基于ERK信号通路抑制星形胶质细胞活化探讨益气活血通络方对糖尿病大鼠神经病理性疼痛的作用  

Yiqi Huoxue Tongluo Prescription Inhibits Activation of Astrocytes to Relieve Diabetic Neuropathic Pain in Rats via ERK Signaling Pathway

作　　者：袁慧伦 程凯 陈前 王璐洁 李家劼 王雨晴 江爱娟[1,2] YUAN Huilun;CHENG Kai;CHEN Qian;WANG Lujie;LI Jiajie;WANG Yuqing;JIANG Aijuan(School of Integrated Traditional Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Institute of Integrated Traditional Chinese and Western Medicine,Anhui Academy of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学中西医结合学院,合肥230012 [2]安徽省中医药科学院中西医结合研究所,合肥230012

出　　处：《中国实验方剂学杂志》2023年第23期36-46,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81874457);安徽省高等学校科学研究项目(2022AH050480);安徽省自然科学基金面上项目(2308085MH307)。

摘　　要：目的:探讨益气活血通络方(YHTP)防治糖尿病神经病理性疼痛(DNP)的作用机制。方法:体内实验将90只SPF级SD雄性大鼠随机分为空白组、模型组、YHTP低剂量组(2.25 g·kg^(-1))、YHTP中剂量组(4.5 g·kg^(-1))、YHTP高剂量组(9 g·kg^(-1))和甲钴胺组(0.175 mg·kg^(-1))。除空白组外,其余5组采用高脂高糖饮食联合小剂量(35 mg·kg^(-1))腹腔注射链脲佐菌素(STZ)构建糖尿病神经病理性疼痛模型。通过神经电生理检测DNP大鼠坐骨神经传导速度,酶联免疫吸附测定法(ELISA)检测大鼠脊髓组织白细胞介素-6(IL-6)、白细胞介素-1β(IL^(-1)β)和肿瘤坏死因子-α(TNF-α)炎症因子水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠脊髓组织中胶质纤维酸性蛋白(GFAP)和细胞外调节蛋白激酶(ERK)的mRNA表达,蛋白免疫印迹法(Western blot)检测大鼠脊髓组织GFAP和磷酸化细胞外调节蛋白激酶(p-ERK)蛋白表达量,免疫荧光测定大鼠脊髓组织GFAP和p-ERK荧光表达水平。体外实验用100 mmol·L^(-1)葡萄糖(GLU)诱导星形胶质细胞(CTX-TNA2)活化,构造高糖环境下神经细胞损伤模型。分为空白组、模型组、含药血清组(10%YQHT)、抑制剂组(10 mol·L^(-1)COR)、含药血清+抑制剂组(10%YHTP+10 mol·L^(-1)COR),用ELISA检测CTX-TNA2细胞的促炎因子TNF-α、IL-1β及抗炎因子白细胞介素-10(IL-10)表达水平,Western blot检测CTX-TNA2细胞的GFAP和p-ERK蛋白表达量。结果:动物实验中,与空白组比较,模型组大鼠机械痛阈值(MWT)、热缩足反应时间(TWL)和神经传导速度显著降低(P<0.01),空腹血糖、炎症因子、GFAP和p-ERK蛋白,以及ERK1、ERK2、GFAP mRNA表达量显著升高(P<0.01);与模型组比较,YHTP各剂量组大鼠的MWT、TWL和坐骨神经传导速度显著提高(P<0.01),IL-1β、TNF-α、IL-6炎性因子含量显著减少(P<0.01),脊髓GFAP、p-ERK蛋白,以及ERK1、ERK2、GFAP mRNA表达水平明显降低(P<0.05,P<0.01)。体外实验中,与空Objective:To investigate the mechanism of Yiqi Huoxue Tongluo prescription(YHTP)in the treatment of diabetic neuropathic pain(DNP).Method:Ninety SPF-grade SD male rats were randomized into blank,model,low-(2.25 g·kg^(-1)),medium-(4.5 g·kg^(-1)),and high-dose(9 g·kg^(-1))YHTP,and mecobalamin(0.175 mg·kg^(-1))groups.Except those in the blank group,the rats in the remaining 5 groups were fed with a high-fat and high-glucose diet and subjected to intraperitoneal injection of low-dose(35 mg·kg^(-1))streptozotocin(STZ)to establish the model of DNP.The sciatic nerve conduction velocity in DNP rats was measured by the neurophysiological method,and the levels of interleukin-6(IL-6),IL-1β,and tumor necrosis factor-α(TNF-α)were measured by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was employed to measure the mRNA levels of glial fibrillary acidic protein(GFAP)and extracellular signal-regulated kinase(ERK)in the spinal cord.Western blot was employed to measure the protein levels of GFAP and phosphorylated ERK(p-ERK),and immunofluorescence staining to measure the fluorescence intensity of GFAP and p-ERK in the spinal cord.In the cell experiments,100 mmol·L^(-1) high glucose was used to induce the activation of astrocytes(CTX-TNA2)for the modeling of nerve cell injury.The cells were randomized into the normal,model,drug-containing serum(10%YQHT),inhibitor[10 mol·L^(-1) corynoxeine(COR)],drug-containing serum+inhibitor(10%YHTP+10 mol·L^(-1) COR)groups.The levels of pro-inflammatory factors(TNF-αand IL-1β)and the anti-inflammatory factor IL-10 in CTX-TNA2 cells were determined by ELISA,and the protein levels of GFAP and p-ERK in CTXTNA2 cells by Western blot.Result:The animal experiments showed that compared with the blank group,the model group presented reduced mechanical withdrawal threshold(MWT),thermal work limit(TWL),and nerve conduction velocity,elevated levels of fasting blood glucose,IL-1β,TNF-α,and IL-6,and up-regulated protein lev

关 键 词：糖尿病神经病理性疼痛 益气活血通络方 细胞外调节蛋白激酶(ERK)信号通路 星形胶质细胞 

分 类 号：R2-0[医药卫生—中医学] R33R587.1R289