胆汁酸对胰腺腺泡细胞的损伤与大黄素调节作用的实验研究
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摘要
背景与目的:
急性胰腺炎(acute pancreatitis,AP)的发生、发展和转归是当今外科领域研究的热点,其发病机理尚未完全阐明。其中急性胆源性胰腺炎(acute biliary pancreatitis,ABP)在我国发病率最高,占AP总数的50-80%。目前普遍认为,ABP是由于胆管炎症、结石、寄生虫、水肿、痉挛等病变使壶腹部发生梗阻,加之胆囊收缩,胆管内压力升高,胆汁通过共同通道反流入胰管,激活胰酶原,导致胰腺自身消化而引起胰腺炎。但其确切的发病机制尚未完全明确。可以认为,随胆汁异常反流入胰管的胆汁酸,造成胰腺腺泡细胞功能与结构损害是大多数ABP发病的一个关键性因素。因此,本研究探讨胆汁酸7种不同成分对胰腺腺泡细胞的损伤作用,检测在各种胆汁酸作用下胰腺腺泡细胞的存活率,凋亡/坏死的改变,观察对腺泡细胞分泌功能的影响,并探究细胞核转录因子的DNA结合活性的变化,研究大黄素对胆汁酸诱导的胰腺腺泡细胞损伤的保护性作用;同时,进一步揭示急性胆源性胰腺炎大鼠发病中,胆汁酸对胰腺的损伤性作用及其机制,观察中药清胰汤的治疗作用,为揭示急性胆源性胰腺炎的发病机制和寻求更为有效的防治措施提供新的实验佐证。
方法:
实验一:以大鼠AR42J胰腺腺泡细胞系为研究对象,应用MTT方法检测7种不同胆汁酸对细胞存活率的影响和剂量与时间依赖性关系,采用光学显微镜和荧光显微镜观察细胞形态学改变与凋亡/坏死的变化,流式细胞术AV/PI双染法检测细胞的凋亡/坏死率。细胞经0.4mM DCA作用15min,30min,1h,4h后分别收集细胞培养液上清检测淀粉酶活性,提取细胞浆和细胞核蛋白,分别检测各组细胞浆淀粉酶的活性,利用Luminex检测40种细胞核转录因子(TF)的DNA结合活性的变化。
实验二:大黄素(Emodin)是治疗急性胰腺炎中药方剂的一种主药,通过体外实验观察其对胆汁酸诱导的胰腺腺泡细胞损伤的保护性作用和影响。以大鼠AR42J胰腺腺泡系为研究对象,分为三组,分别为CON组,0.4mM DCA刺激组和0.4mM DCA+Emodin(20μM)干预组,利用流式细胞术AV/PI双染法检测各组细胞凋亡/坏死率,以上各组进一步按照不同时间点分为4组:15min,30min,1h和4h,于相应时间点分别收集细胞培养液上清检测淀粉酶活性,提取细胞浆和细胞核蛋白,分别检测各组细胞浆淀粉酶的活性,利用Luminex检测40种细胞核转录因子(TF)的DNA结合活性的变化。
实验三:经胆胰管逆行注入不同浓度去氧胆酸钠制备大鼠急性水肿性胰腺炎(AEP)和急性坏死性胰腺炎(ANP)模型。40只SD大鼠随机分为假手术组、AEP组、ANP组及对ANP的清胰汤治疗组(1ml/100g),每组10只。治疗组大鼠于造模后立即一次和造模后12h再次给药。24h后采取标本做病理检查和各项指标检测。光镜下观察各组胰腺标本病理改变,进行组织学评分;采用TUNEL技术检测胰腺腺泡细胞的凋亡,计算凋亡指数;应用分光光度法检测凋亡蛋白酶Caspase-3,8和9的活性变化。
结果:
