A CB2 Agonist Alleviates Liver Fibrosis by Downregulating the ERK1/2 Signalling Pathway in Mouse Hepatic Fibrosis Model

doi:10.21203/rs.3.rs-3705753/v1

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A CB2 Agonist Alleviates Liver Fibrosis by Downregulating the ERK1/2 Signalling Pathway in Mouse Hepatic Fibrosis Model

https://doi.org/10.21203/rs.3.rs-3705753/v1

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Cannabinoid type 2 (CB2) receptors have been reported to display potent anti-fibrogenic properties in mice with carbon tetrachloride (CCL4)-induced hepatic fibrosis.However, the mechanism by which CB2 receptors inhibit fibrogenesis remains unclear. We aimed to study the effects of CB2 receptors in a mouse CCL4-induced hepatic fibrosis model.

Hepatic fibrosis was induced by CCL4 administration in wild-type (WT) and CB2^−/− mice. WT mice received vehicle, CCL4, AM1241 (CB2 receptor agonist) or AM1241 plus AM630 (CB2 receptor antagonist), with the AM1241 and AM1241 plus AM630 treatments injected intraperitoneally on each day of the 16-week CCL4 treatment. Real-time PCR and immunohistochemical staining were used to analyse α-smooth muscle actin (α-SMA) and profibrogenic marker (TGF-β1) expression and extracellular matrix (ECM) deposition (collagen-Ⅰ). HSC apoptosis were performed by TUNEL staining. Extracellular signal-regulated kinase (ERK1/2), cAMP response element-binding protein (CREB), Bcl-2, Bax and cleaved caspase-3 levels were characterized in mouse hepatic tissues via western blotting.

Hepatic fibrosis was enhanced in CB2^−/− mice compared to that in WT mice, and this phenomenon was alleviated in AM1241-treated WT mice. Moreover, the CB2 receptor agonist AM1241 decreased α-SMA, TGF-β1 and collagen-Ⅰ mRNA and protein expression and inhibited ERK1/2-CREB-Bcl-2 signalling pathway activity. Combined AM1241 and AM630 administration reversed the effects of AM1241.

AM1241 inhibited hepatic stellate cell (HSC) activation, downregulated profibrogenic marker expression, decreased ECM deposition and alleviated fibrosis in WT mice. These effects were mediated at least in part by the induction of HSC apoptosis through ERK1/2-CREB-Bcl-2 downregulation. Thus, CB2 agonists are potential therapeutic agents for managing liver fibrosis.

CB2 receptor

AM1241

liver fibrosis

ERK1/2

CREB

Hepatic fibrosis is a dynamic process that results from the wound-healing response to chronic liver injury of various aetiologies and is mainly related to chronic viral hepatitis B or C, nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis, cholestasis and autoimmune hepatitis[1]. As fibrosis progresses, cirrhosis occurs, eventually culminating in hepatocellular carcinoma development. Therefore, hepatic fibrosis is a critical health problem, with a worldwide mortality rate of approximately 1.5 million deaths per year attributable to cirrhosis and primary liver cancer[2]. However, although removal of agents that cause liver fibrosis will regress liver tissue scarring, treatment of advanced cirrhosis is different[3]. In this context, suitable targets for anti-fibrosis therapies are critical to inhibit fibrosis progression.

