BMSC reduces ROS and inflammation levels by inhibiting TLR4/MYD88/NF-κB signaling axis to alleviate dry eye

Abstract

Abstract Objective To investigate the therapeutic effect of Bone marrow mesenchymal stem cells (BMSCs) on dry eye mice, and to investigate the mechanism of TLR4/MYD88/NF-κB signaling pathway on corneal injury repair in dry eye mice. Methods To establish a hypertonic dry eye cell model. Western blot for measureing the protein expressions of caspase-1, IL-1β，NLRP3 and ASC，and Rt-qpcr for mRNA expression. Flow cytometry for detecting the ROS content and apoptosis rate. CCK-8 for detecting the proliferation activity of cells, and ELISA for the levels of inflammation-related factors.The levels of inflammation-related factors were detected by ELISA. The dry eye mouse model of benzalkonium chloride was established. Three clinical parameters used to evaluate ocular surface damage, namely tear secretion, tear film rupture time and corneal sodium fluorescein staining, were measured with phenol cotton thread. Flow cytometry and TUNEL staining are both for he apoptosis rate. Western blot also for detecting the protein expressions of TLR4, MYD88, NF-κB, inflammation-related factors and apoptosis-related factors . The pathological changes were evaluated by HE and PAS staining. Results In vitro, BMSCs and inhibitors of TLR4, MYD88 and NF-κB showed decreased ROS content, decreased inflammatory factor protein level, decreased apoptotic protein level and increased mRNA expression compared with NaCl group. BMSCS partially reversed cell apoptosis induced by NaCl and improved cell proliferation. In vivo, it reduces corneal epithelial defects, goblet cell loss and inflammatory cytokine production, and increases tear production. In vitro, BMSC and inhibitors of TLR4, MYD88 and NF-κB could protect mice from apoptosis induced by hypertonic stress. In terms of mechanism, NACL-induced NLRP3 inflammasome formation, caspase-1 activation and IL-1β maturation can be inhibited. Conclusion BMSCs treatment can reduce ROS and inflammation levels and alleviate dry eye by inhibiting TLR4/MYD88/NF-κBsignaling pathway.