胆汁酸7中主要成分对胰腺腺泡细胞的损伤作用不同,其中DCA,CDCA,LCA和TDCA 4种胆汁酸细胞毒作用明显,呈剂量和时间依赖性诱导腺泡细胞凋亡和坏死,CA,GCA和GDCA则不具有细胞毒作用。胆汁酸DCA和CA对腺泡细胞淀粉酶的合成与分泌功能均没有显著影响,在检测的40种核转录因子(TF)的DNA结合活性中,DCA诱导AR42J胰腺腺泡细胞核内ATF2,AR33,STAT5,NFAT,FKHR和NKX-2.5这6种核转录因子(TF)的DNA结合活性显著升高,而RUNX/AML,NF-Y,MEF2和E2F1这4种TF的DNA结合活性则明显下降,其余30种TF活性没有明显变化。
20μM Emodin可以减少DCA诱导的AR42J胰腺腺泡细胞的凋亡与坏死,对细胞淀粉酶的合成与分泌功能没有明显影响,同DCA诱导的TF活性变化相比,Emodin升高PAX3, E2F1, RUNX/AML, NF-Y, IRF, MEF和NFAT这5种TF活性,降低FKHR的活性。
大鼠体内实验结果表明,AEP组与ANP组腺泡细胞凋亡指数、Caspase-3,8和9的活性均显著高于假手术组,且AEP组腺泡细胞凋亡指数及凋亡蛋白酶的活性水平显著高于ANP组。同时,AEP组与ANP组病理组织学评分、血清淀粉酶、血清促炎性细胞因子等指标均较假手术组显著升高,且ANP组显著高于AEP组(p<0.01)。清胰汤通过诱导凋亡蛋白酶活性升高使ANP组大鼠胰腺腺泡细胞凋亡增多而坏死减轻,从而减轻胰腺组织损害和炎症因子水平。
结论:
胆汁酸主要的7种成分中,DCA,CDCA,LCA和TDCA在0.3-0.4mM的较低浓度,在24h内可导致大鼠AR42J胰腺腺泡细胞存活率显著下降,这种细胞毒作用呈时间和剂量依赖性关系,而CA,GCA和GDCA这3种胆汁酸则在1.0mM浓度内不会损伤腺泡细胞。胆汁酸对腺泡细胞的损伤作用主要表现为凋亡和坏死,对细胞内酶的合成和分泌功能没有明显影响。研究发现,DCA可诱导细胞6种TF的DNA结合活性显著升高和4种TF的DNA结合活性显著下降,从而影响相关基因的表达水平,是其造成细胞损伤的分子生物学基础。因此,本研究为胆汁酸诱导的胰腺腺泡细胞损伤提供有力的实验依据,并揭示了胆汁酸7种不同成分的作用特点和可能机制,将为探讨胆源性胰腺炎的发病机制和防治措施提供参考。
Emodin对胆汁酸诱导的胰腺腺泡细胞损伤有一定的保护性作用,对细胞淀粉酶的合成与分泌功能没有明显影响。通过调节细胞TF的DNA结合活性,从而改变相关基因和蛋白的表达,可能是其发挥保护性作用的分子机制。
急性胆源性胰腺炎时,胆汁酸诱导的胰腺腺泡细胞损伤时疾病发生的重要因素,腺泡细胞损伤主要表现为凋亡和坏死,且凋亡的多少与病情轻重呈负相关的关系,腺泡细胞凋亡可能是急性胰腺炎时机体的保护机制之一。凋亡蛋白酶Caspase依赖的凋亡调控通路参与腺泡细胞的凋亡调节,Caspase-8和9的活性均升高。清胰汤可通过诱导Caspase活性升高促进腺泡细胞凋亡而减轻ANP大鼠胰腺病理损害,降低血清淀粉酶、TNF-α、IL-6水平等,改善病情。如何有效控制AP发病时腺泡细胞的损伤,特别是减少坏死的发生,将会为临床进一步降低ANP的重症程度和病死率提供可能。
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.
