川陈皮素对脓毒症小鼠肝损伤的保护作用及分子机制研究

发布时间：2018-04-26 09:50

本文选题：脓毒症 + 肝损伤　；参考：《重庆医科大学》2016年博士论文

【摘要】：脓毒症是一种由于感染引起的全身炎性反应综合征,是各种严重创伤、烧伤、或大手术后常见并发症,常导致脓毒症休克甚至多器官功能障碍综合征。肝脏不仅是脓毒症时最易受损的靶器官之一,还在脓毒症发生和发展过程发挥着举足轻重的作用。脓毒症发生时,肝脏定居的巨噬细胞-Kupffer细胞被血中内毒素所激活,大量释放各类炎症因子,直接导致肝脏炎症并引发全身炎症反应;Kupffer细胞和其他炎性细胞产生大量氧自由基和脂质过氧化产物如丙二醛等,引起肝细胞膜破坏和线粒体功能受损,导致转氨酶和胆红素等大量释放入血;同时,脓毒症时肝脏多种凝血因子、抗凝血因子以及纤溶抑制物合成减少,肝细胞清除活性因子能力下降,导致机体凝血功能障碍,进一步加剧全身炎症反应和器官损伤。目前认为,早期肝功能障碍是脓毒症患者死亡的一项独立的预警因素,因此对脓毒症肝损伤患者开展早期有效治疗、及时改善肝功能,有助于改善脓毒症患者预后。川陈皮素(Nobiletin,5,6,7,8,3',4'-hexame-thoxy flavone),别名川皮亭,又称蜜橘黄酮,是从芸香科柑桔属橘子Citrus reticulaia Blanco果皮中提取的一种多甲氧基黄酮类化合物。近年来研究显示川陈皮素具有抗炎、抗氧化、抗肿瘤以及神经保护等多种生物活性,其作用可能与抑制NF-kB和激活剂蛋白-1(AP-1)等转录因子的活性有关,并且没有明显的毒副作用。因此,川陈皮素可用于临床治疗多种炎症性疾病和肿瘤等,具有很大的开发前景。然而目前为止,川陈皮素的药理效应和抗炎作用机制并未阐明,能否作为脓毒症肝损伤的治疗用药也需要进一步评价。本研究拟采用内毒素诱导小鼠脓毒症肝损伤的动物模型,观察川陈皮素治疗脓毒症肝损伤后小鼠死亡率的改变、血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBil)的水平、血清和肝组织中TNF-α、IL-1β和IL-6的变化,提取Kupffer细胞并检测炎症介质i NOS和COX-2的含量改变,同时检测NF-κB、MAPK(ERK、JNK和p38MAPK)和Nrf2-HO-1信号转导通路的变化等,旨在阐明川陈皮素治疗脓毒症肝损伤的相关分子机制,为脓毒症肝损伤的预防和临床治疗提供一种新的研究思路。第一部分:川陈皮素对脓毒症肝损伤小鼠的保护作用目的:观察川陈皮素对脓毒症肝损伤小鼠的保护作用。方法:利用内毒素腹腔注射建立脓毒症肝损伤小鼠模型。将75只成年C57BL/6小鼠随机分为五组:正常对照组(15只,磷酸盐缓冲液)、LPS模型组(15只,LPS 10mg/kg)、川陈皮素低剂量组(15只,LPS10mg/kg和川陈皮素50mg/kg)、川陈皮素中剂量组(15只,LPS 10mg/kg和川陈皮素100mg/kg)、川陈皮素高剂量组(15只,LPS 10mg/kg和川陈皮素200mg/kg)。各组于腹腔注射药物后不同时间点(6h、12h、24h)分别处死5只小鼠,收集血液及肝组织标本。HE染色观察肝组织病理形态变化,测定实验各组血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBil)的水平,测定血清和肝组织中TNF-α、IL-1β、IL-6的含量。另外取50只小鼠重复以上分组,注射药物后观察并比较72小时内各组小鼠生存率。结果:与正常对照组相比较,模型组和药物干预组各项指标均发生了显著改变,表明本实验模型构建成功。(1)LPS注射2h后,模型组和药物干预组小鼠均出现精神萎靡、触之不动、呼吸急促、体温下降、不进食水等表现,而NOB干预组小鼠的精神和状态相对较好;(2)LPS注射后24h后,小鼠肝脏出现明显病理改变,组织细胞肿胀、坏死、充血和炎性细胞浸润,但NOB干预组组织损伤明显缓解;(3)模型组血清中ALT、AST和TBil水平较正常对照明显升高,而NOB干预组中以上指标明显下降(P0.05或P0.01);(4)模型组血清中和肝脏匀浆中炎症因子TNF-α、IL-1β和IL-6的含量明显升高,而NOB干预组中以上指标明显下降(P0.05或P0.01);(5)正常对照组、模型组及NOB干预组72h生存率观察发现,正常对照组生存率为100%,模型组小鼠72h生存率为0%,NOB干预组能有效提高脓毒症小鼠72h生存率:其中50mg/kg剂量组72h生存率为40%;100mg/kg剂量组72h生存率为50%;200mg/kg剂量组72h生存率为80%;结论:川陈皮素通过抑制脓毒症全身和肝脏炎症因子释放,减轻肝脏肝细胞水肿、坏死和抑制炎性细胞浸润,减少肝酶的释放,进而对脓毒症肝损伤小鼠发挥保护作用,最终显著提高小鼠的生存率。第二部分:川陈皮素对LPS活化的小鼠Kupffer细胞IkB/NF-kB信号通路的影响目的:通过观察川陈皮素对LPS活化的小鼠Kupffer细胞IkB/NF-kB信号通路的影响,阐明川陈皮素在脓毒症肝损伤保护作用的分子机制。方法:采用离体胶原酶消化、密度梯度离心联合选择性贴壁法分离Kupffer细胞,接种于六孔板,随机分为五组:正常对照组(磷酸盐缓冲液)、LPS模型组(LPS 10μg/ml)、川陈皮素低剂量组(LPS 10μg/ml和川陈皮素10μM)、川陈皮素中剂量组(LPS 10μg/ml和川陈皮素20μM)、川陈皮素高剂量组(LPS 10μg/ml和川陈皮素40μM)。药物刺激后分别在1h收取细胞和12h收取细胞培养上清,进行如下检测:ELISA检测12h时细胞培养上清细胞因子TNF-α、IL-1β、IL-6水平;Western blotting检测1h时Kupffer细胞p-IkB、IkB、p-p65、p65蛋白变化;EMSA检测Kupffer细胞核内NF-kB p65的DNA结合活力。结果:LPS刺激体外培养的Kupffer细胞12h后,细胞培养上清中炎症因子TNF-α、IL-1β、IL-6的含量显著增加,而不同剂量川陈皮素干预后上述炎症因子含量显著下降(P0.05或P0.01);LPS刺激1h后,Kupffer细胞中p-IkB/IkB、p-p65/p65蛋白比例明显增高(P0.05或P0.01),细胞核内NF-kB p65的DNA结合活力也明显增加(P0.05或P0.01),而不同剂量川陈皮素干预后上述指标显著下降(P0.05或P0.01)。结论:川陈皮素对脓毒症肝损伤的保护作用可能与抑制Kupffer细胞IkB/NF-kB信号通路的过度活化、减少相关炎症因子的蛋白表达有关。第三部分:川陈皮素对LPS活化的小鼠Kupffer细胞MAPK和Nrf2-HO-1信号通路的影响目的:通过观察川陈皮素对LPS活化的小鼠Kupffer细胞MAPK(ERK、JNK和p38MAPK)和Nrf2-HO-1信号通路的影响,探索川陈皮素对脓毒症肝损伤保护作用的分子机制。方法:采用离体胶原酶消化、密度梯度离心联合选择性贴壁法分离Kupffer细胞,接种于六孔板,随机分为五组:正常对照组(磷酸盐缓冲液)、LPS模型组(LPS 10μg/ml)、川陈皮素低剂量组(LPS 10μg/ml和川陈皮素10μM)、川陈皮素中剂量组(LPS 10μg/ml和川陈皮素20μM)、川陈皮素高剂量组(LPS 10μg/ml和川陈皮素40μM)。药物刺激后分别在1h、6h、12h收取细胞,Western blotting检测细胞p-ERK、p-JNK、p-p38MAPK、Nrf2、HO-1、i NOS、COX-2表达蛋白变化。结果:LPS刺激体外培养的Kupffer细胞1h后,p-ERK、p-JNK、p-p38MAPK的表达相比正常对照组明显增高(P0.05或P0.01),而不同剂量川陈皮素干预后上述指标显著下降(P0.05或P0.01);LPS刺激6h后,细胞浆HO-1和细胞核Nrf2蛋白表达相比正常对照组轻微升高,而不同剂量川陈皮素干预后上述指标显著升高(P0.05或P0.01);LPS刺激12h后,细胞i NOS和COX-2蛋白表达相比正常对照组明显增高(P0.05或P0.01),而不同剂量川陈皮素干预后上述指标显著下降(P0.05或P0.01)。结论:川陈皮素对脓毒症肝损伤的保护作用可能与抑制Kupffer细胞MAPK(ERK、JNK和p38 MAPK)信号通路过度活化、减少炎症介质i NOS和COX-2的表达和激活Nrf2-HO-1抗氧化通路有关。
