Pathological, immunohistochemical and molecular studies on the curative role of synthetic cannabinoid receptors-2 agonist (AM1241) in induced hepatic and pulmonary fibrosis in male rats

Abstract

Cannabinoids are a group of compounds acting primarily via CB1 and CB2 receptors. Therapies based on cannabinoids including CB2 agonists and CB1 antagonists may be novel therapeutic perspectives for the treatment of chronic liver and lungs diseases. The present study includes three experiments; the first experiment aimed to explore the role of CB2 agonist (AM1241) on suppressing the progress of liver fibrosis induced by bile duct ligation (BDL). AM1241 was administrated to BDL rats in two doses (3 and 6 mg/kg). Forty five adult male Wistar rats were divided into 5 groups, 9 rats each. Group 1; sham operated group; rats exposed to surgery without bile duct ligation and served as control non-treated, they received i.p injection of 0.1 ml of 1:2 solution of DMSO: PBS (vehicle). Group 2; bile duct ligated group; rats exposed to bile duct ligation surgery then they received vehicle as group 1. Group 3; bile duct ligated rats received the standard drug, 100 mg/Kg/day silymarin by gavage. Groups 4 and 5; bile duct ligated rats were i.p injected with the investigated drug; AM1241 at a dose of 3 and 6 mg/kg respectively. All treatments started two weeks post-surgery (post fibrosis induction) and continued for two weeks after induction of fibrosis. When compared AM1241 to BDL and silymarin groups, the results showed that AM1241 administration at both doses ameliorated liver function markers, MDA level, pro-inflammatory cytokines (TNF-α and IL-6) and TLR4 gene expression. Additionally, AM1241 increased GSH content and immunomodulatory cytokine; IL-10. Histologically, AM1241 limited fibroplasia extension, decreased cholongiocytes proliferation, and decreased immune-expression of CD31, α-SMA and NF-κB with strongly expressed CD34. The second experiment aimed to investigate the role of CB2 activation by synthetic analog (AM1241) on revoking the progress of liver fibrosis. Thioacetamide (TAA) was used to induce liver fibrosis in rats. AM1241 was administrated to TAA group in two doses (3 and 6 mg/kg). Fifty-five adult male Wistar rats were randomly allocated into 5 groups, 11 rats each. Group 1: control group; rats received i.p injection of 0.1 ml of 1:2 solution of DMSO: PBS (vehicle). Group 2: TAA group; rats received TAA (200 mg/kg b.wt) by i.p injection twice/week for 6 weeks. Groups 3, 4 and 5; rats administrated TAA as in group 2, but starting from the fourth week (post fibrosis induction), they received different treatments as follows; Group 3 received the standard hepatoprotective drug; silymarin (100 mg/kg b.wt) daily by gavage. Groups 4 and 5 were i.p injected with the investigated drug; AM1241 at doses of 3 and 6 mg/kg b.wt daily respectively. All treatments continued for three weeks concurrently with TAA. Some tests were done at the third week of TAA administration to assure that fibrosis was induced in a previous preliminary study. When compared AM1241 to TAA and silymarin groups, the results showed that AM1241 administration at both doses to TAA rats post liver fibrosis induction, significantly maintained liver function markers and decreased; malondialdehyde, Vimentin, TLR4, TGF-β1, α-SMA and miR-155 genes expression, NFκB p65 immune-expression and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and increased E-Cadherin level. Additionally, AM1241 significantly increased GSH and SOD content, histologically limited fibroplasia extension, broke the itinerary of bridging fibrosis, and decreased immune-expression of NFκB p65.