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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。
Objective: To observe the changes of substance P (SP) and substance P receptor (NK-1R) expression and the activation of astrocytes in L5 ~ S2 spinal dorsal horn in rats with chronic prostatitis pain model. On the basis of murine spinal cord astrocytes, further understand the role of SP on astrocyte activation. Methods: Chronic prostatitis pain model was established by injection of complete Freund’s adjuvant (CFA) in rats. The control group was injected with normal saline. The observation time was 0, 14, and 28 days. The pain model was identified by hot tail flick test. The expression of SP mRNA, NK-1R, glial fibrillary acidic protein (GFAP), tumor necrosis factor alpha (TNF-α) and inducible NO synthase (iNOS) protein in the posterior horn of L5 ~ The rats were randomly divided into control group and two experimental groups. The control group was treated with ITS alone. The SP group was stimulated with SP (SP concentration: 10-9 ~ 10) 6 mol / L, time: 12 h). The changes of TNF-α, IL-1β, NO secretion and NOS activity were measured by ELISA, nitrate reductase and colorimetric assay respectively. SP stimulation (SP concentration: 10-7mol / L, time: 0,24,48,72 h). The changes of GFAP expression were measured by Western blotting. Results: The rat model of chronic prostatitis pain was successfully established. The expression of SP mRNA and NK-1R, GFAP, TNF-α and iNOS in the spinal dorsal horn of L5 ~ S2 was significantly higher than that of their respective Compared with control group (P <0.01), the expression of GFAP at 28 d and 14 d was significantly higher than that at 0 d (P <0.01) , The expression of GFAP in spinal cord astrocytes gradually increased from 0 to 72 h, and the difference was significant compared with the normal control group (P <0.01). SP concentration was in the range of 10-9 ~ 10-6 mol / L IL-1β, NO and NOS increased in a dose-dependent manner (12 h) in spinal cord astrocytes, which was significantly different from the control group (P <0.01 or P <0.05) 1β in SP 10-6mol / L concentration group decreased, with no significant difference (P> 0.05). CONCLUSIONS: Chronic prostatitis pain can cause increased synthesis of pain-inducing SP in the spinal cord of L5 ~ S2, upregulation of the receptor, and activation of astrocytes and increased secretion of inflammatory cytokines, indicating that chronic prostatodynia can be mediated through SP The secondary neuroinflammatory pain that causes central L5 ~ S2 spinal cord center may be closely related to the continuous and generalized pain of prostatitis.