慢性前列腺炎疼痛中P物质作用与L_5～S_2脊髓中枢星形胶质细胞活化的关系

目的:观察大鼠慢性前列腺炎疼痛模型中L5～S2脊段背角P物质(SP)和P物质受体(NK-1R)表达及星形胶质细胞活化的变化,并在体外纯化培养大鼠脊髓星形胶质细胞的基础上进一步了解SP对星形胶质细胞活化的作用。方法:通过前列腺完全弗氏佐剂(CFA)注射制作大鼠慢性前列腺炎疼痛模型,对照组注射生理盐水,观察时间为0、14、28 d,用热甩尾实验进行疼痛模型鉴定,采用RT-PCR、Western印迹检测L5～S2脊段后角中SP mRNA、NK-1R、胶质原纤维酸性蛋白(GFAP)、肿瘤坏死因子α(TNF-α)和诱导型NO合酶(iNOS)蛋白表达的变化,并通过体外培养大鼠脊髓星形胶质细胞,分为对照组和2个实验组,对照组仅加ITS培养液,实验组1施加SP刺激(SP浓度:10-9～10-6mol/L,时间:12 h),分别应用ELISA法、硝酸还原酶及比色法测定TNF-α、白介素-1β(IL-1β)、NO分泌水平及NOS活性的变化;实验组2施加SP刺激(SP浓度:10-7mol/L,时间:0、24、48、72 h),用Western印迹测定GFAP表达的变化。结果:慢性前列腺炎疼痛大鼠模型成功建立,并观察到L5～S2脊段背角中SP mRNA和NK-1R、GFAP、TNF-α、iNOS表达在14 d和28 d均明显高于各自的对照组(P<0.01),28 d和14 d组明显高于0 d组(P<0.01),GFAP 28 d组表达高于14 d组(P<0.05),10-7mol/L SP的作用下,脊髓星形胶质细胞GFAP的表达在0～72 h逐渐升高,与正常对照组相比差异有显著性(P<0.01);SP在10-9～10-6mol/L浓度范围内呈浓度依赖性促进脊髓星形胶质细胞分泌TNF-α、IL-1β、NO和NOS活性增强(12 h),与对照组有显著性差异(P<0.01或P<0.05),但IL-1β在SP 10-6mol/L浓度组下降,与对照组无显著性差异(P>0.05)。结论:慢性前列腺炎疼痛可以引起L5～S2脊段致痛递质SP合成增多,受体上调,并导致星形胶质细胞活化和炎性因子分泌增多,表明慢性前列腺痛可以通过SP的介导引起L5～S2脊髓中枢继发性的神经炎性痛,可能与前列腺炎疼痛的持续和泛化有密切关系。

Correlation Between Activation of L5-S2 Spinal Cord Astrocytes and Effect of Substance P in Chronic Prostatitis Pain

Objective: To observe the expressions of the substance P (SP) mRNA and neurokinin-1 receptor (NK-1R) in the posterior horn of the L_5-S_2 spinal cord in the rat model of chronic prostatitis pain, and to investigate the changes in the activation of astrocytes and influence of SP on this activation in rat spinal cord astrocytes cultured in vitro. Methods : The rat model of chronic prostatitis pain was established by injection of complete Freund's adjuvant (CFA) and assessed by the tail flick threshold test, the control rats injected with sodium chloride and all observed at 0, 14 and 28 days. Changes in the expressions of SP mRNA, NK-1R, glial fibrillary acidic protein (GFAP), tumor necrosis factor-or (TNF-α) and inducible nitric oxide synthase (iNOS) in the posterior horn of the L_5-S_2 spinal cord were detected by RT-PCR and Western blot. Rat spinal cord astrocytes were cultured in vitro and divided into a control group, cultured with ITS cell culture fluid, and two experiment groups, with Group 1 stimulated with SP at the concentration of 10~(-9)-10~(-6) mol/L for 12 hours followed by determination of the expressions of TNF-α, IL-1β, NO and NOS by ELISA and nitrate reductase and colorimetric methods, and Group 2 at 10~(-7)mol/L for 0, 24, 48 and 72 hours followed by detection of the GFAP expression by Western blot. Results : The expressions of SP mRNA, NK-1R, GFAP, TNF-α and iNOS in the posterior horn of the L_5-S_2 spinal cord were obviously higher in the rat prostatitis pain models than in the controls, successively higher at 28 than at 14 and 0 d (P < 0. 01), and so was the expression of GFAP at 28 than at 14 d in the experiment groups (P < 0.05). SP induced a gradual increase at 10~(-7) mol/L in the expression of GFAP in the spinal cord astrocytes at 0-72 h, significantly different from that of the control group (P <0.01), and it promoted the excretion of TNF-α and IL-1β and the activity of NO and NOS at 10~(-9)-10~(-6) mol/L at 12 h in a concentration-dependent manner, with marked differences between the experiment and control groups (P < 0.01, P < 0.05). But a decreased excretion of IL-1β was observed in the 10~(-6) mol/L group, though with no significant difference from the control (P > 0.05).Conclusion : Chronic prostatitis pain could upregnlate the expressions of the excitatory transmitter SP and receptor in the L_5-S_2 spinal cord, and result in the activation of astrocytes and increased excretion of proinflammatory cytokines, which may be associated with the persistence and generalization of prostatitis pain.