肝豆补肾汤通过抑制铁死亡和内质网应激改善肝豆状核变性模型TX小鼠卵巢组织损伤

Gandou Bushen Decoction Alleviates Ovarian Tissue Damage in TX Mice with Hepatolenticular Degeneration by Inhibiting Ferroptosis and Endoplasmic Reticulum Stress

目的观察肝豆补肾汤对肝豆状核变性(hepatolenticular degeneration,HLD)小鼠卵巢损伤的保护作用,并探究其分子机制。方法以TX小鼠作为HLD模型,将其分为HLD组、青霉胺组和肝豆补肾汤组,另设DL同系小鼠作为正常对照组。测量小鼠体质量、卵巢质量、卵巢系数。采用促性腺激素促排卵法观察小鼠的排卵情况。采用苏木精—伊红(hematoxylin-eosin,HE)染色法观察小鼠卵巢的组织形态,透射电子显微镜下观察卵巢组织的超微结构。采用比色法测定血清铁含量,TBA法检测卵巢组织中丙二醛(malondialdehyde,MDA)含量,微量酶标法检测卵巢组织中还原型谷胱甘肽(glutathione,GSH)及氧化型谷胱甘肽(oxidized glutathione,GSSG)水平。采用Western blot法检测小鼠卵巢组织铁死亡相关标志物前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,PTGS2)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)水平,及内质网应激通路相关蛋白[葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、蛋白激酶核糖核酸样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)、磷酸化PERK(phosphorylated PERK,p-PERK)、真核起始因子2α(eukaryotic initiation factor 2 alpha-subunit,eIF2α)、磷酸化eIF2α(phosphorylated eIF2α,p-eIF2α)、活化转录因子4(activating transcription factor 4,ATF4)和C/EBP同源蛋白(C/EBP homologous protein,CHOP)]的表达水平。结果HE染色显示HLD组小鼠卵细胞形态结构受损严重,闭锁卵泡显著增加;透射电子显微镜下HLD组小鼠线粒体皱缩明显,出现内质网肿胀和脱颗粒等内质网应激表现。与正常对照组比较,HLD组小鼠卵巢质量、排卵数均显著降低(P<0.05),血清铁及卵巢组织中MDA、GSSG水平显著升高(P<0.05),卵巢组织中GSH水平、GSH/GSSG显著降低(P<0.05),卵巢组织中PTGS2、GRP78、p-PERK、p-eIF2α、ATF4、CHOP表达水平均显著升高(P<0.05),GPX4表达水平显著降低(P<0.05)。与HLD组比较,肝豆补肾汤组小鼠的卵泡形态、线粒体和内质网结构均显著改善,促排卵后排卵数显著升高(P<0.05),血清铁及卵巢组织中MDA和GSSG水平显著降低(P<0.05),卵巢组织中GSH水平和GSH/GSSG显著升高(P<0.05),卵巢组织中PTGS2、GRP78、p-PERK、p-eIF2α、CHOP表达水平显著降低(P<0.05),卵巢组织中GPX4表达水平显著升高(P<0.05)。结论肝豆补肾汤可减轻TX小鼠铜沉积诱导的卵巢损伤,其机制可能与抑制铁死亡和PERK通路介导的内质网应激有关。

Objective To investigate the protective effect of Gandou Bushen Decoction(GDBSD)against ovarian damage in mice with hepatolenticular degeneration(HLD)and its molecular mechanism.Methods TX mice were used to establish a model of HLD,and then they were divided into HLD group,penicillamine group,and GDBSD group,while DL mice were established as normal control group.Body weight,ovarian weight,and ovarian coefficient were measured.The gonadotropin ovulation induction method was used to observe ovulation.HE staining was used to observe the histomorphology of mouse ovaries,and transmission electron microscopy was used to observe the ultrastructure of ovarian tissue.Colorimetry was used to measure serum iron content,the TBA method was used to measure the content of malondialdehyde(MDA)in ovarian tissue,and ELISA was used to measure the levels of reduced glutathione(GSH)and oxidized glutathione(GSSG)in ovarian tissue.Western blotting was used to measure the levels of the ferroptosis markers prostaglandin-endoperoxide synthase 2(PTGS2)and glutathione per oxidase 4(GPX4)in ovarian tissue,as well as the expression levels of endoplasmic reticulum stress pathway-related proteins,including glucose-regulated protein 78(GRP78),protein kinase RNA-like endoplasmic reticulum kinase(PERK),phosphorylated PERK(p-PERK),eukaryotic initiation factor 2α(eIF2α),phosphorylated eIF2α(p-eIF2α),activating transcription factor 4(ATF4),and C/EBP homologous protein(CHOP).Results HE staining showed that the HLD group had severe damage of oocyte morphology and structure and a significant increase in atretic follicles,and transmission electron microscopy showed obvious mitochondrial wrinkling and the manifestations of endoplasmic reticulum stress such as swelling and degranulation.Compared with the normal control group,the HLD group had significant reductions in ovarian weight(P<0.05)and the number of ovulations(P<0.05),significant increases in serum iron and the levels of MDA and GSSG in ovarian tissue(P<0.05),and significant reductions in GSH and GSH/GSSG(P<0.05),as well as significant increases in the expression levels of PTGS2,GRP78,p-PERK,p-eIF2α,ATF4,and CHOP and a significant reduction in the expression level of GPX4 in ovarian tissue(P<0.05).Compared with the HLD group,the GDBSD group had significant improvements in follicular morphology and the structure of mitochondria and endoplasmic reticulum,a significant increase in the number of ovulations(P<0.05),significant reductions in serum iron and the levels of MDA and GSSG in ovarian tissue(P<0.05),and significant increases in GSH and GSH/GSSG in ovarian tissue(P<0.05),as well as significant reductions in the expression levels of PTGS2,GRP78,p-PERK,p-eIF2α,and CHOP and a significant increase in the expression level of GPX4 in ovarian tissue(P<0.05).Conclusion GDBSD can alleviate ovarian damage induced by copper deposition in TX mice,possibly by inhibiting ferroptosis and endoplasmic reticulum stress mediated by the PERK pathway.