CCl₄ (cat. no. 5623-5) for liver fibrosis induction was obtained from National Pharmaceutical Chemical Reagent (https://www.reagent.com.cn/). TGF-β1 (cat. no. PRT221-0020), used to stimulate LX-2 cell activation, was sourced from Beyotime Institute of Biotechnology. The Rev-erbα agonist GSK4112 (cat. no. 1216744-19-2) and antagonist SR8278 (cat. no. 1254944-66-5) were purchased from MedChemExpress. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were quantified using ALT (cat. no. C009-2-1) and AST (cat. no. C010-2-1) assay kits from Nanjing Jiancheng Bioengineering Institute. For liver tissue staining, Masson's trichrome kit (cat. no. G1006), hematoxylin solution (cat. no. G1077) and diaminobenzidine (DAB) chromogenic kit (cat. no. G1212) were obtained from Wuhan Servicebio Technology Co., Ltd., while the Sirius Red staining kit (cat. no. 2610-10-8) was sourced from ChemicalBook Inc. Xylene (cat. no. 1330-20-7), glycerol (cat. no. 56-81-5), citrate buffer (cat. no. 6132-04-3) and glacial acetic acid (cat. no. 64-19-7), used for immunohistochemistry, were supplied by Sinopharm Chemical Reagent Co., Ltd. Cell culture reagents included DMEM (cat. no. 8119054) and FBS (cat. no. A5670701) from Gibco; Thermo Fisher Scientific, Inc. The CCK-8 cell viability assay kit (cat. no. 40203ES80) was provided by Shanghai Yeasen Biotechnology Co., Ltd. Custom lentiviral transfection reagents for Rev-erbα overexpression and knockdown were obtained from Jikai Biotechnology, and puromycin (cat. no. ST551-50mg) used for selection was purchased from Beyotime Institute of Biotechnology. Total RNA was extracted using a commercial RNA extraction kit (cat. no. 19211ES60) from Shanghai Yeasen Biotechnology Co., Ltd., followed by reverse transcription with a kit from Tiangen Biotech Co., Ltd. (cat. no. KR118). SYBR Green qPCR mix (cat. no. 11201E03) was purchased from Shanghai Yeasen Biotechnology Co., Ltd. Primers targeting α-smooth muscle actin (α-SMA), TGF-β1, Rev-erbα, NLRP3, Caspase-1, apoptosis associated speck (ASC), interleukin (IL)-18, IL-1β and β-actin were designed based on GenBank cDNA sequences and synthesized by Shanghai Shanjing Molecular Biotechnology Co., Ltd. Protein extraction was performed using RIPA lysis buffer (cat. no. WB0102) with protease inhibitors (cat. no. WB0122), both from Shanghai Weiao Biotechnology Co., Ltd. Protein concentration was determined using a BCA assay kit (cat. no. MA-0082-2) from Dalian Meilun Biology Technology Co., Ltd., and chemiluminescent detection was performed with an ECL kit (cat. no. SW-WB012) from Shanghai Weiao Biotechnology Co., Ltd. Primary antibodies against Rev-erbα (cat. no. ab305753), Caspase-1 (cat. no. ab207802), IL-18 (cat. no. ab243091), IL-1β (cat. no. ab254360), α-SMA (cat. no. ab124964), collagen 1 (COL-1) (cat. no. ab34710) and TGF-β1 (cat. no. ab27937) were obtained from Abcam. Antibodies against NLRP3 (cat. no. 15101) and ASC (cat. no. 67824S) were sourced from Cell Signaling Technology, Inc. β-actin (cat. no. GB15003) and HRP-conjugated Affinipure Goat Anti-rabbit IgG (cat. no. GB22303) were provided by Wuhan Servicebio Technology Co., Ltd.

A total of 42 male C57BL/6 mice, SPF grade, 6 weeks (22±2 g) were obtained from Shanghai Jiesijie Experimental Animal Co., Ltd., and housed under standard conditions at the Animal Facility of Shanghai Municipal Hospital of Traditional Chinese Medicine. Mice were kept at a standard temperature of 25–27°C and a humidity of 55–65%, in a light/dark cycle of 12/12 h, and with ad libitum access to sterilized food and water. After a 1-week acclimatization period, animals were randomly assigned to experimental groups as follows: i) A total of 12 mice were divided into a control group (Con) and a CCl₄-induced model group (CCl₄) (n=6 per group). Liver fibrosis was induced by intraperitoneal injection of a 20% CCl₄ solution in olive oil (5 ml/kg), administered twice weekly for 6 weeks (25). Control mice received an equal volume of physiological saline. ii) The remaining 30 mice were randomly allocated into three groups: Control (n=12), GSK4112 (n=12) and SR8278 (n=12). For pharmacological modulation of Rev-erbα, lyophilized GSK4112 and SR8278 were reconstituted in DMSO. Mice in the SR8278 group received daily intraperitoneal injections of SR8278 (25 mg/kg) (26), while those in the GSK4112 group were administered GSK4112 (20 µg/mouse) (27,28). Control mice were treated with equivalent volumes of DMSO. Injections were performed once daily for 14 consecutive days. After treatment, six mice from each group were randomly selected and reassigned to receive CCl₄ treatment as described above, forming the CCl₄ group (n=6), SR8278 + CCl₄ group (n=6) and GSK4112 + CCl₄ group (n=6).

