| Objective: Through controlled experimental studies,observing the effects of Chaihuang Qingyi Huoxue granule on serological indicators,pancreatic histopathological scores and pancreatic acinar cell apoptosis index of SAP model rats.To discuss the therapeutic effect of this drug on SAP model rats and its effects on apoptosis of pancreatic acinar cell.At the same time,by detecting the expression of apoptosis-related protein cleaved Caspase-3 and Survivin in the pancreatic tissue to further studing the possible regulatory mechanism,to provide experimental and theoretical basis for the development of new drugs and wider clinical application of Chaihuang Qingyi Huoxue granule.Methods: Forty-eight SD rats were randomly divided into three groups:a sham-operation group(Sham group),a severe acute pancreatitis model group(SAP group),and a Chaihuang Qingyi Huoxue granule treatment group(CHQY group).Each group of 16.Rat models of SAP group and CHQY group were injected with 3.5% sodium taurocholate(calculated at 0.1ml/100g)via duodenal retrograde pancreaticobiliary duct.Rats in Sham group didn't need to be injected with sodium taurocholate.The abdomen was closed after turning the abdominal organs several times.After anaesthesia recovery,the rats of CHQY group were gavaged with Chaihuang Qingyi Huoxue granule(calculated at 0.2g/100 g,diluted to 1ml/0.2g)every 4 hours,and the rats of Sham group and SAP group were given saline(calculated at 1ml/100g)every 4 hours.Rats ofThree groups were sacrificed at 12 h and 24 h after surgery,8 rats at each time point.5ml of abdominal aortic blood was collected and centrifuged to detect the levels of serumamylase(AMY)and lipase(LIP).Pancreatic tissue of rats was removed completely,part of the pancreatic tissue was fixed in 4% parafor maldehyde.After 24 hours,paraffin embedding,sectioning and HE staining were performed,and pathological scores were obtained.Another part of pancreatic tissue was prepared to paraffin sections,and the apoptosis of pancreatic acinar cells was detected by TUNEL.Then using formula to calculate the Apoptosis Index(AI).Western Blot was used to detect the expression of cleaved Caspase-3 protein and Survivin protein in rest pancreatic tissue.Statistical method: Statistical software SPSS 25.0 is used for statistical analysis.Quantitative data is expressed as meanąstandard deviation((?)ąs).When the data is normally distributed and the homogeneity of variance test is P>0.05,one-way ANOVA is used for comparison between multiple groups,the LSD method is used for further pairwise comparisons,and the t-test is used for pairwise comparisons within the groups;When the data is non-normally distributed or the variance is not uniform,the non-parametric rank sum test is used.P<0.05 indicates that the difference is statistically significant.Results:(1)General observation after laparotomy:There was no obvious fluid accumulation in the abdominal cavity of rats in the Sham group at all time points,and organs were basically normal.A large amount of red and yellow fluid accumulation was observed in the abdominal cavity of the rats in the SAP group at each time point,and the pancreas was swollen,congested,and necrotic.Bowel was swollen and congested.A large number of yellow-white saponification spots could be seen in the pancreas,adjacent tissues and omentum.Bdominal organs adhesion was serious.The amount of peritoneal effusion in the CHQY group was less than that in the SAP group,and the degree of organs damage and organs adhesion was lighter than that in the SAP group.(2)Comparison of each item scores and total scores of pancreatic histopathology:The scores of edema,bleeding,necrosis,inflammation and cavitat ion in SAP group and CHQY group at each time point were all higher than those in Sham group,and the differences were statistically significant(P<0.05).Comparison of total pathology scores of the SAP group at two time points,the total pathology scores at 24 h were significantly higher than those at 12h(t=4.583,P<0.001).The scores of edema,necrosis,and vacuole at 12 h in the CHQY group were significantly lower than those in the SAP group at 12h(Z=-2.128,P=0.033;Z=-2.199,P=0.028;Z=-2.199,P=0.028),but there was no significant difference in the scores of two items of inflammation and bleeding(Z=-0.447,P=0.655;Z=-0.810,P=0.418).The scores of edema,inflammation,and bleeding at 24 h in the CHQY group were lower than those in the SAP group at 24h(Z=-2.640,P=0.008;Z=-2.513,P=0.012;Z=-3.019,P=0.003),while there was no significant difference between the scores of necrosis and cavitation(Z=-1.264,P=0.206;Z=-1.073,P=0.283).The total pathology scores in the CHQY group at 12 h and 24 h were significantly lower than those in the SAP group at corresponding time points(t=4.640,P<0.001;t=9.031,P<0.001).