Animals

Male C57BL/6J mice (6 weeks, 16–18g) were obtained from the Experimental Animal Center of Chongqing Medical University. GPR81^+/- mice on a C57BL/6J background were purchased from Cyagen Biosciences Inc. (Guangzhou, China) and self-mated to generate GPR81^−/− homozygous mice (GPR81 KO) and wild-type control mice. The primer sequences used for genotype identification are listed in Table 1. All mice were housed under specific pathogen-free conditions with controlled temperature and a 12 h light/dark cycle. After all treatment, mice were anesthetized with 5% chloral hydrate and sacrificed. All animal experiments in this study were performed in accordance with and approved by the Ethics Committee of Chongqing Medical University.

CCl₄-induced liver fibrosis

Liver fibrosis was induced in mice by intraperitoneal injection of CCl₄ (1 ml/kg, dissolved in olive oil) or the vehicle (olive oil) twice per week, for 8 weeks. To determine the significance of GPR81, CCl₄-challenged mice received once-daily administration of GPR81 agonist DHBA (30 mg/kg, dissolved in normal saline) or the vehicle (saline). Three days after the last injection of CCl₄, the mice were anesthetized with 5% chloral hydrate and sacrificed. The left lobe of the liver and the blood sample were harvested for further experiments.

Histological examination

Liver tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sliced into 4 μm-thick sections. To determine the histological abnormalities, the sections were stained with hematoxylin and eosin (H&E). To reveal the degree of collagen deposition, liver sections were subjected to Masson's trichrome staining and Sirius red staining using commercial kits from Solarbio (Beijing, China) according to the manufacturer's instructions. The positive area and total dyed area were examined using Image Pro Plus 6.0 software by measuring five randomly chosen, non-overlapping fields at a magnification of 100×, and the average positive area was calculated. The results were represented as a fold change ratio toward the relative control group.

Cell culture and treatment

The human hepatic stellate cell line (LX-2) was purchased from National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured using Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (#11995065, Gibco) supplemented with 5% (v/v) FBS and 1% (v/v) Penicillin–Streptomycin solution and was maintained at 37 ºC in a humidified atmosphere containing 5% CO₂. LX-2 is an immortalized human hepatic stellate cell line prepared by Xu et al. through SV40 T-antigen transformation in 2005. The mycoplasma, bacterial, and fungal tests were performed on the cells before use, and the results were negative.

LX-2 cells were exposed to FBS-free medium overnight, and then the culture media was replaced by fresh media supplemented with recombinant human TGF-β1 (#100-21, PeproTech) at a concentration of 10 ng/ml or the vehicle. To determine the role of GPR81, DHBA was supplemented at a concentration of 1 mM. 24 h later, cells were collected for mRNA or protein extraction.

Determination of biochemical parameters

To evaluate the degree of liver injury, the mice blood samples were collected and centrifuged (5000g, 4 °C, 15 min) to obtain serum for analysis. Serum ALT and AST were determined with the Alanine aminotransferase/Aspartate aminotransferase Assay Kit (Nanjing Jiancheng Biotech) according to the manufacturer’s instructions.

To evaluate the degree of liver fibrosis, the liver tissues were homogenized in PBS and then centrifuged at 8000g for 10 min to collect the supernatant for analysis. The hydroxyproline contents in liver tissues were determined with the Hydroxyproline Assay Kit (Nanjing Jiancheng Biotech) following the manufacturer's instructions.

Determination of cAMP by ELISA

Liver tissues were homogenized and the supernatants were collected. The contents of cAMP in the supernatants were evaluated using the cAMP ELISA Kit (Elabscience Biotechnology, China) according to the manufacturer's instructions. The hepatic content of cAMP was normalized by the total protein concentration of the supernatant, and the data were expressed as the fold change relative to the respective control.

Quantitative real-time PCR analysis (RT-qPCR)

Total RNA was isolated from mice liver samples by using a SteadyPure Quick RNA Extraction Kit (Accurate Biology) and reverse-transcribed to cDNA with a reverse transcriptase kit (Accurate Biology) following the manufacturer’s instruction. Then Real-time PCR was performed with a SYBR Green RT-PCR Kit (Accurate Biology). The primers for GPR81, TNF-α, IL-6, TGF-β1, α-SMA, COL1A1, and Smad7 were synthesized by Accurate Biology (Changsha, China) and their sequences are listed in Table 2. The relative mRNA levels of GPR81, TNF-α, IL-6, TGF-β1, and Smad7 were normalized by that of β-Actin and were expressed as a fold change relative to the respective control.

Immunohistochemistry staining

Liver tissues fixated in paraffin were dewaxed, hydrated, treated with sodium citrate for antigen retrieval, and blocked with non-specific stain inhibitors (#KIT-9710, Maxim) for 10 min. Then, the sections were incubated with the primary antibody against GPR81 (#SAB1300792, Sigma-Aldrich) or α-SMA (#A17910, ABclonal) at 4  overnight, washed with PBS, and incubated at room temperature for 20 min with secondary antibodies. Next, the DAB developer was used for 1 min. Finally, sections were counterstained with hematoxylin for 10 min, dehydrated, and mounted. The positive area was quantified using Image Pro Plus 6.0.

