The Impact Of Cdc42 Deficiency In Macrophages In Caerulein-induced Acute Pancreatitis

Objective: Acute pancreatitis（AP）is one of the common abdominal inflammatory disease.AP is often caused by activated auto-digestion by the enzymes released from the pancreas itself,and often is self-limited with mild edema and necrosis.But severe pancreatitis causes systemic inflammatory response and multiple organ failure.Cell cycle protein 42（Cdc42）belongs to Ras superfarmily,as mall GTPase in Rho protein family,which combines with GTP or GDP to be active or inactive.The activated Cdc42 plays variety of biological functions in regulating cytoskeletal reorganization,cell adhesion,cell migration and cell polarization.In this study,we generated macrophage specific Cdc42 knockout mice to investigate the role of Cdc42 in macrophage in acute pancreatitis and its related mechanism.Methods: 1.8-12 week-old mices including macrophage specific Cdc42 KO（F/F+）and Cdc42^flox/flox mice（F/F-）were divided into two groups,caerulein experimental group（or experimental）and control group.2.The acute pancreatitis（AP）model was induced by the caerulein solution,which is an analogue of cholecystokinin（CCK）and prepared in PBS or saline at 200ug/ml as 20 X stock solution.Eight serial intraperitoneally injections of caerulein（50ug/5ml/kg）were executed at hourly intervals,and control mice were injected with the same volume of PBS or saline.Blood,serum,pancreas,lung,and spleen were obtained from individual mice at 1h,8h,14 h post the last caerulein injection.The HE staining and immunohistochemical staining were used to observe the histology and infiltration of immune cells.At the same time,serum amylase,lipase,the chemotactic factor MCP-1 and inflammatory factors IL-6 and TNF-α were examined with the related manufaturer’s kits.Blood routine examination were performed for routine blood cell counts including neutrophil,thrombocyte and lymphocyte,respectively.3.Peritoneal macrophages were collected from caerulein-administered or control mice.Inflammatory genes expression of those macrophage were detected by RT-q PCR.Meanwhile,we cultured peritoneal macrophages from F/F+ and F/Fcontrol mice or RAW264.7 macrophage cell line（with ML141,the inhibitor of Cdc42 for 2 hours）,were stimulated by 0,10,100 and 1000ng/ml LPS for 24 or 6 hous,respectively.The CCR2 and other related genes expression of macrophage were detected by Western blot.Results: 1.There were significant increment of serum amylase and pro-inflammatory cytokine（IL-6 and TNF-α）in Cdc42 KO mice induced by Caerulein.Cdc42 deficiency in macrophages aggravatd the damage in AP in mice.2.The number of CD11b+ macrophages in pancreas significantly increased in Cdc42 KO mice in Caerulein-induced AP.The infiltrated neutraphils in the lungs（pancreatitis related injury of lung）were significantly increased,but the serum lipase and serum MCP-1 levels were similar between two groups.3.The counts of white blood cell and lymphocyte were significantly reduced in mice at 1h after the final caerulein injection,but the portion of neutrophils and thrombocytes were significantly increased.And they gradually returned towards normal level.There were no significant differences between the two groups.4.The expressions of IL-6,IL-1β and TNFα were significantly higher in peritoneal macrophaes from F/F+AP mice at 1 hour post Caerulein induction.5.Inhition of Cdc42 activities by ML141 increased the CCR2 expression under LPS stimulation in RAW264.7 macrophages,and AMPK 、 NF-κB and ERK phosphorylation expression were significantly increased.Meanwhile,CCR2 expression in deleted Cdc42 of peritoneal macrophage administered by LPS was also significantly increased,and AMPK 、 NF-κB and ERK phosphorylation expression were significantly increased..Conclusion: Specific knockout Cdc42 in macrophage aggravated acute pancreatitis induced by Caerulein in mice.Our study revealed that Cdc42 deletion enhanced the production of pro-inflammatory cytokine（IL-6 and TNF-α）in macrophages and increased CD11b+ cells rention in pancreas under caerulein-induced acute pancreatitis via significantly increasing CCR2 expression.