基于NCOA4/NF-κB信号通路介导的铁自噬探讨祛瘀解毒方对子宫内膜异位症模型大鼠子宫异位组织的影响

Exploring the Effects of Quyu Jiedu Formula(祛瘀解毒方)on the Ectopic Tissues of the Rat Uterus in An Endometriosis Model Based on Iron Autophagy Mediated by the NCOA4/NF-κB Signalling Pathway

目的从核受体共激活因子4/核因子κB(NCOA4/NF-κB)信号通路介导的铁自噬角度探讨祛瘀解毒方治疗子宫内膜异位症的可能作用机制。方法将50只雌性SD大鼠随机分为假手术组、模型组、米非司酮组和祛瘀解毒方低、高剂量组,每组10只。假手术组仅行开腹后关腹操作,其余各组均采用自体移植的方法建立子宫内膜异位症大鼠模型。造模成功后次日,假手术组和模型组予生理盐水2 ml/d灌胃,米非司酮组予米非司酮悬浊液1.05 mg/(kg·d)灌胃,祛瘀解毒方低、高剂量组分别予祛瘀解毒方12.23、48.92 g/(kg·d)灌胃,各组均连续给药4周。4周后假手术组大鼠剪取正常子宫内膜组织,其余4组摘取大鼠全部异位内膜组织。测量模型组、米非司酮组和祛瘀解毒方低、高剂量组大鼠异位病灶体积;HE染色观察各组大鼠子宫内膜/异位内膜组织的病理学改变;检测子宫内膜/异位内膜组织NCOA4、铁蛋白重链(FTH1)、红杉(P62)、微管相关蛋白1轻链3β(LC3B)、核因数(P-NF-κB)蛋白表达及NCOA4、FTH1、LC3B、P62 mRNA表达;检测子宫内膜/异位内膜组织NCOA4、LC3B的共定位情况,游离铁含量,白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平。结果假手术组未见异位病灶。模型组大鼠异位组织出现了明显的病理损伤,而各给药组大鼠异位组织病理损伤不同程度减轻。与模型组相比,米非司酮组及祛瘀解毒方高、低剂量组异位病灶体积均缩小,并且祛瘀解毒方高剂量组和米非司酮组大鼠异位病灶体积明显小于祛瘀解毒方低剂量组(P<0.01)。与假手术组相比,模型组异位组织LC3BⅡ/LC3BⅠ值及NCOA4、P-NF-κB蛋白表达上调,P62、FTH1蛋白表达下调,游离铁含量及IL-6、TNF-α水平升高,NCOA4、LC3B共定位阳性率及共定位细胞密度增加(P<0.05或P<0.01)。与模型组相比,祛瘀解毒方低、高剂量及米非司酮组异位内膜组织LC3BⅡ/LC3BⅠ值及NCOA4、P-NF-κB蛋白表达下调,NCOA4、LC3B共定位阳性率明显减少,游离铁含量及IL-6水平降低(P<0.05或P<0.01)。与米非司酮组相比,祛瘀解毒方高剂量组P62蛋白上调更明显,TNF-α水平降低(P<0.05)。与祛瘀解毒方低剂量组比较,祛瘀解毒方高剂量组大鼠异位组织游离铁含量及IL-6、TNF-α水平降低(P<0.01)。结论祛瘀解毒方治疗子宫内膜异位症机制可能与抑制子宫内膜异位组织NCOA4/NF-κB信号通路介导的铁自噬,从而改善子宫内膜炎症有关。

Objective To investigate the possible mechanism of Quyu Jiedu Formula(祛瘀解毒方)in the treat⁃ment of endometriosis in terms of iron autophagy mediated by nuclear receptor coactivator 4/nuclear factorκB(NCOA4/NF-κB)signalling pathway.Methods Fifty female SD rats were randomly divided into sham surgery group,model group,mifepristone group,low-and high-dose Quyu Jiedu Formula group,with 10 rats in each group.In the sham surgery group,only operation of opening and closing abdomen was performed,and in the remaining groups,the rat with endometriosis was modelled by autotransplantation.On the next day after successful modelling,saline 2 ml/d was given by gavage to the sham surgery group and the model group;mifepristone 1.05 mg(/kg·d)was given by gavage to the mifepristone group;Quyu Jiedu Formula 12.23 g(/kg·d)and 48.92 g(/kg·d)were given to the low-and high-dosage Quyu Jiedu Formula groups,respectively administered for 4 weeks consecutively.In the remaining 4 groups,all ectopic endometrial tissues were removed from the rats.The volume of ectopic lesions was measured in the model group,the mifepristone group,and the low-and high-dose Quyu Jiedu Formula groups,and the pathological changes of endometrial/ectopic tissues were observed by HE staining,and the protein expression and expression of NCOA4,Ferritin Heavy Chain 1(FTH1),Panax quinquefolium(P62),Microtubule-associated Protein 1 Light Chain 3β(LC3B),and P-NF-κB protein expression and NCOA4,FTH1,LC3B,P62 mRNA expression were detected in the endometrium and ectopic tissues;the co-localisation of NCOA4 and LC3B,free iron content,and levels of interleukin 6(IL-6)and tumour necrosis factorα(TNF-α)in endometrial/eutopic endometrial tissues were also detected.Results No ectopic lesions were seen in the sham surgery group.The ectopic tissues of rats in the model group showed obvious pathological damage,while the pathological damage of the ectopic tissues of rats in each admi-nistration group was reduced to different degrees.Compared with the model group,the volume of ectopic lesions was reduced in the mifepristone group and the high-and low-dose Quyu Jiedu Formula groups,and the volume of ectopic lesions in the high-dose Quyu Jiedu Formula group and the mifepristone group was significantly smaller than that in the low-dose Quyu Jiedu Formula group(P<0.01).Compared with the sham surgery group,the ectopic tissues of the model group showed up-regulation of LC3BⅡ/LC3B I values,NCOA4,and P-NF-κB protein expression,downregulation of P62 and FTH1 protein expression,increase in free iron content and IL-6 and TNF-αlevels,and increase in the co-localisation positivity rate and co-localised cell density of NCOA4 and LC3B(P<0.05 or P<0.01).Compared with the model group,the ectopic endothelial tissue LC3BⅡ/LC3BⅠvalues and the expression of NCOA4 and P-NF-κB proteins were down-regulated in the low-and high-dose Quyu Jiedu Formula group and mifepristone group,the colo⁃calisation positivity rate of NCOA4 and LC3B significantly reduced,and the content of free iron and the level of IL-6 decreased(P<0.05 or P<0.01).Compared with the mifepristone group,P62 more obvious up-regulated and TNF-αlevel reduced in the high-dose Quyu Jiedu Formula group(P<0.05).Compared with the low-dose Quyu Jiedu Formula group,the free iron content of ectopic tissues and the levels of IL-6 and TNF-αreduced in the high-dose Quyu Jiedu Formula group(P<0.01).Conclusion The mechanism of endometriosis treatment by Quyu Jiedu Formula may be related to the inhibition of iron autophagy mediated by the NCOA4/NF-κB signalling pathway in endometriotic tissues,which improves endometrial inflammation.