急性胰腺炎(acute pancreatitis,AP)的发生、发展和转归是当今外科领域研究的热点,其发病机理尚未完全阐明。其中急性胆源性胰腺炎(acute biliary pancreatitis,ABP)在我国发病率最高,占AP总数的50-80%。目前普遍认为,ABP是由于胆管炎症、结石、寄生虫、水肿、痉挛等病变使壶腹部发生梗阻,加之胆囊收缩,胆管内压力升高,胆汁通过共同通道反流入胰管,激活胰酶原,导致胰腺自身消化而引起胰腺炎。但其确切的发病机制尚未完全明确。可以认为,随胆汁异常反流入胰管的胆汁酸,造成胰腺腺泡细胞功能与结构损害是大多数ABP发病的一个关键性因素。因此,本研究探讨胆汁酸7种不同成分对胰腺腺泡细胞的损伤作用,检测在各种胆汁酸作用下胰腺腺泡细胞的存活率,凋亡/坏死的改变,观察对腺泡细胞分泌功能的影响,并探究细胞核转录因子的DNA结合活性的变化,研究大黄素对胆汁酸诱导的胰腺腺泡细胞损伤的保护性作用;同时,进一步揭示急性胆源性胰腺炎大鼠发病中,胆汁酸对胰腺的损伤性作用及其机制,观察中药清胰汤的治疗作用,为揭示急性胆源性胰腺炎的发病机制和寻求更为有效的防治措施提供新的实验佐证。
方法:
实验一:以大鼠AR42J胰腺腺泡细胞系为研究对象,应用MTT方法检测7种不同胆汁酸对细胞存活率的影响和剂量与时间依赖性关系,采用光学显微镜和荧光显微镜观察细胞形态学改变与凋亡/坏死的变化,流式细胞术AV/PI双染法检测细胞的凋亡/坏死率。细胞经0.4mM DCA作用15min,30min,1h,4h后分别收集细胞培养液上清检测淀粉酶活性,提取细胞浆和细胞核蛋白,分别检测各组细胞浆淀粉酶的活性,利用Luminex检测40种细胞核转录因子(TF)的DNA结合活性的变化。
实验二:大黄素(Emodin)是治疗急性胰腺炎中药方剂的一种主药,通过体外实验观察其对胆汁酸诱导的胰腺腺泡细胞损伤的保护性作用和影响。以大鼠AR42J胰腺腺泡系为研究对象,分为三组,分别为CON组,0.4mM DCA刺激组和0.4mM DCA+Emodin(20μM)干预组,利用流式细胞术AV/PI双染法检测各组细胞凋亡/坏死率,以上各组进一步按照不同时间点分为4组:15min,30min,1h和4h,于相应时间点分别收集细胞培养液上清检测淀粉酶活性,提取细胞浆和细胞核蛋白,分别检测各组细胞浆淀粉酶的活性,利用Luminex检测40种细胞核转录因子(TF)的DNA结合活性的变化。
实验三:经胆胰管逆行注入不同浓度去氧胆酸钠制备大鼠急性水肿性胰腺炎(AEP)和急性坏死性胰腺炎(ANP)模型。40只SD大鼠随机分为假手术组、AEP组、ANP组及对ANP的清胰汤治疗组(1ml/100g),每组10只。治疗组大鼠于造模后立即一次和造模后12h再次给药。24h后采取标本做病理检查和各项指标检测。光镜下观察各组胰腺标本病理改变,进行组织学评分;采用TUNEL技术检测胰腺腺泡细胞的凋亡,计算凋亡指数;应用分光光度法检测凋亡蛋白酶Caspase-3,8和9的活性变化。
结果:
胆汁酸7中主要成分对胰腺腺泡细胞的损伤作用不同,其中DCA,CDCA,LCA和TDCA 4种胆汁酸细胞毒作用明显,呈剂量和时间依赖性诱导腺泡细胞凋亡和坏死,CA,GCA和GDCA则不具有细胞毒作用。胆汁酸DCA和CA对腺泡细胞淀粉酶的合成与分泌功能均没有显著影响,在检测的40种核转录因子(TF)的DNA结合活性中,DCA诱导AR42J胰腺腺泡细胞核内ATF2,AR33,STAT5,NFAT,FKHR和NKX-2.5这6种核转录因子(TF)的DNA结合活性显著升高,而RUNX/AML,NF-Y,MEF2和E2F1这4种TF的DNA结合活性则明显下降,其余30种TF活性没有明显变化。
20μM Emodin可以减少DCA诱导的AR42J胰腺腺泡细胞的凋亡与坏死,对细胞淀粉酶的合成与分泌功能没有明显影响,同DCA诱导的TF活性变化相比,Emodin升高PAX3, E2F1, RUNX/AML, NF-Y, IRF, MEF和NFAT这5种TF活性,降低FKHR的活性。
大鼠体内实验结果表明,AEP组与ANP组腺泡细胞凋亡指数、Caspase-3,8和9的活性均显著高于假手术组,且AEP组腺泡细胞凋亡指数及凋亡蛋白酶的活性水平显著高于ANP组。同时,AEP组与ANP组病理组织学评分、血清淀粉酶、血清促炎性细胞因子等指标均较假手术组显著升高,且ANP组显著高于AEP组(p<0.01)。清胰汤通过诱导凋亡蛋白酶活性升高使ANP组大鼠胰腺腺泡细胞凋亡增多而坏死减轻,从而减轻胰腺组织损害和炎症因子水平。
结论:
