Ophicordyceps Lanpingensis Polysaccharides Alleviate The Pulmonary Fibrosis Induced By Bleomycin In Mice

Pulmonary fibrosis is the result of persistent lung injury and abnormal lung tissue remodeling which is characterized by inflammatory diffuse alveolar injury and collagen deposition due to fibroblast differentiation.Although the pathogenesis of pulmonary fibrosis has been explored for 20 years,glucocorticoid and immunosuppressant are still the main treatments in clinical practice,which cause toxic and side effects and can not fundamentally reverse pulmonary fibrosis.Therefore,there is an urgent need to find new methods and strategies for the treatment of pulmonary fibrosis.Ophiocordyceps lanpingensis is a new species of the genus Ophiocordyceps discovered in recent years,which is mainly distributed in Lanping County of Yunnan Province.The local minorities have used O.lanpingensis for health care and treatment of diseases,especially for respiratory diseases,but the pharmacological mechanism is still unclear.Our previous studies have shown that Ophiocordyceps lanpingensis polysaccharides(OLP)may be the main active component of Ophiocordyceps lanpingensis.In this study,OLP was used as the experimental drug to explore the relieving effect and mechanism of OLP on bleomycin-induced pulmonary fibrosis in mice.This study provides theoretical support for the development and utilization of O.lanpingensis resources,and also provides reference basis for the clinical treatment of pulmonary fibrosis.The research content of this paper mainly includes the following two aspects:1.50 C57BL/6 male mice were divided into control group,model group,and OLP low-dose/medium-dose/high-dose groups with 10 mice in each group,a total of 5groups.The pulmonary fibrosis model of mice was established by a single intratracheal infusion of 6 mg/kg bleomycin,and the OLP low,medium and high dose groups were given polysaccharides solution with the doses of 0.3 g/kg,0.5 g/kg and 1.0 g/kg every day.The control group was given the same amount of normal saline.After 21 days of administration,the morphological and structural changes of the lung tissue were observed,and the hydroxyproline(HYP)content and oxidative stress indicators in the lung tissue were detected.The lung tissue was taken for HE and Masson staining in order to observe pathological changes.The results showed that OLP significantly alleviated the disease status of fibrotic mice,relieved lung tissue edema and reduced lung coefficient.Pathological section results showed that OLP could effectively reduce alveolitis and inhibit collagen fiber deposition.In addition,OLP reduced the content of HYP in lung tissue to alleviate collagen deposition,increased the content of superoxide dismutase glutathione,enhanced the antioxidant defense mechanism of lung tissue,and reduced the production of malondialdehyde.The above studies confirmed that OLP could effectively alleviate the progression of bleomycin-induced pulmonary fibrosis in mice.2.Study on the mechanism of OLP in alleviating pulmonary fibrosis in miceReal-time fluorescent quantitative PCR was used to detect the m RNA expression levels of monocyte chemokine factor MCP-1 and macrophage marker genes NOS2,CXCL10/IP10,MARCO,ST2.Furthermore,the gene expression levels of inflammatory-related cytokines TNF-α,IL-1β,IL-6,IL-10 and IL-13 secreted by macrophages,the key fibrosis factor TGF-β1,and the multi-effect cytokine OSM that regulates the expression of inflammatory factors were also analyzed.Immunohistochemical staining,immunofluorescence staining and western blotting were used to detect the protein expression levels of macrophage marker CD68~+、IL-6 and fibroblast marker geneα-SMA.The results showed that OLP significantly down-regulated the expression levels of MCP-1,NOS2,CXCL10/IP10,MARCO and ST2;furthermore,the expression levels of TNF-α,IL-1β,IL-6,IL-10,IL-13,OSM and TGF-β1 were also inhibited.The protein expression levels of CD68~+、IL-6 andα-SMA were also restrained by OLP.These results suggested that OLP alleviated the progression of pulmonary fibrosis by reducing the recruitment of macrophages in the lungs and inhibiting the secretion of cytokines.Fluorescence quantitative PCR was used to detect gene expression levels of autophagy-related genes of Beclin1,ATG7,p62 and autophagy negative regulatory pathway PI3K/AKT/m TOR.Protein immunoblotting was used to detect the phosphorylation level of this signaling pathway.In addition,the expression of MMP-2/TIMP-1,MMP-9/TIMP-1,E-Cadherin,Collagen-I and Collagen-III associated with matrix metalloproteinases were detected to clarify their effects on ECM deposition.The results showed that OLP inhibited the expression of PI3K/Akt/m TOR signaling pathway,promoted the expression of Beclin1 and Atg7,and decreased the expression of p62,thus promoting pulmonary autophagy.Meanwhile,OLP increased the expression of E-cadherin,decreased the expression of Collagen-I,Collagen-III,and maintained the balance of MMP-2/TIMP-1 and MMP-9/TIMP-1.These results suggested that the mechanism of OLP against pulmonary fibrosis might be related to the promotion of autophagy and the maintenance of the balance of matrix metalloproteinase system,and finally achieved the objective of reducing the abnormal deposition of ECM and alleviating the progression of pulmonary fibrosis.Based on the above research results,it is shown that OLP could effectively alleviate BLM-induced lung fibrosis in mice.Its pharmacological mechanisms included OLP could inhibit the oxidative stress status and inflammatory response of lung tissue,reduce the recruitment of macrophages in lung tissue,and thereby reduce the release and influence of cytokines.OLP could also promote autophagy,maintain the balance of the matrix metalloproteinase system,and reduce the abnormal deposition of ECM.Together,the above multiple effects finally alleviated the progression of BLM-induced pulmonary fibrosis.