论文标题:当归A_3部位的抗炎作用及机制研究二苯乙烯苷对eNOS的作用及机制研究
Effects and Mechanisms of A_3 on Inflammation Effects and Mechanisms of THSG on eNOS
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 151,论文页数 74页File Size926K
2006-04-01论文网 http://www.lw23.com/lunwen_44411287/
Angelica;; Angelica oil;; A_3 active component;; anti-inflammatory;; cyclooxygenase-2;2,3,5,4’-tetrahydroxystilbene-2-O-beta-D-glucoside(THSG);; human umbilical vein endothelial cells(HUVEC);; endothelial nitric oxide synthase(eNOS)
第一部分当归A_3部位的抗炎作用及其对大鼠子宫Cox-2表达的影响 当归A_3部位(简称A_3)是我系从当归挥发油(CO2超临界法提取)中萃取得到的中性、非酚性部位。实验证明当归挥发油具有抗炎镇痛作用,且其抗炎作用与抑制PGs生成有关。我们前期的研究发现:A_3为当归挥发油中最佳抑制子宫收缩、抗痛经活性部位,其抑制子宫收缩机制可能与抑制PGs生成有关。本部分拟先建立炎症模型研究A_3的抗炎作用,然后进一步研究其对Cox-2表达的影响,探讨其抗炎抗痛经的作用机制,为将其开发成新药提供理论和实验依据。 §1 A_3对二甲苯所致小鼠耳廓肿胀的影响 方法:连续灌胃给药3天(1次/天)后,采用二甲苯制备小鼠耳廓肿胀的炎症模型,观察二甲苯致炎后1 h药物对小鼠耳肿胀的影响。结果:A_3 0.001 g/kg、0.005 g/kg和0.01 g/kg可剂量依赖性地抑制小鼠耳廓肿胀,抑制率分别为10.6%、21.1%和53% ( n=10)。当归挥发油0.1 g/kg的抑制率为51.3%。A_3 0.01 g/kg抑制小鼠耳廓肿胀作用与当归挥发油0.1 g/kg的作用相当。 §2 A_3对角叉菜胶所致大鼠足肿胀的影响 方法:连续灌胃给药4天(1次/天)后,采用角叉菜胶制备大鼠足趾肿胀的炎症模型,观察角叉菜胶致炎后1,2,4,6 h药物对大鼠足趾肿胀的影响。结果:A_3(0.001, 0.005, 0.01 g/kg)可剂量依赖性抑制角叉菜胶所致大鼠足趾肿胀,致炎后1 h足肿胀度由对照0.224±0.032 cm降至0.204±0.060,0.171±0.053和0.126±0.061 cm(n=8, P >0.05,P <0.05,P <0.01)。当归挥发油0.1 g/kg组的足趾肿胀度为0.171±0.041 cm。A_3 0.005 g/kg抑制足趾肿胀作用与当归挥发油0.1 g/kg作用相当。 §3 A_3对大鼠子宫Cox-2 mRNA表达的影响 方法:采用RT-PCR法检测子宫Cox-2 mRNA的表达 结果:LPS 1μg/mL可显著增加离体大鼠子宫Cox-2 mRNA的表达水平。A_3 (10,20,40,80,160,320 mg/L)剂量依赖性地抑制LPS诱导的子宫Cox-2 mRNA表达水平增高,光密度比值分别从0.462±0.164下降至0.408±0.136,0.368±0.126,0.306±0.065,0.250±0.084,0.138±0.016和0.008±0.003(n=8)。§4 A_3对大鼠子宫Cox-2蛋白表达的影响 方法:采用Western blot法检测Cox-2蛋白表达水平 结果:LPS 1μg/mL可显著增加离体大鼠子宫Cox-2蛋白的表达水平。A_3 (10,20,40,80,160,320 mg/L)剂量依赖性地抑制LPS诱导的子宫Cox-2蛋白表达水平增高,灰度值分别从187.8±13.5依次下降至162.6±16.3,155.0±17.0,148.4±14.3,133.6±13.3,125±15.4和119.4±14.4 (n=8) 结论 1. A_3可显著抑制二甲苯所致的小鼠耳廓肿胀和角叉菜胶所致的大鼠足趾肿胀,且A_3 0.01 g/kg抑制耳廓肿胀度和A_3 0.005 g/kg 1 h抑制足趾肿胀度分别与当归挥发油0.1 g/kg抑制度相当,提示A_3具有强大的抗炎作用。 2. A_3可剂量依赖性地抑制LPS诱导的大鼠子宫Cox-2 mRNA及蛋白表达增加,提示A_3可通过下调子宫组织中Cox-2的表达来减少PGs(包括PGF2α)的生物合成,从而发挥抗炎抗痛经作用。 第二部分二苯乙烯苷对HUVEC eNOS表达的影响 二苯乙烯苷(2,3,5,4’-tetrahydroxystilbene-2-O-beta-D-glucoside,THSG)是从中药何首乌中提取的一种活性成分,已证实其对离体血管具有内皮依赖性舒张作用,且与增强一氧化氮合酶(nitric oxide synthase ,NOS)活性和增加一氧化氮(nitric oxide,NO)的含量有关。同时发现THSG也能增强人脐静脉内皮细胞(human umbilical vein endothelial cells ,HUVEC)内皮型一氧化氮合酶(endothelial nitric oxide synthase ,eNOS)活性和增加NO产量,本部分进一步研究THSG对eNOS mRNA和蛋白表达的影响,以期阐述THSG增加NO产量、舒张血管的作用机制。 §1 THSG对HUVEC eNOS mRNA表达的影响 方法:采用RT-PCR法检测HUVEC eNOS mRNA表达 结果:用不同浓度(1,3,10,30,100μmol/L)THSG孵育HUVEC 24 h后,提取细胞总RNA进行RT-PCR。结果显示THSG增强eNOS mRNA表达呈浓度依赖性。孵育24 h后,1μmol/L THSG即可使eNOS mRNA水平增加(P<0.05)。10μmol/L THSG使eNOS mRNA水平显著增加(P<0.01),平均光密度比值为对照组的1.9倍。用10μmol/L THSG孵育HUVEC 12,24,36,48 h后提取RNA进行RT-PCR ,结果显示THSG增强eNOS mRNA表达呈时间依赖性,10μmol/L THSG孵育12 h后,eNOS mRNA表达水平增高即有显著差异(P<0.05),24 h达高峰(P<0.01),48 h仍高于正常(P<0.01)。 §2 THSG对HUVEC eNOS蛋白表达的影响 方法:采用Western blot法检测HUVEC eNOS蛋白表达水平 结果:用不同浓度(1,3,10,30,100μmol/L)THSG孵育HUVEC 48 h后,提取细胞总蛋白进行Western blot。结果显示THSG增强eNOS蛋白表达呈浓度依赖性。孵育48 h后,1μmol/L THSG即可使eNOS蛋白水平增加(P<0.05)。10μmol/L THSG使eNOS蛋白水平显著增加(P<0.01),平均灰度比值为对照组的1.8倍。用10μmol/L THSG孵育HUVEC 12,24,36,48 h后提取蛋白进行Western blot,结果显示THSG增强eNOS蛋白表达呈时间依赖性,10μmol/L THSG孵育12 h后, eNOS蛋白表达水平即有增高趋势(P > 0.05)。24 h出现显著性差异(P<0.01),48 h仍高于正常(P<0.01)。 结论 THSG能增强HUVEC eNOS mRNA和蛋白表达水平,提示THSG可通过上调血管内皮细胞中eNOS表达来增多NO产量,从而发挥舒张血管的作用。