胆汁酸主要的7种成分中,DCA,CDCA,LCA和TDCA在0.3-0.4mM的较低浓度,在24h内可导致大鼠AR42J胰腺腺泡细胞存活率显著下降,这种细胞毒作用呈时间和剂量依赖性关系,而CA,GCA和GDCA这3种胆汁酸则在1.0mM浓度内不会损伤腺泡细胞。胆汁酸对腺泡细胞的损伤作用主要表现为凋亡和坏死,对细胞内酶的合成和分泌功能没有明显影响。研究发现,DCA可诱导细胞6种TF的DNA结合活性显著升高和4种TF的DNA结合活性显著下降,从而影响相关基因的表达水平,是其造成细胞损伤的分子生物学基础。因此,本研究为胆汁酸诱导的胰腺腺泡细胞损伤提供有力的实验依据,并揭示了胆汁酸7种不同成分的作用特点和可能机制,将为探讨胆源性胰腺炎的发病机制和防治措施提供参考。
Emodin对胆汁酸诱导的胰腺腺泡细胞损伤有一定的保护性作用,对细胞淀粉酶的合成与分泌功能没有明显影响。通过调节细胞TF的DNA结合活性,从而改变相关基因和蛋白的表达,可能是其发挥保护性作用的分子机制。
急性胆源性胰腺炎时,胆汁酸诱导的胰腺腺泡细胞损伤时疾病发生的重要因素,腺泡细胞损伤主要表现为凋亡和坏死,且凋亡的多少与病情轻重呈负相关的关系,腺泡细胞凋亡可能是急性胰腺炎时机体的保护机制之一。凋亡蛋白酶Caspase依赖的凋亡调控通路参与腺泡细胞的凋亡调节,Caspase-8和9的活性均升高。清胰汤可通过诱导Caspase活性升高促进腺泡细胞凋亡而减轻ANP大鼠胰腺病理损害,降低血清淀粉酶、TNF-α、IL-6水平等,改善病情。如何有效控制AP发病时腺泡细胞的损伤,特别是减少坏死的发生,将会为临床进一步降低ANP的重症程度和病死率提供可能。
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.
引文
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[18] Kaiser AM, Saluja AK, Sengupta A, Saluja M, Steer ML. Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis. Am J Physiol. 1995. 269(5 Pt 1): C1295-304.
[19]罗文敏,毓江萍.大黄中五种蒽醌衍生物的HPLC测定.药物分析杂志,1989;9(5):259-262.
1. Gukovskaya AS, Perkins P, Zaninovic V, Sandoval D, Rutherford R, Fitzsimmons T, Pandol SJ, and Poucell-Hatton S. Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat. Gastroenterology, 1996, 110: 875–884
2. Kaiser AM, Saluja AK, Sengupta A, Saluja M, and Steer ML. Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis. Am J Physiol Cell Physiol, 1995, 269: C1295–C1304
3. Buja LM, Eigenbrodt ML, and Eigenbrodt EH. Apoptosis and necrosis. Basic types and mechanisms of cell death. Arch Pathol Lab Med, 1993, 117: 1208–1214
4. Majno G and Joris I. Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol, 1995, 146: 3–15
5. Green DR. Overview: apoptotic signaling pathways in the immune system. Immunol Rev. 2003. 193: 5-9.
6. Mazzoni C, Falcone C. Caspase-dependent apoptosis in yeast. Biochim Biophys Acta. 2008. 1783(7): 1320-7.
7. Kaiser AM , Salugi AK , Sengupta A , et al . Relation between severity , necrosis , and apoptosis in five models of experiment acute pancreatitis . Am J Physiol, 1998, 269 (5 part1 ) : C1295-1298.
