| Part 1. Change and significance of serum thymosin β4 levels is in severe and mild acute pancreatitis patients.Objective:To investigate the change and significance of thymosin β4 levels in severe acute pancreatitis (SAP) and mild acute pancreatitis (MAP) patients.Methods:From January 2012 to November 2013,36 patients with SAP and 40 patients with MAP were enrolled in the study at Renmin Hospital of Wuhan University.20 healthy people served as controls. Blood samples were taken from SAP and MAP patients at the day of admission (day 1) and day 14. For healthy people, only a single blood sample was taken. Blood samples were centrifuged at 3000rpm, and serum was stored at -80℃ until analyses. Serum thymosin β4 levels were measured by human thymosin β4 enzyme-linked immunosorbent assay (ELISA). Clinical data were collected and APACHE II scores were evaluated at day 1.Results:Serum thymosin 04 level in acute pancreatitis patients were higher than healthy people at day 1 (P<0.05). Serum thymosin β4 level in the MAP group was significantly higher than the healthy controls (P<0.01), and lower than the SAP patients (P<0.01) at day 1. There was a positive relationship between serum thymosin β4 level and APACHE II score on admission (r=0.419, P<0.01). Patients with higher serum thymosin 04 level were at a higher risk of developing organ dysfunction (P<0.01). Within the MAP group, the thymosin β4 level was not significantly different at dayl and day 14 (P>0.05). Similar trends were observed in the SAP group (P>0.05).Conclusion:Serum thymosin β4 level was increased in AP patients, particularly in SAP patients. Increased thymosin 04 was linked with poor outcomes of SAP, suggesting thymosin β4 might play an important role in the process of acute pancreatitisPart 2 Change and significance of serum thymosin β4 levels in acute pancreatitis in rat modelsObjective:To investigate the relationship between serum thymosin β4 level and the severity of the acute pancreatitis (AP).Methods:Seventy male Sprague-Dawley rats, weighing 180-200g, were randomly divided into three groups:control group (CON group, N=10), sham operated group (SO group, N=30), severe acute pancreatitis (SAP group, N=30). Prior to the experiment, rats were deprived of food for 12hours, while drinking water was available ad libitum. All rats were performed anesthesia induction in a closed chamber with 5% isoflurane in 2L/min of oxygen, and were maintaining anesthesia for surgery by 2-3% isoflurane in 2L/min of oxygen. The SAP rat model was elicited by a standardized retrograde infusion 1mL/kg body weight of 5% sodium taurocholate (STC) solution into the bile-pancreatic duct. Rats of CON group were sacrificed directly. Rats of SO and SAP group were sacrificed at 3,6,12hours after operation (10 rats at each time point). Blood samples were obtained from inferior vena cava. Blood samples were centrifuged at 3000rpm and serum was stored at -80℃ until analyses. Serum amylase (AMY) levels were detected by automatic biochemistry analyzer. Continuous sections of the paraffin embedded pancreas tissue were taken for pathological examination with hematoxylin-eosin (HE) staining. Morphometric documentation for pancreatic sections under light microscope was evaluated. Serum thymosin β4 and interleukin-1β (IL-1β) levels were detected by ELISA. The correlation between the serum thymosin β4 level and AMY, IL-1β and pathological score was also analyzed.Results:Serum AMY levels of SAP group were significantly higher than CON group and SAP group at each time point (P<0.05). Pathological sections in pancreatic tissue of SAP group were observed in the light microscope. Serum thymosin 04 and IL-1β levels and pathological score of SAP group increased gradually within 12 hours. Serum thymosin β4 and IL-1β levels and pathological score of SAP group at each time point were significantly higher than CON group and SO group (P<0.05). Serum thymosin β4 level are related to AMY, IL-1β level and pathological score (P<0.05).Conclusion:Serum thymosin β4 level was increased in the SAP rat model, and was found to be positively related to disease severity. Bile salt-induced acute pancreatitis model could be used as appropriate animal model for further research of thymosin β4 function in acute pancreaitis.Part 3. Protective effect of recombinant thymosin β4 on severe acute pancreatitis in ratsObjective:The aim of our present study was to investigate the effect and mechanism of thymosin β4 on a rat model of severe acute pancreatitis induced by sodium taurocholate.Methods:Fifty-four male Sprague-Dawley rats were randomly divided into sham operation (SO) group, severe acute pancreatitis (SAP) group, thymosin β4 (Tβ4) pretreatment group, and each group included 8 rats. SAP rat model was established by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. Rats in Tβ4 group were administrated with thymosin (34 (6mg/kg) by intraperitoneal injection prior to SAP model establishment. Rats in three groups were killed at 3h,6h and 12h after operation, and there were six rats in respective time points. The serum level of amylase, tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected, and pathological scores of the head of pancreatic tissues were evaluated under light microscope. Pancreatic myeloperoxidase (MPO) activity assay was performed with MPO kit. The activation of nuclear factor-KB (NF-kB) was evaluated using an immunohistochemistry assay. Pancreatic NF-kB p65, IkB a and ICAM-1 expression were detected by the western blot.Results:Pathological scores of pancreas and serum level of amylase, TNF-a, IL-1β, and IL-6 were increased with time in SAP group (P<0.05). These indexes in SAP group were increased at each time point than in SO group (P<0.05). Compared with SAP group, these indexes in Tβ4 group were reduced at all time points. There were significant differences between them (P<0.05). MPO activity of the SAP group increased significantly at each time point compared with the SO group (P<0.05). For prophylactic administration, thymosin β4 reduced pancreatic tissue MPO activity at 6h to 12h compared with the SAP group (P<0.05). NF-kB p65 expression in Tβ4 group is mainly in the nucleus, but decreased significantly than SAP group.The relative expression of pancreatic NF-kB p65 in SO group were significantly lower than SAP group at 12 hours (P<0.05). The relative expression of pancreatic NF-kB p65 in Tβ4 group were also significantly lower than SAP group at 12 hours (P<0.05). The relative expression of pancreatic IkB a in SO group were significantly higher than SAP group at 12 hours (P<0.05). But the relative expression of pancreatic IkB a in SAP group were significantly lower than Tβ4 group at 12hours (P<0.05).Conclusions:Thymosin β4 has the protective effect on SAP rat model, and the mechanism may be associated with inhibition of NF-kB signaling pathway and decreased proinflammatory cytokines. |
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