Immunoblotting

The liver samples were homogenized with RIPA lysis buffer to prepare tissue lysates. The total proteins were extracted after centrifuging for 15 min at 4 °C, and the concentrations were determined using BCA Protein Assay Kit (Thermo Fisher Scientific). The protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After treating with blocking buffer (containing 5% skim milk, 10 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature, the membranes were incubated with the primary antibodies (the information about the antibodies is listed in Table 3) overnight at 4 °C and were incubated with the secondary antibody for 1 h at 37 °C. The bands were visualized with the enhanced chemiluminescence (ECL). Relative quantification of band intensities was carried out using ImageJ software (US National Institutes of Health). For the data shown, the gray value of the target protein was normalized with that of internal control, and the protein relative expression was represented as a fold change ratio toward the control group.

Deletion of GPR81 alleviated CCl₄-induced chronic liver injury

To investigate the potential roles of GPR81 in the development of liver fibrosis, GPR81 knockout (KO) mice were generated (Fig. 2A and B) and exposed to CCl₄. As expected, CCl₄-challenge failed to induce the upregulation of GPR81 in KO mice (Fig. 2C–E). As expected, no difference was detected in the olive oil-treated mice (Fig. 2F and G). However, deletion of GPR81 significantly suppressed CCl₄-induced elevation of ALT and AST in serum (Fig. 2F). Deletion of GPR81 also resulted in alleviated morphological abnormalities of the liver, such as swollen and scattered patches (Supplementary Fig. 2). In addition, CCl₄-induced upregulation of pro-inflammatory cytokines, including TNF-α and IL-6, were inhibited in GPR81 deficient mice (Fig. 2G). These results suggest that GPR81 might be a detrimental factor in CCl₄-induced chronic liver injury.

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Fig. 2

Deletion of GPR81 alleviated CCl₄-induced chronic liver injury. The GPR81 KO mice were established and intraperitoneally injected with olive oil or CCl₄ for 8 weeks to induce liver fibrosis. A Schematic representation of the strategy used to produce GPR81 KO mice. B The genotype of wild-type (WT) and GPR81 knockout (KO) mice. C The hepatic mRNA and D protein levels of GPR81 were detected (n=4). E Representative images of immunohistochemistry (IHC) staining of GPR81 (scale bar: 100 μm) were shown. The positive area was quantified and expressed as a fold change relative to the WT group (n=4). F The serum ALT and AST levels were examined (n=4). G The mRNA levels of TNF-α and IL-6 were detected (n=4). All data were expressed as mean±SD

Deletion of GPR81 attenuated CCl₄-induced fibrogenesis

TGF-β1 is a central profibrogenic factor that plays an essential role in the activation of HSCs (Yang et al. 2023a). The upregulation of α-SMA is a molecular marker for the activation of HSCs, and the activated HSCs are the essential contributors to the synthesis of collagen and the deposition of extracellular matrix in liver fibrosis (Fan et al. 2022). The present study found no difference between olive oil-treated WT and KO mice, but the downregulation of TGF-β1, α-SMA, or COL1A1 in CCl₄-treated GPR81 KO mice (Fig. 3A–C). In addition, deletion of GPR81 was not associated with the elevation of hydroxyproline (Fig. 3D), an amino acid specific to collagen (Udenfriend 1966). Thus, GPR81 deficiency does not induce spontaneous fibrogenesis in liver.

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Fig. 3

Deletion of GPR81 suppressed CCl₄-induced fibrogenesis. WT mice and GPR81 KO mice were intraperitoneally injected with olive oil or CCl₄ for 8 weeks to induce liver fibrosis. A The hepatic mRNA expressions of TGF-β1, B α-SMA, and C COL1A1 were examined (n=4). D The hydroxyproline contents in liver tissues were detected (n=4). E The protein levels of pro-TGF-β1, mature TGF-β1, α-SMA, and COL1A1 were examined (n=4). F Representative images of H&E, Masson and Sirius red staining, and immunohistochemistry (IHC) staining of α-SMA of mice liver sections (scale bar: 50 μm) were shown. The positive area was quantified and expressed as a fold change relative to the WT group (n=4). All data were expressed as mean±SD

In mice that received repeatedly CCl₄ challenge, the mRNA levels of TGF-β1, α-SMA, COL1A1, and the hepatic level of hydroxyproline increased significantly, which were all suppressed in GPR81 deficient mice (Fig. 3A–D). Consistently, immunoblot analysis found that the protein levels of pro-TGF-β1, mature TGF-β1, α-SMA, and COL1A1 were lower in CCl₄-challenged GPR81 KO mice than those in CCl₄-challenged WT mice (Fig. 3E). Additionally, the morphological examinations with H&E staining, Masson staining and Sirius red staining, and immunohistochemistry staining of α-SMA indicated that deletion of GPR81 alleviated liver injury, suppressed collagen deposition, and reduced α-SMA-positive area in CCl₄-challenged mice (Fig. 3F). These results suggest that GPR81 might signal a profibrotic response in liver fibrosis.

