Experimental Study On PGE2 Pathway Relevant To Anti-pulmonary Fibrosis Effect Of Citrus Alkaloid In Mice

Objective:To establish murine model of pulmonary fibrosis by Bleomycin and screen the anti-pulmonary fibrosis activity of Citrus alkaloids and their hydrochlorates in vitro. To explore the therapeutic effect and mechanism of alkaloid via observations of lung pathological changes and prostaglandin E2 (PGE2) signal pathway in vivo.Methods:1. Experiment in vitro:The primary lung fibroblasts were prepared from the mice lung with pulmonary fibrosis induced by Bleomycin. The inhibitory effects of Citrus alkaloids (A: hydroxyphenethylamine, B:synephrine, C:N-methyl-4-methoxy-beta phenethylamine, D: methoxy phenyl ethylamine, E:N-methyl tyramine) and their corresponding hydrochlorates (named as A1 to E1) by MTT (methylthiazol tetrazolium) method in vitro. The cytotoxicity of the tested samples was tested with lactic dehydrogenase (LDH) assay.2. Experiment in vivo:Mice were divided into normal group, control group, alkaloids groups (at three dosages 100 mg/kg/day,50 mg/kg/day and 25mg/kg/day) and prednisone group (6mg/kg/day) randomly. Pulmonary fibrosis model was induced by Bleomycin except for the normal group. After 14 days, each group was orally administrated one time per day (the normal group and control group were given 0.9% saline, the other groups were given with designed doses of alkaloids). Observation on activity, diet, hair and mental state of mice was conducted. Finally, the mice were sacrificed at the 28th day. Pathological lung tissue inflammation score was assess to evaluate the severity of pulmonary fibrosis in mice. Hydroxyproline (HYP), transforming growth factor beta 1 (TGF-β1), and prostaglandin E2 (PGE2) contents were analyzed with corresponding ELISA kits. Western blot method was applied fordetection of TGF-β1, cyclooxygenases-2(COX-2), prostaglandin E2 receptor (EP2) expression levels in lung.Results:1. Compound A1 showed the most potent inhibitory activity among Citrus alkaloids and their hydrochlorates that have been screened in vitro, the inhibition rate were 46.36%, 26.71%,19.53%,5.32%,5.27% respectively, when A1 at 50,25,12.5,6.25 and 3.125μM. The activity of LDH(U/L) were 176.11±14.87、159.30±11.29、152.93±9.45 respectively when it at 50,25 and 12.5μM compared with normal group (148.16±6.38), which revelaed that the inhibitory activity was not due to its cytoptoxicity.2. The weight of mice at high, medium and low dose groups were higher than control group (P<0.01 or P<0.05).3. The alveolitis and pulmonary fibrosis score at dose of 100 mg/kg/day were lower than control group (P<0.05 or P<0.01).4. The TGF-β1 content in serum of mice at dose of 100 mg/kg/day and prednisone group were lower than control group (P<0.01, suggesting that A1 significantly reduced the TGF-β1).5. Compared with control group, HYP contents in lung tissue of 100 mg/kg/day dose group and prednisone group were significantly decreased The result demonstrated that A1 significantly reduce the HYP content.6. Compared with nomal group, the PGE2 level of control group was significantly increased (P<0.01).7. The TGF-β1 protein expression in lung tissue of mice at all doses and prednisone group were lower than control group (P<0.01), suggested that A1 reduced the TGF-β1 protein expression in lung tissue.8. The COX-2 protein expression in lung tissue of mice at high group was higher than model group (P<0.01). High dose of A1 improved the level of COX-2 protein expression.9. The EP2 protein expression in lung tissue of mice at 100 mg/kg/day dose group was also higher than control group (P<0.01). High dose of A1 improved the EP2 protein expression.Conclusion:A1 showed the best inhibitory activity on mice lung fibroblasts proliferation assay, and the activity was not due to the cytotoxicity. A1 at 100 mg/kg·day reduced alveolar inflammation and fibrosis degree, inhibited the TGF-β1 content in serum, HYP content in lung tissue, and TGF-β1 protein expression level. A1 improved the PGE2, EP2, COX-2 protein expressions. Those data revealed that inhibitory effect of A1 on pulmonary fibrosis might be via elevating the level of COX-2, PGE2, EP2, suppressimng the TGF-β1 expression, finally relieving the pulmonary fibrosis process.