| Part One Expression and significance of serum ghrelin and obestatin in patients with acute pancreatitis in early stageObjective To observe the serum levels of ghrelin and obestatin in patients with acute pancreatitis(AP)in early stage and their relationship with disease severity,pathogenesis and clinical features.And then,we evaluate the role of ghrelin and obestatin as a predictor in assessment the severity of AP in early stage.Methods A total of 193 consecutive patients with AP and 24 healthy controls were included.Patients were divided into three groups,namely,mild acute pancreatitis(MAP)group,moderately severe acute pancreatitis(MSAP)group,and severe acute pancreatitis(SAP)group.Serum levels of ghrelin and obestatin were measured on the first,third,and fifth days after patients hospitalization and only one time for healthy controls.The concentrations of ghrelin and obestatin on the first day of hospitalization were analyzed and compared according to the etiology and severity of AP.The predictive value of serum ghrelin and obestatin levels on the first day in AP was examined using receiver-operating characteristic(ROC)curves.Results1.There were 94(48.70%)cases of MAP,70(36.27%)cases of MSAP and29(15.03%)cases of SAP in this study.Based on etiology classification,there were 67 cases(34.72%)of biliary AP,61(31.6%)cases of alcohol related AP,25(12.95%)cases of hypertriglyceridemia AP,40(20.75%)cases with other or unknown etiology AP.The propotions of MAP in the group of biliary AP,alcohol related AP,hypertriglyceridemia AP and other AP were 59.7%,45.9%,20% and 52.5%,respectively.2.The serum consentration of triglyceride were significantly higher in SAP group than that in MAP group or MSAP group(all P<0.05).The level of triglyceride was higher in hypertriglyceridemia group than that in the other three groups(all P<0.01).The serum White blood cell(WBC),neutrophil(N),albumin(ALB),serum calcium(Ca)in AP patients with different etiology or severity of the groups were not significantly different.3.The Ranson score in SAP group was significantly higher than that in MAP group or MSAP group(all P<0.05).The MCTSI score in MAP group was significantly lower than that in MSAP group or SAP group(all P<0.01).The BISAP score in SAP group was significantly higher than that in MAP group(P< 0.05).The BISAP score in the hyperlipidemic group was significantly higher than that in other etiology or etiology of unknown etiology(P<0.05).3.Serum ghrelin levels were dramatically lower in patients with MAP,MSAP,and SAP than those in healthy controls(all P < 0.001).With AP severity increasing,serum ghrelin level decreased and was significantly lower in patients with MAP than in those with MSAP and SAP(P < 0.05 and P < 0.01,respectively).No significant difference in the serum ghrelin level was found between patients with MSAP and SAP.Serum ghrelin gradually increased from the first day to the fifth day.Serum ghrelin levels on the third day were lower in patients with SAP than in those with MAP(P < 0.01);on the third day,no noticeable difference in serum ghrelin level was observed between patients with MSAP and SAP.On the fifth day after treatment,serum ghrelin concentration was found significantly lower in patients with SAP than in those with MSAP or MAP(P < 0.05 and P <0.01,respectively).4.In contrast to ghrelin,patients with AP and healthy controls had similar level of obestatin.No noticeable difference in serum obestatin level was observed among patients with MAP,MSAP,and SAP.However,serum obestatin levels on the first day were significantly lower in patients with AP of hypertriglyceridemic etiology than in those with AP of biliary,alcohol-related,other etiologies and the healthy controls(all P < 0.05).5.Serum obestatin levels on the first day were significantly lower in patients with AP of hypertriglyceridemic etiology than in those with AP of biliary,alcohol-related,or other etiologies.In logistic regression models,serum ghrelin level < 87.83 pg/ml was an independent risk factor for Non-MAP(odds ratio =10.94;95% confidence interval,5.08–23.55;P < 0.01).Conclusions1.Patients with hypertriglyceridemia AP are more likely to develop severe disease.2.Serum ghrelin level in patiens with AP is decreased,and the level I s related with the severity of AP.The concentration of ghrelin may predict the severity of patients with non MAP in the early stage.3.Serum obestatin level is more significantly related with patients with hyperlipidemia AP than other causes of AP.Part two Expression of endogenous ghrelin,obestatin and the status of IKKβ /NF-κB pathway in rats with acute pancreatitisObjectives To observe the expression of serum ghrelin,obestatin and IKKβ /NF-κB signaling pathway in rats with diffenrent severe of AP.Methods Thirty-two male SD rats randomly divided into four model groups: group SAP(n=8),sham operation group(group C-S,n=8),group MAP(n=8),normal saline group(group C-M,n=8).Group SAP was retrograde injected with 4%sodium taurocholic acid into biliary-pancreatic duct inducing the model of acute necrotizing pancratitis.The pancreas and part of intestinal canal of the rats in group C-S were turned light only.Group MAP was intraperitoneal injected of cerulein inducing the model of acute edematous pancreatitis.Group C-M was intraperitoneal injection with saline.All the rats were executed at 24 th hours after the model