The Impact Of Bile Acid To Pancreatic Acinar Cell And The Regulation Function Of Emodin

Background and Objective:Acute pancreatitis is a common, potentially life-threatening disease. In most reports, gallstone disease is the lead-ing cause being responsible for roughly 30-50% of cases of acute pancreatitis. In acute biliary pancreatitis, the reflux bile acid plays important role in the path-ogenesis of disease.To investivate the impact of seven different components of bile acid onpancreatic acinar cell, and detect the survival rate ofpancreatic acinar cell with bile acid, the change of necrosis and apoptosis, and the impact on the secretion function of pancreatic acinar cell, the binding activity of the nuclear transcrition factor to DNA, and the protection effect of Emodin on the acinar cell damage induced by bile acid; at the same time, we further investivate the impact and mechanism of pancreatic damage by bile acid during the development of acute biliary pancreatitis, and the threapy efficiency of Chinese medicine QingYiTang Decoction, to reveal the mechanism and pathological development of acute biliary pancreatitis and provide more information on more effective therapy method.Methods:Part1: By using rat AR42J pancreatic acinar cell line, we detected the impact of seven different bile acid on the cell survival rate and time dependency with MTT assay. The morphological change of the cells and cell apoptosis or necrosis were observed with microscope and fluorescence microscope. The rate of apoptosis or necrosis was deter-mined by AV/PI double staining and cell flowmetry. After treatment of the cells with 0.4mM DCA for 15min,30min,1h or 4h, the medium were collected to detect the activity of amylase. And the cytoplamic and nuclear proteins were extracted to detect the activity of amylase. Then we detected the DNA binding activity of 40 nuclear transcription factor by Luminex.Part 2: Emodin is an important element in Chinese medicine to cure acute pancreatitis. We are to observe the impact and pretection function of Emodin on pancreatic acinar cell damage induced by bile acid. Rat AR42J pancreatic acinar cell lines were divided into three groups: Con group, the group treated wth 0.4 mM DCA treatment and the group treated with 0.4 mM DCA+ Emodin ( 20μg/ml ) . And all these samples were treated with 15min,30min,1h or 4h, and then collect the medium and extract cytoplasmic and nuclear proteins and detect the activity of cytoplasic amylase. We used cell flowmetry and AV/PI double staining to detect the rate of apoptosis and necrosis. Then we detected the DNA binding activity of 40 nuclear transcription factor by Luminex.Part 3: The different severity of acute pancreatitis model of SD rat was established by retrograde injection of different concentration of sodium deoxycholate into the common biliopancreatic duct. Rats (n=40) were divided at random into sham operated group, AEP group(0.75% dose group), ANP group(1.5 dose group), ANP+QingYiTang Decoction group(therapeutic dose: 1ml/100g). All the rats were killed at 24 hours after operation. Pathological examination and indexes of AP were measured. The apoptotic index of acinar cells was derermined by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The activity levels of Caspase-3,8 and 9 were ana- lyzed by spectrophotometry. We also measured the level of serum amylase, TNF-αin blood et al.Results:It was found that the impact effect of the 7 bile acids on pancreatic acinar cell was different, DCA, CDCA, LCA, and TDCA had significant time and dose dependent cytotoxic effect on AR42J pancreatic acinar cell, induce cell apoptosis and necrosis. While, CA, GCA, and GDCA didn't have this cytotoxic effect on dose of 0.1mM-1.0mM. Bile acid DCA and CA both had no significant influence on the amylase synthesis and secretion function of acinar cell. Among the DNA binding activity of the 40 different nuclear transcription factors, 0.4mM DCA up regulated the activity of ATF2, AR33, STAT5, NFAT, FKHR, and NKX-2.5, and down regulated the activity of RUNX/AML, NF-Y, MEF2, and E2F1.20μM Emodin decreased the amount of AR42J pancreatic acinar cell apoptosis and necrosis induced by DCA, while didn't influence the amylase synthesis and secretion function of acinar cell. Compared with the change of TF activity induced by DCA, Emodin up regulated the activity of PAX3, E2F1, RUNX/AML, NF-Y, IRF, MEF, and NFAT, but down regulated the activity of FKHR.The apoptotic index and the activity level of Caspase-3, 8,9 in AEP group and ANP group were all significant higher than sham operated group(p<0.05), and these indexes in AEP group were higher than ANP group(p<0.05). Meanwhile, the indexes such as pathohisto- logy score, serum amylase, and TNF-αin blood both in AEP group and ANP group were all significant higher than sham operated group (p<0.01), and those in ANP group were significant higher than AEP group(p<0.01). Compared with the ANP group, QingYiTang Decoction increased the amount of cell apoptosis, decreased the amount of cell necrosis through up regulated the activity of caspases, and alleviated the patho- logical damage of pancreatic tissues.Conclusions:Among the 7 main different bile acids in human bile, DCA, CDCA, LCA, and TDCA can cause AR42J pancreatic acinar cell damage by apoptosis and necrosis in dose of 0.3mM-0.4mM in 24h, significantly decreased the survival rate of acinar cell with dose and time dependent manner. The other 3 bile acids CA, GCA, and GDCA didn't cause acinar cell damage in dose of 0.1mM-1.0mM. Bile acids didn't influe- nce the amylase synthesis and secretion function of acinar cell. had significant time and dose dependent cytotoxic effect on AR42J panc- reatic acinar cell, induce cell apoptosis and necrosis. Meanwhile, CA, GCA, and GDCA didn't have this cytotoxic effect in dose of 0.1mM-1.0mM. Bile acid DCA and CA both had not significant influence on the amylase synthesis and secretion function of acinar cell. DNA binding activity of several nuclear TFs are inducible by DCA, which maybe the molecular mechanism of cell damage caused by DCA.Emodin has some protection effect on AR42J pancreatic acinar cell damage caused by bile acid DCA. It dosn't influence the amylase synthesis and secretion function of acinar cell. Emodin can induce up or down regulate some nuclear TFs DNA binding activity, which maybe the foundation mechanism of its protection effect on cell damage induced by DCA.In acute biliary pancreatitis, the damage of pancreatic acinar cell is the key factor of the disease. Apoptosis and necrosis are the main forms of acinar damage, and the apoptotic index of pancreatic acinar cells was correlated negatively with the severity of pancreatitis. It is suggested that acinar cell apoptosis may be one of the mechanisms of self-defense. Caspase dependent apoptotic pathway attend the regula- tion pathway of pancreatic acinar cell apoptosis. QingYiTang Decoc- tion can up-regulate the level of caspases activity, increase the apo- ptotic index to lessen the pathological lesion of the pancreas and decrease the level of serum amylase, TNF-αto amendment the patho- genetic condition.