Upon completion of modeling, mice were anesthetized with 1% pentobarbital sodium (50 mg/kg). Livers were harvested, with a 1×1-cm section from the central lobe fixed in 4% paraformaldehyde for histological analysis at ~25°C. The remaining tissue was snap-frozen in liquid nitrogen and stored at −80°C for subsequent experiments. Euthanasia was performed via cervical dislocation (29). All animal procedures were approved by the Ethics Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine (Shanghai, China; approval no. 2022033).

The human HSC LX-2 cell line was obtained from Shanghai Kanglang Biological Technology Co., Ltd., and cultured in DMEM supplemented with 10% FBS under standard conditions (37°C and 5% CO₂).

For fibrogenic induction, LX-2 cells were divided into a control group and a TGF-β1-treated group, with the latter exposed to 5 ng/ml TGF-β1 for 24 h (25).

The Rev-erbα overexpression plasmid and knockdown shRNA plasmid utilized in this study were constructed by Shanghai Jikai Biotechnology, employing a triple-plasmid co-transfection system in 293T cells for lentiviral production; the lentiviral supernatant was concentrated and purified into high-titer stocks within 48–72 h post-transfection. For Rev-erbα overexpression lentivirus, the plasmid combination consisted of the transfer plasmid GV208 carrying the target gene Rev-erbα (4 µg), the packaging plasmid Helper 1.0 (3 µg), and the envelope plasmid Helper 2.0 (1 µg). For Rev-erbα knockdown lentivirus, the plasmids included the transfer plasmid pLKO.1-shRNA (4 µg), the packaging plasmid psPAX2 (3 µg) and the envelope plasmid pMD2.G (1 µg). LX-2 cells were seeded in 24-well plates at 5×10⁴ cells/ml (1 ml/well) and were assigned to the following groups: Con522 (negative control for Rev-erbα overexpression), con313 (negative control for Rev-erbα knockdown), OvRev-erbα (Rev-erbα overexpression; MOI=10) and Rev-erbα sh1/sh2/sh3 (shRev-erbα knockdown groups; MOI=30/20/10, respectively). After adherence to 24-well plates and reaching 20–30% confluency, transduction was performed with Rev-erbα lentiviruses at different multiplicities of infection. The viral volume was calculated using the formula: Virus volume (µl)=(MOI × cell number)/viral titer (TU/ml). After 12 h of incubation at 37°C, the DMEM was replaced with culture medium containing 10% FBS, and cells were cultured for an additional 72 h. Transduction efficiency was assessed via fluorescence inverted microscopy (Olympus IX73; Olympus Coporation) to determine optimal conditions. Puromycin selection (2 µg/ml) was initiated at 72 h post-transduction and maintained for 7–14 days. Successful Rev-erbα modulation was validated by RT-qPCR for mRNA expression and western blotting for protein expression, as performed below, with successfully transfected cells subsequently used for further experiments.

To assess the effect of Rev-erbα on TGF-β1-induced activation, LX-2 cells were categorized into TGF-β1, OvRev-erbα + TGF-β1 and shRev-erbα + TGF-β1 groups. Based on preliminary findings, OvRev-erbα + TGF-β1 was transduced with Rev-erbα at MOI=10 and shRev-erbα + TGF-β1 received interference at MOI=20. The TGF-β1 group was treated with DMEM without viral particles. All groups were subsequently treated with 5 ng/ml TGF-β1 for 24 h at 37°C and in 5% CO₂.

For serum collection, blood samples from each mouse group were left at room temperature (25°C) for 4 h, centrifuged at 3,000 rpm and 4°C for 15 min, and serum was harvested. AST and ALT levels were measured using the aforementioned commercially available detection kits, following the manufacturer's protocols.

