论文标题:二苯乙烯苷的抗炎作用及其机制研究
Anti-inflammatory Effects and Mechanisms of 2, 3, 5, 4"-tetrahydroxystilbene-2-O-β-D-glucoside
论文作者
论文导师 王嘉陵,论文学位 硕士,论文专业 药理学
论文单位 华中科技大学,点击次数 105,论文页数 55页File Size731K
2006-04-01论文网 http://www.lw23.com/lunwen_530509107/
2;;3;;5;;4"-tetrahydroxystilbene-2-O-β-D-glucoside;; Polygonum multiflorum Thunb;; Dimethylbenzene;; Carrageenin;; Lipopolysaccharide;; Cyclooxygenase-2;; Prostaglandin E2;; Inflammation
2,3,5,4’-四羟基二苯乙烯-2-O-β-D葡萄糖苷(2,3,5,4’- tetrahydroxystilbene-2-O -β-D- glucoside, THSG)是从传统中药何首乌中提取的一种水溶性有效成分。经研究表明,THSG是何首乌发挥多种药理作用的主要成分,何首乌乙醇提取物明显抑制二甲苯、角叉菜胶等所致的炎性反应,并有一定的镇痛作用;本课题组已经证实THSG具有抗动脉粥样硬化及抗氧化等作用,这些作用可能与THSG抗炎作用有关。而且THSG化学结构与白藜芦醇(resveratrol, RES)很类似,都属于二苯乙烯类化合物。文献报道,从法国红葡萄酒中提取的白藜芦醇在细胞水平上抑制COX-2(cyclooxygenase-2, COX-2)基因表达且直接抑制COX-2酶活性,这促使我们推测THSG在细胞水平上有抑制COX-2酶活性及与之相关的作用。 本学位论文由二苯乙烯苷的抗急性炎症作用和其抗炎作用机制研究两部分组成,旨在探讨THSG的抗炎作用及机制,为将其开发成新药提供理论和实验依据。 第一部分:二苯乙烯苷的抗急性炎症作用 方法:连续三天,每天一次,灌胃给予THSG后,采用二甲苯(dimethylbenzene)诱导小鼠耳廓局部肿胀的炎症模型,观察二甲苯致炎后一小时THSG对小鼠耳廓肿胀程度的作用;连续三天,每天一次,灌胃给予THSG后,采用角叉菜胶(carrageenin)诱导大鼠足趾肿胀的炎症模型,于角叉菜胶致炎后1, 2, 4, 6小时观察THSG对大鼠足趾肿胀程度的作用。 结果:①在小鼠炎症模型中,二甲苯外涂一小时后可以明显诱导小鼠耳廓肿胀,其使耳廓重量由正常值7.1±0.7 mg增至10.7±2.9 mg (P < 0.05),THSG 2.3, 4.6, 9.2 mg?kg-1均能浓度依赖性地抑制耳廓肿胀,有明显抗炎作用,肿胀度从生理盐水组4.6±1.5 mg分别下降至3.4±1.0,1.0±0.4,0.6±0.2 mg (P < 0.05, P < 0.01)。THSG 9.2 mg?kg-1的抑制率为86.95 %,阳性对照药吲哚美辛(indomethacin, Indo)13 mg?kg-1的抑制率为89.13 %。 ②在大鼠炎症模型中,给予角叉菜胶后大鼠足趾肿胀,随着时间的延长,肿胀度增加。4、6小时作用比较明显,其使大鼠右后足趾肿胀度在6小时由正常值21.59±0.49 mm增至25.66±1.03 mm (P < 0.01)。在4小时THSG 3.2, 6.4, 12.8 mg?kg-1抑制大鼠足趾肿胀,肿胀度从生理盐水组2.60±1.07 mm下降至1.47±0.54 mm, 1.56±0.38 mm (P < 0.05),1.40±0.53 mm (P < 0.01);6小时三个剂量都表现出显著性差异,且呈浓度依赖性,肿胀度从生理盐水组4.07±0.92 mm分别下降至2.28±1.03 mm,2.02±0.25 mm,1.81±0.46 mm (P < 0.01)。且在6小时THSG 12.8 mg?kg-1的抑制率达到57 %,吲哚美辛9 mg?kg-1的抑制率是57.49 %。 第二部分:二苯乙烯苷的抗炎作用机制 §1二苯乙烯苷对细胞存活率的影响 方法:采用MTT法分析巨噬细胞存活率。 结果:①RAW264.7细胞加入LPS 1μg?ml-1作用6小时后,细胞存活率从100 %到110.47±8.19 %,经统计学比较没有差异(P > 0.05),LPS 1μg?ml-1和THSG 1, 10, 100μmol?l-1共同处理细胞8小时,对细胞存活率没有统计学意义上的改变(LPS + THSG组vs LPS(+)组, P > 0.05),存活率分别为112.24±7.44 %,108.49±9.61 %,108.01±7.01 %,可见THSG对细胞存活率没有影响。 §2二苯乙烯苷对PGE2产量的作用 方法:酶联免疫法(enzyme-line immunosorbnent assay, ELISA)检测前列腺素E2 (prostaglandin E2, PGE2)产量。 结果: LPS 1μg?ml-1作用于细胞6小时,能够改变内源性花生四烯酸(arachidonic acid, AA)代谢,增加PGE2产量,从空白组44.87±2.48 pg?ml-1增至69.07±5.73 pg?ml-1(P < 0.05)。LPS 1μg?ml-1和THSG 1, 10, 100μmol?l-1共同处理细胞8小时,能够浓度依赖性地减少PGE2产量,分别为57.87±3.20 pg?ml-1,38.12±4.73 pg?ml-1,34.51±3.34 pg?ml-1(P < 0.05, P < 0.01)。THSG 100μmol?l-1显著性地抑制PGE2产量达到50 %,阳性对照药NS-398 100μmol?l-1的抑制率是71 %。 §3二苯乙烯苷对COX-2和COX-1 mRNA表达的影响 方法:采用半定量逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)检测COX-2和COX-1(cyclooxygen-ase-1, COX-1)的mRNA表达。 结果:单独用LPS 1μg?ml-1处理细胞6小时,COX-2 mRNA表达量较对照组明显增高,COX-2条带灰度/β-actin条带灰度的比值明显深于对照组,灰度比值分别为0.152±0.016, 0.734±0.015 (P < 0.01),但是COX-1 mRNA表达量较对照组没有改变,条带灰度比值基本一致。LPS 1μg?ml-1和THSG 1, 10, 100μmol?l-1共同处理细胞,COX-2表达量较LPS处理组低,条带灰度比值依次降低,且具有剂量依赖性,灰度比值分别为:0.651±0.021,0.498±0.016,0.189±0.016 (P <0.05, P < 0.01),但是COX-1 mRNA表达量较LPS处理组没有改变,条带灰度比值基本一致。 §4二苯乙烯苷对COX-2和COX-1蛋白表达的影响 方法:采用免疫印迹法(Western blot)检测COX-2和COX-1蛋白表达。 结果:培养细胞经药物处理8小时后,采用Western blot方法分析不同组细胞中COX-2和COX-1的表达量。单独用LPS 1μg?ml-1处理细胞6小时,COX-2蛋白表达量较对照组明显增高,条带灰度明显深于对照组,灰度值从对照组65.14±3.6增至103.01±9.2 (P < 0.01),但是COX-1蛋白表达量较对照组没有改变,条带灰度基本一致。LPS 1μg?ml-1和THSG 1, 10, 100μmol?l-1共同处理细胞,COX-2表达量较LPS处理组低,条带灰度依次降低,灰度值分别为:98.22±5.85,90.49±8.12,88.82±7.24 (P < 0.05, P < 0.01),具有剂量依赖性,但是COX-1蛋白表达量较LPS处理组没有改变,条带灰度基本一致。 §5二苯乙烯苷对COX-2酶活性的影响 方法:加入外源性AA采用酶联免疫法检测PGE2产量,分析COX-2酶活性。 结果:COX-2酶催化外源性AA代谢生成PGE2,PGE2产量可以反映COX-2酶活性。PGE2产量高COX-2酶活性强,PGE2产量低COX-2酶活性弱。LPS 1μg?ml-1作用于细胞6小时,能够明显地诱导COX-2酶活性,增加PGE2生成量,从59.25±8.17 pg?ml-1到79.95±5.53 pg?ml-1 (1×105细胞/ 96孔板, P < 0.05)。LPS 1μg?ml-1和THSG 1,10,100μmol?l-1共同处理细胞8小时,能够浓度依赖性地减少外源性AA生成的PGE2产量,从79.95±5.53 pg?ml-1分别降至66.32±1.83 pg?ml-1,48.13±4.65 pg?ml-1,34.78±0.96 pg?ml-1 (P< 0.05, P < 0.01),从而抑制COX-2酶活性。THSG 100μmol?l-1和NS-398 100μmol?l-1的抑制率分别为56 %和68 %。 结论: ①THSG有抗小鼠耳廓急性炎症作用,此作用可能与抑制急性期小鼠耳局部血管扩张和毛细血管通透性增加的非特异性炎症反应有关。 ②THSG有抗大鼠足趾肿胀的作用,此作用可能与抑制大鼠足趾局部炎症介质如前列腺素和5-羟色胺的释放有关。 ③经过MTT法检测THSG在100μmol?l-1时对细胞存活率没有明显作用,表明THSG对细胞没有毒性。 ④THSG 1, 10, 100μmol?l-1能够浓度依赖性地减少内源性AA代谢生成的PGE2产量,表明THSG能够直接抑制PGE2产量,此作用可能与抑制基因水平COX-2表达或者是直接抑制COX-2酶活性有关。
2,3,5,4"-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is an active water solubility compound extracted from the roots of traditional Chinese drug Polygonum multiflorum Thunb. The documents suggested that THSG was a major component of Polygonum multiflorum Thunb pharmacological actions. The documents suggested that Ethanol extracted components of Polygonum multiflorum Thunb inhibited inflammatory responses induced by dimethylbenzene and carrageenin (CGN) and afforded analgesic effects. It had also been reported that THSG afford anti-atherosclerotic and anti-oxidation effect. These effects of THSG may be related to anti-inflammatory effects in experimental animals. The chemical structure of THSG and resveratrol was very similar and belonged to hydroxystilbene compounds. Resveratrol, which extracted from red wine in France, suppressed the activation of cyclooxygenase-2 (COX-2) gene expression and directly inhibits the COX-2 enzyme activity. On the basis of previous work, we wonder whether THSG suppressed COX-2 activity and expression. The following two sections were investigated in this thesis:①Anti-inflammation effects of THSG on experimental animals,②Anti-inflammation mechanisms research of THSG in LPS-stimulated mouse RAW264.7 macrophage cells. To demonstrate these effects of THSG, It provides the theory and experiment documents for developing THSG into a novel drug. PartⅠ: Anti-inflammatory effects THSG on experimental animals Methods: In mouse ear edema model, after 3 days by oral administration once a day, dimethylbenzene-induced mouse ear edema model was prepared, it was observed to the effet of THSG on mouse ear edema in an hour of dimethylbenzene-induced. In rat paw edema model, after 3 days by oral administration once a day, the CGN-induced rat was determined, it was observed to the effet of THSG on