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目的:建立癌痛动物模型寻找有效缓解疼痛的方法,进一步探讨芷辛方的镇痛作用及对骨癌痛大鼠模型中脊髓胶质细胞蛋白[胶质原纤维酸性蛋白(GFAP)及CD11b]表达以及Toll样受体4(TLR4)和核因子κB(NF-κB)的影响。方法:70只雄性SD大鼠随机分为7个组:空白对照组,骨癌痛模型组,西药对照组(盐酸曲马多0.01g/kg),芷辛方高、中、低剂量治疗组(9、4.5、2.25g/kg生药),中药五灵止痛胶囊对照组(0.06g/kg),每组10只。造模14d后开始灌胃,每日灌胃1次,连续7d。第21天取脊髓L4-L5部位,分别应用免疫组化、RT-PCR及Western Blot等方法检验星形胶质细胞的标志性蛋白GFAP、小胶质细胞的标志性蛋白CD11b、髓内TLR4和NF-κB的变化。结果:1免疫组化检测骨癌痛模型组的髓内星形胶质细胞的标记蛋白GFAP显著高于空白对照组及芷辛方中、高剂量治疗组。骨癌痛发生时,星形胶质细胞增生肥大,芷辛方对其具有抑制镇痛作用。2PCR检测小胶质细胞标志性蛋白CD11b,骨癌痛模型组的CD11b m RNA显著高于芷辛方中、高剂量治疗组(P<0.01)。表明骨癌痛时,小胶质细胞表现为增殖活化,芷辛方对其有抑制镇痛的作用。3芷辛方干预治疗的骨癌痛后中枢神经系统免疫活性重要因子TLR4 m RNA与骨癌痛模型组出现显著性差异(P<0.01,P<0.05)。骨癌痛模型组的NF-κB/GAPDH的蛋白水平与正常组和芷辛方高剂量治疗组的表达出现显著性差异(P<0.05)。结论:中枢疼痛TLR-NF-κB信息通路被激活,芷辛方通过对TLR的抑制从而达到对其下游通路的抑制和治疗作用,进而达到止痛的效果。
OBJECTIVE: To establish an effective pain relief model for animal models of cancer pain, to further explore the analgesic effect of Zhixin Prescription and to investigate the effects of Zhixin Prescription on glial cellular protein [glial fibrillary acidic protein (GFAP) and CD11b] Expression and the effects of TLR4 and NF-κB. Methods: Seventy male Sprague-Dawley rats were randomly divided into seven groups: control group, model group of bone cancer pain, western medicine control group (tramadol hydrochloride 0.01g / kg), Zhixinfang high, medium and low dose treatment group (9,4.5,2.25g / kg crude drug), Chinese medicine Wulingzhitong capsule control group (0.06g / kg), 10 rats in each group. 14d after the start of intragastric administration, intragastric administration 1 times a day for 7d. The spinal cord L4-L5 sites were obtained on the 21st day. The expression of astrocyte marker protein GFAP, microglial marker protein CD11b, intramedullary TLR4, and intralesional TLR4 were detected by immunohistochemistry, RT-PCR and Western Blot respectively Changes in NF-κB. Results: 1 Immunohistochemical detection of bone marrow pain model group intramedullary astrocyte marker protein GFAP was significantly higher than the blank control group and Zhixinfang medium and high dose treatment group. When bone cancer pain occurs, astrocyte hyperplasia and hypertrophy, Chih-Fang has inhibitory effect on it. 2PCR microglial marker protein CD11b, bone cancer pain model group CD11b m RNA was significantly higher than the Zhixinfang medium and high dose treatment group (P <0.01). That bone pain, microglial cells showed proliferation and activation, Chih-Fang Fang on its inhibition of analgesic effect. There was a significant difference (P <0.01, P <0.05) between TLR4 mRNA and bone cancer pain model group after the treatment of Zhixin Fang intervention of bone cancer pain central nervous system immune activity important factor. The protein level of NF-κB / GAPDH in bone cancer pain model group was significantly different from that in normal group and Zhuxinfang high-dose treatment group (P <0.05). CONCLUSION: TLR-NF-κB signaling pathway is activated in central pain. Zhi-sheng Fang can inhibit and treat downstream pathway by inhibiting TLR, so as to achieve analgesic effect.