| Objective: To establish the rat model of SAP and inhibit the release of HMGB1 by ethyl pyruvate(EP)and investigate the mechanism of HMGB1 mediated apoptosis in pancreatic acinar cells and explore its possible mechanism of the protective effect of Qingyi Ⅱ on pancreatic acinar cell apoptosis.Methods: SD rats were randomly(either male or female,200-250 g,n=96)divided into four groups: Sham group(group A,n=24),SAP model group(group B,n=24),SAP+EP group(group C,n=24)and SAP+QY-Ⅱ group(group D,n=24).The rats in sham operation group were only flipped the intestinal canal,the rats in SAP model group were induced by retrograde injection of 5% sodium taurocholate into bilio-pancreatic duct,the rats in group C was given intraperitoneal injection of EP solution(40mg/kg,Once every 6 hours)after the rats were recovered,to fill the stomach with QY-Ⅱ to establish the QY-Ⅱ treated group.Each group was divided into 3 subgroups in 6 hours,12 hours and 24 hours,there were 8 rats in each subgroup.The venous blood and pancreatic tissue were collected at three time points.Light microscope was used to observe the pancreas pathological changes and the pathological score was evaluated by Schimidt scoring standard;HMGB1 concentration in serum was measured by ELISA,real-time quantitative reverse transcription polymerase chain reaction(RT-PCR)to detect the expression of Fasl m RNA and Bcl-2 m RNA in pancreatic tissue,flow cytometry to measure pancreatic acinar cell apoptosis rate,Western blot method was used to detect the content of HMGB1,TLR-4 and NF-κB in pancreatic tissue.Finally,the indexes of each group were statistically analyzed.Results: The pathological score of pancreatitis in SAP model group was higher than the sham operation group(P<0.05).Compared with group A,serum HMGB1 concentration,HMGB1,TLR4 and NF-κB protein expression in pancreatic tissue were increased in group B(P<0.05).The apoptotic rate of acinar cells and the expression of Fas Lm RNA in pancreas were increased at 6h and 12 h time points in group B(P<0.05).The pancreas pathological score and serum HMGB1 concentration increased in group B as time went on(P<0.05).The apoptosis rate of pancreatic acinar cells decreased gradually(P<0.05)and the expression of HMGB1,TLR-4,NF-κB proteins in pancreatic tissues increased from 6h to 12h(P<0.05)and decreased from 12 h to 24h(P<0.05).Compared with group B,the apoptosis of acinar score,serum HMGB1 content,apoptosis rate of acinar cells,the expression of Bcl-2m RNA in pancreatic tissue and pancreatic tissues of HMGB1,TLR4 and NF-κB protein were all decreased in C and D group(P<0.05).The apoptotic rate of Fas Lm RNA and acinar cells in group C and group D(except 6h in group D)increased at the corresponding time points(P<0.05).Group C compared with group D,apoptosis rate of pancreatic acinar cells at 12 hours,expression of HMGB1 protein in pancreas tissues at 6 h and 24 h,and expression of TLR4 protein in pancreas tissues at 12 h and 24 h were higher than group D(P<0.05).The expression of NF-κB protein in pancreas of group D(6h and 12h)and the TLR4 protein in pancreatic tissue were higher than group C(P<0.05);Each time point of pancreatitis pathological score and pancreatic tissue of Bcl-2m RNA and Fas Lm RNA expression,serum concentration of HMGB1,apoptosis rate of pancreatic acinar cells at 6h and 24 h,the expression of HMGB1 protein in pancreatic tissue at 12 h and expression of NF-κB protein in pancreatic tissue at 24 h were all showed no significant difference(P>0.05).Conclusion: Under the expermental conditions,QY-Ⅱ can increase the apoptosis of pancreatic acinar cells and reduce pancreatic tissue injury.The possible mechanism is reducing the concentration of HMGB1 in serum,Prevent HMGB1 from combining with TLR4 which reduced the protein expression of NF-κB,the expression and expression of apoptotic gene Fas L and the decrease of anti-apoptotic gene Bcl-2. |
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