8.张肇达,严律南,刘续宝.急性胰腺炎. 2004,122-124
9.尚东,关凤林,陈海龙,等.茵陈承气汤对大鼠急性出血坏死性胰腺炎腺泡细胞凋亡及调控基因的影响.中国中西医结合外科杂志,2002, 8(2):70-73
10. Kaiser AM, Saluja AK, Lu L, Yamanaka K, Yamaguchi Y, Steer ML. Effects of cvcloheximide on pancreatic endonuclease activity, apoptosis, and severity of acute pancreatitis. Am J Physiol, 1996, 271:C982-989
11. Hahm KB, Kim丁H, You B14, Kim YS, Cho SW, Yim H,Ahn B0, Kim WB. Induction of apoptosis with an extract of artemisia asiatica attenuates the severity of cerulean-induced pancreatiti:in rats. Pancreas 1998, 17 (2):153-157
12. Bhatia M, Wallig MA, Hofbauer B, Lee HS, Hrossard JL, Steer ML, Saluja AK. Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreas- titis. Biochem Biophys Res Common 1998, 246 (2):476-483
13. Ashok Salu ja, Hofbauer B, Yamaguchi Y, Yamanaka K, Steer ML Induction of apoptosis reduces the severity of caerulein-induced pancreatttis in mice. Biochem and Biophys Res Common, 1996, 220:876-878
14. Gukovskaya AS, Gukovsky I, Jung Y, Mouria M, and Pandol SJ. Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis. J Biol Chem, 2002, 277: 22595–22604
2. Bhatia M, Wallig MA, Hofbauer B, Lee HS, Frossard JL, Steer ML, Saluja AK. Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis. Biochem Biophys Res Commun, 1998;246:476-483
3. Saluja A, Hofbauer B, Yamaguchi Y, Yamanaka K, Steer ML. Induction of apoptosis reduces the severity of caerulein-induced pancreatitis in mice. Biochem Biophys Res Commun, 1996;220:875-878
4. Kaiser AM, Saluja AK, Lu L, et a1. Effects of eyeloheximide on pancreatic endonuclease activity, apoptosis, and severity of acute pancreatitis. Am J Physiol, 1996;271:982-993
5. He zJ, Podkletnova I, Alho H, Sand J, Nordback I. Apoptosis in acute pancreatitis.Ann Chir Gynaecol, 2000;89:65-67
6 Green DR. Overview: apoptotic signaling pathways in the immune system. Immunol Rev, 2003;193: 5-9
7 Yasuda H, Kataoka K,Ichimura H, Mitsuyoshi M, Iida T, Kita M, Imanishi J. Cytokine expression and induction of acinar cell apoptosis after pancreatic duct ligation in mice.J Interferon Cytokine Res, 1999;19:637-644
8 Jeyarajah DR, Kielar M, Gokaslan ST, et a1. Fas deficiency exacerbates cerulein-induced pancreatitis. J Invest Surg, 2003;l6(6):325-333
9李震东,马清涌,徐军,李明. Fas和FasL在急性胰腺炎中的共表达及其与凋亡的关系.南方医科大学学报, 2006;26:25-29
10 Gallagher SF, Yang J, Baksh K, et a1. Acute pancreatitis induces FasL gene expression and apoptosis in the liver. J Surg Res, 2004;122;201-209
11 Beil M, Leser J, Lutz MP, et a1. Caspase 8-mediated cleavage of plectin precedes F-actin breakdown in acinar cells during pancreatitis. Am J Physiol Gastrointest Liver Physiol, 2002;282: 450-460
12 Gomez G,Lee HM,He Q,Englander EW,Uchida T,Greeley GH Jr.Acute pancreatitis signals activation of apoptosis-associated and survival genes in mice.Exp Biol Med(Maywood), 2001;226:692-700
13 Gukovskaya AS, Gukovsky I, Jung Y, et al, Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis. J Biol Chem, 2002; 277: 22595-22604
14 Petersen OH, Burdakova N. The specificity of Ca2+ signalling. Acta Physiol Hung, 2002;89:439-450
15 Yu JH, Kim H, Kim KH. Calcium-dependent apoptotic gene expression in cerulein-treated AR42J cells. Ann N Y Acad Sci, 2003;1010:66-69
16 Masamune A, Sakai Y, Satoh A, Fujita M, Yoshida M, Shimosegawa T. Lysophosphatidylcholine induces apoptosis in AR42J cells. Pancreas, 2001;22:75-83
17张喜平,林谦.急性胰腺炎时炎症介质与细胞凋亡关系的研究进展.世界华人消化杂志, 2005;13:2773-2777
18 Frossard JL, Rubbia-Brandt L, Wallig M A, et a1. Severe acute pancreatitis and reduced acinar cell apoptosis in the exocrine pancreas of mice dificient for the Cx32 gene. Gastroenterology, 2003;124:481-493