induction.Pancreatic pathological were routinely observed and scored.The expression of p-p65 and p-IKKβ proteins in pancreatic tissue were evaluated by immunohistochemistry.The Serum amylase(AMS)concentration was detected by automatic biochemical analyzer.Serum ghrelin,obestatin,TNF-α and IL-1β were determined by ELISA.Results1.Pancreatic pathological score,serum AMS,TNF-α and IL-1β concentration in group SAP were significantly higher than that in group C-S or group MAP(all P<0.05).Pancreatic pathological score,serum AMS,TNF-α and IL-1βconcentration in group MAP were significantly higher than that in group C-M(all P < 0.05).2.The concentration of serum ghrelin in SAP group was significantly higher than that in group C-S or group MAP(all P < 0.05),the level of ghrelin in group MAP than group C-M had no significant difference.3.The concentration of serum obestatin in SAP group was significantly lower than that in group C-S or in group MAP(all P < 0.05).However,such level in group MAP and group C-M was similar.4.The expressions of p-p65 and p-IKKβ protein in pancreas in group SAP and group MAP were significantly higher than that in corresponding control group(all P < 0.05),the expressions of p-p65 and p-IKKβ protein in pancreas in group SAP were also significantly higher than that in group MAP(P < 0.05).Conclusions1.The level of serum ghrelin and obestatin may associate with severity of AP in rat.2.The activity of IKKβ /NF-κB signaling pathway was associated with severity of AP in rat.Part three The effect of regulation of ghrelin gene on IKKβ/NF-κB pathway in cell acute pancreatitis modelObjective To observe the effect of endogenous ghrelin and obestatin expression on the IKKβ/NF-κB pathway in acute pancreatitis acinar cells,and to evaluate the effects of ghrelin and obestatin protein on AP.Methods The overexpression and interference vector of ghrelin gene were constructed and packaged into lentivirus vector.Then,ghrelin overexpression and interference lentiviral vector were transfected into rat pancreatic acinar AR42 J cell,respectly,and those infected with mock vector were work as control groups.The transfection efficiency was determined by fluorescence intensity,gene sequencing,q RT-PCR and Western blot.After AR42 J with stable ghrelin interference and overexpression were successfully constructed,the cells were divided into two groups.One group of ghrelin gene interference group,including ghrelin interference stable AR42 J cells(AR42J+ghrelin sh RNA),empty plasmid transfected AR42 J cells(NC),and AR42 J cells.Another group of ghrelin gene overexpression in the experimental group,including ghrelin overexpression of stable AR42 J cells,empty plasmid transfected AR42 J cells(NC)and AR42 J cells.The cells were incubated caerulein inducing AP cells model.The expression of protein p65,p-p65,IKKβ and p-IKKβ protein of IKKβ/ NF-κB pathway and ghrelin were detected by Weston blot.The expression of obestatin,TNF-α and IL-1β protein were detected by ELISA.Results1.q RT-PCR and WB results confirmed that the expression of ghrelin gene and ghrelin protein was up-regulated and down regulated in overexpression and interference AR42 J cells than NC cells.ELISA results showed that the level of obestatin was similar between NC cell with ghrelin overexpression or interference cell.2.In the ghrelin interference experiments group: The levels of IKKβ/NF-κB pathway,ghrelin,TNF-α and IL-1β protein were similar in NC and AR42 J cell regardless of whether the intervention of caerulein,those proteins in those cells with intervention of caerule were significantly increased than unintervention of caerulein(P<0.05).The levels of IKKβ/NF-κB pathway,TNF-α and IL-1β protein were signigicantly increased in ghrelin interference cells than that in NC cells regardless of whether the intervention of caerulein(all P<0.05).The level of ghrelin protein were significantly decreasd in ghrelin interference cells than NC cell regardless of whether the intervention of caerulein(all P<0.05).The consentration of obestatin in cerulein intervention group had no significant difference than those in the untreated group.3.In the ghrelin overexpression experiments group: The levels of IKKβ/NF-κB pathway,ghrelin,TNF-α and IL-1β protein were similar in NC and AR42 J cell regardless of whether the intervention of caerulein,those proteins in those cells with intervention of caerule were significantly increased than unintervention of caerulein(P<0.05).The levels of IKKβ/NF-κB pathway,TNF-α and IL-1βprotein were signigicantly decreased in ghrelin overexpression cells than that in NC cell regardless of whether the intervention of caerulein(all P<0.05).The level of ghrelin protein were significantly increasd in ghrelin overexpression cells than that in NC cell regardless of whether the intervention of caerulein(all P<0.05).The consentration of obestatin in cerulein overexpression group than those in the untreated group had no significant difference.Conclusion1.The inhibition of the expression of ghrelin increased the level of proinflammatory factor TNF-α and IL-1β and the activation of IKKβ/NF-κB signaling pathway in AP cell model.2.The up-regulated ghrelin expression decrease the levels of proinflammatory factor TNF-α and IL-1β and the activation of IKKβ/NF-κB signaling pathway in AP cell model. |
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