Arsenic Trioxide Prevents Bleomycin-induced Rat Pulmonary Fibrosis Via MiR-98 Overexpression

This paper mainly explored the mechanism of arsenic trioxide(As2O3)-induced overexpression of miR-98 in preventing bleomycin(BLM)-induced pulmonary fibrosis, which is divided into two parts:(a) studying the roles of As2O3 and miR-98 in preventing BLM-induced pulmonary fibrosis in vivo; (b) investigating the molecular mechanisms of As2O3 and miR-98 anti-fibrotic effects in vitro.1. Studying the roles of As2O3 and miR-98 in preventing BLM-induced pulmonary fibrosis in vivo.(1) Constructing BLM-induced pulmonary fibrosis models in rats.Forty rats were randomly divided into BLM model groups (0d,14d,28d), normal saline(NS) model control group and As2O3-treated group. Rats were given BLM(5mg/ml-kg BW) through intratracheal injection to build fibrotic models, while the NS model control group given saline(1ml/kg BW) through intratracheal injection. As2O3-treated group was received As2O3 (0.4mg/kg/d) intervention via tail vein injection for 14 days since the 14th day after BLM modeling. BLM model groups were sacrificed on the same time with modeling and on day 14th and 28th after modeling respectively. NS model control group and As2O3-treated group were killed on day 28 after modeling. Morphologically, the lung of rats was observed by HE staining, while the expression of collagen in lung was examined through Masson staining. We detected collagen content of lung tissue via hydroxyproline(HYP) detection, western blotting was used for detection the expression of α-smooth muscle actin (α-SMA) and E-cadherin in each group. A comprehensive analysis was used to evaluate the effects of As2O3 on the development of pulmonary fibrosis. Results suggested that pulmonary interstitial was gradually widened and lung alveolar structural get disordered with the time after BLM modeling, accompanied by collagen deposition in lung interstitial and collagen content of lung tissue gradually increasing. Above changes in BLM-induced rats were not obvious compared with that in NS model control group. Lung interstitial in As2O3-treated group was thinner compared 28d BLM model group. We assumed rat pulmonary fibrosis models were constructed successfully and As2O3 can reduce bleomycin-induced pulmonary fibrosis injury.(2) Detection of miR-98 expression in pulmonary fibrosis rats.miR-98 expression was detected by in the lung tissues of rats, our results showed that the expression of miR-98 is gradually reduced with the development of pulmonary fibrosis, As2O3 treatment group significantly increased the expression of miR-98 compared with the 28d BLM model group, which suggests that As2O3 probably plays a therapeutic role in pulmonary fibrosis by miR-98 overexpression.(3) Detection of the downstream signaling factors(Jak-Stat3 and Bax/Bcl-2) of miR-98.Our study found that both Stat3 and p-Stat3 expression in rats lung were significantly increased with BLM modeling, and the ratio of Bax/Bcl-2 in lung increased consistently. On the contrary, As2O3 treatment could suppress Jak-Stat3 signaling pathway and decrease the ratio of Bax/Bcl-2. We proposed that As2O3 maybe play an anti-fibrotic effects via up-regulating miR-98, thus suppressed Jak-Stat3 signaling pathway.2. Investigate the molecular anti-fibrotic mechanisms of As2O3 and miR-98 anti-fibrotic in vitro.(1) We built a cell model of fibrosis by TGF-β1 inducing A549 cells epithelial qualitative transdifferentiation (EMT).A549 cell made morphological changes gradually after TGF-β1 being added in cell culture medium, we observed that A549 cell gradually changed from cubic or triangular to spindly or fusiform. Immunofluorescence observation showed the epithelial marker protein E-Cadherin when interstitial marker protein a-SMA expression increased after TGF-β1 treatment, supporting that TGF-β1 can induce A549 cells EMT.(2) The effects of As2O3 intervention and miR-98-transfected A549 cells on TGF-β1-induced EMT.We observed that As2O3 intervention and miR-98-transfected A549 cells can reduce TGF-β1-induced morphological changes of A549 cells. Immunofluorescence detection displayed that, As2O3 intervention and miR-98 treatment can promote expression of epithelial cell marker protein E-Cadherin, while reduce expression of interstitial marker protein a-SMA.(3) As2O3 intervention and miR-98 treatment can inhibit the Jak-Stat3 signaling pathway activation and expression of Bax/Bcl-2.Western Blotting results suggested that miR-98 and As2O3 intervention could effectively inhibit EMT by reducing the expression of Stat3 and p-Stat3 and ratio of Bax/Bcl-2.