芍药甘草汤通过JAK2/STAT3信号通路对神经病理性疼痛大鼠的镇痛及对免疫调节机制影响

Analgesic Effect and Immunomodulatory Mechanism of Shaoyao Gancao Decoction on Neuropathic Pain Rats through JAK2/STAT3 Signaling Pathway

目的探寻芍药甘草汤对神经病理性疼痛(NP)的改善作用及其机制。方法采用坐骨神经分支选择性损伤(SNI)法建立NP大鼠模型,模型制备成功后予以芍药甘草汤灌胃治疗14 d。治疗前后监测大鼠疼痛行为学指标变化,TUNEL染色检测脊髓背角细胞凋亡情况,免疫荧光组织化学法测定p-STAT3在脊髓中与Iba1的共定位情况,RT-PCR检测CD68、SOCS3、Arg-1的mRNA表达,ELISA检测白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)表达,Western blotting检测JAK2/STAT3通路蛋白表达。结果与假手术组比较,模型组大鼠机械缩足阈值(MWT)、热缩足潜伏期(TWL)明显降低(P<0.01),脊髓背角中凋亡细胞显著增加(P<0.05),IL-1β、1L-6和TNF-α的表达显著增加(P<0.01),JAK2、p-JAK2、STAT3、p-STAT3的表达显著增加(P<0.01),术后7、14 d的CD68、SOCS3 mRNA表达显著增加(P<0.01),Arg-1 mRNA表达显著降低(P<0.01)。与模型组比较,芍药甘草汤组大鼠治疗后MWT、TWL明显上升(P<0.01),脊髓背角中凋亡细胞减少,IL-1β、IL-6、TNF-α的水平显著减少(P<0.01),JAK2、p-JAK2、STAT3、p-STAT3蛋白表达水平明显降低(P<0.01),CD68、SOCS3 mRNA表达显著减少(P<0.01),Arg-1 mRNA表达水平显著增加(P<0.01)。结论芍药甘草汤可能通过调控JAK2/STAT3通路调控小胶质细胞极化状态进而发挥改善NP的作用。

Objective:To explore the analgesic effect of Shaoyao Gancao Decoction(SGD)on neuropathic pain(NP)and its mechanism of action.Methods:Spared nerve injury(SNI)was adopted to establish a NP model in rats,which were treated with SGD gavage for 14 days.The behavioral changes in rats from each group were detected before and after treatment.The apoptosis of spinal dorsal horn(SDH)was detected by TUNEL staining,and immunofluorescence staining was used to determine the co-localization of p-STAT3 and Iba1.The expression of markers associated with different phenotypes of microglia(CD68,SOCS3,Arg-1)was examined by real-time RTPCR.The expression of IL-1β,IL-6 and TNF-αin SDH were evaluated by ELISA.The protein expressions of JAK2、p-JAK2,STAT3,p-STAT3 in SDH were determined by Western blotting.Results:Compared with the sham group,the model group showed significant decreases on the mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)in the right hind paw(P<0.01),the ratio of apoptotic cells in SDH increased significantly(P<0.05),the expressions of IL-1β,IL-6,TNF-αwere significantly increased(P<0.01),JAK2,pJAK2,STAT3,p-STAT3 protein expression increased significantly(P<0.01),CD68,SOCS3 m RNA expression was increased(P<0.01),while Arg-1 mRNA expression was decreased on the postoperative 7th and 14th days(P<0.01).Compared with the model group,the SGD group showed significant increases on the MWT and TWL in the right hind paw(P<0.01),the ratio of apoptotic cells in SDH decreased with no statistical difference,the expressions of IL-1β,IL-6,TNF-αwere significantly decreased(P<0.01),JAK2,p-JAK2,STAT3,p-STAT3 protein expression decreased significantly(P<0.01),CD68,SOCS3 mRNA expression was decreased(P<0.01),while Arg-1m RNA expression was increased on the postoperative 7th and 14th days(P<0.01).Conclusion:SGD may partici-pate in the regulation of microglial polarization in NP rats by regulating the JAK2/STAT3 signaling pathway,thusexerting its analgesic action.