The cannabinoid (CB) system consists of CB receptors, endocannabinoids, and the enzymes involved in CB receptor biosynthesis and degradation; these components of the CB network are involved in a range of physiological functions, including neuroprotection, pain, motor function, energy balance, food intake, cardiovascular homeostasis, immune and inflammatory responses, and cell proliferation[4, 5]. CB receptors include cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), which are different types of G protein-coupled receptors. CB1, a central cannabinoid receptor, is highly expressed in the brain and central neurons[6], whereas CB2, which is considered a peripheral cannabinoid receptor, is mainly expressed in haematopoietic cells and immune cells[7]. An increasing number of studies suggest that CB1/CB2 receptors play a significant role in liver disease pathogenesis[8, 9]. CB1 receptors have been shown to mediate profibrogenic effects in the liver and have also been implicated in the pathogenesis of alcoholic liver disease, NASH, and liver fibrosis[10–12]. Although CB1 receptor antagonists prevent liver disease progression, the development of CB1 receptor antagonists has recently been suspended due to the high incidence of central side effects. Notably, CB2 receptors are beneficial in patients with liver diseases, and CB2-selective agonists that do not affect CB1 mediate psychoactive effects because of their predominantly peripheral distribution. Hence, CB2 receptors have gained attention as very promising therapeutic targets. Several studies have demonstrated that CB2 receptors alleviate hepatic fibrosis in carbon tetrachloride (CCL4)-induced mouse models[13], reduce hepatocyte apoptosis and promote liver regeneration in response to acute injury[14]and ameliorate hepatic ischaemia/reperfusion injury[15], concanavalin-A-induced hepatitis[16]and alcoholic liver disease[17]. Together, these findings have led to the generally accepted idea that CB2 receptors may possess therapeutic potential in the context of liver disease pathology. However, the detailed mechanisms underlying these hepatoprotective effects of CB2 receptors remain unclear.

2.1. Animals

WT and CB2^−/− male mice on a C57BL/6 background were used at 8–12 weeks of age. WT rodents were purchased from the animal centre of Guizhou Medical University (approval number SCXKC (Guizhou) 2002-0001, Guiyang, China), and CB2^−/−mice (B6.129P2-Cnr2tm1Dgen 005786) were purchased from Jackson Laboratory (Bar Harbour, ME, USA). Animals were housed in temperature- and humidity-controlled rooms, kept on a 12-hour light-day cycle and provided unrestricted amounts of food and water. All animal experiments were approved by the Guizhou Medical University Animal Care and Use Committee.

2.2. Fibrosis models

The experiment was divided into two phases. In the first phase, WT and CB2^−/− male mice were divided into the following groups: control group-WT (olive oil); CCL4 group-WT; control group-CB2^−/− (olive oil) and CCL4 group-CB2^−/−. WT male mice were divided into the following groups in the second phase: the control group (olive oil); CCL4 group; AM1241-3 mg/kg group (CCL4 plus the CB2 receptor agonist AM1241 at 3 mg/kg/day, intraperitoneal (IP) injection); AM1241-9 mg/kg group (WT, CCL4 plus AM1241 at 9 mg/kg/day, IP) and AM630 + AM1241-3 mg/kg group (CCL4 plus AM1241 and the CB2 receptor antagonist AM630 at 3 mg/kg/day, IP). Fibrosis was induced by IP administration of CCL4 (C131583; Aladdin, China) mixed with olive oil (3/7 vol/vol) at 5 ml/kg body weight three times per week for 16 weeks. AM1241 (S1544; Selleck, USA) and AM630 (10006974; Cayman, USA) were administered at the beginning of the fibrosis induction protocol. Sham CCL4 animals received olive oil. After 16 weeks, the mice were starved overnight and euthanized 48 hours after the last CCL4 injection. Blood was collected for biochemical detection. Some liver samples were harvested for histology, and some were frozen and stored at -80°C until further use.

2.3. Histological assessment of stained liver tissue

Fibrotic scarring was assessed by haematoxylin/eosin (H&E) staining, and collagen deposition was assessed by Sirius Red staining in mouse liver tissue. Relative fibrosis area (expressed as a percentage of the total liver area) was assessed by analysing 5 fields from Sirius Red-stained liver sections per animal. Each field was acquired at 10× magnification and analysed using ImageJ.