Fresh liver specimens (~1×1 cm) were fixed in 4% paraformaldehyde at room temperature for 24 h. After paraffin embedding, tissue sections were stained using Masson's trichrome and Sirius red protocols. For liver tissues, sections of 5–7 µm in thickness were used. Collagen fiber staining was performed at room temperature, with Masson's trichrome staining for 10 min and Sirius red staining for 30 min. Histopathological alterations were assessed using a light microscope at ×200 magnification (BX41, Beijing Ruike Zhongyi Technology Co., Ltd.).

For immunofluorescence, cells from the TGF-β1, OvRev-erbα + TGF-β1 and shRev-erbα + TGF-β1 groups were fixed with 4% paraformaldehyde at room temperature for 15 min and subjected to 10% FBS blocking at 37°C for 30 min. The TGF-β1, OvRev-erbα + TGF-β1 and shRev-erbα + TGF-β1 groups were incubated with an α-SMA antibody (1:200) at 4°C for 16 h, followed by secondary antibody incubation at room temperature for 50 min. After PBS washing (pH 7.4), nuclei were counterstained with DAPI at 25°C for 10 min in the dark. Slides were rinsed, spin-dried and mounted using an anti-fade glycerol medium. Fluorescence imaging was performed with a fluorescence inverted microscope (Olympus IX73 microscope). Quantification of positive staining was carried out using ImageJ 1.8.0 software (National Institutes of Health), and statistical analyses of protein expression levels were performed using GraphPad Prism 8.3.0 (Dotmatics).

Following fixation (4% paraformaldehyde at room temperature for 24 h), liver tissues were dehydrated at room temperature for 30 min and embedded in paraffin (melting point ~60°C). Paraffin blocks were sectioned into 5-µm slices, rinsed and air-dried prior to staining. The procedure included the following steps: i) At room temperature, dewaxing with xylene for 30 min and graded ethanol hydration (100, 95, 80 and 75%) for 20 min; ii) rehydration through a descending ethanol series followed by distilled water rinses; iii) nuclear staining with hematoxylin at room temperature for 10 min; iv) rinsing in distilled water; v) antigen retrieval by heating sections in 200 ml 0.01Μ citrate buffer (pH6.0) at 70°C for 10 min; vi) washing in PBS three times (5 min each), then incubating in 3% hydrogen peroxide at room temperature for 10 min to quench endogenous peroxidase activity; vii) blocking with 2% FBS in PBS at 37°C for 2 h; viii) incubation with primary antibodies, α-SMA (1:500), COL-1 (1:600) and TGF-β1 (1:800), at 4°C for 16 h; ix) after PBS washes, incubation with HRP-conjugated goat anti-mouse secondary antibody (1:200) at 37°C for 2 h; x) DAB chromogenic development for 10 min; xi) rinsing with distilled water; and xii) clearing with xylene for 10 min, followed by mounting with neutral resin and drying at 70°C. Microscopic images were captured using a Horiba BX41 light microscope (Beijing Ruike Zhongyi Technology Co., Ltd.) from five randomly selected fields per section at magnifications of ×100 and ×400. Quantitative analysis of antibody-positive staining areas was performed using ImageJ 1.8.0 software (National Institutes of Health). Statistical analysis of target protein expression levels was conducted using GraphPad Prism 8.3.0 (Dotmatics).

Cell viability was assessed using the CCK-8 assay. A suspension of 2×10³ cells in 100 µl complete culture medium containing 10% FBS was seeded into each well of a 96-well plate and incubated under standard conditions (37°C and 5% CO₂) for 6 h. Subsequently, 10 µl CCK-8 solution were added to each well. After 2 h of incubation, optical density (OD) was measured at 450 nm using a microplate reader (BioTek Epoch2; Agilent Technologies, Inc.). OD values were used to calculate cell viability, and results were presented in histogram form for comparative analysis.

For RT-qPCR analysis, 40–50 mg of liver tissue was collected from each group, and total RNA was extracted using TRIzol^® (Thermo Fisher Scientific, Inc.) and precipitated in ethanol. Reverse transcription was performed using the FastKing gDNA Dispelling RT SuperMixJun (cat. no. KR118; Tiangen Biotech Co., Ltd.), and the resulting cDNA was diluted 10-fold with ddH₂O for amplification. qPCR was performed using SYBR Green PCR Mastermix (cat. no. 11201E03). The results were analyzed on an ABI StepOnePlus real-time PCR system (Applied Biosystems) using the 2^−ΔΔCq method (30). Values were normalized to β-actin. The reaction conditions were as follows: 95°C predenaturation once for 5 min, followed by denatured at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 72°C for 20 sec, for 40 cycles. Primer sequences are provided in Table SI.

