【作者】 孙道；

【导师】 齐海智；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 第一部分小白菊内酯对枯否细胞分泌功能的影响目的：研究小白菊内酯对枯否细胞释放炎性因子的影响并阐明其作用机制。方法：采用胶原酶原位灌注法分离及提纯枯否细胞。将细胞分为3组：A组为对照组(Control group),培养正常大鼠KCs: B组为LPS刺激组(LPS group),在开始培养KCs时,培养液中加入终浓度为10mg/1LPS；C组为LPS+PTN组(PTN group),培养液中先加入5μmol/l PTN培养1h,然后再加入lOmg/1LPS。各组分别于培养3h后获取标本作相应指标检测,重复8次实验后取其均值(n=8)。将各组细胞置于倒置的显微镜下观察其形态变化,台盼蓝拒染实验判断细胞活力,抗ED-2免疫组化染色检测纯度,ELISA法检测细胞上清液中IL-1和TNF-a的水平,EMSA法检测NF-κB DNA结合活性。结果：新分离的KCs在倒置的显微镜下呈球形,悬浮于培养基中;约6h后,大部分细胞贴壁呈扁圆形,少数细胞伸出伪足；24h后大部分细胞伸出伪足,呈典型星形和多角形,细胞体积明显变大；各组枯否细胞分离纯度均为>90%,活性>96%。与A组比较,B组IL-1和TNF-a水平明显增高(P<0.05)；与B组比较,C两组IL-1和TNF-a水平明显较低(P<0.05)；与A组比较,B组NF-κB DNA结合活性显著增高(P<0.05)；与B组比较,C组NF-κB DNA结合活性显著降低(P<0.05)。结论：1、在LPS的刺激下,肝脏枯否细胞活化而释放大量炎性因子；2、小白菊内酯可能通过下调NF-kB活性,抑制肝脏枯否细胞的活化,减少炎性因子的大量释放。第二部分小白菊内酯对大鼠肝脏缺血再灌注损伤的作用目的：研究小白菊内酯在大鼠肝脏缺血再灌注损伤中的作用及其相关机制。方法：将健康雄性SD大鼠随机分为四组(n=18)：假手术组(A)、对照组(B)、小白菊内酯小剂量干预组(C)和小白菊内酯大剂量干预组(D)。缺血前10min分别给予小剂量干预组和大剂量干预组大鼠腹腔注射250和500μg/kg的小白菊内酯,建立肝左、中叶70%部分肝缺血再灌注模型,假手术组给予同样体积的0.1%DMSO。肝脏缺血90min后对缺血肝叶进行再灌注,分别于再灌注后2、6、24h收集血液及缺血肝叶组织标本,每个时点n=6。检测血清ALT、AST水平,肝组织HE染色后置光镜下观察病理形态学变化,并根据Suzuki’s评分标准进行病理损伤半定量评分,ELISA检测肝组织中MPO活力和MDA含量检测。结果：与对照组相比,小剂量和大剂量小白菊内酯干预组血清ALT、AST水平均明显下降(P<0.05),大剂量干预组较小剂量组更明显(P<0.05)；与对照组相比,小白菊内酯干预后肝脏组织病理学表现明显改善,Suzuki’s评分明显降低(P<0.05),大剂量干预组较小剂量组更明显(P<0.05)；小白菊内酯干预后肝组织MPO活力和MDA含量较对照组明显下降(P<0.05),大剂量干预组较小剂量干预组更明显(P<0.05)。结论：1、小白菊内酯干预能有效改善大鼠肝脏缺血再灌注损伤引起的肝功能和肝脏病理损害；2、小白菊内酯能抑制氧自由基的损伤作用,减轻脂质过氧化引发的组织损伤；3、小白菊内酯干预能够减少大鼠肝脏缺血再灌注损伤后中性粒细胞的聚集浸润和活化,从而减轻炎症反应。第三部分小白菊内酯在大鼠肝脏缺血再灌注损伤中对IKK/NF-kB信号通路的影响目的：通过观察小白菊内酯对肝脏缺血再灌注损伤中IKK/NF-kB信号通路的影响,探讨其缺血再灌注保护作用的分子机制。方法：将健康雄性SD大鼠随机分为三组(n=6)：假手术组(A)、对照组(B)、小白菊内酯干预组(C)。缺血前10min给予小白菊内酯干预组大鼠腹腔注射500μg/kg的小白菊内酯,建立肝左、中叶70%部分肝缺血再灌注模型,假手术组给予同样体积的0.1%DMSO。肝脏缺血90min后对缺血肝叶进行再灌注,于再灌注后6h收集血液及缺血肝叶组织标本。ELISA检测血清中TNF-α、ICAM-1及MIP-2水平；RT-PCR检测肝组织TNF-α、ICAM-1及MIP-2 mRNA的表达；EMSA法检测肝组织NF-kB DNA结合活性；Werstern blotting测定肝组织细胞核内NF-kB p65和肝组织中p-IKKser180/βser181和IkBα蛋白的表达。结果：与对照组(B)比较,小白菊内酯干预后血清TNF-α、ICAM-1及MIP-2水平和肝组织TNF-α、ICAM-1及MIP-2 mRNA的表达均明显下降(P<0.05)；与对照组(B)比较,小白菊内酯干预组(B)肝组织NF-kB DNA结合活性和肝组织细胞核内NF-kB p65蛋白及肝脏组织p-IKKser180/βser181蛋白水平较对照组(B)显著降低(P<0.05),肝脏组织IKBa含量明显升高(P<0.05)。结论：小白菊内酯可能通过抑制IKK/NF-kB信号通路减少肝脏缺血再灌注损伤中炎性相关基因和蛋白表达。第四部分JAK2/STAT3信号通路在大鼠肝脏缺血再灌注损伤的作用及小白菊内酯对JAK2/STAT3信号通路的影响目的：探讨JAK/STAT信号通路在肝脏缺血再灌注损伤中的活化规律及作用机制,并观察小白菊内酯对JAK2/STAT3信号通路的影响,深入探讨其对缺血再灌注保护作用的分子机制。方法：将健康雄性SD大鼠随机分为四组(n=6)：假手术组(A)、对照组(B)、AG490干预组(C)、小白菊内酯干预组(D)。缺血前10min分别给予AG490干预组大鼠腹腔注射500μg/kg的AG4901mg/kg和小白菊内酯干预组大鼠腹腔注射500μg/kg的小白菊内酯,建立肝左、中叶70%部分肝缺血再灌注模型,假手术组给予同样体积的0.1%DMSO。肝脏缺血90min后对缺血肝叶进行再灌注,于再灌注后6h收集血液及缺血肝叶组织标本。免疫组织化学染色和Werstern blotting检测p-JAk2和p-STAT3蛋白表达；RT-PCR检测肝组织TNF-amRNA的表达；ELISA检测血清中TNF-a水平。结果：与假手术组(A)比较,对照组(B)肝组织p-JAk2和p-STAT3蛋白水平和TNF-amRNA的表达水平及血清中TNF-a水平明显升高(P<0.05)；与对照组(B)比较,AG490干预组(C)肝组织p-JAk2和p-STAT3蛋白水平和TNF-amRNA的表达水平及血清中TNF-a水平明显降低(P<0.05)；与对照组(B)比较,小白菊内酯干预组(D)肝组织p-JAk2和p-STAT3蛋白水平和TNF-amRNA的表达水平及血清中TNF-a水平明显降低(P<0.05)。结论：1、JAK2/STAT3信号通路在肝脏缺血再灌注损伤中激活；2、阻断JAK2/STAT3信号通路的异常激活可能抑制肝脏缺血再灌注损伤中炎性相关基因和蛋白表达；3、小白菊内酯可能通过抑制JAK2/STAT3信号通路减少肝脏缺血再灌注损伤中炎性相关基因和蛋白表达。更多还原

