Deletion Of Cdc42 Gene In Macrophages Aggravates The Inflammation Induced By LPS In Mice

Objective: Inflammation is a defensive reaction of the organism to external stimulus.It is involved in the pathological process of obesity,atherosclerosis and other metabolic diseases and cardiovascular diseases.Macrophages are present in the tissue where they provide a first line of defense against pathogens and help to maintain homeostasis by the specific functions.Cdc42 is a member of the Rho GTPase family.As a molecular switch,Cdc42 plays a role in the transformation between an inactive GDP-bound form and an active GTP-bound form in response to diverse signals.The downstream effectors of Cdc42 have revealed a number of kinases such as PAKs,MLKs,and MRCKs.The effectors activated downstream of Cdc42 set in motion a variety of signaling cascades initiating changes in cellular processes including cell polarity,cytoskeletal remodeling,proliferation,migration,adhesion.Although it has been reported that Cdc42 affects the inflammation,the effect of macrophage-specific knockout of Cdc42 on inflammation is unknown.The aim of this study is to investigate the effect of Cdc42 deficiency in macrophages on LPS-induced inflammation,and explore its molecular mechanisms.Methods and Results: 1.Preparation and identification of macrophage-specific Cdc42 knockout mice: the Cdc42/Flox mice and Lyz-cre mice were backcrossed to generate macrophage-specific Cdc42 knockout mice.The PCR results showed that the macrophage-specific Cdc42 knockout mice were successfully prepared.In addition,the western blot analysis showed that the Cdc42 protein in macrophages was reduced to 10% of the macrophages in control mice.2.Preparation of the shock mouse models induced by LPS and detection: the shock mouse models were generated by injection of lipopolysaccharide(LPS,20mg/kg)intraperitoneally with both macrophage-specific Cdc42 knockout mice and wild type mice.The results showed that the mortality of the macrophage-specific Cdc42 knockout mice induced by LPS was significantly increased,and at 108 th hours after administration of LPS,the mortalities of the null mice group and control group were 77.8% and 44.4%,respectively.The injury of organs including liver,lung,kidney and spleen from the macrophage-specific Cdc42 knockout mice was more serious compared with wild type mice after mice after injection of LPS 6h by HE staining,the ratio of spleen weight and body weight in the Cdc42 knockout mice was significantly higher than the control group.The blood examination showed that the numbers of white blood cells and lymphocytes in Cdc42 specific knockout mice were significantly higher than that of control mice.3.Effects of Cdc42 deletion on LPS-induced cytokine releases in mouse macrophage: three or 4 ml of 4% sulfur colloid solution were injected into each mouse intraperitoneally and the macrophages were collected after injection of 5days.The mouse peritoneal macrophages were treated for 12 hours with 100 ng/ml LPS and the expressions of cytokines including TNF-alpha,IL-1 beta,IL-6,IL-10 were examined by QPCR.In addition,the possible signaling pathways were determined in the cells.The results showed that the expressions of inflammatory factors in macrophages of the Cdc42 conditional knockout mice induced by 12 hours of LPS stimulation were significantly higher than the control group.In addition,the expressions of P-P38,P-NF-kappa B protein in the primary macrophages of Cdc42 knockout mice after 16 hours of LPS stimulation were higher than that of the control group,and the expressions of the P-ERK and P-PI3 K and P-AKT proteins were lower than the control group.Conclusion: 1.Macrophage-specific Cdc42 deletion aggravated the inflammatory injury induced by LPS in mice.2.The mechanism of Cdc42deletion-mediated aggravation of the LPS-induced inflammatory injury may be related with activations of P38 and NF-?B pathways in macrophages.