基于神经生长因子探讨温经活血汤治疗寒凝血瘀证原发性痛经的作用机制

Explore the Mechanisms of Wenjing Huoxue Decoction in the Treatment of Primary Dysmenorrhea with Cold Coagulation and Blood Stasis Syndrome Based on Nerve Growth Factor

目的基于神经生长因子(nerve growth factor,NGF)及酪氨酸激酶受体A(TrkA)、p75营养因子受体(p75NTR)揭示原发性痛经(primary dysmenorrhea,PD)的发病机理,探讨温经活血汤治疗原发性痛经的作用机制。方法将48只SPF级雌性Wistar大鼠随机分为正常组(8只)和模型复制组(40只)。采用寒冷刺激联合苯甲酸雌二醇和缩宫素复制寒凝血瘀证痛经模型,模型复制成功后将大鼠随机分为模型组、西药布洛芬组(0.06 g·kg^(-1))和温经活血汤低、中、高剂量组(6.55、13.09、26.18 g·kg^(-1)),每组各8只。自模型复制第7天起所有大鼠分别给予温经活血汤、布洛芬和蒸馏水灌胃处理,每天1次,连续6 d。观察大鼠注射缩宫素后的扭体潜伏时间和30 min内扭体次数。提取大鼠子宫组织,蛋白质免疫印迹(Western Blot)法检测大鼠子宫组织中NGF、TrkA和p75NTR的蛋白水平;实时荧光定量PCR(Real-time PCR)法检测大鼠子宫组织中NGF、TrkA和p75NTR的mRNA表达。结果与正常组比较,模型组NGF、TrkA、p75NTR蛋白水平及mRNA表达均明显升高(P<0.01)。与模型组比较,各治疗组大鼠扭体潜伏时间均有所延长,但差异无统计学意义(P>0.05),扭体次数明显减少(P<0.01),NGF、TrkA、p75NTR蛋白水平及NGF、TrkA mRNA表达均明显降低(P<0.01),西药布洛芬组和温经活血汤中、高剂量组p75NTR mRNA表达降低(P<0.01)。结论温经活血汤治疗寒凝血瘀证原发性痛经的作用机制可能与其下调NGF、TrkA、p75NTR的表达有关。

Objective To reveal the pathogenesis of primary dysmenorrhea(PD)based on nerve growth factor(NGF)and tyrosine kinase receptor A(TrkA),p75 neurotrophin receptor(p75NTR)and explore the mechanisms of Wenjing Huoxue decoction in the treatment of PD.Methods A total of 48 SPF female Wistar rats were randomly divided into normal group(8 rats)and model group(40 rats).Dysmenorrhea rat model with cold coagulation and stasis syndrome was established by cold stimulation combined with estradiol benzoate and oxytocin.After successful modeling,rats were randomly divided into model group,western medicine group(ibuprofen,0.06 g·kg^(-1)),TCM low dose group,middle dose group and high dose group(Wenjing Huoxue decoction,6.55,13.09,26.18 g·kg^(-1)).There were eight rats in each group.All rats were treated with Wenjing Huoxue decoction,ibuprofen and distilled water respectively once a day at the 7th day of modeling,and for 6 days consecutively.We observed the time of writhing latency and writhing times of rats within 30 minutes after oxytocin injection.The proteins of rat uterus were extracted to detect the levels of NGF,TrkA and p75NTR by Western Blot.mRNA expression of NGF,TrkA and p75NTR was also examined by Real-time PCR.Results The protein level and mRNA expression of NGF,TrkA and p75NTR were significantly increased in the model group compared with the normal group(P<0.01).Compared with the model group,the time of writhing latency of rats in each treatment group was extended,but the difference was not statistically significant(P>0.05).The writhing times of rats were reduced significantly(P<0.01).The protein levels of NGF,TrkA,p75NTR and the mRNA expressions of NGF and TrkA were reduced significantly(P<0.01),and the mRNA expressions of p75NTR in the western medicine group,medium-and high-dose of Chinese medicine group were reduced significantly(P<0.01).Conclusion The mechanism of Wenjing Huoxue decoction in the treatment of PD with cold coagulation and blood stasis may be related to the down-regulation of NGF,TrkA and p75NTR.