(3)Comparison of serum amylase level:The serum amylase level of SAP group and CHQY group at 12 h and 24 h was significantly higher than that of Sham group,and there was statistical difference(P<0.05).There was no significant difference in serum amylase level between CHQY group and SAP group at 12h(Z=-1.575,P=0.115).The serum amylase level in CHQY group at 24 h was significantly lower than that in SAP group at 24h(Z=-2.521,P=0.012).(4)Comparison of serum lipase level:The serum lipase level of SAP group and CHQY group at 12 h and 24 h was higher than that of Sham group,and the difference was statistically significant(P<0.05).The serum lipase level in CHQY group at 12 h was not significantly different from that in SAP group(Z=-1.680,P=0.093);The serum lipase level in CHQY group at 24 h was lower than that in SAP group at 24 h,which was statistically different(Z=-3.151,P= 0.002).(5)Comparison of pancreatic acinar cell apoptosis indexes:Apoptosis indexes of SAP group and CHQY group were all higher than those of Sham group at 12 h and 24 h,with statistically significant difference(P<0.05).Comparison within SAP group,the apoptosis indexs of acinar cells at 24 h were significantly lower than those at 12h(t=3.388,P=0.004).The apoptosis indexs of acinar cells in CHQY group at 12 h and 24 h were significantly higher than those in SAP group at corresponding time points(t=11.795,P<0.001;T=10.209,P<0.001).(6)Comparison of the expression level of cleaved Caspase-3 protein of pancreatic tissue: Cleaved Caspase-3 protein of pancreatic tissue in Sham group was low expressed at all time points.The expression level of cleaved Caspase-3 protein in SAP group and CHQY group was significantly higher than that in Sham group at each time point,and the difference was statisticallysignificant(P<0.05).In SAP group,the expression level of cleaved Caspase-3 protein at 24 h was significantly lower than that at 12h(t=2.209,P=0.044).The expression level of cleaved Caspase-3 protein in CHQY group was higher than that in SAP group at 12 h and 24 h,and the difference was significant(t=3.826,P=0.001;T=3.981,P=0.001).(7)Comparison of the expression level of Survivin protein of pancreatic tissue: Survivin protein of pancreatic tissue in Sham group was lowly expressed at each time point.The expression level of Survivin protein of pancreatic tissue in SAP group and CHQY group was significantly higher than that in Sham group,and the difference was statistically significant(P<0.05).Comparison within SAP group,the expression level of Survivin protein of pancreatic tissue at 24 h was significantly higher than that at 12h(t=2.665,P=0.018).The expression level of Survivin protein of pancreatic tissue in CHQY group at 12 h and 24 h was lower than that in SAP group at corresponding time points,and the difference was significant(t=4.671,P =0.001;t=4.740,P=0.001).Conclusion:(1)The severity of SAP model rats is inversely related to the degree of apoptosis of pancreatic acinar cells.(2)Chaihuang Qingyi Huoxue Granule can increase the occurrence of pancreatic acinar cell apoptosis in SAP model rats,reduce pathological damage and inflammatory response of pancreatic tissues.The therapeutic effects of Chaihuang Qingyi Huoxue Granule on SAP model rats can be achieved by inducing apoptosis of pancreatic acinar cells.(3)The expression level of cleaved Caspase-3 protein in pancreatic tissue is positively correlated with the degree of apoptosis of pancreatic acinar cells,and the expression level of Survivin protein is negatively correlated with the degree of apoptosis in pancreatic acinar cells;The apoptosis of pancreatic acinar cells induced by Chaihuang Qingyi Huoxue Granule in SAP model rats may be related to up-regulation of cleaved Caspase-3 protein expression in pancreatic tissue and inhibition of Survivin protein expression. |
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