【作者】 赵雷；

【导师】 张淑玲；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 目的:柯里拉京(corilagin)是从叶下珠中提取的有效药理成分。本研究探讨柯里拉京(corilagin)对脂多糖(LPS)刺激的RAW 264.7小鼠巨噬细胞瘤细胞分泌的促炎细胞因子和介质的干预作用。方法:MTT法检测柯里拉京(corilagin)对RAW 264.7小鼠巨噬细胞瘤细胞的最大无毒浓度,采用LPS刺激的RAW 264.7小鼠巨噬细胞瘤细胞,同时用柯里拉京(corilagin)进行干预, ELISA法检测细胞上清中TNF-α,IL-1β,IL-6,Il-10的含量,Griess法检测细胞上清液中的NO,Real-time PCR法检测细胞中TNF-α,COX-2,iNOS,HO-1 mRNA的表达情况,Western-blotting法检测细胞内COX-2和HO-1蛋白的表达。结果:MTT法显示柯里拉京(corilagin)对RAW 264.7细胞株的最大无毒浓度为0.5mg/L。LPS刺激的RAW264.7细胞经柯里拉京(corilagin)干预24小时后,细胞上清液中的TNF-α,IL-1β和IL-6水平明显比模型组要低(p<0.01)。柯里拉京(corilagin)可以明显降低经LPS刺激的RAW264.7细胞内COX-2蛋白的表达(p<0.01)。柯里拉京(corilagin)不仅可以明显降低细胞上清液中一氧化氮的释放(p<0.05),而且可以明显下调细胞中一氧化氮合酶mRNA的表达(p<0.05 or 0.01),并且对IL-10有抑制作用,可促进HO-1基因和蛋白的表达(p<0.01)。柯里拉京(corilagin)可以明显阻止LPS刺激的RAW264.7细胞内NF-κB的核转位(p<0.01)。结论:柯里拉京(corilagin)对促炎细胞因子和炎性介质TNF-α,IL-1β,IL-6,COX-2,NO均有不同程度的抑制作用,并可以阻止NF-κB的核转录,说明柯里拉京(corilagin)对炎症的起始阶段有明显的抑制作用。在炎症的修复阶段,柯里拉京(corilagin)对HO-1的表达有明显的促进作用,说明此成分有促进炎症恢复的功效。柯里拉京(corilagin)对IL-10有抑制作用的同时没有地塞米松诱导凋亡的作用,其机制还需要进一步探讨。总之,从本实验可以看出,柯里拉京(corilagin)在对LPS刺激下的RAW264.7细胞分泌的炎性因子和介质有不同程度的抑制作用,可以初步说明柯里拉京(corilagin)具有抗炎的作用。第二部分柯里拉京(corilagin)抗乙肝病毒的作用研究目的:明确柯里拉京(corilagin)是否有抗乙肝病毒的作用。方法:MTT法检测柯里拉京(corilagin)对HepG2.2.15细胞的最大无毒浓度。采用柯里拉京(corilagin)干预HepG2.2.15细胞,实时荧光定量PCR法检测细胞上清液中HBV-DNA的分泌量,EIA法检测细胞上清中HBsAg和HBeAg。结果:处理9d后,80mg/L柯里拉京(corilagin)组OD值与正常HepG2.2.15细胞组相比有显著性差异(p<0.05),9d时柯里拉京(corilagin)对HepG2.2.15细胞的半数抑制浓度(IC50)为75.85mg/L。各实验剂量的柯里拉京(corilagin)组分别在3d、6d、9d上清液中分泌的HBV-DNA与正常细胞组相比无显著性差异(p>0.05)。各实验剂量的柯里拉京(corilagin)组分别在3d、6d、9d分泌的HBsAg与正常细胞组相比无显著性差异(p>0.05)。各实验剂量的柯里拉京(corilagin)组分别在3d、6d、9d分泌的HBeAg与正常细胞组相比无显著性差异(p>0.05)。结论:从结果来看,实验浓度的柯里拉京(corilagin)对HepG2.2.15细胞没有毒性,在此浓度作用下HepG2.2.15细胞分泌的HBsAg、HBeAg、HBV-DNA的量没有受到明显影响,说明柯里拉京(corilagin)没有明显的抗乙肝病毒的作用。目的:研究叶下珠柯里拉京(Corilagin)提取物对肝内胆汁淤积的干预作用。方法:给予SD大鼠ANIT造成急性淤胆型肝炎的动物模型,采用叶下珠柯里拉京(Corilagin)提取物灌胃,以地塞米松和熊去氧胆酸(UDCA)作为对照,检测肝功能,观察动物生活质量,病理切片检测肝组织病理学变化,实时荧光定量PCR法检测核因子Egr-1,趋化因子CINC-1和MIP-2,western-blot法检测粘附分子ICAM-1,免疫组织化学法检测核因子NF-κB转录,诱导型一氧化氮合酶iNOS表达,ELISA法检测肝组织和血清中TNF-α和IL-6的水平,采用硝酸还原酶法检测组织中一氧化氮(NO),硫代巴比妥酸比色法检测组织中丙二醛(MDA),黄嘌呤氧化酶法检测组织中超氧化物歧化酶(SOD),比色法检测组织中髓过氧化物酶(MPO)。结果:经柯里拉京(corilagin)处理的淤胆型肝炎大鼠的淤胆指数明显降低,柯里拉京(corilagin)对AKP,γ-GT和TBA在高峰期或者消退期有作用,但对转氨酶无明显影响。对急性淤胆型肝炎动物模型的生存状况也有明显改善,对肝脏组织学变化起到降低损害强度和缩短进程的作用。对核因子Egr-1和NF-κB在高峰期或消退期有作用,对趋化因子CINC-1和MIP-2在高峰期或消退期有明显抑制作用,对粘附分子ICAM-1的表达有降低作用,对肝组织内促炎细胞因子TNF-α和IL-6有一定影响,对肝组织iNOS和NO的表达有明显促进的作用,对肝组织氧化应激的指标有明显恢复作用。UDCA在急性淤胆型肝炎时有保护细胞膜和促进胆汁排泄,改善肝功能的作用,但出现的时间较晚,对急性淤胆型肝炎动物模型的生存状况也有明显改善,对肝脏组织学变化起到降低损害强度和延缓损害进程的作用。