19 Song JY, Lim J W, Kim H, Morio T, Kim KH. Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells. J Biol Chem, 2003;278:36676-36687
20 O’Neill S, O’Neill AJ, Conroy E, et a1. Altered caspase expression results in delayed neutrophil apoptosis in acute pancreatitis. J Leukoc Biol, 2000;68:15-20
21 Sandoval D, Gukovskaya A, Reavey P, et al. The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis. Gastro-enterology, 1996;111:1081-1091
22 Fujimoto K, Hosotani R, Doi R, et a1. Role of neutrophils in cerulein-induced pancreatitis in rats possible involvement of apoptosls. Digestion, 1997;58:421-430
23 Rau B, Paszkowski A, Esber S, et a1. Anti-ICAM-1 antibody modulates late onset of acinar cell apoptosis and early necrosis in taurocholote-induced experimental acute pancreatitis. Pancreas, 2001;23:80-88
24 Gukovskaya AS, Gukovsky I, Zaninovic V, et al. Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-alpha. Role in regulating cell death and pancreatitis. J Clin Invest, 1997;100:1853-1862
25 Sandoval DA, Gukovskaya P, Reavey S, Gukovsky A, Sisk P, Braquet S, Pandol, S. Poucell-Hatton. The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis. Gastroenterology, 1996;111: 1081-1091
26 Fujimoto K, Hosotani R, wada M, Lee J, Koshiba T, MiyamotoY, Doi R, Imamura M. Ischemia-reperfusion injury on the pancreas in rats: identi- fication of acinar cell apoptosis. J Surg Res, 1997;71:127-136
27 Sebastian Gaiser, Astrid Ahlerm, Felix Gundling, Marie-Luise Kruse, Vuk Savkovic, Lena Selig, Niels Teich, Richard Tomasini, Jean-Charles Dagorn, Joachim Mossner, Volker Keim, Hans Bodeker. Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells. Biochem Biophys Res Commun, 2005;334:721-728
28 Ji Hoon Yua, Joo Weon Lima, Kyung Hwan Kima, Tomohiro Moriob, Hyeyoung Kim. NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. Free Radical Biology & Medicine, 2005;39: 590-602
29 Enrico Lupia, Alberto Goffi, Paolo De Giuli, Ornella Azzolino, Ornella Bosco, Enrico Patrucco, Maria Cristina Vivaldo, Marco Ricca, Matthias P. Wymann, Emilio Hirsch, Giuseppe Montrucchio, Giorgio Emanuelli. Ablation of Phosphoinositide 3-Kinase- Reduces the Severity of Acute Pancreatitis. American Journal of Pathology, 2004;165:2003-2011
30 Tachibana I, Imoto M, Adjei PN, Gores GJ, Subramaniam M, Spelsberg T, Urrutia R. Overexpression of the TGFβ-regulated zinc finger encoding gene, TIEG, induces apoptosis in p ancreatic epithelial cells.J Clin Invest, 1997;99:2365-2374
31 Gerasimenko JV, Gerasimenko OV, Palejwala A, Tepikin AV, Petersen OH, and Watson AJ. Menadione-induced apoptosis: roles of cytosolic Ca2+ elevations and the mitochondrial permeability transition pore. J Cell Sci, 2002;115: 485-497
32 Hahm KB, Kim JH, You BM, Kim YS, Cho SW, Yim H, Ahn BO, and Kim WB. Induction of apoptosis with an extract of Artemisia asiatica attenuates the severity of cerulein-induced pancreatitis in rats. Pancreas, 1998, 17: 153-157
33 Cosen-Binker LI, Binker MG, Cosen R, Negri G, Tiscornia O. Relaxin prevents the development of severe acute pancreatitis. World J Gastroenterol, 2006; 12: 1558-1568
34 Chao KC, Chao KF, Chuang CC, et al. Blockade of interleukin 6 accelerates acinar cell apoptosis and attenuates experimental acute pancreatitis in vivo. Br J Surg, 2006,93:332-338
35尚东,关凤林,陈海龙,辛毅.茵陈承气汤对大鼠急性出血坏死性胰腺炎腺泡细胞凋亡及调控基因的影响.中国中西医结合外科杂志, 2002;8:70-73
36邵宏伟,齐清会.紫杉醇诱导胰腺腺泡细胞凋亡与减轻急性胰腺炎严重度的研究.中华普通外科杂志, 2003;18: 307-308
37汪琳,武征,赵维中,高锟.芸香苷静脉输注对大鼠急性胰腺炎腺泡细胞凋亡的影响及机制.中国药理学通报, 2006; 22:44l-446
38潘亮,袁耀宗,章永平,乔敏敏,翟祖康.大黄素诱导急性胰腺炎胰腺细胞凋亡机制的实验研究.胰腺病学, 2002;2:214-217
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[1]黄莛庭.急性胰腺炎细胞内早期事件的再认识.中华肝胆外科杂志. 2006. (02): 75-76.
