Liver samples were collected by trans-parietal puncture from 11 healthy individuals and 11 patients with liver fibrosis. Written informed consent was obtained from all patients, and the study was approved by the Medical Ethics Committee of First Teaching Hospital of Tianjin University of Traditional Chinese Medicine (Tianjin, China).

Primary HSCs were isolated as described previously (11). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), L-glutamine (4 mmol/l; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), penicillin (100 IU/ml; Sigma-Aldrich; Merck KGaA), and streptomycin (100 µg/ml; Sigma-Aldrich; Merck KGaA) at 37°C in a humidified 5% CO₂ atmosphere.

Small-interfering RNA targeting Fstl1 (si-Fstl1) and non-targeting control siRNA (scramble) were synthesized by Guangzhou Ribo Bio Co., Ltd. (Guangzhou, China). HSCs at a density of 5×10⁴ cells/well were seeded in each cell of a 24-well micro-plate, grown for 24 h to reach 30–50% confluence, and then transfected with si-Fstl1 or scramble using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Total RNA of quiescent HSCs and activated HSCs, as well as normal and hepatic fibrosis tissues, was extracted and purified using an RNeasy Mini kit according to the instructions of the manufacturer (Qiagen, Inc., Valencia, CA, USA). Up to 5 µg of the total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Sigma-Aldrich; Merck KGaA). RT-qPCR was performed with the Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR Green Real-Time PCR Master Mix kit (Takara Biotechnology Co., Ltd., Dalian, China). The following primers were used: Fstl1, 5′-CGAGGTGGAGTTGACGAGAAAC-3′ (sense), 5′-AGGACTGGATCATCATGACGTTCT-3′ (antisense); and β-actin 5′-CCGTGAAAAGATGACCCAGATC-3′ (sense), 5′-CACAGCCTGGATGGCTACGT-3′ (antisense). The relative levels of transcript were determined by using the2^−∆∆Cq method (12) and normalized by β-actin.

Total protein was extracted from hepatic fibrosis tissues or HSCs using RIPA Cell Lysis buffer (Takara Biotechnology Co., Ltd.). Lysates were sonicated for 5 sec on ice and centrifuged at 6,000 × g for 5 min at 4°C. Supernatants were collected and the protein concentration was detected using a Bio-Rad Protein Assay kit II (cat. no. 500-0002; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The cell lysates (30 µg/lane) were subjected to SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane. Then, nonspecific binding was blocked by incubating with 5% non-fat milk in TBS containing 0.1% Tween-20 at room temperature for 1 h. The blots were incubated with primary antibodies: anti-Fstl1 (1:1,000; cat. no. sc-80408), anti-collagen I (1:2,000; cat. no. sc-59772), anti-α-SMA (1:2,000; cat. no. sc-130616), anti-Smad3 (1:2,000; cat. no. sc-101154), anti-p-Smad3 (1:2,000; cat. no. sc-11769) or anti-GAPDH (1:5,000; cat. no. sc-400163; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. sc-2789; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Following washing, the blots were visualized using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Chalfont, UK). Protein expression was analyzed using BandScan software version 5.0 (Glyko; BioMarin Pharmaceutical, Inc., San Rafael, CA, USA). All experiments were repeated ≥3 times.

Cell proliferation was determined using the Cell Counting kit-8 assay. Briefly, infected HSCs were plated at a density of 1×10⁴ cells/well in a 96-well culture plate and treated with or without TGF-β1 for 24 h. Then, 10 µl CCK-8 reagent was added (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) to each well. Following 1 h of incubation at 37°C, the absorbance was measured with a microplate reader (Bio-Rad Laboratories, Inc.) at a wavelength of 490 nm.

All statistical analyses were performed using the SPSS software (version, 13.0; SPSS, Inc., Chicago, IL, USA). Results were presented as mean ± standard deviation for three experiments. Statistical comparisons were performed using one-way analysis of variance followed by the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The authors initially measured the expression of Fstl1 in human hepatic fibrosis tissues. As indicated in Fig. 1A, the mRNA expression levels of Fstl1 were significantly upregulated in human hepatic fibrosis tissues, as compared with the normal liver tissues. Moreover, the authors observed that the expression levels of Fstl1 at both mRNA and protein were higher in activated HSCs than that of in the quiescent cells (Fig. 1B and C).

In order to investigate the effect of Fstl1 on HSC proliferation, HSCs were transfected with si-Fstl1 or scramble, respectively. The efficiency of transfection was confirmed by RT-qPCR and western blotting. The results of RT-qPCR analysis indicated that the mRNA expression of Fstl1 was obviously decreased in HSCs transfected with si-Fstl1 (Fig. 2A). Similarly, knockdown of Fstl1 greatly downregulated the protein expression level of Fstl1 in HSCs (Fig. 2B). Then, the authors performed the CCK-8 assay to detect the effect of Fstl1 on HSC proliferation. As indicated in Fig. 2C, TGF-β1 treatment markedly promoted the proliferation of HSCs, compared with the control group. However, knockdown of Fstl1 significantly inhibited HSC proliferation in TGF-β1-induced HSCs.

The authors next investigated whether si-Fstl1 attenuated ECM expression in HSCs. The results of western blotting analysis demonstrated that TGF-β1 obviously induced the protein expression levels of α-SMA and type I collagen, as compared with the control group. Meanwhile, knockdown of Fstl1 significantly blunted TGF-β1-induced α-SMA and type I collagen expression in HSCs (Fig. 3).

To determine the molecular mechanism of Fstl1 regulates HSCs activation described above, the effect of Fstl1 on the activation of Smad3 signaling pathway in HSCs was examined. Western blotting indicated that TGF-β1 led to a marked increase in Smad3 phosphorylation; however, knockdown of Fstl1 remarkably decreased the phosphorylation level of Smad2/3 in TGF-β1-induced HSCs (Fig. 4).