对核转录因子Egr-1无明显作用,但淤胆后期对NF-κB有一定抑制作用,对趋化因子CINC-1和MIP-2没有明显抑制作用,对粘附分子ICAM-1的表达有一定的降低作用,对肝组织内促炎细胞因子TNF-α和IL-6,以及血清TNF-α无明显作用,仅后期对血清IL-6有一定影响,对肝组织iNOS有轻微的促进表达作用,但不影响肝组织中的NO,对肝组织中中性粒细胞的聚集无明显影响,对肝组织中氧化应激的指标也无明显影响。经地塞米松处理的淤胆型肝炎大鼠的淤胆指数和碱性磷酸酶出现降低外,其他指标如转氨酶,γ-谷氨酰基转移酶和总胆汁酸均上升,说明地塞米松导致的免疫抑制可能造成肝细胞的继发性损伤。并且,地塞米松处理的大鼠生活质量明显变差,体重急剧下降,凝血功能失常,出现出血倾向,肝组织病理学改变明显加重,对核因子Egr-1无影响,早期可以抑制NF-κB,中后期反而加重,甚至还有加强趋化因子CINC-1和MIP-2的作用,对粘附分子ICAM-1的表达也是早期抑制,中后期反而促进,对iNOS和NO在上升期和消退期有一定作用,有阻止组织和血清TNF-α和血清IL-6达到高峰并促进恢复的作用,对MPO反而有促进作用,但对氧化产物MDA和抗氧化酶SOD有一定的调节作用。结论:柯里拉京(corilagin)对淤胆型肝炎的保护作用上,对肝细胞膜没有明显的保护作用,但对促进胆红素的排泄,缓解肝脏的炎症反应和氧化应激损伤方面均有不同程度的作用:以促进胆红素排泄,抑制白细胞向肝脏的募集和趋化,缓解肝脏的氧化应激最为明显,且在整个急性淤胆型肝炎的发展演变过程中,以发病的中后期效果最好,起到降低炎症反应的强度和加快炎症反应的过程,促进炎症的恢复三个方面的作用。UDCA可能是通过调节肝细胞和胆管细胞膜上的受体,促进胆汁排泄,减轻胆汁酸的损害起到保护肝脏的作用,其自身对肝脏的炎症并没有很明显的保护作用。而地塞米松虽然具备一定的退黄作用,但同时也具备加重肝脏损害,促进中性粒细胞趋化和聚集,粘附,导致内环境紊乱,使病情加重的危险。因此,柯里拉京(corilagin)与UDCA比较,主要体现在通过抑制炎症和抗氧化达到护肝,退黄的作用,并且没有地塞米松的副作用。更多还原

【Abstract】 Aim: Corilagin is a novel member of the tannin family which has been discovered from many medicinal plants and has been confirmed many pharmacological activities. However, the purified Corilagin that was used in experiment is rare, and the anti-inflammatory mechanism of Corilagin has not been investigated clearly. This study is to explore the inner anti-inflammatory mechanism of Corilagin.Methods: Inflammatory cellular model was established by lipopolysaccharide (LPS) interfering on RAW264.7 cell line. Levels of TNF-α, IL-1β, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-α, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, translocation of NF-κB were assayed by ELISA or Griess method, real-time quantitative PCR, western-blot and immunocytochemistry method, respectively.Results: Corilagin could significantly reduce production of pro-inflammatory cytokines and mediators TNF-α, IL-1β, IL-6, NO (iNOS) and COX-2 on both protein and gene level by blocking NF-κB nuclear translocation. Meanwhile Corilagin could notably promote release of anti-inflammatory factor HO-1 on both protein and gene level, but suppress the release of IL-10.Conclusion: the anti-inflammatory effects of Corilagin are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-κB activation. Corilagin also can promote HO-1 production to induce regression of inflammation but can inhibit IL-10 production like Dexamethasone. Corilagin possesses a potential anti-inflammatory effect by not only abating inflammatory impairment but also promoting regression of inflammation and