Section One: Effect of angelica A_3 active component on inflammation and isolated rat uteruses cyclooxygenase-2 expression Angelica A_3 active component (A_3), extracted from Angelica oil(AO), is a neutral and nonphenol component. Experiments have revealed that AO had anti-inflammatory effects and it might be related to its inhibitory effects on inhibiting PGs. Our prophase research found that A_3 was the most effective component on the inhibition of the myometrium contraction and dysmenorrheal which was related to inhibiting PGs. In this study, the effects of A_3 on anti-inflammatory and Cox-2 were investigated to supply experimental evidences for developing it into a novel drug. §1 Effect of A_3 on dimethylbenzene-induced mice ear edema Method:After peroral A_3 3 days, the mice ear edema models induced by dimethylbenzene were prepared. The mice ear edema was tested at 1 h. Results:Peroral A_3 (0.001 g/kg、0.005 g/kg and 0.01 g/kg) could dose-dependently inhibit dimethylbenzene-induced mice ear edema. The ratio of inhibition was 10.6%、21.1% and 53% respectively. The inhibition ratio of AO(0.1 g/kg) was 51.3%. The effect of A_3(0.01 g/kg) was correspondent to that of AO(0.1 g/kg). §2 Effect of A_3 on carrageenin(CGN)-induced rats paw edema Method:After peroral A_3 4 days, the rats paw edema models induced by CGN were prepared. The rat paw edema was tested at 1, 2, 4, 6 h. Results:Peroral A_3 (0.001 g/kg、0.005 g/kg and 0.01 g/kg) could dose-dependently inhibit CGN-induced rats paw edema from 0.224±0.032 cm to 0.204±0.060,0.171±0.053 and 0.126±0.061 cm(n=8, p>0.05,P<0.05,P<0.01)at 1 h respectively. The edema degree of AO(0.1 g/kg) was 0.171±0.041 cm. The effect of A_3(0.005 g/kg) was correspondent to that of AO(0.1 g/kg) at 1 h. §3 Effect of A_3 on isolated rat uteruses Cox-2 mRNA expression Method:RT-PCR was used to analyze the uteruses Cox-2 mRNA expression level Results:LPS 1μg/mL could significantly increase the level of Cox-2 mRNA expression. A_3 (10,20,40,80,160,320 mg/L) could concentration-dependently inhibit Cox-2 mRNA overexpression induced by LPS from 0.462±0.164 to 0.408±0.136,0.368±0.126,0.306±0.065,0.250±0.084,0.138±0.016 and 0.008±0.003 respectively. §4 Effect of A_3 on isolated rat uteruses cox-2 protein expression Method:Western blot was used to analyze the uteruses Cox-2 protein expression level. Results:LPS 1μg/mL could significantly increase the level of Cox-2 protein expression. A_3 (10,20,40,80,160,320 mg/L) could concentration-dependently inhibit Cox-2 protein overexpression induced by LPS from187.8±13.5 to 162.6±16.3,155.0±17.0,148.4±14.3,133.6±13.3,125±15.4,119.4±14.4 respectively. Conclusion 1. A_3 could significantly inhibit dimethylbenzene-induced mice ear edema and CGN-induced rats paw edema. So A_3 possessed much stronger anti-inflammatory effects. 