EGCG对小鼠肝缺血再灌注损伤的保护作用及机制

发布时间：2018-07-07 17:10

本文选题：EGCG + 肝缺血再灌注　；参考：《广西医科大学》2015年硕士论文

【摘要】：目的：观察表没食子儿茶素没食子酸酯[(-)-Epigallocatechin-3-gallate, EGCG]对Balb/c小鼠肝缺血再灌注损伤的保护性作用,研究其作用与血红素氧合酶-1(Hemeoxygenase-1,HO-1)、Toll样受体-4(Toll-like receptor4, TLR4)/核因子kappa B P65 (Nuclear fator-κB P65, NF-κB P65)及相关信号分子的关系,探讨其作用机制。方法：健康雄性Balb/c小鼠18只,随机分3组(n=6),分别为：假手术组(Sham组)、缺血再灌注组(I/R组)和I/R+EGCG处理组(EGCG组)。各组分别于再灌注末摘除眼球取血样,处死小鼠、收集肝组织。测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)水平；缺血再灌注肝组织苏木素-伊红(Hematoxylin-Eosine, HE)染色后光镜下观察其病理学改变；免疫组织化学测定TLR4、HO-1和NF-κB P65蛋白的表达量；Western Blot进一步验证TLR4、HO-1和NF-κB P65蛋白水平表达的变化; Real time-PCR检测EGCG作用后小鼠左外叶肝组织中TLR4、HO-1、IL-1β、IL-6及TNF-α等信号分子mRNA表达水平的变化。结果：经EGCG预处理后,EGCG组血清ALT水平较I/R组均明显降低[(823.00-±200.25)vs(1143.33±268.66),(715.33±159.15)vs(1295.00±214.39),P0.05],与Sham组比较,EGCG组血清AST、 ALT水平均显著升高[(823.00±200.25)vs(349.00±39.24),(715.33±159.15)vs(205.00±28.35),P0.01]。从病理学上观察,根据Suzuki评分标准,EGCG组(6.50±1.05)显著高于Sham组(0.43±0.38)(P0.01),I/R组(8.17±1.47)明显高于EGCG组(6.50±1.05)(P0.05),EGCG组血再灌注肝组织损伤程度明显小于IR组,但较Sham组显著增大。依据Soslow评分标准对免疫组化结果进行评分：Sham组、EGCG组、I/R组TLR4指标得分分别为2.83±0.98分、5.67±1.51分、8.33±2.25分,Sham组、I/R组、EGCG组指标得分分别为3.00±0.98分、6.67±1.97分、9.00±1.55分,Sham组、I/R组、EGCG组HO-1指标得分分别为2.92±0.85分、6.56±1.44分、9.43±2.31分,与I/R组比较,EGCG组TLR4、NF-kB P65蛋白表达量明显降低(P0.05),EGCG组HO-1蛋白表达量明显升高(P0.05)；与Sham组比较,EGCG组TLR4、NF-kB P65蛋白表达量显著增高(P0.01),EGCG组HO-1蛋白表达量明显增高(P0.01)。以β-actin为内参照,Western Blot结果示,EGCG组HO-1蛋白相对表达量明显高于I/R组[(0.90±0.06)vs(0.71±0.06),P0.05],同时I/R组HO-1蛋白的相对表达量明显高于Sham组[(0.71±0.06)vs(0.52±0.05),P0.05]；I/R组TLR4、NF-κB P65蛋白的相对表达量明显高于EGCG组[(1.24±0.09)vs(0.98±0.07),(1.56±0.06)vs(1.27±0.06),P0.05],且EGCG组TLR4、NF-κB P65蛋白相对表达量明显高于Sham组[(0.98±0.07)vs(0.60±0.07),(1.27±0.06)vs(0.59±0.05),P0.05]。Real time-PCR结果示,HO-1 mRNA的相对表达水平EGCG组(5.86±0.94)的较I/R组(3.32±0.823)明显增高(P0.05),I/R组较Sham组(1.00±0.00)显著增高(P0.01); TLR4、TNF-α、IL-6及IL-lb mRNA的相对表达水平I/R组[(5.86±0.78)、(4.43±1.39)、(5.06±1.43)、(6.53±1.62)]较EGCG组[(3.82±1.42)、(2.74±0.73)、(2.93±0.80)、(4.36±1.19)]明显增高(P0.05),EGCG组较Sham组(1.00±0.00)显著增高(P0.01)。结论：EGCG可通过抑制炎症反应,减少肝组织缺血再灌注损伤,其机制：一方面可能与EGCG下调TLR4/NF-κB P65信号通路及通路下游TNF-α、IL-1β、IL-6等炎症信号分子表达有关,减少炎症介质介导的炎症反应对肝缺血再灌的损伤；另一方面其机制可能是EGCG抑制TLR4的表达,从而上调HO-1的表达,发挥对肝缺血再灌注损伤的保护作用。
[Abstract]:Objective: To observe the protective effect of epigallocatechin gallate (-) (-) -Epigallocatechin-3-gallate, EGCG] on hepatic ischemia reperfusion injury in Balb/c mice, and to study the role of epigallocatechin gallate (EGCG]) and the role of heme oxygenase (Hemeoxygenase-1, HO-1), Toll like receptor -4 (Toll-like receptor4, TLR4) / nuclear factor kappa. The relationship between kappa B P65) and the related signal molecules. Methods: 18 healthy male Balb/c mice were randomly divided into 3 groups (n=6), respectively: sham operation group (group Sham), ischemia reperfusion group (group I/R) and I/R+EGCG treatment group (EGCG group). Each group was removed from the end of reperfusion and removed the blood sample of the eyeball, killed the mice and collected the liver tissue. The serum was collected and serum was measured. The level of middle glutamic pyretransaminase (ALT) and glutamic oxaline aminotransferase (AST); the pathological changes were observed under the light microscopy of hematoxylin eosin (Hematoxylin-Eosine, HE) staining