The Impact Of 1G6-D7,the Anti-eHsp90α Monoclonal Antibody,on BLM-induced Pulmonary Fibrosis

Background:Pulmonary fibrosis is a kind of difussed lung disease with unknown pathogenesis.It is a refractory disease with poor prognosis and its incidence is increasing sharply.Nevertheless,effective treatment for this disease is still not available.Hence,it’s urgent that we keep on exploring the mechanism involved in pulmonary fibrosis and developing novel and safer treatment.Damage of alveolar epithelium,severe inflammation,persistent activation and migration of lung fibroblasts,pathological deposition of collagen pathological deposition are important processes of pulmonary fibrosis.So far,researches about the mechanism of lung fibrosis are focused on fuctional change of lung fibroblast.Heat shock protein 90(Hsp90)participates in the tissue wound-healing process.Hsp90 inhibitors is reported to effectively attenuate various kinds of organ fibrosis,but its severe side effects hamper its accessibility to clinical utility.In recent years,it is revealed that,extracellular Hsp90a(eHsp90a),a new form of Hsp90,could be secreted by cells under various stress attack and plays an irreplaceable role in promoting wound healing,tumor invasion and metastasis via binding to LRP-1 outside.However,the role of eHsp90a in pulmonary fibrosis remains unclear.Our research group has the monoclonal antibody of eHsp90a-1G6-D7,which was generously contributed by Wei Li from university of southern California.Therefore,We aim to explore:1.the expression of eHsp90a in lung fibrosis,whether eHsp90a plays a role in BLM-induced pulmonary fibrosis and the mechanism involved;2.whether 1G6-D7 ameliorates lung fibrosis.Method:1.Animal experimentsC57BL/6 mice were divided into four groups:Control(Ctrl)group,BLM group,1G6-D7 group,1G6-D7+BLM group.Mice in BLM group and 1G6-D7+ BLM group were intratracheal injected with BLM sulfate,while those in control group were injected with equal volume of saline.Mice in the 1G6-D7+BLM group and 1G6-D7 group received intranasal inhalation of 1G6-D7 for 5 days consecutively every week.Three weeks later,all the mice were sacrificed.Blood and Bronchial alveolar lavage fluid(BALF)sample was collected for Elisa detection of serum eHsp90a,BALF eHsp90a,BALF IL-6,BALF IL-4 and BALF IFN-y.H&E staining,Masson’s trichrome staining and Ashcroft score were used to assessed the degree of fibrosis.Immunohistochemistry and Western blot were used to detect expressions of a-SMA and collagen I.Expressions of LRP-1,p-ERK/ERK,p-Akt/Akt,p-P38/P38 were detected by Western blot.2.Cell experimentsHuman lung fibroblasts(HFL-1)were cultured in F12K medium supplemented with 10%fetal bovine serum.When HFL-1 reached the confluence of 80%-90%,they were stimulated with TGF-β1 with or without pretreatment of 1G6-D7.The condition medium of HFL-1 stimulated with TGF-β1 were collected and then concentrated with ultrafiltration device to detect secretion of eHsp90a.And the cell lysates were prepared for Western Blot analysis of a-SMA,collagen I,and LRP-1,p-ERK/ERK,p-Akt/Akt and p-P38/P38.Results:1.Pulmonary fibrosis murine model and TGF-β1 induced HFL-1 secreted elevated extracellular Hsp90a:Both BALF eHsp90a and serum eHsp90a were upregulated in BLM-induced mice.Western blot of condition medium also showed TGF-β1 caused increased secretion of Hsp90a in HFL-1 cells.2.1G6-D7 ameriolated lung structure damage and collagen deposition induced by BLM:pathological staining showed that 1G6-D7 ameliorated alveolar septum thickening,alveolar epithelium injury and infiltration of inflammatory cells,prevented formation of fibroblastic foci and inhibited collagen secrection induced by BLM.Immunohistochemistry revealed that,a-SMA positive cells in the lung were increased and gathered into fibroblastic foci,and expression of collagen I in both the alveolar epithelial cell and lung fibroblast were upregulated in BLM group.Similar results were observed by Western Blot.Whereas,treatment with 1G6-D7 greatly inhibited expressions of a-SMA and collagen I,which were observed both in vivo and in vitro experiments.3.1G6-D7 could partially restore inflammatory cytokines disorder in BLM-induced mice:BALF IL-6 and BALF IL-4 were upregulated in BLM-induced mice,1G6-D7 interfered the increase of BALF IL-6,BALF IL-4 induced by BLM.However,BALF IFN-y was downregulated in BLM group and 1G6-D7 partially restored this.4.1G6-D7 blocked activation of downst:ream fibrotic signaling proteins such as p-Akt,p-ERK,p-P38 induced by BLM in vivo and TGF-β1 in vitro.Conclusion:1.eHsp90α probably participates in development of pulmonary fibrosis by promoting lung fibroblast activation and extracellular matix secrection.2.1G6-D7 effectively attenuates BLM-induced pulmonary fibrosis,which may be through inhibiting lung fibroblast activation and ECM synthesis via blocking activation of LRP-1 and downstream fibrotic pathway induced by eHsp90α.