PLCε1 mediates one-lung ventilation injury by regulating the p38/RhoA/NFκB activation loop

Background: Phospholipase C epsilon-1 (PLC epsilon 1) might be a novel and potential target in treating inflammatory conditions. In the present study, we aimed to clarify whether PLC epsilon 1 is involved in lung injury caused by one-lung ventilation (OLV) and to elucidate the potential molecular mechanism of PLC epsilon 1-mediated signaling pathway on OLV induced inflammatory response and injury. Methods: Male Sprague-Dawley (SD) rats were divided into wide-type (PLC epsilon 1-WT) group and PLC epsilon 1-KO group, and were treated with OLV for 0.5 h, 1 h, and 2 h respectively. Observation of lung tissue injury in rats was performed by Hematoxylin and eosin (HE) staining and Wet/dry (W/D) radios. In addition, pulmonary microvascular endothelial cells (PMVECs) transfected with PLC epsilon 1-si RNA, were stimulated by lipopolysaccharide (LPS). To explore the possible roles of PLC epsilon 1 in the OLV induced inflammatory injury and the involved pathway underlying, the lung tissue and bronchoalveolar lavage fluids (BALF) of OLV rats, as well as the PMVECs were prepared for further analysis. Enzyme-linked immunoassay (ELISA) was used to detect the expression of proinflammatory factors. The activities of related pathway proteins (NF-kappa B, phospho-p38, p38, phospho-ERK1/2, ERK1/2, RhoA and ROCK) were also detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Results: Compared to the PLC epsilon 1-WT rats, PLC epsilon 1-KOrats exhibited marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, decreases in the expressions of pro-inflammatory mediators, and declines in the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid (BALF). Moreover, the increased expressions of RhoA and NF-kappa B p65 mRNA induced by OLV were significantly inhibited in PLC epsilon 1-KO rats. In LPS treated PMVECs, PLC epsilon 1-si RNA transfection ones also showed the decrease expression of proinflammatory mediators, reduction in p38 phosphorylation levels and downregulation of RhoA/ROCK signaling activation. Co-cultured with PLC epsilon 1-si RNA and BTRB796 (p38 inhibitors) in LPS-stimulated PMVECs resulted in a significant reduction in RhoA and NF-kappa B activity. In addition, treatment with either ROCK inhibitor (Y-27632) or dominant negative mutant of RhoA (RhoT19 N) significantly reduced the expression of NF-kappa B in PLC epsilon 1-si RNA treated PMVECs. Conclusion: The results indicated that PLC epsilon 1 played an important role in the inflammatory response induced by OLV. Moreover, through promoting p38/RhoA/ROCK activation loop, PLC epsilon 1 promoted NF-kappa B activation and thereby increased the expressions of inflammatory mediators, which induced the PMVECs inflammation and subsequent injury. The results of this study provide a potential therapeutic target for the reduction of inflammatory response in patients with OLV.