Cav1.2 And Cav2.2 Expressions Are Regulated By Different Endogenous Ghrelin Levels In Pancreatic Acinar Cells During Acute Pancreatitis

CHARPTER ONECHANGES OF SERUM GHRELIN AND ITS RELATIONSHIP WITH CAV1.2 AND CAV2.2 EXPRESSION OF PANCREATIC TISSUE IN ACUTE PANCREATITIS RATSObjective To observe the changes of serum ghrelin in rats with acute pancreatitis and its relationship with Cav1.2(a subtype of L-type calcium channel)and Cav2.2(N-type calcium channel)expressions of pancreatic tissue in acute pancreatitis rats.Methods Thirty Sprague Dawley rats were randomly divided into five groups: the normal group,an acute edematous pancreatitis(AEP)group,an AEP-control group,an acute necrotizing pancreatitis(ANP)group and an ANP-control group.AEP was induced by the administration of 50μg/kg of caerulein with intraperitoneal injections seven times per day in one-hour intervals,and the same volume of normal saline was injected intraperitoneally inthe AEP control rats.ANP was induced by the injection of 4% sodium taurocholate into a biliopancreatic duct.The pancreas and duodenum treatments were switched as in the ANP-control group.All the rats were sacrificed at 24 hour after AP induction.Pathological changes in pancreatic tissue were observed and scored.Cav1.2 and Cav2.2 expressions were assessed by immunohistochemistry in the pancreatic tissues of rats;ghrelin,interleukin 1 beta(IL-1β)and tumor necrosis factor alpha(TNF-α)serum levels were detected using ELISA.Results(1)In AEP group,pancreatic edema and infiltration of inflammatory cells were observed under light microscope,The pathological score was higher than that in AEP-control group and normal group(P<0.05);In ANP group,pancreatic necrosis was observed in rats,and pancreatic edema,inflammatory cell infiltration,hemorrhage and necrosis were observed under light microscope,Pathological scores were higher than those in the other groups(P<0.05);(2)The serum ghrelin level of ANP group was significantly higher than that of other groups(P<0.05),and the serum ghrelin level of AEP group was higher than that of AEP-control group and normal group(P<0.05);(3)The serum IL-1β level of ANP group was significantly higher than that of other groups(P<0.05),there was no difference between the other groups;(4)The serum TNF-α of group ANP was higher than that of other groups except AEP group(P<0.05);(5)The IRS of Cav1.2 and Cav2.2 were higher in the ANP group compared with the other groups(P<0.05),IRS scores in the AEP rats were higher than those obtained for the AEP-control and normal rats(P<0.05);(6)Serum ghrelin levels were positively correlated with pancreatic histopathological score,serum IL-1β,as well as pancreatic tissue Cav1.2 IRS and Cav2.2 IRS.Conclusions The levels of serum ghrelin,IL-1β,TNF-α and the expressionof Cav1.2,Cav2.2 increased in ANP rats,and serum ghrelin levels were positively correlated with pancreatic histopathological score,serum IL-1β,as well as the expressions of Cav1.2 and Cav2.2 in pancreatic tissue.Meanwhile,serum ghrelin levels might be related to the severity of AP.CHARPTER TWO EFFECTS OF GHRELIN ON CAV1.2 AND CAV2.2EXPRESSIONS IN PANCREATIC ACINAR CELLSObjective Stably transfected AR42 J cell clones with ghrelin overexpression or knockdown were established in order to study the effect of different ghrelin gene expression on calcium channels and intracellular calcium concentration in AR42 J cells.Methods(1)AR42J cells were transfected with ghrelin-overexpressing vector,knockdown ghrelin short hairpin RNA(sh RNA)vector or blank vector(negative control,NC).Lentiviral vector encoding human ghrelin was generated by cloning ghrelin PCR fragments(full sequence)into a p MD19-T simple vector.The ghrelin sh RNA was cloned into a p Lenti6.3-IRES2-EGFP vector.Then,cells were infected with lentiviral.Stable ghrelin overexpression or knockdown cell lines were then isolated and verified using western blot.Finally,stably transfected AR42 J cell clones with ghrelin overexpression or knockdown were chosen for subsequent experiments;(2)AR42J cells were divided into five groups:AR42J+ghrelin group、AR42J+ghrelin NC group、AR42J group、AR42J+ghrelin shRNA NC group、AR42J+ghrelin shRNA group.Expression of ghrelin protein in each group was detected by Western Blot.[Ca2+]i in each group was determined by calcium fluorescence probe Fluo-4-am loading combined with laser scanning confocal microscope(LSCM).Results(1)Stable ghrelin knockdown in AR42 J cells resulted in low ghrelin protein expression,whereas cells transfected with an empty vector(ghrelin sh RNA NC)had similar ghrelin expression as that detected for control untransfected AR42 J cells(p<0.05).Additionally,the stable ghrelin overexpression in AR42 J cells resulted in high ghrelin expression,whereas the cells transfected with an empty vector(ghrelin NC)had a low ghrelin expression that was similar to the control untransfected AR42 J cells(p<0.05);(2)Compared with the control untransfected AR42 J cells,Cav1.2 and Cav2.2 expressions decreased in AR42 J cells with ghrelin knockdown,whereas the expressions of these calcium channels were increased in AR42 J cells with ghrelin overexpression(p<0.05);[Ca2+]i showed the same trend as the expressions of these two calcium channels in AR42 J cells.Conclusions The [Ca2+]ilevels mediated by Cav1.2 and Cav2.2 expressions were regulated by different ghrelin expression in pancreatic acinar cells.