目的:人蛋白类似物(HNG)对大鼠宫腔手术引起的宫腔粘连(IUAs)有治疗作用吗?
结论:补充HNG可以减轻子宫内膜纤维化和IUAs的发展,提高生育率,并通过抑制IUAs大鼠子宫内膜铁蛋白表达来调节子宫内膜纤维化。
背景:IUAs,以子宫内膜纤维化为特征,是女性不孕的常见原因。Humanin(大鼠中的rattin)是一种线粒体衍生的肽,在多种组织中广泛表达。S14G-humanin(HNG)是一种HNG,据报道具有抗心肌纤维化的保护作用。
方法:测试了3名IUA患者和3名对照患者的子宫内膜组织的humanin表达。使用两种动物模型来评估IUA的建模效果和HNG对IUA的预防效果。在第一个模型中,40只大鼠随机均分为对照组和第7、14、21天组,建立IUA模型。在第二个模型中,66只大鼠随机分为对照组,IUA,和IUA+人蛋白类似物(HNG)组。Erastin用于在Ishikawa细胞系中诱导铁凋亡。
方法:用手术刮铲刮除子宫内膜,结合脂多糖治疗,建立IUAs大鼠模型。从手术当天开始,连续21天腹膜内注射5mg/kg/天的HNG,以评估对IUA的治疗效果。使用苏木精-伊红和Masson三色染色来评估子宫内膜形态并评估纤维化。铁凋亡相关标记,即核因子E2相关因子2(Nrf2),酰基辅酶A合成酶长链家族成员4(ACSL4),血红素加氧酶-1(HO-1),溶质载体家族7成员11(SLC7A11),谷胱甘肽过氧化物酶4(GPX4),和铁蛋白,通过免疫组织化学和蛋白质印迹法进行测量,以确定铁凋亡是否参与IUA的发展,并评估HNG对铁凋亡的减弱作用。此外,雌性大鼠与正常生育力的雄性大鼠交配以评估生育力。
结果:Humanin在子宫内膜细胞中广泛表达,包括上皮细胞和基质细胞,在人类和老鼠身上。Humanin表达水平在患有IUA的患者和大鼠的子宫内膜中相对于对照的子宫内膜下调。子宫内膜刮除后第7、14、21天子宫内膜厚度和腺体数量均明显低于对照组(均P<0.05)。而纤维化面积显著增加(P<0.05)。在测试的铁死亡标志物中,子宫内膜刮除后,Nrf2、SLC7A11和GPX4的表达水平显著下调,ACSL4、HO-1和铁蛋白的表达水平与对照组相比显著上调(均P<0.05)。控制中的交配率,IUA,IUA+HNG组为100%(10/10),40%(4/10),和80%(8/10),分别。IUA大鼠的胚胎数量(平均值±SD:1.6±2.1)显着少于对照组的胚胎数量(11.8±1.5)。补充HNG可显着减轻植入胚胎数量的减少(6.3±4.5)(P<0.01)。进一步的结果表明,HNG显着减弱了IUA大鼠子宫内膜中与铁凋亡有关的蛋白质表达水平的改变。此外,体外实验表明,HNG显着减弱了erastin诱导的Ishikawa细胞系活力降低,并且还减弱了活性氧产生的增加和GPX4的下调。
方法:无。
结论:这项研究的结果表明,HNG在IUAs大鼠模型中抑制了铁细胞凋亡并减少了纤维化。然而,我们无法确定铁死亡与IUA发展之间的因果关系。
结论:HNG可能有效缓解IUAs发展过程中的纤维化,抑制铁细胞凋亡是IUA治疗的一个有前途的新策略。
背景:本研究得到了国家自然科学基金(No.82171647);云南省“1000人才计划”(编号:RLQN20200001);以及云南省基础研究项目--杰出青年基金会(编号202101AW070018)。作者宣布没有竞争的经济利益。
Does a humanin analogue (HNG) have a therapeutic effect on intrauterine adhesions (IUAs) caused by uterine cavity surgery in a rat model?