has a good prospect to be used in many inflammation- related diseases.Part 2: Exploration of Corilagin on the effect of anti-hepatitis B virusAim: To explore the antiviral effect of Corilagin on hepatitis B.Methods: The max non-cytotoxic concentration of Corilagin was determined by MTT assay. The HepG 2.2.15 cell line was interfered by Corilagin. The amount of HBV-DNA in supernatant was measured by real-time PCR assay. The HBsAg and HBeAg in supernatant were detected by EIA.Results: The max non-cytotoxic concentration of Corilagin was 80mg/L after 9d persistent intervention. There were no differences between Corilagin group and control group on HBV-DNA, HBsAg and HBeAg in supernantant.Conclusion: Corilain has no effect on inhibiting hepatitis B virus.Part 3: Exploration of Corilagin to treat alpha-naphthylisothiocyanate- induced chelostatic hepatitis via anti-inflammation pathwayAim: This study is to disclose the mechanism of Corilagin to treat chelostatic hepatitis via anti-inflammation pathway. Methods: Rats were divided into Corilagin, ursodeoxycholic acid, Dexamethasone, model and blank control groups with treatment of respective agent after administration of alpha-naphthylisothiocyanate. At 24h, 48h and 72h time points after administration those rats were examined liver function, pathological changes of hepatic tissue, tumor necrosis factor (TNF)-α, interleukin (IL)-6, myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), cytokine-induced neutrophil chemoattractant (CINC)-1, monocyte inflammatory protein (MIP)-2, intercellular adhesion molecule (ICAM)-1, nuclear factor (NF)-κB and early growth response (Egr)-1, nitric oxide (NO) and inducible nitric oxide synthase (iNOS).Result: Compared to the controls, Corilagin had a notable effect on rat’s living condition, pathological manifestation of hepatic tissue, total bilirubin, direct bilirubin, (P<0.05), and little effect on ALP, GGT and total bile acid. With Corilagin intervention, levels of TNF-α, IL-6, MPO, MDA, CINC-1, MIP-2, ICAM-1, Egr-1 and translocation of NF-κB was decreased to different extent, and levels of SOD, NO and iNOS was markedly increased.Conclusion: Corilagin has a protective effect on liver function and a restoring activity on chelostatic hepatitis by anti-inflammation. The effects are mainly due to antagonizing pro-inflammatory cytokines and mediators, inhibiting oxidative damage, improving hepatic microcirculation, reducing impairment signals, and controlling nutrophil infiltration.更多还原