2. A_3 could concentration-dependently inhibit uteruses Cox-2 mRNA and protein overexpression induced by LPS, These results indicated that anti-inflammatory and anti-dysmenorrheal effecs of A_3 may be related to inhibiting Cox-2 expression. Section Two: Effects of THSG on HUVEC eNOS mRNA and protein expression 2,3,5,4’-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG), extracted from Polygonum multiflorum roots, has been verified to have an endothelia-dependent vasodilative effect in a dose-dependent manner in vitro, which is relative to increasing NOS activity and NO content in the vessels. Some evidences showed that THSG increased eNOS activity and NO content in HUVEC. In this section, the effects of THSG on eNOS expression were investigated to provide the theory for developing THSG into a novel drug. §1 Effect of THSG on HUVEC eNOS mRNA expression Method:RT-PCR was used to analyze the HUVEC eNOS mRNA expression level Results: Incubation of HUVEC with THSG(1,3,10,30,100μmol/L) for 24 h, the level of eNOS mRNA was measured by RT-PCR. THSG increased eNOS mRNA level in a concentration- dependent manner. 1μmol/L THSG incubating for 24 h could increase eNOS mRNA level significantly (P<0.05). The eNOS mRNA was increased 1.9 fold by 10μmol/L THSG compared with the control. Incubation of HUVEC with 10μmol/L THSG for different periods of time(12,24,36,48 h), the level of eNOS mRNA was measured by RT-PCR. THSG increased eNOS mRNA level in a time-dependent manner. The eNOS mRNA significantly increased after incubation with THSG for 12 h (P<0.05). The highest effect appeared at 24 h (P<0.01). §2 Effect of THSG on HUVEC eNOS protein expression Method:Western blot was used to analyze the HUVEC eNOS protein expression level. Results:Incubation of HUVEC with THSG(1,3,10,30,100μmol/L) for 48 h, the level of eNOS protein was measured by Western blot. THSG increased eNOS protein level in a concentration- dependent manner. Incubation with 1μmol/L THSG for 48 h could increase eNOS protein level significantly (P<0.05). The eNOS protein increased 1.8 fold by 10μmol/L THSG compared with the control. Incubation of HUVEC with 10μmol/L THSG for different periods of time(12,24,36,48 h), the level of eNOS protein was measured by Western blot. THSG increased eNOS protein level in a time-dependent manner. The eNOS protein had an increase trend after incubation with THSG for 12 h ( P>0.05). After incubation for 24 h, the level of eNOS mRNA increased significantly ( P<0.01). Conclusion THSG could upregulate eNOS mRNA and protein expression, which may resulte in increasing NO production and relaxation of blood vessels.
|
