亚急性瘤胃酸中毒病牛瘤胃组胺对瘤胃上皮细胞炎性通路的影响

发布时间：2018-07-15 09:56

【摘要】：患有亚急性瘤胃酸中毒的反刍动物，瘤胃处于低pH环境，由于发酵功能紊乱，，使其代谢产物如组胺、内毒素、乳酸等在瘤胃内大量蓄积，损伤瘤胃上皮及其屏障功能，使组胺等有害物质通过瘤胃壁进入血液，使血液内组胺的含量增高，引发瘤胃炎。NF-κB信号通路是细胞内主要炎性信号通路，组胺能否激活瘤胃上皮细胞NF-κB通路引起炎性损伤还不是很清楚。因此，本实验通过正常健康育肥牛和亚急性瘤胃酸中毒病牛的部分血液生化指标，评价患亚急性瘤胃酸中毒病牛的炎性水平。通过体外培养牛瘤胃上皮细胞，在不同pH下，添加不同浓度的组胺和NF-κB通路抑制剂PDTC，检测组胺对NF-κB信号通路及其下游炎性细胞因子的影响，探讨亚急性瘤胃酸中毒时组胺对瘤胃上皮细胞炎性损伤的机制。通过检测正常健康育肥牛和亚急性瘤胃酸中毒病牛血清中脂多糖（LPS）、脂多糖结合蛋白（LBP）、组胺、葡萄糖（GLU）、丙氨酸氨基转移酶（ALT）、门冬氨酸氨基转移酶（AST）、直接胆红素（DBIL）、总胆红素（TBIL）、间接胆红素（IBIL）、总蛋白（TP）、白蛋白（ALB）、球蛋白（GLO）、尿素（BUM）、肌酐（CREA）和β-羟丁酸（BHBA）等血液生化指标的浓度，实验结果表明，亚急性瘤胃酸中毒病牛的生化指标与正常健康牛相比显著升高，但葡萄糖的指标除外。说明亚急性瘤胃酸中毒牛存在不同程度的炎性反应。体外培养牛瘤胃上皮细胞，添加不同浓度的组胺、NF-κB通路抑制剂PDTC，观察NF-κB信号通路关键分子的变化及对其下游炎性因子的影响。通过组胺时间梯度实验，组胺作用瘤胃上皮细胞6h时，IκB α磷酸化水平最高，所以把6h作为最佳作用时间点。分别添加不同浓度的组胺（0、0.5、2.5、12.5和62.5μmmol/L）、10μM PDTC、10μM PDTC+12.5μmmol/L组胺作用瘤胃上皮细胞6h。结果显示，组胺可以增加IκB α和NF-κB p65基因mRNA表达水平及蛋白磷酸化水平，且呈现剂量依赖性。检测NF-κB p65转录因子的入核与转录活性，显示组胺可以增加转录因子NF-κB p65的入核与转录活性，并进一步增加炎性细胞因子TNFα、IL-6和IL-1β等的表达和与释放，从而引起瘤胃上皮细胞的炎性损伤。但添加NF-κB的抑制剂PDTC后，IκB α和NF-κB p65蛋白磷酸化水平降低，NF-κB p65转录活性均降低，并降低组胺引起的炎性因子的表达。根据以上实验结果可知，高浓度的组胺能够激活瘤胃上皮细胞的NF-κB信号通路，促进炎性细胞因子TNF-α、IL-6和IL-1β的释放，从而引起牛瘤胃上皮细胞的炎性损伤。
[Abstract]:In a ruminant with subacute gastric acid poisoning, the rumen is in a low pH environment. Due to the disorder of fermentation, the metabolites such as histamine, endotoxin, lactic acid, etc. are accumulated in the rumen, damaging the rumen epithelium and its barrier function, causing histamine and other harmful substances to enter the blood through the rumen wall, making the content of histamine in the blood increased and triggering a tumor. The.NF- kappa B signal pathway in gastritis is the main inflammatory signaling pathway in the cells. Whether histamine activates the NF- kappa B pathway of the rumen epithelial cell causes inflammatory damage is not clear. Therefore, this experiment was conducted to evaluate the inflammation of cattle with subacute tumor and gastric acidosis by the biochemical indexes of normal healthy fattening cattle and subacute gastric acid poisoning cattle. The effects of histamine and NF- kappa B pathway inhibitor PDTC on the NF- kappa B signaling pathway and its downstream inflammatory cytokines were added to the cultured bovine rumen epithelial cells in vitro, and the effects of histamine on the NF- kappa B signaling pathway and its downstream inflammatory cytokines were examined to explore the mechanism of histamine on the inflammatory damage of the ruminal epithelial cells in subacute gastric acid poisoning.
By detecting lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP), histamine, glucose (GLU), alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bilirubin (TBIL), indirect bilirubin (IBIL), total protein (TP), albumin (ALB), albumin (ALB), ball in the bovine serum of normal healthy fattening cattle and subacute gastric acidosis. The concentration of protein (GLO), urea (BUM), creatinine (CREA) and beta hydroxybutyric acid (BHBA), and so on. The experimental results showed that the biochemical indexes of subacute gastric acid poisoning cattle were significantly higher than those of normal healthy cattle, except for the glucose index.
The bovine rumen epithelial cells were cultured in vitro, adding different concentrations of histamine and NF- kappa B pathway inhibitor PDTC to observe the changes of key molecules of the NF- kappa B signaling pathway and the effect on the downstream inflammatory factors. The highest phosphorylation level of I kappa B a was achieved by histamine time gradient experiment and 6h in the ruminal epithelial cells of histamine, so 6h was used as the best effect. Adding different concentrations of histamine (0,0.5,2.5,12.5 and 62.5 mu mmol/L), 10 M PDTC, 10 mu M PDTC+12.5 mmol/L histamine in ruminal epithelial cells, the results showed that histamine could increase the level of I kappa B A and NF- kappa B. With the transcriptional activity, histamine can increase the nuclear and transcriptional activity of the transcription factor NF- kappa B p65, and further increase the expression and release of inflammatory cytokines, such as TNF alpha, IL-6 and IL-1 beta, and thus cause inflammatory damage in the rumen epithelial cells. But the phosphorylation level of I kappa B alpha and NF- kappa B is reduced after the addition of NF- kappa B inhibitor PDTC. The transcriptional activity of B p65 was decreased and the expression of inflammatory factors induced by histamine was reduced.
According to the above results, it is known that high concentration of histamine can activate the NF- kappa B signaling pathway in the rumen epithelial cells and promote the release of inflammatory cytokines TNF- a, IL-6 and IL-1 beta, thus causing inflammatory injury in the bovine rumen epithelial cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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