2.4. Real-time reverse transcription polymerase chain reaction

Total RNA was extracted from frozen liver samples using an RNeasy Mini kit (Axygen, USA). cDNA was synthesized from 1 µg of total RNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). Quantitative real-time PCR was carried out on a ViiA 7 system (Applied Biosystems, USA) in 20-µl reactions containing 2 µl of cDNA (1:10 dilution) and 400 nM primers (all primers were obtained from Sangon, China) and PowerUp SYBR Green master mix (Applied Biosystems, USA) according to the manufacturer’s instructions. The data are expressed as the ratio of the expression level of the target gene to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the treated group relative to the sham group and were quantified using the comparative cycle threshold Ct method (2^−∆∆CT). The gene primer sequences were as follows: CB2, sense 5^׳-ACGGTGGCTTGGAGTTCAAC-3 ^׳and antisense 5^׳-GCCGGGAGGACAGGAT -AAT-3^׳; α-smooth muscle actin (α-SMA), sense 5^׳-GCCATCTTTCATTGGGATG- GA-3^׳ and antisense 5^׳-CCCCTGACAGGACGTTGTTA-3^׳; TGF-β1, sense 5^׳-GCTG -AACCAAGGAGACGA-3^׳ and antisense 5^׳-ATGTCATGGATGGTGCCCAG-3^׳; Col-Ⅰ, sense 5^׳-ACGCCATCAAGGTCTACTGC-3^׳ and antisense 5^׳-ACTCGAACG -GGAATCCATCG-3^׳; and GAPDH, sense 5^׳-AGGTCGGTGTGAACGGATTTG-3^׳ and antisense 5^׳-TGTAGACCATGTAGTTGAGGTCA-3^׳.

2.5. Immunohistological staining

Immunohistological examinations were carried out with primary rabbit monoclonal or polyclonal antibodies and secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies to detect α-SMA (1:500, Abcam, USA), collagen-Ⅰ (1:800, Abcam, USA), and TGF-β1 (1:700, Boster Biological Technology, China) expression. The expression of α-SMA and Col-Ⅰ is expressed as a percentage of total liver area and was determined using ImageJ, and TGF-β1 was assessed via integrated optical density (IOD) using Image-Pro Plus after analysis of 5 fields from stained liver sections per animal.

2.6.TUNEL Staining

The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Trevigen, USA) was used to assess apoptosis in mouse liver tissue. Sections of 6µm was collected onto slides pretreated and permeabilized with Proteinase K Solution for 45 min at room temperature. After incubating in 1×TdT Labeling Buffer for 5 min, the sections were treated with Labeling Reaction Mix for 60 min at 37℃ in a dark humidified atmosphere, followed by converter-DAB for 5 min. After washing with PBS three times for 5 min each, the specimens were counterstained in Methyl xylenes for 10 min.

2.7. Western blot analysis

Total protein was isolated from liver tissues, and protein concentrations were quantified using a BCA protein estimation kit. For each sample, 50–80 micrograms of protein was loaded. The proteins were transferred onto PVDF membranes using the wet transfer method. Membranes were blocked in 5% non-fat milk in Tris-buffered saline/Tween 20 for 2 hours at room temperature and then incubated with antibodies specific for pERK1/2 (1:2000, CST, USA), ERK1/2 (1:500, Wanlei, China), p-CREB (1:400, Cell Signaling Technology (CST), USA), CREB (1:250, Wanlei, China), Bcl-2 (1:250, Wanlei, China), Bax (1:250, Wanlei, China), cleaved caspase-3 (1:500, CST, USA), and CB2 (1:400, Abcam, USA). Blots were probed with anti-GAPDH (1:500, Wanlei, China) and anti-lamin B (1:500, Wanlei, China) antibodies as the loading controls. An anti-rabbit HRP-conjugated secondary antibody (1:20,000, Lianke Technology, China) was then applied. Immunoblots were developed using the Immobilon ECL method (Millipore, Billerica, Massachusetts, USA).

2.8. Statistical analysis

The results are expressed as the mean ± SEM and were analysed by one-way ANOVA followed by Tukey's post hoc test, using GraphPad Prism 5 software. Comparisons between two groups were analysed with t-tests. P values of < 0.05 were considered statistically significant.

