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目的观察肿瘤坏死因子α(tumor necrosis factorα,TNF-α)对内毒素休克血管钙敏感性的影响及其与Rho激酶和蛋白激酶C(protein kinase C,PKC)的关系。方法取正常和内毒素休克家兔肠系膜上动脉(superior mesenteric artery,SMA),采用离体血管环张力测定技术,用去极化状态下(120mmol/LKCl)SMA血管环对梯度浓度钙的收缩力反映血管的钙敏感性。用ELISA法测定血浆炎性因子TNF-α的水平,观察TNF-α对SMA血管环钙敏感性的影响及与PKC和Rho激酶的关系。结果家兔LPS(1mg/kg)静脉注射后,早期(即LPS注射后30min和1h)SMA血管环对钙的反应性升高,量-效曲线左移,最大收缩力(Emax)升高(P>0.05);随着时间的延长,SMA血管环对钙的反应性逐渐下降,到LPS注射后6h明显降低,其量-效曲线明显右移,最大收缩力(Emax)明显降低(P<0.01)。家兔LPS(1mg/kg)静脉注射后2h,血清中TNF-α的水平逐渐升高,4h达到高峰,6h有所降低但仍高于正常组(P<0.05,P<0.01),其变化趋势与血管钙敏感性变化呈一定负相关关系(r=-0.6753)。低浓度的TNF-α(20ng/ml)孵育可明显升高SMA对钙的反应性,高浓度的TNF-α(200ng/ml)可明显降低SMA对钙的反应性(P<0.05,P<0.01)。Rho激酶特异性抑制剂Y-27632(1×10-5mol/L)可明显降低低浓度的TNF-α升高SMA对钙的反应性(P<0.05,P<0.01);而PKC的抑制剂Staurosporine(1×10-7mol/L)无拮抗作用(P>0.05)。结论TNF-α对内毒素休克血管的钙敏感性有重要的调节作用,其调节作用可能主要与Rho激酶有关,与PKC关系不大。
Objective To investigate the effects of tumor necrosis factor α (TNF-α) on the calcium sensitivity of endotoxin-induced shock and its relationship with Rho kinase and protein kinase C (PKC). Methods The normal mesenteric artery and endotoxin shock rabbit superior mesenteric artery (SMA) were harvested and the contractile force of gradient calcium concentration was measured by using detached vascular ring tension measurement (120 mmol / L KCl) Reflects the calcium sensitivity of blood vessels. The level of plasma TNF-α was measured by ELISA. The effect of TNF-α on the calcium sensitivity of SMA and the relationship with PKC and Rho kinase were observed. Results After intravenous injection of LPS (1 mg / kg) in rabbits, the response to calcium increased at early (30 min and 1 h after LPS injection), the dose-response curve shifted to the left and the maximal contractile force (Emax) P> 0.05). With the extension of time, the reactivity of calcium ring to SMA ring decreased gradually and decreased significantly 6h after LPS injection. The volume-response curve shifted to right and the maximal contractile force (Emax) decreased significantly (P < 0.01). The level of TNF-α in serum increased gradually at 2h after intravenous injection of LPS (1mg / kg) in rabbits, reached the peak at 4h, decreased at 6h but remained higher than that of normal group (P <0.05, P <0.01) The trend was negatively correlated with the changes of vascular calcium sensitivity (r = -0.6753). Incubation with low concentration of TNF-α (20ng / ml) significantly increased the reactivity of SMA to calcium. High concentrations of TNF-α (200ng / ml) significantly decreased the reactivity of SMA to calcium (P < 0.01). Rho kinase-specific inhibitor Y-27632 (1 × 10-5mol / L) could significantly decrease the reactivity of SMA with low concentration of TNF-α (P <0.05, P <0.01) Staurosporine (1 × 10-7mol / L) had no antagonism (P> 0.05). Conclusion TNF-α may play an important regulatory role in the calcium sensitivity of endotoxin-shocked vessels, and its regulation may be mainly related to Rho kinase, but not to PKC.