The Effect And Mechanism Of CB2 Receptor Agonist JWH133 On Pulmonary Fibrosis In Mice

Objective:By establishing an animal model of pulmonary fibrosis induced by bleomycin in mice,CB2 receptor agonist JWH133 and CB2 receptor antagonist AM630 were used to intervene in pulmonary fibrosis mice.The protective effect of JWH133 on pulmonary fibrosis was studied and analyzed,and whether its protective effect was related to the regulation of the ERK1/2-RSK1 signaling pathway was explored to find new targets for treating pulmonary fibrosisMethods:A mouse pulmonary fibrosis model was prepared by intratracheal instillation of bleomycin hydrochloride(5mg/kg).Twenty four C57BL/6J male mice were randomly divided into four groups:control group,model group,JWH133 intervention group,and JWH133+AM630 antagonistic group,with six mice in each group.Starting from the second day,the JWH133 treatment group mice were injected with a daily dose of JWH133 2.5mg/kg intraperitoneally,while the JWH133+AM630 antagonist group mice were injected with 2.5 mg/kg of JWH133 and AM630 intraperitoneally.The control group and model group mice were injected with the same volume of physiological saline intraperitoneally every day for 28 days.After 28 days,mouse serum and lung tissue samples were collected for research.The lung tissue was sectioned and then stained with HE,Masson and immunohistochemistry to observe the histopathology changes of the lung and determine the content of collagen I(Col-Ⅰ),enzyme-linked immunosorbent assay(ELISA)to detect the levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)in the serum of mice,and determine the content of hydroxyproline(HYP)in the lung tissue of mice,Real-time PCR was used to detect the expression levels of collagen Ⅲ(ColⅢ),α-smooth muscle actin(α-SMA),Col-Ⅰ in mouse lung tissue,and Western blot was used to detect the protein expression levels of Col Ⅲ,α-SMA,Col-Ⅰ,extracellular signal regulated kinase(ERK1/2),ribosome S6 kinase type 1(RSK1),phosphorylated extracellular signal regulated kinase(p-ERK1/2),and phosphorylated ribosome S6 kinase type 1(p-RSK1).Results:1.In HE staining and Masson staining,compared with the control group,the model group mice showed disordered lung tissue morphology and structure,increased infiltration of inflammatory cells,deposition of blue collagen,increased alveolar inflammation score(P<0.05),and increased Ashcroft score(P<0.05);Compared with the model group,the JWH133 intervention group showed improved alveolar structure,reduced infiltration of inflammatory cells,reduced blue collagen,decreased alveolar inflammation score(P<0.05),and decreased Ashcroft score(P<0.05)in mice;Compared with the JWH133 intervention group,the JWH133+AM630 antagonistic group mice reversed the above results(P<0.05).2.Immunohistochemistry showed that the positive expression of Col-Ⅰ in the model group was enhanced compared to the control group,and the average optical density value increased(P<0.05);Compared with the model group,the JWH133 intervention group showed a decrease in Col-Ⅰ positive expression and a decrease in average optical density(P<0.05);Compared with the JWH133 intervention group,the JWH133+AM630 antagonistic group mice showed an increase in Col-Ⅰ positive expression and an average increase in optical density(P<0.05).3.In the experiment of measuring inflammatory factors and hydroxyproline,it was found that compared with the control group,the content of IL-1β,TNF-α,IL-6,and HYP in the model group mice increased(P<0.05);Compared with the model group,the JWH133 intervention group mice showed a decrease in IL-1β,TNF-α,IL-6,and HYP levels(P<0.05);Compared with the JWH133 intervention group,the JWH133+AM630 antagonistic group mice reversed the above results(P<0.05).4.WB and PCR showed that compared with the control group,the model group mice had higher levels of Col-Ⅲ,α-SMA,Col-Ⅰ mRNA and protein,while the expression of p-ERK1/2 and p-RSK1 proteins increased(P<0.05);Compared with the model group,the JWH133 intervention group showed a decrease in the levels of Col-Ⅲ,α-SMA,Col-Ⅰ mRNA and protein,as well as a decrease in the expression of p-ERK1/2 and p-RSK1 proteins,with a statistically significant decrease(P<0.05);Compared with the JWH133 intervention group,the JWH133+AM630 antagonistic group mice reversed the above results(P<0.05)Conclusions:The CB2 receptor agonist JWH133 can significantly inhibit inflammatory response,improve extracellular matrix deposition,and alleviate bleomycin induced pulmonary fibrosis in mice.Its mechanism may be related to the inhibition of the ERK1/2-RSK1 signaling pathway.