[Abstract]:Sepsis is a systemic inflammatory response syndrome caused by infection, which is a common complication of severe trauma, burns, or major surgery, often leading to septic shock and even multiple organ dysfunction syndrome. The liver is not only one of the most vulnerable target organs in sepsis, but also plays a foot in the pathogenesis and development of sepsis. When sepsis occurs, the macrophage -Kupffer cells that are settled in the liver are activated by the endotoxin in the blood and release a large number of inflammatory factors, which directly cause inflammation of the liver and cause systemic inflammatory reactions; Kupffer cells and other inflammatory cells produce a large number of oxygen free radicals and lipid peroxidation products, such as malondialdehyde, which cause liver fine. The destruction of the membrane and the damage of the mitochondrial function caused the release of aminotransferase and bilirubin into the blood. At the same time, the synthesis of a variety of coagulant factors, anticoagulant factors and fibrinolytic inhibitors in the liver decreased, the ability to scavenging active factors of liver cells decreased, causing the body coagulation dysfunction and further aggravating systemic inflammatory response and organ damage. It is now considered that early liver dysfunction is an independent early warning factor for the death of sepsis, so early effective treatment for patients with sepsis and liver injury, timely improvement of liver function, can help improve the prognosis of sepsis patients. Nobiletin, 5,6,7,8,3', 4'-hexame-thoxy flavone, other name Chuan Ting, also known as tangerine flavone A poly (methoxy) flavonoids extracted from the peel of oranges Citrus reticulaia Blanco of the oranges of rutfamily. In recent years, it has been studied that it has many biological activities, such as anti-inflammatory, antioxidant, anti-tumor and neuroprotection, and its effect may be associated with the inhibition of the activity of transcription factors such as NF-kB and activator protein -1 (AP-1). So far, the pharmacological effects and anti inflammatory mechanisms of the pericarin have not been elucidated, but it is also necessary to further evaluate the therapeutic effect of the drug as a treatment for the liver injury of sepsis. The animal model of lipopolysaccharide induced liver injury in mice was used to observe the changes in the mortality of mice after the treatment of septic liver injury, the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), the changes of TNF- a, IL-1 beta and IL-6 in serum and liver tissues, and the extraction of Kupffer cells and detection of Kupffer cells. The changes in the content of I NOS and COX-2 in the inflammatory mediators were measured, and the changes of NF- kappa B, MAPK (ERK, JNK and p38MAPK) and Nrf2-HO-1 signal transduction pathway were detected. The purpose of this study was to clarify the molecular mechanism of the treatment of liver injury in sepsis, and to provide a new research idea for the prevention and treatment of liver injury in sepsis. Objective: To observe the protective effect of peptide on sepsis liver injury in mice. Methods: a mouse model of sepsis liver injury was established by intraperitoneal injection of endotoxin. 