亞急性瘤胃酸中毒病牛瘤胃組胺對瘤胃上皮細胞炎性通路的影響
發(fā)布時間：2018-07-15 09:56
【摘要】：患有亞急性瘤胃酸中毒的反芻動物，瘤胃處于低pH環(huán)境，由于發(fā)酵功能紊亂，，使其代謝產(chǎn)物如組胺、內(nèi)毒素、乳酸等在瘤胃內(nèi)大量蓄積，損傷瘤胃上皮及其屏障功能，使組胺等有害物質(zhì)通過瘤胃壁進入血液，使血液內(nèi)組胺的含量增高，引發(fā)瘤胃炎。NF-κB信號通路是細胞內(nèi)主要炎性信號通路，組胺能否激活瘤胃上皮細胞NF-κB通路引起炎性損傷還不是很清楚。因此，本實驗通過正常健康育肥牛和亞急性瘤胃酸中毒病牛的部分血液生化指標，評價患亞急性瘤胃酸中毒病牛的炎性水平。通過體外培養(yǎng)牛瘤胃上皮細胞，在不同pH下，添加不同濃度的組胺和NF-κB通路抑制劑PDTC，檢測組胺對NF-κB信號通路及其下游炎性細胞因子的影響，探討亞急性瘤胃酸中毒時組胺對瘤胃上皮細胞炎性損傷的機制。 通過檢測正常健康育肥牛和亞急性瘤胃酸中毒病牛血清中脂多糖（LPS）、脂多糖結(jié)合蛋白（LBP）、組胺、葡萄糖（GLU）、丙氨酸氨基轉(zhuǎn)移酶（ALT）、門冬氨酸氨基轉(zhuǎn)移酶（AST）、直接膽紅素（DBIL）、總膽紅素（TBIL）、間接膽紅素（IBIL）、總蛋白（TP）、白蛋白（ALB）、球蛋白（GLO）、尿素（BUM）、肌酐（CREA）和β-羥丁酸（BHBA）等血液生化指標的濃度，實驗結(jié)果表明，亞急性瘤胃酸中毒病牛的生化指標與正常健康牛相比顯著升高，但葡萄糖的指標除外。說明亞急性瘤胃酸中毒牛存在不同程度的炎性反應。 體外培養(yǎng)牛瘤胃上皮細胞，添加不同濃度的組胺、NF-κB通路抑制劑PDTC，觀察NF-κB信號通路關(guān)鍵分子的變化及對其下游炎性因子的影響。通過組胺時間梯度實驗，組胺作用瘤胃上皮細胞6h時，IκB α磷酸化水平最高，所以把6h作為最佳作用時間點。分別添加不同濃度的組胺（0、0.5、2.5、12.5和62.5μmmol/L）、10μM PDTC、10μM PDTC+12.5μmmol/L組胺作用瘤胃上皮細胞6h。結(jié)果顯示，組胺可以增加IκB α和NF-κB p65基因mRNA表達水平及蛋白磷酸化水平，且呈現(xiàn)劑量依賴性。檢測NF-κB p65轉(zhuǎn)錄因子的入核與轉(zhuǎn)錄活性，顯示組胺可以增加轉(zhuǎn)錄因子NF-κB p65的入核與轉(zhuǎn)錄活性，并進一步增加炎性細胞因子TNFα、IL-6和IL-1β等的表達和與釋放，從而引起瘤胃上皮細胞的炎性損傷。但添加NF-κB的抑制劑PDTC后，IκB α和NF-κB p65蛋白磷酸化水平降低，NF-κB p65轉(zhuǎn)錄活性均降低，并降低組胺引起的炎性因子的表達。 根據(jù)以上實驗結(jié)果可知，高濃度的組胺能夠激活瘤胃上皮細胞的NF-κB信號通路，促進炎性細胞因子TNF-α、IL-6和IL-1β的釋放，從而引起牛瘤胃上皮細胞的炎性損傷。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23
本文編號：2123694
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23
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