基于NF-κB信号通路探讨通管助孕方治疗输卵管阻塞性不孕症大鼠的作用机制

Explore the mechanism of Tongguan Zhuyun Decoction in the treatment of tubal obstructive infertility rats based on NF-κB signal pathway

关键词:  通管助孕方  输卵管阻塞性不孕症  NF-κB  IκB  COX-2  PGE2

Key words:  Tongguan Zhuyun Decoction  tubal obstructive infertility  NF-κB  IκB  COX-2  PGE2

摘要:

[目的] 基于核转录因子-κB（NF-κB）信号通路探讨通管助孕方治疗输卵管阻塞性不孕症的作用机制。[方法] 将140只雌性SD大鼠随机分为正常组、模型组、中成药组、西药组及通管助孕方高、中、低剂量组，每组20只。除正常组外，其余各组采用金黄色葡萄球菌建立输卵管阻塞性不孕症大鼠模型，造模后第7天，各组分别给予生理盐水、丹黄祛瘀胶囊混悬液、人胎盘组织液、通管助孕方高、中、低剂量治疗21 d后，药效组大鼠取材处理，生殖组大鼠进行雌雄合笼实验。采用酶联免疫吸附（ELISA）法检测各组大鼠血清中NF-κB、核因子κB抑制蛋白（IκB）、环氧合酶2（COX-2）、前列腺素E2（PGE2）的水平，采用蛋白免疫印迹（Western Blot）法和实时定量荧光聚合酶链反应（RT-PCR）法分别检测输卵管组织中NF-κB、IκB、COX-2、PGE2的蛋白和基因表达情况。采用肉眼观察以及光镜下观察输卵管组织病理形态学变化。观察并记录生殖组大鼠受孕及产仔情况。[结果] 1）与正常组比较，模型组大鼠NF-κB、COX-2、PGE2蛋白和基因表达升高，IκB蛋白和基因表达降低（P＜0.05）；与模型组比较，通管助孕方高、中、低剂量组NF-κB、COX-2、PGE2蛋白和基因表达降低，IκB蛋白和基因表达升高（P＜0.05）；与中成药组及西药组比较，通管助孕方高剂量组中NF-κB、COX-2、PGE2蛋白和基因表达降低，IκB蛋白和基因表达升高（P＜0.05）。2）通过肉眼观察及光镜观察后，与模型组比较，各给药组输卵管组织形态均有不同程度改善，尤以通管助孕方高剂量组改善最为明显，基本接近正常。3）对生殖功能的影响：模型组妊娠率为0%，正常组妊娠率为100%，两组比较差异有统计学意义（P＜0.05），表明金黄色葡萄球菌接种法可以成功建立输卵管阻塞性不孕症大鼠模型。通管助孕方高、中、低剂量组妊娠率及平均产仔数与模型组比较，差异有统计学意义（P＜0.05），说明通管助孕方对输卵管阻塞性不孕症有确切疗效。[结论] 通管助孕方可以抑制输卵管阻塞性不孕症模型大鼠中促炎因子NF-κB、COX-2、PGE2的表达，促进抗炎因子IκB的表达，发挥抗炎抗粘连作用，其治疗作用机制可能与阻断NF-κB信号传导通路有关。

Abstract:

[Objective] To explore the mechanism of Tongguan Zhuyun Decoction in the treatment of tubal obstructive infertility rats based on NF-κB signal pathway. [Methods] The 140 female SD rats were randomly divided into normal group,model group,Chinese patent medicine group,Western medicine group and Tongguan Zhuyun Decoction high,medium and low dose groups,with 20 rats in each group. Except the normal group,the other groups were injected with staphylococcus aureus to establish the rat model of tubal obstructive infertility. On the 7th day after modeling,each group was given physiological salt water,Danhuang Quyu Decoction suspension,human placental tissue fluid,Tongguan Zhuyun Decoction high,medium and low dose were treated for 21 d,the rats in the pharmacodynamic group were sampled and treated,and the rats in the reproductive group were used for the male female cage experiment. ELISA was used to detect the levels of NF-κB,IκB,COX-2 and PGE2 in serum of rats in each group. The protein and gene expression levels of NF-κB,IκB,COX-2 and PGE2 were detected by Western blot and RT-PCR. The pathomorphological changes of fallopian tube were observed by naked eye and light microscope. Observe and record the pregnancy and delivery of rats in the reproductive group. [Results] 1) Compared with the normal group,the expression levels of NF-κB,COX-2,PGE2 protein and gene in the fallopian tube of the model group increased significantly,the expression levels of IκB protein and gene decreased significantly(P<0.05);compared with the model group,the expression levels of NF-κB,COX-2,PGE2 protein and gene in rat fallopian tube tissue of Tongguan Zhuyun Decoction high,medium and low dose groups decreased significantly,the expression levels of IκB protein and gene increased significantly(P<0.05);compared with Chinese patent medicine group and Western medicine group,the expression levels of NF-κB,COX-2,PGE2 protein and gene in rat fallopian tube tissue of Tongguan Zhuyun Decoction high-dose group decreased significantly,the expression levels of IκB protein and gene increased significantly(P<0.05). 2) After visual observation and light microscope observation,compared with the model group,the tissue morphology of fallopian tube in each administration group was improved in varying degrees,especially in the high-dose group of Tongguan Zhuyun Decoction,which was basically close to normal. 3) Effect on reproductive function:the pregnancy rate was 0% in the model group and 100% in the normal group, there is a statistical difference between the two groups(P<0.05). This shows that the method of staphylococcus aureus inoculation can successfully establish the rat model of oviduct obstructive infertility. The pregnancy rate and average litter size of the high,middle and low dose groups of Tongguan Zhuyun Decoction are statistically different from those of the model group(P<0.05). This shows that Tongguan Zhuyun Decoction has a definite effect on infertility caused by fallopian tube obstruction. [Conclusion] Tongguan Zhuyun Decoction can inhibit the expression of the pro-inflammatory factor NF-κB,COX-2 and PGE2 in SOI model rats,promotes the expression of anti-inflammatory factor IκB playing an anti-inflammatory and anti-adhesion role. The mechanism of treatment may be related to blocking NF-κB signal transduction pathway.