Effects And Mechanisms Of A₃ On Inflammation Effects And Mechanisms Of THSG On ENOS

Section One: Effect of angelica A₃ active component on inflammation and isolated rat uteruses cyclooxygenase-2 expressionAngelica A₃ active component (A₃), 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 A₃ 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 A₃ on anti-inflammatory and Cox-2 were investigated to supply experimental evidences for developing it into a novel drug.§1 Effect of A₃ on dimethylbenzene-induced mice ear edemaMethod:After peroral A₃ 3 days, the mice ear edema models induced by dimethylbenzene were prepared. The mice ear edema was tested at 1 h. Results:Peroral A₃ (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 A₃(0.01 g/kg) was correspondent to that of AO(0.1 g/kg).§2 Effect of A₃ on carrageenin(CGN)-induced rats paw edemaMethod:After peroral A₃ 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 A₃ (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 A₃(0.005 g/kg) was correspondent to that of AO(0.1 g/kg) at 1 h. §3 Effect of A₃ 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. A₃ (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 A₃ 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. A₃ (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.Conclusion1. A₃ could significantly inhibit dimethylbenzene-induced mice ear edema and CGN-induced rats paw edema. So A₃ possessed much stronger anti-inflammatory effects.2. A₃ 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 A₃ may be related to inhibiting Cox-2 expression.Section Two: Effects of THSG on HUVEC eNOS mRNA and protein expression2,3,5,4'-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 expressionMethod: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 expressionMethod: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). ConclusionTHSG could upregulate eNOS mRNA and protein expression, which may resulte in increasing NO production and relaxation of blood vessels.