pilosula Franch (0.56 g), Pinellia temata Breitenbach (0.56 g), Glycyrrhiza glabra L. (0.56 g), Zingiber officinale Roscoe (0.56 g), and Zizyphus vulgaris Lam. (0.56 g) and excipients (5.14 g) (Pharmacopeia Committee of China, 2015). Baicalin is the index of content determination of Xiaochaihu granule. The content of baicalin was detected by high performance liquid chromatography (HPLC) and the results (24.307 mg/10 g) met the requirement of “No less than 20 mg/10 g” (Pharmacopeia Committee of China, 2015; Supplementary Figure S1).

Animal experiment was performed in accordance with the Guide for the Care and Use of Laboratory Animals. Sprague-Dawley rats weighing 180–220 g (Experimental Animal Centre of the Third Military Medical University) were used. Rats were randomly divided into three groups: control group (n = 20), XCHT treatment groups (5 g/kg, n = 20), and Silybin treatment group as positive drug group (50 mg/kg, n = 20). Serum pharmacology as a new experiment method in in vitro pharmacology research, is gradually widely used in the field of traditional Chinese Medicine compound pharmacodynamics (Guo et al., 2017). According to the clinical application of dosage (Su et al., 2014) and the dose conversion from human to rat, the dosage of Xiaochaihu granule was 5 g/kg (equivalent to 1.5 g/kg of Xiaochaihutang). After treatment for 6 days, all rats were sacrificed and the blood were obtained. Standing at room temperature for 2 h, the blood was centrifuged at 860 × g for 15 min. The serum was separated and mixed with the serum of the same group. The serum was inactivated by a water bath at 56°C for 30 min, filtered through a 0.22 μm microporous membrane filter, and stored at -20°C refrigerator (Iwama et al., 1987).

Hepatic stellate cell line T6 (HSCT6) was derived from rat normal liver (KunMing Cell Bank, Chinese Academy of Sciences. KunMing Cell Bank number: KCB200703YJ). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM/HIG GLUCOSE, HyClone, Utah, United States) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Australia), 100 μg/mL streptomycin (Sigma-Aldrich, United States), 100 U/mL penicillin (Sigma-Aldrich, United States), at 37°C in a humidity atmosphere of 5% (v/v) CO2.

There were four groups: XCHT compound serum group (XCHT group), Silybin compound serum group (Silybin group), Control-serum group (Control group), and the vehicle control group. The vehicle control group was cultured in DMEM supplemented with 10% (v/v) fetal bovine serum.

Hepatic stellate cell line T6 cells with a density of 5 × 10³ cells/well were evenly spread and incubated in a 96-well plate for 24 h and treated with different concentrations of XCHT compound serum [2.5, 5, and 10% (v/v)] for a further 24, 36, 48, and 60 h. The Cell counting Kit-8 (CCK8, Beyotime, China) was used to test the viability of HSCT6 cells. The CCK8 assay was used as originally described by the instruction. Briefly, the cell cultures were in-cubated with CCK8 (10 μL/well) for 1 h at 37°C, and absorbance of the lysates was measured spectro-photometrically at 450 nm. The results were referred to the absorbance of samples not treated by any agent, which was taken as 100% viability value. Under the incubation conditions used in the experiments as usual, none of the compounds added to the cell cultures affected the outcome of the CCK8 assay (data not shown).

Hepatic stellate cell line T6 cells with a density of 2 × 10⁵ cells/well were evenly spread and incubated in a six-well plate for 24 h and treated with 10% (v/v) concentrations of XCHT compound serum for a further 60 h. The amount of collagen-I in the supernatant of HSCT6 cells were measured with commercial kits (Jianglaibio, Shanghai, China).

Hepatic stellate cell line T6 cells were cultured on glass coverslips for 24 h and treated with XCHT compound serum for a further 24 h. Coverslips were wash with PBS, fixed in 4% (v/v) paraformaldehyde for 20 min at room temperature, washed, prior to detergent extraction with 0.3% (v/v) Triton X-100 for 10 min at 4°C. Coverslips were saturated with PBS containing 5% (v/v) Normal goat serum for 1 h at room temperature. Next, cells were incubated with the specific primary antibody for α-SMA for 1 h, washed, and incubated with secondary antibody. Finally, cells were stained for 30 min at room temperature with 4,6-diamidino-2-phenylindole (DAPI). Slides were viewed with OLYMPUS IX73 microscope.

Hepatic stellate cell line T6 cells with a density of 2 × 10⁵ cells/well were evenly spread and incubated in a six-well plate for 24 h and treated with 10% (v/v) concentrations of XCHT compound serum for a further 60 h. The total RNA of HSCT6 Cell was also isolated by using Trizol (TAKARA, Dalian, China). The nucleotide sequences of the primers were synthesized by TAKARA Biological Engineering Company. The nucleotide sequences of the primers used in this experiment were listed in Table 1. The results were analyzed by relative quantification of gene expression method (Xu et al., 2012).