HNG supplementation attenuated the development of endometrial fibrosis and IUAs, improved fertility, and contributed to the regulation of endometrial fibrosis by inhibiting endometrial ferroptosis in rats with IUAs.
IUAs, which are characterized by endometrial fibrosis, are a common cause of female infertility. Humanin (rattin in rats) is a mitochondrial-derived peptide that is widely expressed in multiple tissues. S14G-humanin (HNG) is an HNG that has been reported to have a protective effect against myocardial fibrosis.
Endometrial tissues from three patients with IUAs and three controls were tested for humanin expression. Two animal models were used to evaluate the modelling effect of IUAs and the preventive effect of HNG against IUAs. In the first model, 40 rats were equally randomized to control and Day 7, 14, and 21 groups to establish the IUA model. In the second model, 66 rats were equally randomized to the control, IUA, and IUA + humanin analogue (HNG) groups. Erastin was used to induce ferroptosis in the Ishikawa cell line.
The endometrium was scraped with a surgical spatula, combined with lipopolysaccharide treatment, to establish the rat model of IUAs. Rats were intraperitoneally injected with 5 mg/kg/day HNG for 21 consecutive days beginning from the day of operation to evaluate the therapeutic effect on IUAs. Haematoxylin-eosin and Masson\'s trichrome staining were used to assess endometrial morphology and evaluate fibrosis. Ferroptosis-related markers, namely nuclear factor E2-related factor 2 (Nrf2), acyl-CoA synthetase long-chain family member 4 (ACSL4), haeme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and ferritin, were measured by immunohistochemistry and western blotting to determine whether ferroptosis was involved in the development of IUAs and to assess the attenuative effect of HNG on ferroptosis. Additionally, the female rats were mated with male rats with normal fertility to assess fertility.
Humanin was widely expressed in endometrial cells, including epithelial and stromal cells, in both humans and rats. Humanin expression levels were downregulated in the endometria of patients and rats with IUAs relative to the endometria of controls. Endometrial thickness and the number of glands were significantly decreased on Day 7, 14, and 21 after endometrial scraping when compared with the controls (all P < 0.05), whereas the fibrotic area was significantly increased (P < 0.05). Among the tested ferroptosis markers, the expression levels of Nrf2, SLC7A11, and GPX4 were significantly downregulated and those of ACSL4, HO-1, and ferritin were significantly upregulated after endometrial scraping relative to their expression levels in controls (all P < 0.05). The mating rates in the control, IUA, and IUA + HNG groups were 100% (10/10), 40% (4/10), and 80% (8/10), respectively. The number of embryos in rats with IUAs (mean ± SD: 1.6 ± 2.1) was significantly less than the number in the controls (11.8 ± 1.5). HNG supplementation significantly attenuated this decrease in the number of implanted embryos (6.3 ± 4.5) (P < 0.01). Further results showed that HNG significantly attenuated the altered expression levels of proteins involved in ferroptosis in the endometria of rats with IUAs. Moreover, in vitro experiments showed that HNG significantly attenuated the erastin-induced decrease in the viability of the Ishikawa cell line and also attenuated the increase in reactive oxygen species production and the downregulation of GPX4.
None.
The findings of this
study showed that HNG inhibited ferroptosis and reduced fibrosis in a rat model of IUAs. However, we could not establish a causal relationship between ferroptosis and the development of IUAs.
HNG may be effective at alleviating fibrosis during the development of IUAs, and the inhibition of ferroptosis is a promising new strategy for IUA therapy.
This
study was supported by the National Natural Science Foundation of China (No. 82171647); the \'1000 Talent Plan\' of Yunnan Province (No. RLQN20200001); and the Basic Research Project of the Yunnan Province-Outstanding Youth Foundation (No. 202101AW070018). The authors declare no competing financial interests.