The Early Intervention Effect Of Citrus Alkaloid On PF Via PGE2 Signaling Pathway

Objective:To study bleomeycin(BLM)-induced pulmonary fibrosis(PF) model in C57BL/6 mice, and separate the lung fibroblasts by primary culture.To select the most active with MTT method, and explore the early intervention effect of Citrus alkaloids on pulmonary fibrosis in C57BL/6 mice via PGE2 signaling pathway.Methods:1. C57BL/6 mice were randomly divided into normal group and model group,10 mice per group.The bleomycin-induced pulmonary fibrosis C57BL/6 mice model was used, and the fibroblasts were isolated from the fibrotic lung for the primary culture.2. In order to find out the most active Citrus alkaloid, mice lung fibroblasts in the logarithmic growth phase were used in the experiment; Citrus alkaloids at 20 μM were screened, and then IC50 of the alkaloid with potent activity was measured with5 concentration,50μM,25μM,12.5μM,6.25μM,3.125μM; The lactate dehydrogenase (LDH) test was applied for cytotoxicity.3. C57BL/6 mice were divided into normal group, model group (bleomycin group), Citrus alkaloid groups (at doses of 100,50,25 mg/kg/day), positive control group (prednisone). Except the normal group, the mice of other groups were intratracheally instilled with a single dose of bleomycin (5mg/kg); On the next day of BLM administration,the nomal and the BLM groups received the 100mg/kg of sterilized saline,Citrus alkaloid groups (100,50,25mg/kg) and prednisone group (6mg/kg) wereorally administered once per day for 28 days. Mice were sacrificed on the 28st day posttreatment, and the lung tissues were taken for HE staining and Masson staining to observe the pathological changes. HYP content of lung tissue wasdetermined by kit according to the manufacturer’s instructions; Transforming growth factor betal (TGF-betal) in serum and prostaglandin E2 in lung tissue (PGE2) were determined by ELISA; The protein expressions of TGF-β1, COX-2 and EP2 in lung tissue were detected by western blot.Results:1. Under the optical microscope, mice lung fibroblasts were fusiform or polygonal, parallel growth; After the vimentin and asmooth muscle protein (a-SMA) immunocytochemical staining, Vimentin and a-SMA protein were expressed in cytoplasm in yellow brown, which confirmed that the cultured cells were lung fibroblasts; The MTT method was used to measure the growth curve of lung fibroblast; after 48h culture, the fibroblasts entered the logarithmic growth phase,72-96h were in the logarithmic growth phase,120h reached to after growth in the peak, and then the cell growth into the platform period.2. In vitro experiment:Citrus alkaloids and its hydroxychloratesinhibited the proliferation of the lung fibroblast; Among them, the inhibitory effect of the p-hydroxyphenylethylamine salt is the best. LDH experiments showed that the inhibitory effect of p-hydroxyphenylethylamine hydroxychlorateon the proliferation of lung fibroblast cells was activity rather than the cytotoxicity.3. The weight change of mice:(1)The speed of body weights growth of mice in model group were significantlys lower than those innomal group, and the body weigts were significantly decreased compared with normal group(P<0.01);(2)Compared with model group, the speed of body weights growth of mice in all treatment and positive control groups were increased,but no statistical significance was observed.4. The alveolitis and fibrosis score:(1) The alveolitis and fibrosis score of model group was significantly higher than that in nomal group(P<0.01);(2) Compared with model group, the alveolitis score and fibrosis score of high dose group and prednisone group were markedly reduced(P<0.05orP<0.01),however,in middle and low dose groups, only the alveolitis score was significantly relieved(P<0.05).5. HYP content in lung tissue:Compared with nomal group, HYP content in lung tissue of model group was significantly increased(P<0.01); Compared with model group, HYP contents in lung tissue of high dose group, middle dose group and positive group were significantly decreased(P<0.05orP<0.01).6. TGF-β1 content in serum:Compared with nomal group, TGF-β1 level of serum was significantly higher than that in modelgroup(P<0.01); Compared with modelgroup, TGF-β1 levels of serum inhigh, middle dose groups and positive group were significantly decreased(P<0.01orP<0.05).7. The PGE2 level of lung tissue:Compared with nomal group, the PGE2 level of model group was significantly increased(P<0.01); Compared with model group,the PGE2 level inhigh dose group and prednisone group were significantly increased(P<0.05).8. The results of Western blot:The ratio of TGF-β1 in BLM-induced lung tissue were significantly increased compared with the nomal group(P<0.05).The protein expression of COX-2 and EP2 of model groupin lung tissue were significantly increased compared with normal group(P<0.05); The protein expression of TGF-β1 in lung tissue of high and middle dose groups was lower than model group(P<0.05); The protein levels of COX-2 and EP2 in lung tissue of p-hydroxyphenylethylamine hydrochlorateat high dose was elevated, the protein expressions of EP2 in lung tissue was also significantlyincreased(P<0.05).Conclusion:1. Using trypsin digestionthe lung fibroblasts were isolated, the cells met experiment needs, while was time-consuming; And the cultured lung fibroblasts have the characteristics of muscle fibroblasts, could be used to study the mechanism of pulmonary fibrosis.2. Citrus alkaloid significantly inhibited the proliferation of pulmonary fibroblasts in BLM-induced mice, p-hydroxyphenylethylamine hydrochlorate showed the most potent activity, and its inhibitory activity was not due to its cytotoxicity.3. p-Hydroxyphenylethylaminehydrochlorate improved the symptom and increased the weight of the mice.4. p-Hydroxyphenylethylamine hydrochlorate relieved alveolitis and fibrosis of BLM-induced pulmonary fibrosis mice.5. p-Hydroxyphenylethylamine hydrochlorate decreased HYP content in lung tissue, and TGF-β1 level in serum and lung tissueofpulmonary fibrosis mice, which displayed a early intervention effect on pulmonary fibrosis; p-Hydroxyphenylethylamine hydrochlorate increased the protein expressions of COX-2 and EP2, suggesting the mechanism of early interventionon pulmonary fibrosis migh trelate PGE2 signaling pathway.6. This experiment demonstrated a potential that p-hydroxyphenylethylamine hydrochlorate maight be effective for earlyintervention for IPF.