【Abstract】 Part 1:Effects of parthenolide on the secretion function of Kupffer cellsObjective:To explore the effects of parthenolide on the inflammatory cytokines expression in kupffer cells and to elucidate the probable mechanism of the effects.Methods:Kupffer Cells were isolated and purified by situ collagenase perfusion. The isolated and purified cells were then divided into 3 groups:Group A, control group in which kupffer cells were cultured normally; Group B, LPS group in which 10mg/l LPS was added into the nutrient solution; Group C, PTN group in which 5μmol/l parthenolide was added into the nutrient solution 1h before 10mg/l LPS was added. After 3h stimulating to each group, the morphology of kupffer cells were measured by an inverted microscope, the activity of kupffer cell was measured by trypan blue staining, the purity was measured by anti ED-2 immunohistochemical staining, the concentration of IL-1 and TNF-a in the supernatant were measured by ELISA, the NF-kB DNA binding activity were measured by EMSA. Treatments in each group were repeated for 8 times to obtain the mean value of each index as the final result.Results:The shape of newly isolated kupffer cells were sphere like, suspending in the nutrient solution; Most of them became flat and adhered to the wall, pseudopodium formation occurred in few cells; After about 24h, pseudopodium formation occurred in most of the cells and all cells were extended totally with bigger size in star-shape or polygonal shape; The purity ratio of each group was 90%, and the activity ratio was more than 96%. Concentration of TNF-a and IL-1 of the nutrient solution in group B is much higher than that of group A (p＜0.05), Concentration of TNF-αand IL-1 of the nutrient solution in group C is much lower than that of group A,Group B was higher in DNA binding activity of NF-kB than that of group A (P＜0.05), Group C was lower in DNA binding activity of NF-kB than that of group B (P＜0.05).Conclusions:(1) LPS can activate the kupffer cells and as a result induce its ability of releasing cytokines.(2) Parthenolide can inhibit the activation of NF-kB in kupffer cells and as a result reduce its ability of releasing cytokines.Part 2:Effect of parthenolide on hepatic ischemia-reperfusion injuryObjective:To investigate the effect and related mechanisms of parthenolide on liver function and hepatic tissues in the liver ischemia-reperfusion injury.Methods:healthy male SD rats were randomized into 4 groups (n=18):sham-operated group(A), model control group(B), low dosage of parthenolide treatment group(C) and large dosage of parthenolide treatment group(D).10 minutes before Ischemia, treatment group rats were intraperitoneally injected 250 or 500μg/kg parthenolide.Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatie lobe, were established.The I/R model control group and were administered with the same volume of DMSO.After 90 min ischemia,6 cases rats of each group were killed at 2h,6h and 24h after residual liver reperfusion respectively, blood and liver tissue samples were collected from the experimental groups. Serum levels of ALT and AST were measured, part of the liver tissues were made into paraffin-embedded specimens to detect rat liver histological change and grade hepatic IRI(Suzuki’s score), the content of MDA and the activity of MPO in liver tissues were detected by ELISA.Results:Compared with the model control group,serum ALT and AST in both treatment group of the low and large dosage parthenolide were significantly decreased(P<0.05),even lower in the large dosage group than in the low dosage group(P<0.05). Hepatic pathology performance was improved and Suzuki’s scores was decreased significantly in both dosage group (P<0.05), the large dosage group is more significantly. The content of MDA and the activity of MPO in liver tissues were significantly decreased in both treatment groups when compared with the I/R model control group(P<0.05), also lower in the large dosage group than in the low dosage group(P<0.05).Conclusion:(1) Treatment with parthenolide can effectively improve the rat liver function and liver pathology damage induced by liver ischemia-reperfusion injury;(2) Intervention with parthenolide can inhibit the effection of oxygen free radicals, and reduce the tissues damage induced by lipid peroxidation;(3) Intervention with parthenolide can reduce the aggregation, infiltration and activation of neutrophil in the rat liver induced by ischemia-reperfusion injury,thereby reducing inflammatory response.Part 3:Effects of parthenolide on IKK/NF-κB signaling pathway in the rat hepatic ischemia-reperfusion injuryObjective:To study the molecular mechanism of the effect of parthenolide inhibiting the inflammation signaling pathway of IKK/ NF-κB in the rat hepatic ischemia-reperfusion injury.Methods:healthy male SD rats were randomized into 3 groups (n=6):sham-operated group(A), model control group(B), parthenolide treatment group(C).10 minutes before Ischemia, treatment group rats were intraperitoneally injected 500μg/kg parthenolide.Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatie lobe, were established.The I/R model control group and were administered with the same volume of DMSO.After 90 min ischemia and 6h reperfusion,6 cases rats of each group were killed respectively, blood and liver tissue samples were collected from the experimental groups. Serum levels of TNF-a, MIP-2 and ICAM-1were measured by ELISA,TNF-a, MIP-2 and ICAM-1mRNA of liver were measured by RT-PCR, the NF-kB DNA binding activity were measured by EMSA, the level of protein of NF-kB p65 in caryon and p-IKKser180/βser181 and IkBαin the liver tissues were measured by Western blot.Results:Compared with the model control group, the levels of TNF-a, MIP-2 and ICAM-1 in serum and mRNA of liver in parthenolide treatment group were significantly