The quantity of whole-protein extracts from cells and livers of mice, extracted using RIPA lysis buffer (cat. no. WB0102) with protease inhibitors (cat. no. WB0122), were determined using the BCA assay. Equal amounts of total protein (20–30 µg) were resolved by 7.5% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated with specific primary antibodies for 16 h at 4°C [Rev-erbα (1:1,000), Caspase-1 (1:1,000), IL-18 (1:1,000), IL-1β (1:1,000), α-SMA (1:1,000), collagen 1 (1:1,000), TGF-β1 (1:1,000), NLRP3 (1:1,000), ASC (1:1,000) and β-actin (1:3,000)], followed by incubation with HRP-conjugated Affinipure Goat Anti-rabbit IgG (1:5,000) at room temperature for 2 h. Signal detection was performed using a chemiluminescence imaging system (Amersham Imager 600; Cytiva), and band intensities were semi-quantified using ImageJ 1.8.0 software.

Statistical analyses were performed using GraphPad Prism 8.3.0 (Dotmatics). Comparisons between two groups were performed using unpaired, two-tailed t-tests, while multiple-group comparisons were evaluated by one-way ANOVA. When comparing ≥3 groups (e.g., shRev-erbα 1, shRev-erbα 2 and shRev-erbα3) requiring assessment of all possible pairwise comparisons (shRev-erbα 1 vs. shRev-erbα 2, shRev-erbα 1 vs. shRev-erbα 3 and shRev-erbα 2 vs. shRev-erbα 3), Tukey's test was employed for post-hoc evaluation. When analyzing ≥3 groups (e.g., CCl₄, GSK4112 + CCl₄ and SR8287 + CCl₄) focusing on specific pairwise comparisons between predetermined groups (CCl₄ vs. GSK4112 + CCl₄ and CCl₄, vs. SR8287 + CCl₄), Bonferroni's correction was applied for post-hoc statistical assessment. Data are expressed as the mean ± SEM. P<0.05 was used to indicate a statistically significant difference.

To investigate the expression dynamics of the circadian clock gene Rev-erbα in liver fibrosis, a mouse model was established using CCl₄ administration. Compared with those in the Con group, serum ALT and AST levels were significantly elevated in the CCl₄-treated mice, indicating substantial hepatic injury (Fig. 1A and B). Histological analysis via Masson's trichrome and Sirius red staining revealed well-preserved hepatic architecture in the control group, whereas CCl₄-treated mice exhibited marked collagen deposition and expanded fibrotic areas, consistent with progressive fibrosis (Fig. 1C). In parallel, mRNA and protein expression levels of TGF-β1 and α-SMA were significantly upregulated in the livers of CCl₄-treated mice compared with levels in the controls (Fig. 1D-F), confirming successful model induction. Notably, Rev-erbα mRNA and protein levels were significantly reduced in the liver tissue of CCl₄ group mice relative to the controls (Fig. 1G and H). This downregulation was further corroborated in vitro, where TGF-β1-stimulated LX-2 cells showed significant upregulation of fibrotic markers (α-SMA and TGF-β1) (Fig. 1I-K) and a concomitant decrease in Rev-erbα expression at both the transcript and protein levels (Fig. 1L and M). These results suggest that Rev-erbα may act as a negative regulator in the progression of liver fibrosis, with its reduced expression potentially facilitating fibrogenic and pro-inflammatory responses in hepatic tissue.

Rev-erbα serves as a critical transcriptional regulator of metabolic, circadian and inflammatory pathways, rendering it a promising therapeutic target for metabolic disorders, malignancies, epilepsy, inflammatory conditions and neurodegenerative diseases (22,31). To evaluate its role in liver fibrosis progression, C57BL/6 mice received intraperitoneal injections of the Rev-erbα agonist GSK4112 (25 mg/kg) or the antagonist SR8278 (20 µg/mouse). Western blot analysis confirmed successful pharmacological modulation, with Rev-erbα expression significantly reduced in the SR8278 group and significantly elevated in the GSK4112 group (Fig. 2A).

