Effect Of Chaihuang Qingyi Huoxue Granule On Microcirculation Disturbance In Rats With Severe Acute Pancreatitis

Objective:Preparation of severe acute pancreatitis by taurocholic acid sodium in rats model.Changes in pancreatic histopathological score before and after the intervention of chaihuangqing yishuoxue granules were detected.Changes in serum related indicators,levels of nitric oxide,endothelin,thromboxineA₂ and prostaglandinI₂ in pancreatic tissues,and expression of vascular endothelial growth factor in pancreatic tissues.To investigate the effect of Chaihuang Qingyi Huoxue Granule on pancreatic microcirculation disturbance in rats and its possible mechanism.Methods:The 48 SD rats were divided randomly into three groups:sham operation group（group A）,SAP model group（group B）,and Chaihuang Qingyi Huoxue Granule treatment group（group C）.In group A,abdominal organs were turned around after laparotomy,and the abdomen was closed after 5min.Group B and group C were injected with 3.5%sodium sulfocholate（0.1ml/100g,0.1ml/min）through retrograde cholangiopancreatography through duodenal papillary duct to prepare the rat SAP model.The concentration of prepared Chaihuang Qingyi Huoxue Granule was 0.2g/ml.In group C,the prepared Chaihuang Qingyi Huoxue Granule were gavaged 10ml/kg once every 4 hours（the gavage was stopped from 0 to 6 o’clock）,and in group A and group B,the same amount of normal saline was gavaged once every 4 hours（the gavage was stopped from 0 to 6 o’clock）.After 12 hours and 24 hours of modeling,8 rats were killed for each group,the ascites,saponification spots and necrosis of the pancreas were observed in the abdominal cavity.5ml of blood was taken from the abdominal aorta and the serum amylase（anylase,AMY）level was detected with the supernatant after centrifugation.The rats were killed and the pancreatic tissues were rapidly extracted.A part of pancreatic tissue were cut,some of which were stored in 4%paraformaldehyde solution for HE staining and pathological scoring.Another of the pancreas was mashed with normal saline and centrifugated at 3000 RPM for 10min to obtain the supernatant.The levels of ET,NO,TXB₂ and 6-keto-PGF1αin the pancreatic tissues of the rats were determined by ELISA.The expression of VEGFmRNA in the pancreatic tissues of each group was detected by RT-PCR.Protein expression of VEGF in pancreatic tissue was detected by Western Blot.Results:（1）General observation after laparotomy:In group A,the pancreatic tissues were uniformly gray and white.No edema,congestion or necrosis were observed,and no saponification spots,but partial intestinal adhesions were observed in the abdominal cavity.In group B,obvious intestinal adhesion and gastrointestinal flatulence,obvious hyperemia and edema in pancreatic tissues,lamellar hemorrhage,and large amount of saponification plaques in retroperitoneum,greater omentum and mesenteric roots were observed.Compared with group B,intestinal adhesion,gastrointestinal flatulence,pancreatic tissue congestion,edema and bleeding in group C were significantly reduced,with scattered saponification spots in retroperitoneum and greater omentum.Pancreatic histopathological score:the pancreatic histopathological score of group B and group C at 12h and 24h was significantly higher than that of group A（P<0.05）,and the pancreatic histopathological score of group C was significantly lower than that of group B at corresponding time points（P<0.05）.There was no difference among the three groups（t=1.027,p=0.322;t=0.197,p=0.846;t=1.998,p=0.335）.（2）the serum amylase level comparison:group B and group C at 12 h,24h serum amylase level higher than that corresponding point of group A was statistically significant（p<0.05）,group C at 12 h the serum amylase level compared with group B at 12h there was no significant difference（p>0.05）,the level of serum amylase in group C was significantly lower than that in group B at 24h（p<0.05）.Compared each time point in groups,group A and group C is no difference in the serum amylase group（t=0.672,P=0.214;t=0.730,p=0.447）,and the serum amylase level in group B was significantly higher at 24h than12h（t=2.249,P=0.041）.