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[13]焦河玲,黄兆胜,贾建功.大黄素对四氯化碳损伤原代培养大鼠肝细胞的保护作用.河南中医. 2000. (05): 20-22.
[14]楼恺娴,龚自华,袁耀宗,涂水平,翟祖康,徐家裕.大黄素对急性胰腺炎胰腺组织TGF_(β1)表达的影响.中国中西医结合杂志. 2001. (06): 433-436.
[15]陈德昌,景炳文,杨兴易等.大黄对危重症患者胃肠道的保护作用.中国危重病急救医学. 2000. (02): 87-90.
[16] Brahmachary M, Schonbach C, Yang L, et al. Computational promoter analysis of mouse, rat and human antimicrobial peptide-coding genes. BMC Bioinformatics. 2006. 7 Suppl 5: S8.
[17] Gukovskaya AS, Perkins P, Zaninovic V, et al. Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat. Gastroen- terology. 1996. 110(3): 875-84.
[18] Kaiser AM, Saluja AK, Sengupta A, Saluja M, Steer ML. Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis. Am J Physiol. 1995. 269(5 Pt 1): C1295-304.
[19]罗文敏,毓江萍.大黄中五种蒽醌衍生物的HPLC测定.药物分析杂志,1989;9(5):259-262.
1. Gukovskaya AS, Perkins P, Zaninovic V, Sandoval D, Rutherford R, Fitzsimmons T, Pandol SJ, and Poucell-Hatton S. Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat. Gastroenterology, 1996, 110: 875–884
2. Kaiser AM, Saluja AK, Sengupta A, Saluja M, and Steer ML. Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis. Am J Physiol Cell Physiol, 1995, 269: C1295–C1304
3. Buja LM, Eigenbrodt ML, and Eigenbrodt EH. Apoptosis and necrosis. Basic types and mechanisms of cell death. Arch Pathol Lab Med, 1993, 117: 1208–1214
4. Majno G and Joris I. Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol, 1995, 146: 3–15
5. Green DR. Overview: apoptotic signaling pathways in the immune system. Immunol Rev. 2003. 193: 5-9.
6. Mazzoni C, Falcone C. Caspase-dependent apoptosis in yeast. Biochim Biophys Acta. 2008. 1783(7): 1320-7.
7. Kaiser AM , Salugi AK , Sengupta A , et al . Relation between severity , necrosis , and apoptosis in five models of experiment acute pancreatitis . Am J Physiol, 1998, 269 (5 part1 ) : C1295-1298.
8.张肇达,严律南,刘续宝.急性胰腺炎. 2004,122-124
9.尚东,关凤林,陈海龙,等.茵陈承气汤对大鼠急性出血坏死性胰腺炎腺泡细胞凋亡及调控基因的影响.中国中西医结合外科杂志,2002, 8(2):70-73
10. Kaiser AM, Saluja AK, Lu L, Yamanaka K, Yamaguchi Y, Steer ML. Effects of cvcloheximide on pancreatic endonuclease activity, apoptosis, and severity of acute pancreatitis. Am J Physiol, 1996, 271:C982-989
11. Hahm KB, Kim丁H, You B14, Kim YS, Cho SW, Yim H,Ahn B0, Kim WB. Induction of apoptosis with an extract of artemisia asiatica attenuates the severity of cerulean-induced pancreatiti:in rats. Pancreas 1998, 17 (2):153-157
12. Bhatia M, Wallig MA, Hofbauer B, Lee HS, Hrossard JL, Steer ML, Saluja AK. Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreas- titis. Biochem Biophys Res Common 1998, 246 (2):476-483
13. Ashok Salu ja, Hofbauer B, Yamaguchi Y, Yamanaka K, Steer ML Induction of apoptosis reduces the severity of caerulein-induced pancreatttis in mice. Biochem and Biophys Res Common, 1996, 220:876-878
14. Gukovskaya AS, Gukovsky I, Jung Y, Mouria M, and Pandol SJ. Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis. J Biol Chem, 2002, 277: 22595–22604