中药芷辛方对骨癌痛大鼠胶质细胞蛋白表达以及TLRs和NF-κB的影响

Effect of Zhixin Formula on Gliocyte Protein Expressions,TLRs and NF-κB in Bone Cancer Pain Model Rats

目的通过建立癌痛动物模型,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）、CD11b]表达以及Toll受体（Toll-like receptors,TLRs）和核因子κB（NF-κB）的影响。方法将20只SD雄性大鼠随机分为空白组（10只）、骨癌痛模型组（10只）,通过左胫骨内接种含有3×10~3个Walker256嫁接的腹水瘤细胞液造模,并对这两组的大鼠进行行为学检测、X光片检测及HE染色的检测证实造模成功。依照此法将70只雄性SD大鼠随机分为7组,每组10只。空白组（10只）、骨癌痛模型组（简称模型组,10只）、西药盐酸曲马多对照组（简称西药对照组,10只）、芷辛方高、中、低剂量组（每组各10只）、中药五灵止痛胶囊对照组（简称中药对照组,10只）。造模14天后开始灌胃,芷辛方高、中、低剂量组分别给予芷辛方水煎浓缩制剂（9、4.5、2.25 g/kg生药）。每天灌胃1次,连续7天。第21天取脊髓L4~L5段背角,分别应用免疫组化、逆转录-聚合酶链反应（RT-PCR）及蛋白质印迹法（Western blot）的方法,分别检验星形胶质细胞的标志性蛋白GFAP的变化,小胶质细胞的标志性蛋白CD11b表达变化以及髓内TLRs（TLR2、TLR4）和NF-κB的影响。结果与空白组比较,模型组大鼠体重增加迟缓甚至呈下降趋势（P〈0.01）;自发性运动疼痛评分（SAPS）明显上升（P〈0.01）;触诱发痛阈值（PWT）明显下降（P〈0.05,P〈0.01）;手术侧胫骨X线影像以及HE染色示：骨结构发生明显改变和破坏。免疫组化检测髓内星形胶质细胞的标记蛋白GFAP明显升高;NF-κB蛋白水平升高（P〈0.01）。与模型组比较,CD11b、TLR2、TLR4及NF-κB水平芷辛方中、高剂量组均明显下降（P〈0.05,P〈0.01）。结论芷辛方可抑制脊髓水平胶质细胞的增殖活化和TLRs（TLR2、TLR4）、NF-κB蛋白的激活,这可能是其镇痛机制之一。

Objective To explore the analgesic effect of Zhixin Formula（ZF） and its effects on spinal glial fibrillary acidic protein（glial fibillary acidic protein,GFAP,a marker of astrocyte）,CD11b（a maker of microglia）,Toll like receptors（TLR2 and TLR4） and nuclear factor kB（NF-kB） in bone cancer pain model rats.Methods Totally 20 male SD rats were randomly divided into the blank control group and the bone cancer pain group,10 in each group.The bone cancer pain model was induced by injecting ascites tumor fluid containing 3×10~3 Walker256 cell line from the left tibia.Ethological tests,X-ray test,and HE staining were performed to confirm a successful modeling.After model was successfully established,70 male SD rats were randomly divided into seven groups,10 in each group：the blank control group,the bone cancer pain group（as the model group）,the Western medicine（WM） group（Tramadol Hydrochloride）,the high dose ZF group,the middle dose ZF group,the low dose ZF group,and the Chinese medicine（CM）group（Wulin Zhitong Capsule）.Fourteen days after modeling,rats in the high,middle,and low dose ZF groups were administrated by gastrogavage with 9,4.5,and 2.25 g/kg ZF water condensed preparation respectively,once a day for seven consecutive days.On day 21 MS typical protein expressions including GFAP,CD11 b,TLR（2,4） and NF-κB from cornu dorsal medullae spindis L4-L5 were detected by immunohistochemistry,reverse transcription-polymerase chain reaction（RT-PCR） and Western blot.Results Compare with the blank control group,increased weight in the model group was slow and showed a decreasing trend（P〈0.01）,spontaneous ambulatory pain score（SAPS） obviously increased（P〈0.01）,paw withdrawal threshold（PWT） obviously decreased in the model group（P 〈0.05,P 〈0.01）.Results of lateral tibial X-ray and HE staining showed obvious changes and damage occurred in bone structures of rats in the model group.Immunohistochemistry showed that GFAP expression significantly increased in the model group（P〈0.05）.Protein levels of NF-kB also significantly increased in the model group（P〈0.05）.Compared with the model group,CD11 b expressions obviously decreased in the middle and high dose ZF groups（P 〈0.01）.Meanwhile,protein expressions of TLR2 and TLR4,as well as NF-κB also obviously decreased（P 〈0.05）.Conclusion ZF had analgesic effect,which might be probably related to inhibiting proliferation and activation of gliocytes,as well as activation of TLRs and NF-κB.