基于铁死亡探究益气解毒方对小鼠睾丸辐射损伤的防护作用

Effectof Yiqi Jiedu Decoction on Radiation-Induced Acute Testicular Injury:Based on Ferroptosis

目的探究2 Gy^(60) Coγ射线对小鼠睾丸的损伤作用,并基于铁死亡(Ferroptosis)分析益气解毒方对小鼠睾丸急性辐射损伤的防护作用。方法将150只SPF级BALB/c雄鼠随机分为空白组(NC)、模型组(IR)、Liproxstatin-1预防组(LP)、Liproxstatin-1治疗组(LT)以及益气解毒方组(YQJD),每组各30只;每个组又分3个批次,各10只。YQJD组每日以益气解毒方水煎液0.2 ml/10 g体重灌胃,其余各组以去离子水灌胃,均1次/d,持续10 d。然后除NC组外,其余各组小鼠采用2 Gy^(60) Coγ射线一次性全身照射,复制急性睾丸辐射损伤模型。LP组在照射前30 min予以小鼠腹腔注射0.2 ml/10 g Liproxstatin-1;LT组于照射后30 min,予以小鼠腹腔注射0.2 ml/10 g Liproxstatin-1,其后1次/d,连续给药至小鼠取材前1 d,照射后其余组小鼠不再给药。分别于照射后第1、3、7天取材,检测睾丸系数、MDA含量、SOD活力、GSH含量以及Fe含量、HE染色观察睾丸形态结构,并用Johnsen评分分析生精小管的损伤情况、免疫组化检测GPX4蛋白表达情况,并进行半定量分析。结果2 Gyγ射线照射后第1天起,睾丸组织中MDA明显升高(P<0.05),Fe离子含量升高(P<0.05),GPX4表达量明显降低(P<0.05),而益气解毒方于照射后第1天起明显降低小鼠睾丸组织中MDA和Fe含量(P<0.05),提升SOD活力。第3天起,模型组小鼠睾丸生精细胞的数量及层数明显降低(P<0.05),生精小管萎缩,小鼠睾丸Johnsen评分也明显下降(P<0.05),伴随SOD活力下降。于照射后第7天,各照射组小鼠睾丸系数均明显下降(P<0.05),而益气解毒方明显升高生精上皮的层数、生精细胞的数量以及睾丸系数(P<0.05),并在一定程度上缓解了GPX4蛋白表达下降的趋势。结论电离辐射后,小鼠睾丸内氧化应激水平激增,铁代谢紊乱,睾丸组织结构损伤。而益气解毒方能够缓解辐射后睾丸内氧化应激水平,并且通过调控铁死亡减轻睾丸急性辐射损伤。

Objective To explore the acute damage of 2 Gy^(60)Coγray tomouse testisand to analyze the effect of Yiqi Jiedu Decoction(YQJD)against the damage based on ferroptosis.Methods A total of 150 male SPF BALB/cmice were randomized into the control(NC)group,model(IR)group,Liproxstatin-1 prevention(LP)group,Liproxstatin-1 treatment(LT)group,and YQJD groupwith 30 mice each.Each group was divided into three batches with 10 mice in each batch.YQJD group was given the decoction at 0.2 ml/10 g body weight every day,and other groups the deionized water,once a day for 10 days.The mice(whole body),except for NC group,were irradiated with 2 Gy^(60)Coγray to induce acute testicular injury.Mice in LP group received(ip)Liproxstatin-1 at 0.2 mL/10 g body weight 30 minbefore irradiation,and those in LT group were injected with Liproxstatin-1 at 0.2 ml/10 g body weight 30 min after irradiation.For the two groups,the administration lasted to the day before sampling at 1 time/day.Other mice stopped the administration after exposure to irradiation.Samples were collected on the 1^(st),3^(rd),and 7^(th) day after exposure,and the testicular index,activity of superoxide dismutase(SOD),content of malondialdehyde(MDA),glutathione(GSH)content,and Fe content in the testis were detected.Testicular morphology and structure were observed based on hematoxylin and eosin(HE)staining,and immunohistochemistry was used to detect the expression of glutathione peroxidase 4(GPX4)protein.The injury of seminiferous tubules was evaluated with Johnsen score.Results From the first day after irradiation,mouse testes showed increase in the MDA content(P<0.05)and Fe ion content(P<0.05)and decrease in GPX4 expression(P<0.05).However,YQJD decreased the content of MDA and Fe in mouse testis(P<0.05)and improved SOD activity from the first day after irradiation.From the third day,mouse testes demonstrated decrease in number and layers of spermatogenic cells(P<0.05),atrophy of seminiferous tubules,and decline in Johnsen score of the testes(P<0.05)and SOD activity.On the 7^(th) day after irradiation,the testicular index of mice in each irradiation group decreased(P<0.05),while YQJD raised the number of layers of spermatogenic epithelium,the number of spermatogenic cells,and testicular index(P<0.05),alleviating the downward expression of GPX4 protein.Conclusion After irradiation,mouse testis showed sharp increase in oxidative stress,disorder of iron metabolism,and testicular injury.YQJD relieved the oxidative stress in the testisand alleviated the radiation-induced acute testicular injury by regulating ferroptosis.