in ischemia reperfusion liver tissue; the expression of TLR4, HO-1 and NF- kappa B P65 protein was measured by immunohistochemistry; Western Blot further demonstrated the expression of TLR4, and the level of the protein level. Change; Real time-PCR detected the change of mRNA expression level of TLR4, HO-1, IL-1 beta, IL-6 and TNF- alpha in the left outer lobe liver of mice after EGCG action. Results: after the EGCG pretreatment, the serum ALT level of EGCG group was significantly lower than that of the Group [(1143.33 + 268.66), (715.33 + 159.15), 1295 + 214.39). In group am, serum AST and ALT levels in EGCG group were significantly higher [(823 + 200.25) vs (349 + 39.24), (715.33 + 159.15) vs (205 + 28.35), P0.01]. was observed from pathology. According to Suzuki scoring standard, EGCG group (6.50 + 1.05) was significantly higher than Sham group (0.43 + 0.38) (P0.01). I/R group was significantly higher than that of the group. The degree of hepatic tissue injury in blood reperfusion was significantly smaller than that of the IR group, but it was significantly higher than that in the Sham group. According to the Soslow score, the scores of the immunohistochemical results were 2.83 + 0.98, 5.67 + 1.51, 8.33 + 2.25, Sham, I/R, and EGCG groups were 3 + 0.98, 6.67 + 1.97, 9., respectively. The scores of HO-1 index in group Sham, group I/R and group EGCG were 2.92 + 0.85, 6.56 + 1.44 and 9.43 + 2.31 respectively. Compared with I/R group, EGCG group TLR4, NF-kB P65 protein expression decreased significantly (P0.05), EGCG group HO-1 protein expression increased significantly. The expression of HO-1 protein was significantly higher (P0.01). The results of Western Blot showed that the relative expression of HO-1 protein in group EGCG was significantly higher than that of I/R Group [(0.90 + 0.06) vs (0.71 + 0.06), P0.05], and the relative expression of HO-1 protein in I/R group was significantly higher than that of the Group [(0.71 + 0.06) (0.52 + 0.05). The relative expression was significantly higher than that in the EGCG Group [(1.24 + 0.09) vs (0.98 + 0.07), (1.56 + 0.06) vs (1.27 + 0.06), P0.05], and TLR4 in EGCG group, and the relative expression of NF- kappa B P65 protein was significantly higher than that of Sham Group [(0.98 + 0.07) vs (0.60 + 0.07). Compared with group I/R (3.32 + 0.823), group I/R was significantly higher than that of group Sham (1 + 0) (P0.01), TLR4, TNF- a, IL-6 and IL-lb mRNA were significantly higher than EGCG group (5.86 + 0.78), (4.43 + 1.39), (5.06 + 1.43), (6.53 + 1.62)]. Group (1 + 0) significantly increased (P0.01). Conclusion: EGCG can reduce the liver tissue ischemia reperfusion injury by inhibiting the inflammatory response, its mechanism may be related to EGCG down the TLR4/NF- kappa B P65 signaling pathway and the downstream TNF- a, IL-1 beta, IL-6 and other inflammatory signals, and reduce inflammatory mediators mediated inflammatory reaction to liver ischemia On the other hand, the mechanism may be that EGCG inhibits the expression of TLR4, thereby upregulates the expression of HO-1, and plays a protective role in hepatic ischemia-reperfusion injury.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3

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