GPR81 activator aggravated CCl₄-induced liver fibrosis

DHBA is an activator of GPR81 which has been extensively used in experimental studies (Yili et al. 2023; Liu et al. 2012; Madaan et al. 2017). To validate the detrimental roles of GPR81 in the development of liver fibrosis, CCl₄-exposed mice were treated with DHBA. In contrast to the result from GPR81 KO mice, supplementation with DHBA aggravated the morphological abnormalities of CCl₄-insulted liver, increased the levels of ALT and AST in serum, and upregulated the induction of TNF-α and IL-6 in liver (Supplementary Fig. 3A–C), suggesting that CCl₄-induced hepatic inflammation and liver injury were exacerbated by DHBA. In line with the aggravated liver injury, supplementation with DHBA increased the levels of TGF-β1, α-SMA, COL1A1, and hydroxyproline in CCl₄-exposed mice (Fig. 4A–E). The histological examinations also found that supplementation with DHBA aggravated CCl₄-induced histological lesions, enhanced CCl₄-induced deposition of collagen, and increased α-SMA-positive area in CCl₄-insulted liver (Fig. 4F). Interestingly, supplementation with DHBA failed to increase the level of ALT/AST, TNF-α/IL-6, or TGF-β1/α-SMA/COL1A1 in CCl₄-challenged GPR81 KO mice (Supplementary Fig. 4A–C), suggesting that the detrimental effects of DHBA on CCl₄-induced liver fibrosis depend on GPR81.

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Fig. 4

Treatment with GPR81 activator enhanced CCl₄-induced fibrogenesis. WT mice with CCl₄-induced liver fibrosis were supplemented with or without DHBA for 8 weeks. A The hepatic mRNA expressions of TGF-β1, B α-SMA, and C COL1A1 were examined (n=4). D The hydroxyproline contents in liver tissues were detected (n=4). E The protein levels of pro-TGF-β1, mature TGF-β1, α-SMA, and COL1A1 were examined (n=4). F Representative images of H&E, Masson and Sirius red staining, and immunohistochemistry (IHC) staining of α-SMA of mice liver sections (scale bar: 50 μm) were shown. The positive area was quantified and expressed as a fold change relative to the CCl₄ group (n=4). All data were expressed as mean±SD

GPR81 modulated CREB/Smad7 pathway

Several previous studies have found that GPR81 couples to Gi and activation of GPR81 reduces the level of cAMP (Ahmed et al. 2010; Khatib-Massalha et al. 2020), which functions as a major repressor of fibrotic responses (Delaunay et al. 2019). In agreement with these reports, the present study found that treatment with the GPR81 activator DHBA resulted in the reduction of cAMP (Fig. 5A). PKA/CREB pathway is essential for the anti-fibrotic activities of cAMP (Li et al. 2019). The present study found that treatment with DHBA also resulted in suppressed phosphorylation of CREB (Fig. 5B). Smad3 phosphorylation is an essential molecular event downstream of TGF-β1-induced fibrotic response (Zhang et al. 2023), but CREB-driven expression of Smad7 inhibits the phosphorylation of Smad3 (Gifford et al. 1979). Expectedly, supplementation with DHBA suppressed the expression of Smad7 but enhanced the phosphorylation of Smad3 (Fig. 5C and D). These results suggest that the detrimental effects of DHBA on liver fibrosis might be associated with suppressed CREB/Smad7 pathway.

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Fig. 5

GPR81 modulated CREB/Smad7 pathway. A–D WT mice with CCl₄-induced liver fibrosis were supplemented with or without DHBA for 8 weeks. A The hepatic contents of cAMP were detected by ELISA kit and were expressed as a fold change relative to the CCl₄ group (n=4). B The phosphorylation and total protein levels of CREB were determined (n=4). C The mRNA levels of Smad7 were examined (n=4). D The protein levels of Smad7, phosphorylated-Smad3 (p-Smad3), and total Smad3 (Smad3) were determined (n=4). E–H WT mice and GPR81 KO mice were treated with CCl₄ for 8 weeks to induce liver fibrosis. E The hepatic contents of cAMP were detected by ELISA kit and were expressed as a fold change relative to the WT group (n=4). F The phosphorylation and total protein levels of CREB were determined (n=4). G The mRNA levels of Smad7 were examined (n=4). H The protein levels of Smad7, phosphorylated-Smad3 (p-Smad3), and total Smad3 (Smad3) were determined (n=4). All data were expressed as mean±SD

On the contrary, the alleviated liver fibrosis in CCl₄-insulted GPR81 deficient mice was associated with increased level of cAMP and enhanced phosphorylation of CREB (Fig. 5E and F). In addition, the hepatic level of Smad7 in GPR81 KO mice was higher than that in WT mice, while the hepatic level of phosphorylated Smad3 in GPR81 KO mice was lower than that in WT mice (Fig. 5G and H). These results further support that the pro-fibrotic activities of GPR81 might result from the inhibition of the anti-fibrotic CREB/Smad7 pathway.

Not applicable.