The Protective Effect Of Citrus Alkaline Extract On Mice With Pulmonary Fibrosis And Its Effect On Apoptosis Of Pulmonary Fibroblasts

Objective:To investigate the prevention role of citrus alkaline extract(CAE)in bleomycin-induced pulmonary fibrosis mice and explore the functioning mechanism of CAE in pulmonary fibrosis treatment by promoting pulmonary fibroblasts apoptosisMethods:1.42 C57BL/6 male mice were assigned randomly to the normal group,model group,low(16mg/kg),medial(32mg/kg)and high(64mg/kg)CAE dosage groups,prednisone group(6mg/kg)and pirfenidone group(100mg/kg),respectively.The pulmonary fibrosis mice models in any other groups were constructed by disposable intratracheal instillation except the normal group.One day after model construction,intragastric administration was applied to the mice once a day for 28 days and then killed.10 mg/Kg normal saline solution were administrated to the normal group and model group,while other groups were medicated by corresponding treatments.Mice body weights were recorded.Mice pulmonary tissues were stained by HE and Masson's methods,and then pulmonary alveoli as well as pulmonary fibrosis was scored.The levels of pulmonary hydroxyproline(HYP),prostaglandin E2(PGE2)and interleukin-17(IL-17)in serum and bronchoalveolar fluid(BALF)were tested by the ELISA method.The expression of pulmonary proteins collagen ?,collagen ? and pro-surfactant protein C(Pro-SPC)was tested immunohistochemically.The expression of pulmonary Vimentin was checked by the immunofluorescence method,and pulmonary tissue apoptosis was determined by the Tunel staining method.2.The pulmonary fibroblasts of normal mice and bleomycin-induced pulmonary fibrosis mice(model mice)were collected.The expression of Vimentin and a-SMA was tested immunohistochemically;the inhibition role of CAE was determined with Methylthiazol tetrazolium(MTT)method,and IC50 calculated.Lactate dehydrogenase(LDH)reagent kit was used to test the toxicity of CAE to the pulmonary fibroblasts.The inhibitory role of CAE on pulmonary fibroblasts were examined with flow cytometer and fluorescence staining method.Expression of Cleaved-Caspase3,Cleaved-PARP,Cleaved-Caspase8,Fas,Fas Ligand(FasL),Cyclooxygenase-2(COX-2),prostaglandin E receptor 2(EP2)and phosphorylation of p38(p-p38)was examined by Western Blot assay.The oxidative induction level of CAE on pulmonary fibroblasts were tested with the oxidative induction test reagent kit.Results:1.The pulmonary alveolitis and pulmonary fibrosis scores in the dosed mice groups decreased to some extent compared to the model mice group.In particular,the pulmonary alveolitis score and pulmonary fibrosis score in the CAE treatment was reduced more significantly(P<0.05 or P<0.01)?,2.The expression of collagen I,collagen III and HYP in the model mice group increased significantly compared to the normal group(P<0.01).After CAE treatment,the expression of collagen I,collagen III and HYP was decreased(P<0.05or P<0.01).3.The level of PGE2 in pulmonary tissue after CAE treatment were increased significantly compare to the model group(P<0.05 or P<0.01).Meanwhile,IL-17 levels in the serum and BALF of the CAE teratmen groups,were reduced significantly(P<0.05or P<0.01).4.The expression of Pro-SPC in the CAE treatment group was markedly increase compare to the model group(P<0.05or P<0.01).Meanwhile,after CAE,treatment,the expression of in pulmonary tissue was decreased to a certain extent(P<0.05 or P<0.01).5.Apoptotic cell counts in the model mice group were markedly increased relative to the normal group(P<0.01).In contrast,apoptotic cell counts in mice pulmonary tissues of the medial and high CAE groups,pirfenidone group and prednisone group were significantly reduced(P<0.01).6.The expression intensity of Viementin in pulmonary fibroblasts in model mice was higher than the normal mice(P<0.01).Meanwhile,the positive expression rate and expression intensity of a-SMA in the pulmonary fibroblasts of model mice was higher than the normal mice(P<0.01).7.When both kinds of pulmonary fibroblasts were treated by different concentrations of CAE,the MTT test results showed that CAE had inhibited the proliferation of both kinds of pulmonary fibroblasts(P<0.05 or P<0.01),and IC50 value showed a better effect of CAE on the pulmonary fibrocytes of the model mice.8.The results of flowcytometer suggested that CAE intervention had increased significantly the apoptosis of pulmonary fibroblasts in the model mice in a dosage-dependent manner.Results of fluorescence staining also agreed with this conclusion(P<0.01).Western Blot results showed that after CAE treatment the expression level of Cleaved-Caspase3,Cleaved-PARP,Cleaved-Caspase8,Fas,and FasL was also increased markedly(P<0.05 or P<0.01).The expression of COX-2 and EP2 increased gradually with the growth in drug concentration(P<0.05 or P<0.01).This suggesting that CAE-induced cell apoptosis is probably related to Fas-medicated membrane receptor apoptosis.9.Results of oxidation-induced flurorescence test showed that,after CAE intervention,the oxidative induction level increased significantly(P<0.05 or P<0.01).Western Blot assay also found a significantly increased p-p38 level,suggesting that the CAE probably regulate COX-2 in a P38 phosphorylation dependent on oxidative induction.Conclusion:1.CAE could effectively delay the advancement of bleomycin-induced pulmonary fibrosis in mice,which is probably attributed to inhibition of collagen secretion,prevention of inflammatory response,protection of pulmonary epithelial cells,and inhibited proliferation of pulmonary fibroblasts.2.The pulmonary fibroblasts of model mice have a higher research value than those of normal mice.CAE could effectively induce the apoptosis of mice pulmonary fibroblasts,and the potential functioning mechanism is probably related to the p38/COX-2 signaling pathway dependent on oxidative induction.