Role Of MLCK In Hypertriglyceridemia-associated Acute Pancreatitis In Rats And Interaction With NF-κB,JNK Pathway

CHAPTER ONE STUDY ON THE MODEL OF HYPERTRIGLYCERIDEMIA-ASSOCIATED ACUTE PANCREATITIS IN RATSObjective We designed the project to explore how to establish hypertriglyceridemia-associated acute pancreatitis (HTGP) model.Methods Fifty-six healthy male Sprague Dawley rats were selected and randomly divided into group A (normal fat diet feeding, 16 rats) and group B(high fat diet feeding, 40 rats). When raised four weeks, blood was sampled from retroorbital venous plexus to measure the serum triglyceride (TG) level.Then, group A was randomly divided into two subgroups (each of eight rats), i.e.,the group C (eaqual saline as control) and the group AP (50ug/kg caerulein treatment), And group B was also randomly divided into five subgroups (each of eight rats), i.e., the group HTG+AP1 (50ug/kg caerulein treatment), the group HTG+AP2 (40ug/kg caerulein treatment), the group HTG+AP3 (30ug/kg caerulein treatment), the group HTG+AP4 (20ug/kg caerulein treatment), the HTG group (eaqual saline as control). The pancreas was carefully removed for microscopic examination with Hematoxylin-Eosin Stain and serum amylase was assayed respecrtively in different groups.Results Serum TG levels in group B significantly increased than group A(P<0.05); Compared with the AP group, histological score of the pancreas and serum amylase level in the group HTG+AP1, HTG+AP2 significantly increased(P<0.05), but there were no difference in the group HTG+AP3 and HTG+AP4.Conclusions HTGP may exacerbate the severity of pancreas damage than AP with normal TG levels.CHAPTER TWO ROLE OF MLCK IN HYPERTRIGLYCERIDEMIA-ASSOCIATED ACUTE PANCREATITIS IN RATS AND INTERACTION WITH NF-κB, JNK PATHWAYObjective To observe the changes of pathological scores of pancreas,serum amylase, ultrastructure and tight junction (TJ), and detect the expressions of MLCK, p-JNK, IKKβ, NF-κB p65, NF-κB p-p65 in pancreas in HTGP.Besides, the aims were to investigate the role of MLCK and interaction with TJ,NF-κB and JNK pathway in HTGP.Methods Forty-eight male SD rats were randomly divided into group D(normal diet feeding) and Group E (high-fat diet feeding). When raised for four weeks, blood was harvested from retroorbital venous plexus to measure serum triglyceride (TG) level. Then group D was randomly divided into three subgroups: C, AP, AP+ML-7, and the group B was also randomly divided into three subgroups: HTG,HTG+AP, HTG+AP + ML-7. All rats were sacrificed twenty-four hours after the last injection of cerulein. The pancreas was carefully removed for HE staining and transmission electron microscope to observe the morphological change, ultrastructure and tight junction (TJ). Blood was obtained to measure the serum amylase level. The expression of MLCK, IKK(3,NF-κB p65 were detected by western blot, and the expression and localization of MLCK, p-JNK, NF-κB p65, NF-κB p-p65 in the pancreas were detected by immunohistochemistry. Bisides, serum TNF-α and IL-1-β levels were tested by ELISA.Results Compared with group D, serum TG level was significantly increased in group E after fed by high fat diet (P<0.05). The pathologic scores of pancreas and serum AMY level were significantly increased in group HTG+AP than group AP (P<0.05). Compared with group AP, the expressions of MLCK, NF-κB p65, IKKα, NF-κB p-p65, p-JNK of pancreas were significantly elevated in group HTG+AP (P<0.05), that were in consistent with pathologic scores of pancreas; The ultrastructural of pancreas in group HTG+AP and AP were damaged and the TJ was broadened, in which the TJ of the group HTG+AP was the widest. Bisides, serum TNF-a and IL-1-β levels were remarkably up-regulated in group HTG+AP, compared with group AP (P<0.05);ML-7, an inhibitor of MLCK, notably ameliorated the pathologic signs of pancreas and down-regulated AMY level (P<0.05), especially more decrease in group HTG+AP, and also improved the TJ. In addition, it also improved the TJ ,decreased the expression of NF-κB p65, IKKβ, p-p65, p-JNK, and down-regulated serum TNF-a and IL-1-β levels (P< 0.05),especially more decrease in group HTG+AP.Conclusions MLCK may exacerbate the severity of damage of pancreas in HTGP. Meanwhile, it may accelerate HTGP by broadened cell-cell TJ and activation of NF-κB, JNK pathway.