3.1. Liver injury and fibrosis are more severe in CB2-deficient mice than in WT mice after chronic CCL4 exposure, and CB2 receptor expression levels are increased in a model of CCL4-induced liver fibrosis in WT mice.

We first investigated whether the CB2 receptor plays a protective role in a CCL4-induced liver fibrosis model. Chronic CCL4 treatment for 16 weeks (3 times/week) induced a significant degree of liver fibrosis in CB2^−/− mice compared to that in WT mice, characterized by inflammation, necrosis and collagen deposition as shown by H&E and Sirius Red staining (Fig. 1A-B, D). CB2 receptor mRNA and protein expression was examined via real-time reverse transcription polymerase chain reaction and western blotting. CB2 receptors exhibited a low expression level in normal WT mouse livers. In contrast, CB2 receptors exhibited a high expression level in the CCL4-induced WT mouse liver fibrosis model (Fig. 1C), indicating that the CB2 receptor plays a protective role in CCL4-induced liver fibrosis.

3.2. CB2 receptor activation ameliorates CCL4-induced chronic liver injury and fibrosis in WT mice

To determine whether CB2 receptor activation in WT mice contributes to CCL4-induced fibrosis resistance, we used AM1241, a CB2 receptor agonist. As anticipated, treatment of mice with the CB2 receptor agonist AM1241 markedly attenuated CCL4-induced hepatic injury and fibrosis in WT mice, as evidenced by H&E staining (Fig. 2A) and decreased collagen composition by Sirius Red staining (Fig. 2B-C). All the abovementioned changes were clearly abrogated by the CB2 receptor antagonist AM630. Altogether, these results demonstrate that CB2 receptor activation alleviated CCL4-induced fibrosis in WT mice.

3.3. CB2 receptor activation decreases profibrogenic marker (TGF-β1) expression.

To provide evidence of the antifibrotic effects of CB2 receptor activation, we detected the expression levels of fibrosis-associated genes in fibrotic liver samples. Real-time PCR and immunohistological staining results clearly demonstrated that treatment with the CB2 receptor agonist AM1241 downregulated TGF-β1 mRNA and protein expression (Fig. 3A-B). Western blotting further confirmed that treatment with the CB2 receptor agonist AM1241 reduced TGF-β1 protein expression in fibrotic tissues (Fig. 3C).

3.4. CB2 receptor activation inhibits HSC activation and decreases Col-Ⅰ deposition.

To provide further evidence of the antifibrotic properties of CB2 receptor activation, we next detected the expression levels of α-SMA, a protein marker of hepatic stellate cell (HSC) activation, and Col-I in fibrotic liver samples. Real-time PCR results clearly demonstrated that treatment with the CB2 receptor agonist AM1241 downregulated α-SMA and Col-I mRNA expression. Immunohistological staining further confirmed that treatment with the CB2 receptor agonist AM1241 reduced α-SMA and Col-I protein expression in fibrotic areas (Fig. 4A-D).

3.5. CB2 receptor activation promoted HSC apoptosis.

HSC apoptosis was investigated via TUNEA staining. Results clearly demonstrated that treatment with the CB2 receptor agonist AM1241 promoted HSC apoptosis(Fig. 5A-B).

3.6. CB2 receptor activation suppresses ERK1/2-CREB-Bcl-2 signalling pathway activity levels in CCL4-induced liver fibrosis

To explore the possible anti-fibrogenic mechanism of CB2 receptor activation in hepatic fibrosis, we examined the effects of the CB2 receptor agonist AM1241 on the ERK1/2-CREB-Bcl-2 pathway. Western blot analysis revealed that the relative expression of p/T-ERK1/2, p/T-CREB, and Bcl-2/GAPDH was enhanced and that of Bax/GAPDH and cleaved caspase-3/GAPDH was decreased in the livers of mice exposed to CCL4 compared to that in the control mouse livers. However, administration of the CB2 receptor agonist AM1241 markedly prevented the upregulation of p/T-ERK1/2, p/T-CREB, and Bcl-2/GAPDH and increased Bax/GAPDH and cleaved caspase-3/GAPDH levels in CCL4-induced fibrotic livers. Interestingly, the expression levels of all the above proteins showed the opposite patterns in the AM630 + AM1241 group. These results demonstrated that CB2 receptor activation promotes HSC apoptosis, which may be related to ERK1/2-CREB-Bcl-2 signalling pathway inhibition (Fig. 6A-B).