（3）changes in NO and ET contents and ET/NO ratio（E/N for short）in pancreatic tissues:the contents of ET and NO in pancreatic tissues in group B and group C were significantly higher than those in group A（p<0.05）.The contents of ET and NO in pancreatic tissues at each time point in group C decreased significantly compared with group B corresponding time points（p<0.05）.The contents of ET and NO in the pancreatic tissues of group A were always at low level,with no significant difference between groups（t=0.95,p=0.347;t=0.950,p=0.347）,There was no difference in ET and NO content in pancreatic tissues of group B（t=0.466,p=0.656;t=0.466,p=0.656）.The content of ET and NO in the pancreatic tissues of group C increased significantly at 24h compared with 12h（t=6.544,p<0.001;t=2.745,p=0.029）.The E/N ratio of group B and group C was significantly higher at the time points of 12 and 24 hours than that of group A（p<0.05）.The E/N ratio at each time point in group C was significantly lower than that at the corresponding time point in group B（p<0.05）.Compared each time point in groups,There was no significant difference in E/N ratio in group A（t=1.248,p=0.252）,and the E/N ratio at 24h in group B and group C was higher than that at 12h,with statistically significant difference（t=3.029,p=0.019;t=2.642,p=0.033）.（4）At 12and 24h The content of TXB₂ and 6-keto-PGF1αin pancreatic tissues of group B and group C increased significantly compared with the corresponding time points of group A（p<0.05）,while the content of 6-keto-PGF1αin pancreatic tissues of group C had no significant difference from that of group B at each time point（p>0.05）,and the content of TXB₂ decreased significantly compared with that of group B（p<0.05）.Compared each time point in groups,the content of TXB2 and 6-keto-PGF1αin pancreatic tissues of group A showed no significant difference（t=0.107,P=0.918;t=2.226,p=0.061）.The content of TXB₂ and 6-keto-PGF1αin pancreatic tissues of group B increased significantly at 24h compared with 12h（t=14.639,P<0.001;t=5.072,p=0.001）,The content of TXB₂ in pancreatic tissue of group C was significantly higher than that of group C at 24h（t=11.982,P<0.001）,and there was no significant difference between the 6-keto-PGF1αgroups（t=1.543,P=0.167）.The T/P ratio of group B and group C at 12 and 24h was significantly higher than that of group A（P<0.05）.The T/P ratio of each time point in group C was significantly lower than that in group B（P<0.05）.Comparing two time points within the group,there was no significant difference of T/P ratio between groups A（t=0.456,p=0.662）,The T/P ratio at 24h in group B and group C was significantly higher than that at 12h（t=4.026,p=0.005;t=4.428,p=0.003）.（5）The expression levels of VEGFmRNA and VEGF in pancreatic tissues in group B and group C were significantly higher than those in group A（P<0.05）.The expression levels of VEGFmRNA and VEGF in pancreatic tissues at each time point in group C were significantly lower than those in group B（P<0.05）.Compared with the three groups,the expression levels of VEGFmRNA VEGF in group A were always at a low level,with no significant difference between groups（t=0.883,P=0.392;t=0.334,P=0.743）.The expression levels of VEGFmRNA and VEGF in group B were significantly increased at 12h,and increased gradually with time,the difference between 24h and 12h was significant（t=2.840,P=0.013;t=2.272,P=0.039）,VEGF and VEGFmRNA in group C showed the same changes,the expression level increased significantly at 12h,and increased gradually with time,the difference between 24h and 12h was significant（t=2.265,P=0.040;t=2.262,p=0.040）.Conclusion:1 Chaihuang Qingyi Huoxue Granule can improve the pathological damage degree of pancreatic tissue in SAP model rats.2Chaihuang Qingyi Huoxue Granule may inhibit the expression of VEGF protein in pancreatic tissue and down-regulating the ratio of ET/NO,TXA₂/PGI₂ of vasoactive substances in pancreatic tissues,and finally improve the pancreatic microcirculation disorder of SAP model rat.