decreased(P<0.05);Compared with the model control group, the NF-kB DNA binding activity, the level of protein of NF-kB p65 in caryon, the level of protein of p-IKKser180/βser181 in parthenolide treatment group were significantly decreased(P<0.05), the level of protein of IkBbαwere significantly increased(P<0.05).Conclusion:Parthenolide reduces the expression of inflammatory gene and protein in hepatic ischemia-reperfusion injury probablely by suppressing IKK/NF-kB signal pathway activation.Part 4:Effects of JAK2/STAT3 signal pathway on the rat hepatic ischemia-reperfusion injury and effects of parthenolide on JAK2/STAT3 signaling pathwayObjective:To identify the role of the activated JAK2/STAT3 in rat hepatic ischemia-reperfusion injury and investigate the mechanisms of JAK2/STAT3 signal pathway causing hepatic damage,to study the molecular mechanism of the effect of parthenolide on the JAK2/STAT3 signaling pathway in the rat hepatic ischemia-reperfusion injury.Methods:healthy male SD rats were randomized into 4 groups (n=6):sham-operated group(A), model control group(B), AG490 treatment group(C) and parthenolide treatment group(D).10 minutes before Ischemia, AG490 treatment group and parthenolide treatment group treatment group rats were intraperitoneally injected lmg/kg AG490 and 500μg/kg parthenolide respectively.Then the hepatic ischemia-reperfusion model of 70 percent of liver, including the left and middle hepatie lobe, were established.The I/R model control group and were administered with the same volume of DMSO. After 90 min ischemia,6 cases rats of each group were killed at 6h after residual liver reperfusion respectively, blood and liver tissue samples were collected from the experimental groups. The level of protein of p-JAk2 and p-STAT3 were measured by immunohistochemical method and Western blot, TNF-amRNA of liver were measured by RT-PCR, Serum levels of TNF-αwere measured by ELISA.Results:Compared with the sham-operated group, the levels of protein of pJAk2 and pSTAT3, TNF-amRNA of liver, serum levels of TNF-a in model control group(B)were significantly increased(P<0.05); compared with the model control group(B), the levels of protein of p-JAk2 and p-STAT3, TNF-amRNA of liver, serum levels of TNF-a in AG490 treatment group(C) were significantly increased(p<0.05); compared with the model control group(B), the levels of protein of p-JAk2 and p-STAT3, TNF-amRNA of liver, serum levels of TNF-a in parthenolide treatment group(D) were significantly increased(p<0.05).Conclusion:(1) JAK2/STAT3 signal pathway were activated in hepatic ischemia-reperfusion injury;(2) suppressing JAK2/STAT3 signal pathway activation probablely reduce the expression of inflammatory gene and protein in hepatic ischemia-reperfusion injury;(3) Parthenolide reduces the expression of inflammatory gene and protein in hepatic ischemia-reperfusion injury probablely by suppressing JAK2/STAT3 signal pathway activation.更多还原