Hepatic stellate cell line T6 cells with a density of 2 × 10⁵ cells/well were evenly spread and incubated in a six-well plate for 24 h and treated with 10% (v/v) concentrations of XCHT compound serum for a further 60 h. Cytoplasmic and nuclear protein of each group were extacted by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo-Scientific, Rockford, United States). The cells were homogenized in RIPA buffer. Equal amounts of protein (15 μg) were separated on 8% SDS-polyacrylamide gel. The blots were incubated over-night with each protein anti-body (1:1000), washed, incubated with anti-rabbit IgG (1:1.000), anti-mouse IgG (1:1.000), and developed using ECL Western Blotting Substrate. Western blot signals were quantified using imager (Fusion-Fx-7 with BD-Software, Peqlab, Erlangen, Germany).

Nuclear factor erythroid 2-related factor 2-siRNA was designed and synthesized by Guangzhou RiboBio corporation. The Nrf2-siRNA sequences are shown in Table 2.

Cell viability assay, immunofluorescence assay, enzyme-linked immune assay, western blot analysis, and immune fluorescence assay of transfected cells were measured by the same method above.

All Data were expressed as mean ± standard deviation. The significance of differences between groups was evaluated by one-way ANOVA and P < 0.05 was considered as statistically significant differences.

In order to investigate the effect of XCHT compound serum in HSCT6 cells proliferation, cell viability was assessed with CCK8. Compared to the control group, 2.5% (v/v) concentration of XCHT compound serum treatment didn’t decrease cell viability (Figure 1A). However, 5 and 10% (v/v) concentration of XCHT compound serum treatment inhibited the proliferation of HSCT6 cells in 24, 48, and 60 h (Figures 1B,C). The highest inhibition rate of XCHT compound serum was 20.30% (Figure 1D). Therefore, 10% (v/v) concentration of XCHT compound serum were used for all subsequent experiments. These results suggested that XCHT compound serum could inhibit HSCT6 cells proliferation.

In order to investigate the effect of XCHT compound serum on HSCT6 cells activation, concentration of collagen-I in the supernatant of each group were assessed with ELISA (Enzyme-linked immunosorbent, Jianglai bio, Shanghai, China) assay. The ELISA assay results indicated that 10% (v/v) concentration of XCHT compound serum treatment for 60 h decreased collagen-I secretion compared to control group (Figure 2).

Immunofluorescence (IF) experiment was used to evaluate the impact of XCHT compound serum on the expression of α-SMA. HSCT6 cells were fully stained with α-SMA in the control group (Figure 3). After treatment with XCHT compound serum for 24 h, the expression of α-SMA was reduced (Figure 3).

There was no obvious change in Nrf2 gene expression after treated with XCHT compound serum or Silybin compound serum (Figure 4A). This illustrated that XCHT Compound serum couldn’t affect the transcription of Nrf2. But XCHT Compound serum could affects the translation and post-translational modification or protein localization of Nrf2 protein. The results of our Western Blot showed that XCHT increased Nrf2 protein levels in HSCT6 cell nucleus (Figure 4B) and Cytoplasm (Figure 5).

The mRNA expression of GCLC, GCLM, and Nqo-1 was significantly increased after treatment with XCHT compound serum for 60 h (Figure 5A). Similarly, the mRNA expression of GCLC and GCLM was significantly increased after treatment with Silybin compound serum for 60 h (Figure 5A). However, there was no distinct difference observed on the mRNA expression of Keap-1 and HO-1 mRNA among these groups.

After treatment with XCHT compound serum or Silybin compound serum for 60 h, α-SMA protein expression was significantly decreased in HSCT6 cells (Figure 5B) XCHT compound serum increased Nrf2 protein level in HSCT6 cell cytoplasm. The protein level of GCLC, GCLM, and Nqo-1 was significantly increased in XCHT group (Figure 5B). However, there was no difference between the groups in the expression of Keap-1 and HO-1 protein. These results suggested that XCHT compound serum could activate Nrf2 pathway.

There were three Nrf2-siRNA sequences (Table 2). These sequences were transfected into HSCT6 cells using riboFECTTMCP. After transfected for 24 h, the level of Nrf2 mRNA in siRNA001 group was reduced to 49.98% of NC group (Figure 6A). After transfected for 60 h, the level of Nrf2 protein in siRNA001 group was reduced to 48.65% of NC group (Figure 6B). Therefore, we chose siRNA001 for the subsequent experiments.

The results showed that protein levels of Nrf2, Nqo-1, GCLC, and GCLM were significantly decreased when compared to NC group (Figure 6E). This result indicates that siRNA001 downregulated Nrf2 protein level. Compared to NC group, the protein level of α-SMA was significantly increased after treated with XCHT compound serum (Figure 6E).

Compared to the NC group, XCHT compound serum treatment for 60 h increased cell viability in transfected cells (Figure 6C).

The result showed that XCHT compound serum decressed the secretion of Collagen-I in HSCT6 cells (Figure 2). In transfected cells, however, XCHT compound serum increased the amount of Collagen-I compared with NC group (Figure 6D).

The result showed that XCHT compound serum reduced α-SMA fluorescent staining (Figure 3). In transfected cells, however, XCHT compound serum increased α-SMA fluorescent staining compared with NC group (Figure 7).