基于IL-6/STAT3通路调控Th17/Treg平衡探讨大黄蛰虫丸对子宫内膜异位症血瘀证模型大鼠的影响

Based on the regulation of Th17/Treg balance by IL-6/STAT3 pathway, the effect of Dahuang Zhechong Pill on endometriosis rats with blood stasis syndrome was discussed

关键词:  子宫内膜异位症  大黄蛰虫丸  血瘀证  白细胞介素-6(IL-6)/信号转导和转录激活因子3(STAT3)信号通路  Th17/Treg平衡

Key words:  Endometriosis  Dahuang Zhechong Pills  blood stasis syndrome  interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway  Th17/Treg balance

摘要:

目的:从白细胞介素-6(Interleukin-6, IL-6)/信号转导及转录激活因子3(signal transducer and activator of transcription, STAT3)通路调控异位子宫内膜组织Th17/Treg平衡探讨大黄蛰虫丸治疗血瘀证子宫内膜异位症的潜在分子机制。方法:70只雌性SD大鼠按照随机数字表法分为假手术组、模型对照组、阳性对照(地诺孕素)组、大黄蛰虫丸高、中、低剂量组及通路抑制剂(紫草素)+大黄蛰虫丸高剂量组,每组10只。采用自体移植复合注射盐酸肾上腺素(0.6 mg·kg-1·d-1)的方法建立EMs血瘀证大鼠模型。阳性对照组大鼠ig地诺孕素混悬液0.21 mg·kg-1·d-1,大黄蛰虫丸高、中、低剂量组大鼠分别ig大黄蛰虫丸混悬液2.16、1.08及0.54 g·kg-1·d-1,通路抑制剂+大黄蛰虫丸高剂量组大鼠ig紫草素8 mg·kg-1d-1+大黄蛰虫丸混悬液2.16 g·kg-1·d-1,空白组及模型对照组大鼠ig蒸馏水1mL·100g-1,连续14 d。给药14 d后,观察各组大鼠的一般状况并处死。记录异位组织体积、苏木素-伊红染色观察异位组织病理变化、全自动血细胞分析仪检测血常规指标、血流变分析仪检测血液流变学指标、全自动凝血分析仪检测凝血四项、酶联免疫吸附测定法检测血清性激素及炎症因子水平,蛋白免疫印迹法检测各组大鼠异位组织中IL-6、STAT3及磷酸化信号转导和转录激活因子3(Phosphorylated STAT3, p-STAT3)蛋白表达量,免疫组化检测异位组织孤独核受体(Retinoid related orphan receptor γt, RORγt)、叉头状转录因子(ForkheadboxP3, Foxp3)蛋白表达量,实时荧光定量聚合酶链式反应检测IL-6、STAT3、RORγt、Foxp3 mRNA表达量。结果:与假手术组比较,模型对照组大鼠的异位组织体积显著增大(P<0.01),组织病理学结果符合异位子宫内膜组织病理学特征,全血黏度高切、中切、低切及血浆黏度值均显著升高(均P<0.01),凝血四项PT、APTT及TT值均显著降低(均P<0.01),FIB值显著升高(P<0.01),血清E2、TNF-α、IL-17、IL-6含量均显著升高(均P<0.01),IL-10、TGF-β1含量均显著降低(均P<0.01),异位内膜组织IL-6、STAT3、p-STAT3、RORγt蛋白表达水平均显著升高(均P<0.01)、Foxp3蛋白表达显著降低(P<0.01),异位组织IL-6、STAT3、RORγt mRNA表达水平均显著升高(P<0.05,P<0.01),Foxp3 mRNA表达水平显著降低(P<0.01)。与模型对照组比较,大黄蛰虫丸高、中剂量组,阳性对照组及通路抑制剂+大黄蛰虫丸高剂量组大鼠异位内膜组织体积显著减小(P<0.01),病理损伤不同程度改善,炎性细胞浸润较少、血管生成减少,其中大黄蛰虫丸高、中剂量组及通路抑制剂+大黄蛰虫丸高剂量组大鼠全血黏度高切、中切、低切及血浆黏度值均显著降低(均P<0.01),PT、APTT及TT值均显著升高(均P<0.01),FIB值显著降低(P<0.01),血清E2、TNF-α、IL-17、IL-6含量均显著降低(均P<0.01),IL-10、TGF-β1含量均显著升高(均P<0.01),异位组织IL-6、STAT3、p-STAT3、RORγt蛋白表达水平均显著降低(均P<0.01)、Foxp3蛋白表达显著升高(P<0.01),异位组织IL-6、STAT3、RORγt mRNA表达水平显著降低(P<0.05,P<0.01),Foxp3 mRNA表达水平显著升高(P<0.01)。结论:大黄蛰虫丸可能通过干预IL-6/STAT3信号通路关键蛋白,调控异位子宫内膜组织Th17/Treg平衡,对血瘀证子宫内膜异位症起到治疗作用。