Following model establishment, liver fibrosis was induced using CCl₄. Histological staining with Masson's trichrome and Sirius red revealed pronounced pseudolobule formation, extensive collagen accumulation and expanded fibrotic areas in the CCl₄ + SR8278 group. By contrast, the CCl₄ + GSK4112 group exhibited more preserved hepatic architecture, reduced fibrous proliferation and an absence of steatosis compared with the CCl₄ group (Fig. 2B). Immunohistochemical analysis further demonstrated that expression levels of α-SMA, COL-1 and TGF-β1 were significantly upregulated in the CCl₄ + SR8278 group, whereas these markers were significantly downregulated in the CCl₄ + GSK4112 group (Fig. 2C). These results indicate that Rev-erbα suppression exacerbates fibrogenesis, while its activation attenuates hepatic inflammation, collagen deposition and fibrotic remodeling. Lentiviral transduction was employed to establish LX-2 cell lines with stable Rev-erbα knockdown or overexpression for subsequent in vitro investigations. Fluorescence microscopy confirmed that Rev-erbα sh1 (MOI=20) achieved the most effective knockdown (Fig. 2D), with significantly reduced mRNA and protein expression levels of Rev-erbα in LX-2 cells (Fig. 2E and F). This condition was subsequently selected for downstream experiments. Fluorescence microscopy confirmed efficient overexpression of Rev-erbα in the MOI=10 group (Fig. 2D), accompanied by a significant increase in both mRNA and protein levels of Rev-erbα in LX-2 cells (Fig. 2G and H). LX-2 cells were then allocated into three groups: TGF-β1, OvRev-erbα + TGF-β1 and shRev-erbα + TGF-β1. Following lentiviral transduction at the defined MOIs, cells were stimulated with 5 ng/ml TGF-β1 for 24 h to induce activation. CCK-8 assays revealed a significant reduction in cell viability in the OvRev-erbα + TGF-β1 group, while the shRev-erbα + TGF-β1 group exhibited a significant increase in viability, compared with the TGF-β1 group (Fig. 2I). Immunofluorescence analysis further demonstrated that α-SMA expression was substantially downregulated in the OvRev-erbα + TGF-β1 group, whereas notable upregulation was observed in the shRev-erbα + TGF-β1 group (Fig. 2J). These results suggest that decreased Rev-erbα expression promotes HSC activation and fibrogenic responses, whereas its overexpression exerts a suppressive, anti-fibrotic effect.

The NLRP3 inflammasome consists of ASC, caspase-1 and NLRP3, with ASC serving as a key adaptor that promotes the recruitment and activation of caspase-1 and NLRP3. Caspase-1 processes the inactive precursors of IL-1β and IL-18 into their mature, bioactive forms, thereby initiating pyroptosis and inflammatory responses that play central roles in the progression of alcoholic liver disease, viral hepatitis and liver fibrosis (32,33).

In the present study, following CCl₄ induction in three groups of mice, Rev-erbα protein expression was assessed. Compared with those in the CCl₄ group, the protein levels of Rev-erbα in the GSK4112 + CCl₄ group were significantly elevated and those in the SR8278 + CCl₄ group were significantly decreased (Fig. 3A). To examine whether the anti-fibrotic effect of Rev-erbα is mediated through modulation of the NLRP3 inflammasome, liver fibrosis models were generated in C57BL/6 mice subjected to CCl₄ combined with either GSK4112 or SR8278 treatment. Expression levels of NLRP3, caspase-1, ASC and downstream proinflammatory cytokines IL-18 and IL-1β were analyzed in liver tissue. Relative to the CCl₄ group, mRNA expression of NLRP3, caspase-1, ASC, IL-18 and IL-1β was significantly reduced in the GSK4112 + CCl₄ group, whereas significant increases were observed in the SR8278 + CCl₄ group (Fig. 3B), with protein expression showing consistent trends (Fig. 3C). To further substantiate these findings, validation was performed in three groups of LX2 cells. Following TGF-β1 stimulation, Rev-erbα protein expression was assessed. Compared with that in the TGF-β1 group, Rev-erbα expression was significantly upregulated in the OvRev-erbα + TGF-β1 group and markedly downregulated in the shRev-erbα + TGF-β1 group (Fig. 3D). Subsequent analysis focused on the regulatory effect of Rev-erbα on the NLRP3 inflammasome in LX2 cells. RT-qPCR and western blotting results demonstrated that, relative to the TGF-β1 group, mRNA and protein levels of NLRP3, caspase-1, ASC and the downstream cytokines, IL-18 and IL-1β, were significantly suppressed in the OvRev-erbα + TGF-β1 group, whereas marked increases were observed in the ShRev-erbα + TGF-β1 group (Fig. 3E and F). These results indicate that Rev-erbα knockdown promotes inflammatory responses during liver fibrosis by enhancing NLRP3 inflammasome activation in HSCs, thereby elevating the expression of IL-18 and IL-1β. By contrast, overexpression of Rev-erbα reverses these effects.

Collectively, the data support a role for Rev-erbα as an upstream negative regulator of the NLRP3 inflammasome, contributing to its anti-fibrotic function by attenuating inflammasome-mediated inflammation in liver fibrosis and activated HSCs.