基于NF-κB通路探讨温经汤合琥珀散对寒凝血瘀型子宫内膜异位症大鼠的影响

Investigating the effects of Wenjing Decoction combined with Hupo Powder on cold-congealed blood stasis in endometriosis rats,using the NF-κB pathway

关键词:  子宫内膜异位症  温经汤合琥珀散  核转录因子-κB信号通路  寒凝血瘀

Key words:  endometriosis  Wenjing Decoction combined with Hupo Powder  NF-κB signalling pathway  cold-congealed blood stasis

摘要:

[目的] 基于核转录因子-κB（NF-κB）通路及相关蛋白探讨温经汤合琥珀散对子宫内膜异位症（EMs）大鼠的影响。[方法] 将60只SPF级雌性SD大鼠随机分为假手术组、模型组、孕三烯酮组与温经汤合琥珀散低、中、高剂量组，每组10只。除假手术组外，其余各组大鼠均采用冰水浴联合盐酸肾上腺素和自体内膜移植法建立寒凝血瘀证EMs大鼠模型，假手术组只开腹但不进行内膜移植。假手术组和模型组给予纯水；孕三烯酮组给予孕三烯酮（0.25mg/kg）每周灌胃2次（周一、周四），灌胃4周；温经汤合琥珀散组分别给予低（9.69 g/kg）、中（19.38 g/kg）、高（38.75 g/kg）剂量的温经汤合琥珀散，连续灌胃4周。酶联免疫吸附（ELISA）法检测各组大鼠血清肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）水平，检测各组大鼠的血液流变学、凝血功能。蛋白免疫印迹法检测各组大鼠子宫内膜组织中核转录因子-κB p65（NF-κB p65）、核转录因子-κB抑制蛋白α（IkBα）、核转录因子-κB激酶亚基β抑制因子（IKKβ）蛋白表达。[结果] 与假手术组比较，模型组大鼠血清TNF-α、IL-1β含量升高（P＜0.05或P＜0.01），血液流变学各项指标升高（P＜0.05或P＜0.01）；凝血功能方面，模型组大鼠部分凝血活酶时间（TT）降低（P＜0.05），纤维蛋白原（FIB）含量升高（P＜0.01）；蛋白免疫印迹法检测结果显示，NF-κB p65、IKKβ升高（P＜0.01），IkBα表达降低（P＜0.05）。与模型组比较，温经汤合琥珀散部分剂量组大鼠血清TNF-α、IL-1β表达降低（P＜0.05或P＜0.01），温经汤合琥珀散部分剂量组及孕三烯酮组大鼠血液流变学各项指标降低（P＜0.05或P＜0.01）；凝血功能方面，温经汤合琥珀散部分剂量组及孕三烯酮组大鼠TT升高（P＜0.05或P＜0.01），FIB降低（P＜0.05或P＜0.01）；蛋白免疫印迹法检测结果显示，温经汤合琥珀散部分剂量组大鼠NF-κB p65表达降低（P＜0.05或P＜0.01），温经汤合琥珀散部分剂量组及孕三烯酮组大鼠IKKβ表达降低（P＜0.05或P＜0.01），温经汤合琥珀散部分剂量组及孕三烯酮组大鼠IkBα表达升高（P＜0.05或P＜0.01）。[结论] 温经汤合琥珀散可能通过阻断或抑制NF-κB信号通路活化及阻断异位组织增殖，从而缩小异位灶，调控EMs的发展，且可改善寒凝血瘀大鼠相关症状，疗效优于孕三烯酮。

Abstract:

[Objective] To explore the effects of Wenjing Decoction combined withHupoPowderon cold-congealed blood stasis endometriosis(EMs) rats based on the nuclear transcription factor-κB(NF-κB) pathway and related proteins. [Methods] Sixty SPF-grade female SD rats were randomly divided into sham-operated group,model group,gestrinone categories(GC),and low,medium,and high dose groups(9.69,19.38 and 38.75 g/kg) of Wenjing Decoction combined with Hupo Powder group,with 10 rats in each group. Except for the sham-operated group,the rat model of EMs with cold-congealed blood stasis was established by using ice-water bath combined with epinephrine hydrochloride and autologous endothelial transplantation in all groups of rats. Sham-operation group with only open abdomen without endometrial transplantation. The sham-operated and model groups were given pure water. The progesterone group was given progesterone(0.25 mg/kg) by gavage twice a week(every Monday and Thursday) dissolved in pure water for four weeks. The group of Wenjing Decoction with Hupo Powder was given low dose of Wenjing Decoction with Hupo Powder(9.69 g/kg),medium dose of Wenjing Decoction with Hupo Powder(19.38 g/kg),and low dose of Wenjing Decoction with Hupo Powder(38.75 g/kg) by gastric gavage for four consecutive weeks,respectively. Serum tumor necrosis factor-α(TNF-α) and interleukin 1β(IL-1β) levels were measured using enzyme-linked immunosorbent assay(ELISA) in each group of rats and the blood rheology(plasma viscosity,erythrocyte aggregation index,erythrocyte rigidity index,erythrocyte deformation index,erythrocyte electrophoresis time),coagulation[prothrombin time(PT),prothrombin time international normalized ratio(PT-INR),partial thromboplastin time(TT),fibrinogen(FIB)] was tested in each group of rats. Detection of NF-κB p65,IkBα,and IKKβ protein expression in endometrial tissues of rats in each group using protein immunoblotting. [Results] Compared with the sham-operated group,the serum TNF-α and IL-1β levels were significantly higher in the model group(P<0.05 or P<0.01),and all the indices of blood rheology were significantly higher(P<0.05 or P<0.01). For coagulation,TT was significantly lower(P<0.05) and FIB were significantly higher(P<0.01). The results of protein expression blotting assay showed that NF-κB p65 and IKKβ were significantly elevated(P<0.01),and the expression of IkBα was significantly reduced(P<0.05). Compared with the model group,the expression of serum TNF-α and IL-1β was significantly reduced in the western medicine and part of traditional Chinese medicine(TCM) dose groups(P<0.05 or P<0.01),as well as all the indexes of blood rheology(P<0.05 or P<0.01). For coagulation,TT were significantly higher(P<0.05 or P<0.01) and FIB were significantly lower(P<0.05 or P<0.01). The results of protein expression blotting assay showed that the expression of NF-κB p65 were decreased(P<0.05 or P<0.01),the expression of IKKβ in the western medicine group and part of TCM dose groups were significantly decreased(P<0.05 or P<0.01),the expression of IkBα in the western medicine and part of TCM dose groups were increased(P<0.05 or P<0.01). [Conclusion] Wenjing Decoction combined with Hupo Powder may shrink the ectopic foci and regulate the development of endometriosis by blocking or inhibiting the activation of the NF-κB signalling pathway and inhibiting the proliferation of ectopic tissues,and it can improve the symptoms of cold agglutination and blood stasis,with a therapeutic efficacy superior to that of pregnenolone.