旨在探讨不同浓度积雪草酸(AA)对脂多糖(LPS)诱导肉鸡心肌氧化应激和铁死亡的影响.选取50只1日龄肉鸡适应性饲养至7日龄,并随机分为5组,即对照组(Control)、LPS组(LPS)、低剂量AA组(LPS+AA 15 mg·kg-1)、中剂量 AA 组(LPS+AA 30 mg·kg-1)、高剂量 AA 组(LPS+AA 60 mg·kg-1).所有 AA 组用相应剂量的AA连续灌胃14 d.在16、18和20日龄时,所有LPS组肉鸡腹腔注射0.5 mg·kg-1 LPS以建立急性心肌损伤(AMI)模型并于21日龄时处死肉鸡并采集心肌样品.通过苏木精-伊红(HE)染色观察心肌组织病理学变化;使用谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)试剂盒检测心肌组织氧化应激指标;通过实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)检测心肌组织氧化应激和铁死亡相关基因和蛋白的表达情况;通过免疫组织化学法检测心肌组织中Nrf2和SLC7A11的阳性表达率;通过免疫荧光法检测心肌组织中GPX4的阳性表达率.结果发现,AA预处理可以减轻LPS诱导的心肌病理损伤;与LPS组相比,中剂量AA预处理后心肌中GSH-Px的活性显著升高(P＜0.01),AA预处理均显著降低MDA的含量(P＜0.05);此外,AA预处理降低了LPS 诱导的心肌 Keap1、HMGB1 和 PTGS2 的 mRNA 表达(P＜0.05),促进了 Nrf2、HO1、NQO1、GCLC、GCLM、SLC7A11、FTH1和GPX4的mRNA表达(P＜0.05),同时,也降低了 Keap1、HMGB1的蛋白表达,促进了 Nrf2、HO1、NQO1、SLC7A11、FTH1和GPX4的蛋白表达(P＜0.05);免疫组织化学和免疫荧光结果表明,AA预处理可促进Nrf2(P＜0.01)、SLC7A11(P＜0.01)和GPX4(P＜0.05)的表达.本结果首次表明,AA可通过调节氧化应激和铁死亡缓解LPS诱导的急性心肌损伤(AMI).AA有望成为未来预防LPS诱导肉鸡AMI的潜在食品添加剂.
Effect of Asiatic Acid Alleviating Myocardial Injury Caused by Lipopolysaccharide through Inhibiting Oxidative Stress and Ferroptosis in Broilers
This experiment was conducted to investigate the effects of different concentrations of asiatic acid(AA)on lipopolysaccharide(LPS)-induced acute myocardial injury(AMI)in broilers.All 50 broilers were selected to be reared to 7 days of age,and then divided into 5 groups,namely Control group,LPS group,LPS+AA 15 mg·kg-1 group,LPS+AA 30 mg·kg-1 group,LPS+AA 60 mg·kg-1 group.At 7 days of age,all the AA groups were pretreated with corresponding doses of AA by gavage for 14 consecutive days.At 16,18 and 20 days of age,all the LPS groups were intraperitoneally injected with 0.5 mg·kg-1 LPS to induce AMI.Myocardial samples of broilers were collected at the age of 21 days.Histopathological changes of myocardium were observed by hematoxylin-eosin(HE)staining;Detection of markers of oxidative stress in myocardial tissue u-sing glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)kits;Real-time fluorescence PCR(qRT-PCR)and Western blot were used to detect the expression of genes and proteins related to oxidative stress and ferroptosis in myocardial tissues.The positive expression rate of Nrf2 and SLC7A11 in myocardial tissues was detected by immunohistochemistry.The positive expression rate of GPX4 in myocardial tissues was detected by immunofluorescence.The results showed that AA pretreatment alleviated the myocarial injury induced by LPS.Compared with LPS group,medium-dose AA pretreatment increased GSH-Px activity while all AA pretreatment decreased MDA content in myocardium significantly(P＜0.05 or P＜0.01).Importantly,AA pretreat-ment decreased the mRNA expression of Keap1,PTGS2 and HMGB1,and increased the expres-sion of Nrf2,HO-1,NQO1,FTH1,SLC7A11,GPX4,GCLC and GCLM(P＜0.05 or P＜0.01).AA pretreatment also decreased the protein expression of Keap1 and HMGB1,promoted the protein expression of Nrf2,HO-1,NQO1,FTH1 and GPX significantly(P＜0.05).Immuno-histochemistry and immunofluorescence results showed that AA pretreatment could significantly increased the expression of Nrf2(P＜0.01),SLC7A11(P＜0.01)and GPX(P＜0.01).The results of the current study showed for the first time that AA could alleviate LPS-induced AMI by regulating oxidative stress and ferroptosis.AA is expected to be a potential food additive for preventing LPS-induced AMI in broilers in the future.
万方数据
维普