75 adult C57BL/6 mice were randomly divided into five groups: normal control group (15, phosphate buffer solution), and LPS model group (15 mice, LPS 10mg /kg), low dose group (15, LPS10mg/kg and 50mg/kg), medium dose group (15, LPS 10mg/kg and LPS10mg/kg), high dose group (15, LPS 10mg/kg, and citpericin 200mg/kg). Each group was killed at different time points (6h, 12h, 24h), respectively, to kill 5 mice, respectively, to collect blood and collect blood. The pathological changes of liver tissue were observed by.HE staining, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) were measured in the serum and serum and liver tissue, and the content of TNF- alpha, IL-1 beta and IL-6 in the serum and liver tissues was measured. In addition, 50 mice were repeated and observed and compared for 72 hours after injection. Results: compared with the normal control group, the indexes of the model group and the drug intervention group were significantly changed, which showed that the model was successfully constructed. (1) after LPS injection of 2h, the model group and the drug intervention group were all depressed, touched, shortness of breath, hypothermia, no water and so on, and NOB dry. The spirit and state of the pretreated mice were relatively good. (2) after 24h after LPS injection, there were obvious pathological changes in the liver of mice, tissue cell swelling, necrosis, congestion and inflammatory cell infiltration, but the tissue injury in the NOB intervention group was significantly relieved; (3) the level of ALT, AST and TBil in the serum of the model group was significantly higher than that in the normal control group, and the above finger in the NOB intervention group was marked. Significantly decreased (P0.05 or P0.01); (4) the levels of inflammatory factors TNF- a, IL-1 beta and IL-6 in the serum and liver homogenate of the model group were significantly increased, and the above indexes in the NOB intervention group were significantly decreased (P0.05 or P0.01). (5) the normal control group, the model group and the NOB intervention group found that the survival rate of the normal control group was 100%, the model group mice 72h raw. The survival rate was 0%. The NOB intervention group could effectively improve the 72h survival rate of sepsis mice: the survival rate of 72h in 50mg/kg dose group was 40%, the survival rate of 72h in 100mg/kg dose group was 50%, and 72h survival rate in 200mg/kg dose group was 80%. Inflammatory cells infiltration, reduce the release of liver enzymes, and then play a protective role in sepsis liver injury mice, and ultimately improve the survival rate of mice. Second: the second part: the effect of citrine on the IkB/NF-kB signaling pathway of Kupffer cells activated in mice: by observing the IkB/NF-kB signal of Kupffer cells activated by LPS in mice. The molecular mechanism of the protective effect of Chen piperine in the liver injury of sepsis was explained. Methods: Kupffer cells were separated by collagenase digestion, density gradient centrifugation and selective adherence method, and inoculated into six hole plates and divided into five groups randomly: normal control group (phosphate buffer solution), LPS model group (LPS 10 g/ml), and low Chen piperinin The dose group (LPS 10 mu g/ml and Sichuan Chen piperine 10 u M), the middle dose group (LPS 10 mu g/ml and citpericin 20 micron), the high dose group (LPS 10 mu g/ml and citpericin 40 M). After the drug stimulation, the cell culture and the cell culture supernatant were collected in 1H and 12h, respectively. Alpha, IL-1 beta, IL-6 levels, and Kupffer cells p-IkB, IkB, p-p65, p65 protein changes when 1H was detected by Western blotting. After 1h, the proportion of p-IkB/IkB, p-p65/p65 protein in Kupffer cells increased significantly (P0.05 or P0.01), and the DNA binding activity of NF-kB p65 in the nucleus increased significantly (P0.05 or P0.01) after LPS stimulation of 1H (P0.05 or P0.01). Conclusion: the protective effect of Chen piperin on hepatic injury of sepsis may be related to inhibiting the overactivation of IkB/NF-kB signaling pathway in Kupffer cells and reducing the protein expression of related inflammatory factors. Third part: the effect of the effect of Chen piperin on the MAPK and Nrf2-HO-1 signaling pathway of LPS activated Kupffer cells in mice: by observing the effects of Chen piperine on LP The effect of S activated Kupffer cells MAPK (ERK, JNK and p38MAPK) and Nrf2-HO-1 signaling pathway to explore the molecular mechanism of the protective effect of citalinin on the liver injury of sepsis. Methods: the isolated collagenase digestion, density gradient centrifugation and selective adherence method were used to separate Kupffer cells and be inoculated into five groups randomly. Group (phosphate buffer), LPS model group (LPS 10 mu g/ml), low dose group of Chen piperin (LPS 10 mu g/ml and 10 mu of Chen piperin), middle dose group of Chen piperin (LPS 10 mu g/ml and citpericin 20 micron), high dose group of Chen piperin (LPS 10 mu g/ml and Chen piperine 40 mu M). P-ERK, p-JNK, p-p38MAPK, Nrf2, HO-1, I NOS, COX-2 expression protein changes. Results: LPS stimulation in vitro Kupffer cell 1H, p-ERK, the expression was significantly higher than the normal control group. The expression of -1 and the expression of Nrf2 protein in the nucleus was slightly higher than that in the normal control group, but the above indexes were significantly increased (P0.05 or P0.01) after different doses of the dried tangerinin. After LPS stimulation of 12h, the expression of I NOS and COX-2 protein in the cells was significantly higher than that in the normal control group (P0.05 or P0.01), and the above indexes were significantly decreased after the different doses of the dried tangerinin (P0) (P0). .05 or P0.01) conclusion: the protective effect of Chen piperin on liver injury in sepsis may be related to the inhibition of the overactivation of MAPK (ERK, JNK and p38 MAPK) signaling pathways in Kupffer cells, reducing the expression of I NOS and COX-2 in inflammatory mediators and activating the Nrf2-HO-1 antioxidant pathway.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7

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