rat paw edema in 1, 2, 4 or 6 hour of CGN-induced. Results:①In mouse edema model, dimetheylbenzene could significantly induce mouse ear edema in 1 hour. The ear edema weight was increased from 7.1±0.7 mg (as control) to 10.7±2.9 mg (P < 0.05). THSG 2.3, 4.6 and 9.2 mg?kg-1 by oral administration could inhibit mouse ear edema and had obvious anti-inflammatory effects in concentration-dependent manner. Edema value was decreased from 4.6±1.5 mg to 3.4±1.0, 1.0±0.4, 0.6±0.2 mg (P < 0.05, P < 0.01). The percentage of inhibition of THSG 9.2 mg?kg-1 was 86.95 % and Indomethacin 13 mg?kg-1, a reference compound, show 89.13 % inhibition. ②In rat edema model, CGN could evoke rat paw edema and had obvious effect at 4 or 6 hour. The paw edema circumference was increased from 21.59±0.49 mm to 25.66±1.03 mm (P < 0.01). At 4 hour, THSG 3.2, 6.4 and 12.8 mg?kg-1 could inhibit rat paw edema and had obvious anti-inflammatory effects. Edema value was decreased from 2.60±1.07 mm to 1.47±0.54 mm, 1.56±0.38 mm (P < 0.05), 1.40±0.53 mm (P < 0.01); At 6 hour, THSG 3.2, 6.4 and 12.8 mg?kg-1 could inhibit rat paw edema and had obvious anti-inflammatory effects at dose-dependent manner. Edema value was decreased from 4.07±0.92 mm to 2.28±1.03 mm, 2.02±0.25 mm, 1.81±0.46 mm (P < 0.01); At 6 hour the percentage of inhibition of THSG 12.8 mg?kg-1 was 56 %; Indomethacin 9 mg?kg-1, a reference compound, showed 57 % inhibition in paw edema model. PartⅡ: Anti-inflammation mechanisms of action of THSG in LPS-stimulated mouse RAW264.7 macrophage cells §1 Effects of THSG on cell viability Methods: RAW264.7 macrophage cells were determined by MTT assay. Results: RAW264.7 cells were treated with lipopolysaccharide (LPS) 1μg?ml-1 for 6 hours. The viability of cells afforded no change from 100 % to 110.47 % (P > 0.05). After treatment with THSG 1, 10, 100μmol?l-1 for 2 h, no obvious potential effect could be found (LPS + THSG group vs LPS (+)group, P > 0.05) and cell viability was 112.24±7.44 %, 108.49±9.61 %, 108.01±7.01 %, respectively, so THSG had no effects on cell viability. §2 Effects of THSG on PGE2 production Methods: Prostaglandin E2 (PGE2) production was investigated by Enzyme-Line Immunosorbnent Assay (ELISA). Results: LPS 1μg?ml-1 to RAW264.7 cells for 6 h could change endogenous arachidonic acid (AA) and then significantly increase the production of PGE2 from 44.87±2.48 pg?ml-1 (control level) to 69.07±5.73 pg?ml-1(P < 0.05). THSG 1, 10, 100μmol?l-1 dose-dependently decreased LPS-induced PGE2 production and PGE2 production was 57.87±3.20 pg?ml-1, 38.12±4.73 pg?ml-1, 34.51±3.34 pg?ml-1(P < 0.05, P < 0.01), respectively. THSG 100μmol?l-1 decreased PGE2 production by 50%. NS-398 100μmol?l-1, a selective COX-2 inhibitor was used as a positive control in this assay showed 71% inhibition. §3 Effects of THSG on COX-2 and cyclooxygenase-1 (COX-1) mRNA expressions Methods: COX-2 and cyclooxygenase-1 (COX-1) mRNA expressions were assayed by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR). Results: LPS 1μg?ml-1 for RAW 264.7 cells for 6 h clearly induced COX-2 mRNA, COX-2/β-actin density ratio exceeded control group from 0.152±0.016 (as control) to 0.734±0.015 (P < 0.01) and COX-1/β-actin density ratio had no effect, compared with control group. LPS 1μg?ml-1 and THSG 1, 10, 100μmol?l-1 for RAW 264.7 cells, COX-2 density ratio was less than LPS group, THSG 1, 10, 100μmol?l-1 could concentration-dependently suppressed COX-2 mRNA expression and density ratio was 0.651±0.021, 0.498±0.016, 0.189±0.016 (P < 0.05, P < 0.01), respectively; THSG 1, 10, 100μmol?l-1 had no effect on COX-1 mRNA and density ratio was almost the same. §4 Effects of THSG on COX-2 and cyclooxygenase-1 (COX-1) protein expressions Methods: COX-2 and COX-1 protein expression were assayed by Western blot method. Results: LPS 1μg?ml-1after treatment for 6h clearly induced COX-2 protein expression from 65.14±3.6 (as control) to 103.01±9.2 (P < 0.01) and had no effect on COX-1 protein. LPS 1μg?ml-1 and THSG 1, 10, 100μmol?l-1 for RAW 264.7 cells, COX-2 density ratio was less than LPS group, THSG 1, 10, 100μmol?l-1 could concentration-dependently suppressed LPS-stimulated COX-2 protein expression and optical density value is 98.22±5.85, 90.49±8.12, 88.82±7.24 ( P < 0.05, P < 0.01), respectively; THSG 1, 10, 100μmol?l-1 had no effect on COX-1 protein and density value was almost the same. §5 Effects of THSG on COX-2 enzyme activity Methods: COX-2 enzyme activity was investigated by ELISA. Results: COX-2 enzyme catalyzed exogenous AA; metabolite PGE2 production could represent COX-2 enzyme activity. PGE2 production was much and enzyme activity could be high. LPS 1μg?ml-1for RAW264.7 cells for 6 hour dramatically increased PGE2 production from 59.25±8.17 pg?ml-1 (the basal level) to 79.95±5.53 pg?ml-1 (1×105 cells in a 96-well plate, P<0.05). THSG 1, 10, 100μmol?l-1 could concentration -dependently decreased PGE2 production from AA and PGE2 production was decreased from 79.95±5.53 pg?ml-1 to 66.32±1.83 pg?ml-1, 48.13±4.65 pg?ml-1, 34.78±0.96 pg?ml-1 (P< 0.05, P< 0.01), respectively. The percentage of inhibition of THSG or NS-398 100μmol?l-1 was 56 % and 68 %, respectively. Conclusion: ①THSG could afford anti-inflammatory effect in mouse acute inflammatory model, this effect may be related to inhibit mouse ear capillary vessel edema. ②THSG could afford anti-inflammatory effect in rat inflammatory model, this effect may be related to inhibit inflammation mediator (prostaglandins, 5-hydroxytryptamine). ③THSG 100μmol?l-1 did not show any cytotoxicity judged by MTT assay, indicating that the inhibition of PGE2 production by THSG was not associated with its cytotoxicity. ④THSG could dose-dependently reduce the PGE2 production, from endogenous AA in LPS-activated RAW 264.7 cells, indicating that THSG could directly decrease PGE2 production, this effect was related to inhibit COX-2 gene expression or directly inhibit COX-2 enzyme activity.
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