ERp57 Deficiency Protects Mice From Bleomycin-induced Pulmonary Fibrosis By Regulating Fibroblast Differentiation

Background: Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive,and fibrotic interstitial lung disease caused by unknown causes.Its clinical features are mainly manifested as progressive dyspnea,restrictive ventilatory disorders,and eventually respiratory failure and death.The prognosis of IPF patients is extremely poor with limited treatment options,and the median survival time after diagnosis is only 2-4 years.The pathological manifestations of IPF are mainly characterized by alveolar structural disorder and interstitial fibrosis,and its mechanism is closely related to the repeated damage of alveolar epithelial cells and the proliferation and activation of fibroblasts.Fibroblasts,as the main effector cells,play an important role in the formation of pulmonary fibrosis.In recent years,studies have found that endoplasmic reticulum stress can participate in the occurrence and development of pulmonary fibrosis by regulating the differentiation of fibroblasts.As an endoplasmic reticulum stress chaperone protein,ERp57 can be involved in endoplasmic reticulum stress and correct folding and quality control of newly synthesized glycoproteins,however,the role of ERp57 in IPF disease remains still unclear.Therefore,it is of great scientific significance to explore the mechanism of ERp57 function on the activation and proliferation of fibroblasts and the synthesis and secretion of extracellular matrix.Objective: To investigate the expression and distribution of ERp57 in the lung tissues of IPF patients and bleomycin-induced mice;To investigate the effect of ERp57 inhibitor(Nitazoxanide)on bleomycin-induced lung injury and fibrosis in wild type mice.To explore the effect of ERp57 deficiency or inhibition of ERp57 function on fibroblast activation,in order to initially reveal the role and mechanism of ERp57 in the occurrence and development of pulmonary fibrosis.Methods: Firstly,the expression and distribution of ERp57 in IPF patients and normal lung tissues were detected by Western Blot and immunofluorescence staining.Bleomycin(BLM)was intratracheally injected to induce pulmonary fibrosis of mice,and Western Blot and immunofluorescence staining were used to detect the expression level of ERp57 in mice.In addition,to further explore the effect of Nitazoxanide on BLM induced pulmonary fibrosis in mice,wild-type(WT)mice were divided into four groups:Saline group,DMSO group,BLM+DMSO group,and BLM+Nitazoxanide group.On the 21 st day after BLM treatment,mice were sacrificed.The severity of pulmonary fibrosis was evaluated by H&E,Sirius Red,Masson staining and Ashcroft scores,respectively.Furthermore,the fibrosis related indicators(collagen I,fibronectin)in lung tissues were detected by Western Blot and q PCR.In vitro,primary fibroblasts from WT mice and normal people were extracted.After 12,24 and 48 h of TGFβ1 stimulation,the expression level of ERp57 and α-SMA were detected by Western Blot.At the same time,we used ERp57 si RNA or ERP57 inhibitor to inhibit the function of ERp57 and further detect the effect of deficiency of ERp57 function on fibroblast activation.Results: Compared with normal controls,the expression of ERp57 was significantly upregulated in IPF patients and BLM-induced mice,which was mainly distributed in fibroblasts.Compared with BLM+DMSO group,pathological staining,Western Blot,and q PCR results showed that Nitazoxanide can attenuate BLM-induced lung injury and fibrosis in mice,with reduced accumulation of extracellular matrix and collagen.At the same time,Western Blot and q PCR showed that the protein and m RNA expression levels of collagen I and fibronectin were also significantly decreased in BLM plus Nitazoxanide treatment group.In mouse and human primary fibroblasts,the expression of ERp57 and α-SMA was steadily increased after TGF-β1 stimulation.In addition,ERp57 knockdown or inhibition of ERp57 function could significantly reduce the expression levels of fibrosis related indicators: collagen I,fibronectin and α-SMA,and inhibit the secretion of extracellular matrix.Conclusion: The deficiency of ERp57 function may alleviate bleomycin-induced lung injury and fibrosis in mice through inhibiting the activation of fibroblasts and the synthesis and secretion of extracellular matrix,thereby participating in the pathogenesis of IPF.