Although the underlying mechanisms of hepatic fibrosis formation have been greatly elucidated during the past decade, this understanding has not resulted in effective clinical therapeutic approaches. CB2 receptors are known to be upregulated and exert protective effects in liver disease[18–20]. In the present study, the role of CB2 receptors was evaluated following chronic CCL4 treatment for 16 weeks in CB2^−/− mice and their WT counterparts. We found that compared with naive mice, CB2^−/− mice presented with an enhanced inflammatory response, as evidenced by H&E staining and collagen staining with Sirius Red, and the results indicated that CB2^−/− mice developed more severe fibrosis than WT mice. However, CB2 receptors were highly expressed in CCL4-induced liver fibrosis in WT mice compared to those in normal WT mice. These data demonstrate that CB2 receptors ameliorate liver injury and have anti-fibrogenic properties in a mouse model of CCL4-induced liver fibrosis, and these findings are consistent with a previous report[13]. In CCL4-induced mouse liver fibrosis, the mechanism by which CB2 receptors prevent fibrosis development remains unknown. Therefore, we used the selective CB2 receptor agonist AM1241 (3 mg/kg and 9 mg/kg, IP, daily) to intervene in mouse fibrogenesis during chronic CCL4 exposure. We demonstrated that CB2 receptors, which are continuously activated during AM1241 treatment, play a protective role and prevent CCL4-induced liver fibrosis by significantly alleviating histological damage and reducing collagen deposition in the liver (as determined by H&E staining and Sirius Red staining of collagen). In addition, to further survey whether CB2 receptors are activated by AM1241 and consequently mediate these anti-fibrogenic effects, the CB2 receptor antagonist AM630 was used in combination with AM1241, which completely reversed the above results and increased liver fibrogenesis in a mouse model of CCL4-induced liver fibrosis.

Following repeated liver tissue injury, HSCs attempt to repair tissue by differentiating into contractile, proliferating, fibrogenic myofibroblasts that express α-SMA. Myofibroblasts produce a large amount of ECM, which is deposited in impaired regions of the liver, resulting in scar formation[21, 22]. TGF-β1 is the most potent profibrogenic cytokine; it is produced by activated HSCs[23] and contributes to continuous HSC activation via the autocrine system, which stimulates synthesis of ECM components, primarily Col-Ⅰ. The transcription level of Col-I can also be upregulated by TGF-β1 in HSCs[24]and promote Col-Ⅰ deposition. In brief, activated HSCs are transformed into myofibroblasts, which have profibrogenic properties and secrete TGF-β1, α-SMA and Col-Ⅰ. Our results revealed that liver TGF-β1, α-SMA and Col-Ⅰ expression decreases in mice with CCL4-induced liver fibrosis after treatment with AM1241. Treatment with AM630 plus AM1241 substantially upregulated the TGF-β1, α-SMA and Col-Ⅰ expression levels. These findings indicate that AM1241 may mediate anti-fibrogenic effects by reducing the number of activated HSCs through promoting HSC apoptosis.