Abstract:

objective Exploring the potential molecular mechanism of Dahuang Zhechong Pill in treating endometriosis with blood stasis syndrome by regulating Th17/Treg balance in ectopic endometrial tissue through the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway. Methods Seventy female SD rats were randomly divided into a sham operation group, a model control group, a positive control group (denogestant), high, medium, and low-dose groups of Dahuang Zhechong Pill, and a high-dose group of pathway inhibitor (shikonin)+Dahuang Zhechong Pill, with 10 rats in each group. A rat model of EMs blood stasis syndrome was established by using autologous transplantation combined with injection of adrenaline hydrochloride (0.6 mg·kg-1·d-1). The positive control group of rats received 0.21 mg·kg-1·d-1 suspension of dienogest, while the high, medium, and low dose groups of rats received 2.16, 1.08, and 0.54 g·kg-1·d-1 suspension of Dahuang Zhechong Pill, respectively. The high-dose group of rats receiving pathway inhibitors and Dahuang Zhechong Pill received a 2.16 g·kg-1·d-1 suspension of purpurin, while the blank group and model control group received 1mL·100g-1 suspension of distilled water for 14 consecutive days. After 14 days of administration, observe the general condition of each group of rats and euthanize them. Record the volume of ectopic tissue, observe pathological changes of ectopic tissue with hematoxylin eosin staining, detect blood routine indicators with a fully automated hematology analyzer, detect blood rheology indicators with a blood rheology analyzer, detect coagulation parameters with a fully automated coagulation analyzer, detect serum hormone and inflammatory factor levels with enzyme-linked immunosorbent assay, detect IL-6, STAT3, and phosphorylated signal transduction and transcription activation factor 3 (p-STAT3) protein expression in ectopic tissue of rats in each group with protein immunoblotting, and detect the protein expression of Retinad related orphan receptor gamma t (RORγt) and forkhead transcription factor P3 (Foxp3) in ectopic tissue with immunohistochemistry. Real time fluorescence quantitative polymerase chain reaction was used to detect the mRNA expression levels of IL-6, STAT3, RORγt, and Foxp3. Results Compared with the sham operation group, the volume of ectopic endometrial tissue in the model control group was significantly increased (P<0.01). The histopathological results were consistent with the histopathological characteristics of ectopic endometrial tissue. The whole blood viscosity at high, medium and low shear rates and plasma viscosity were significantly increased (all P<0.01). The values of PT, APTT and TT were significantly decreased (all P<0.01), the FIB value was significantly increased (P<0.01), and the serum levels of E2, TNF-α, IL-17 and IL-6 were significantly increased (all P<0.01). The levels of IL-10 and TGF-β1 were significantly decreased (all P<0.01). The expression levels of IL-6, STAT3, p-STAT3 and RORγt protein in ectopic endometrial tissues were significantly increased (all P<0.01), and the expression of Foxp3 protein was significantly decreased (P<0.01). The expression levels of IL-6, STAT3 and RORγt mRNA in ectopic tissues were significantly increased (P<0.05,P<0.01), and the expression level of Foxp3 mRNA was significantly decreased (P<0.01). Compared with the model control group, the volume of ectopic endometrial tissue in the high and middle dose groups of Dahuang Zhechong Pills, the positive control group and the pathway inhibitor + Dahuang Zhechong Pills high dose group was significantly reduced (P<0.01), the pathological damage was improved to varying degrees, the inflammatory cell infiltration was less, and the angiogenesis was reduced. Among them, the high, middle dose groups of Dahuang Zhechong Pills and the pathway inhibitor + Dahuang Zhechong Pills high dose group rats whole blood viscosity high shear, middle shear, low shear and plasma viscosity values were significantly reduced (all P<0.01), PT, APTT and TT values were significantly increased (all P<0.01), FIB values were significantly reduced (P<0.01). The levels of serum E2, TNF-α, IL-17 and IL-6 were significantly decreased (all P<0.01), and the levels of IL-10 and TGF-β1 were significantly increased (all P<0.01). The expression levels of IL-6, STAT3, p-STAT3 and RORγt protein in ectopic tissues were significantly decreased, and the expression of Foxp3 protein was significantly increased. The expression levels of IL-6, STAT3 and RORγt mRNA in ectopic tissues were significantly decreased (P<0.05,P<0.01), and the expression level of Foxp3 mRNA was significantly increased (P<0.01). Conclusion Dahuang Zhechong Pills may regulate the balance of Th17/Treg in ectopic endometrial tissue by interfering with the key proteins of IL-6/STAT3 signaling pathway, and play a therapeutic role in endometriosis with blood stasis syndrome.