ERK1/2 belongs to the mitogen-activated protein kinase (MAPK) family and plays an important role in cell proliferation, apoptosis, cycle regulation, differentiation and development[25]. The ERK1/2 pathway is activated by various stimuli, such as oxidative stress, ionic imbalance, and glutamate receptor and growth factor activation[26]. In several studies, CB2 receptor stimulation has been shown to counteract the ability of morphine to upregulate protein kinase B or Akt and ERK1/2 activation induced by lipopolysaccharide (LPS), thus reducing nitric oxide (NO) and pro-inflammatory cytokine release[27]; CB2 receptor agonists prevent neuronal injury during neuroinflammation via MAPK upregulation, which results in ERK1/2 inhibition[28]; and CB2 receptor stimulation specifically reduces iNOS production via ERK1/2 phosphorylation inhibition in microglia during central nervous system (CNS) inflammation[29]. CB2 receptor agonists can also inhibit ERK1/2 phosphorylation in immune cells, induce apoptosis and relieve the development of autoimmune diseases[30]. These findings are relevant to the anti-inflammatory effects of CB2 receptors through regulation of ERK1/2 activation in the brain and the control of neuroinflammation. However, the regulatory effects of CB2 receptors on ERK1/2 activation in mouse CCL4-induced fibrosis models have not been reported. CREB regulates gene transcription and participates not only in synaptic plasticity, memory and survival[31]but also in tissue injury pathophysiology[32, 33]. Our data showed that AM1241 inhibits ERK1/2 phosphorylation, which affects p-CREB expression. Phosphorylated CREB reportedly promotes anti-apoptotic Bcl-2 protein expression and decreases Bax expression, both of which are members of the Bcl-2 protein family and are associated with apoptosis[34, 35]. According to our observations, CB2 receptors might inhibit the ERK1/2-CREB signalling pathway, suppressing Bcl-2 formation, promoting Bax and cleaved caspase-3 expression, and increasing activated HSC degeneration and apoptosis. Moreover, combined use of AM630 and AM1241 produced the opposite effects on these signalling proteins.

CB2 receptors have protective effects in mice with CCL4-induced liver fibrosis. These effects may be associated with CB2 receptor-induced downregulation of the ERK1/2-CREB signalling pathway, which results in Bcl-2 suppression and Bax upregulation, which promotes apoptosis of activated HSCs.

CB1: cannabinoid receptor 1, CB2: cannabinoid receptor 2, CB2-/-: CB2 knockout, α-SMA: α-smooth muscle actin, TGF-β1: transforming growth factor beta 1, ECM: extracellular matrix

Acknowledgments

This work was financially supported by National Natural Science Foundation of China (81860118, 81460125), Guizhou Provincial Health Commission Science and Technology Fund（gzwkj2021–119), Guizhou Provincial Department of Education Innovation Group Project (Qiangjiaohe KY word [2021]016), Science and technology plan project of Guizhou Province (Qianke He Platform Talents [2019–5610]).

Authors’ contributions

Yuping Wang: Funding acquisition, Supervision, Writing- Review&Editing. Ping He: Performed the experiments, Formal analysis, Writing Original Draft. Yafeng Wu and Cuizhen Long : Performed the experiments. Jianchao Shun: Supervision, Correcting the draft. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Disclosures

The authors have nothing to disclose.

Date Availability

Original data generated and analyzed during this study are included in this manuscript.

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A CB2 Agonist Alleviates Liver Fibrosis by Downregulating the ERK1/2 Signalling Pathway in Mouse Hepatic Fibrosis Model

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Animals

2.2. Fibrosis models

2.3. Histological assessment of stained liver tissue

2.4. Real-time reverse transcription polymerase chain reaction

2.5. Immunohistological staining

2.6.TUNEL Staining

2.7. Western blot analysis

2.8. Statistical analysis

3. Results

3.2. CB2 receptor activation ameliorates CCL4-induced chronic liver injury and fibrosis in WT mice

3.3. CB2 receptor activation decreases profibrogenic marker (TGF-β1) expression.

3.4. CB2 receptor activation inhibits HSC activation and decreases Col-Ⅰ deposition.

3.5. CB2 receptor activation promoted HSC apoptosis.

3.6. CB2 receptor activation suppresses ERK1/2-CREB-Bcl-2 signalling pathway activity levels in CCL4-induced liver fibrosis

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

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