积雪草酸通过调控细胞凋亡和自噬缓解脂多糖诱导肉鸡急性肾损伤的研究

doi:10.11843/j.issn.0366-6964.2024.02.037

摘要/Abstract

摘要： 旨在探讨积雪草酸(AA)对脂多糖(LPS)诱导肉鸡肾细胞凋亡和自噬的影响。将40只1日龄肉鸡适应性饲养至7日龄,并随机分为对照组(Con)、LPS组(LPS)、低剂量AA组(LPS+AA 15 mg·kg^-1)和高剂量AA组(LPS+AA 30 mg·kg^-1)。AA预处理组的肉鸡连续14 d每日灌胃相应剂量AA。除Con组,其余组肉鸡在第16、18和20日龄时腹腔注射0.5 mg·kg^-1 剂量的LPS构建急性肾损伤(AKI)模型;第21日龄时处死肉鸡并采集肾组织样品。苏木精-伊红(HE)染色观察肾组织病理学变化;计算肾脏系数和检测肾组织抗氧化酶水平;实时荧光定量(qRT-PCR)和蛋白免疫印迹(Western blot)分别检测凋亡和自噬相关基因及蛋白的表达;免疫组织化学和免疫荧光检测细胞色素c(Cytc)和微管相关蛋白1A/1B-轻链3(LC3)蛋白在肾组织的表达与分布;TUNEL染色检测肾细胞凋亡率。结果发现,AA可减轻LPS诱导的肉鸡肾病理损伤,显著降低肾脏系数和肾组织丙二醛(MDA)含量,显著升高谷胱甘肽过氧化物酶(GSH-Px)、超氧化物酶(SOD)和过氧化氢酶(CAT)的活性(P<0.05)。AA显著降低了LPS诱导的AKI中P53、BAX和Caspase3的mRNA表达,显著促进了LPS诱导的AKI中BCl2、Beclin1、ATG5、LC3-Ⅰ和LC3-Ⅱ的mRNA表达(P<0.05);此外,AA预处理显著降低了LPS诱导的AKI中P53、Bak1、BAX和Cleaved-Caspase3的蛋白表达(P<0.05),显著促进了LPS诱导的AKI中Beclin1、ATG5、LC3Ⅱ/Ⅰ的蛋白表达(P<0.05)。免疫组织化学和免疫荧光结果表明,AA预处理显著降低了LPS诱导的AKI中Cytc蛋白的表达与分布(P<0.001),促进了LC3蛋白在LPS诱导的AKI中的表达与分布(P<0.001)。TUNEL结果表明,AA显著降低了LPS诱导的肉鸡肾细胞凋亡率(P<0.001)。总之,AA通过抑制氧化应激、促进细胞自噬和降低细胞凋亡,缓解了LPS诱导的肉鸡AKI。此结果为AA成为潜在饲料添加剂和预防LPS诱导肉鸡AKI提供了理论依据。

关键词: 凋亡, 自噬, 积雪草酸, 脂多糖, 急性肾损伤, 肉鸡

Abstract: This study aimed to investigate the effect of Asiatic acid (AA) on lipopolysaccharide (LPS)-induced apoptosis and autophagy in the kidneys of broilers. Forty broilers (one day old) were fed adaptively until seven days old and then were randomly divided into the control group (Con), LPS group (LPS), low dose AA group (LPS+AA 15 mg·kg^-1) and high dose AA group (LPS+AA 30 mg·kg^-1). The broilers in the AA pretreatment group were gavaged with corresponding doses of AA daily for 14 consecutive days. Except for the control group, broilers in other groups were intraperitoneally injected with 0.5 mg·kg^-1 LPS at the age of 16, 18 and 20 days to establish an acute kidney injury (AKI) model. All broilers were sacrificed at the age of 21 days, and kidney samples were collected. Hematoxylin-eosin (HE) staining was used to observe the histopathological changes in the kidney tissue. The kidney coefficient was calculated and the levels of antioxidant enzymes in kidney tissue were detected. Real-time fluorescence quantitative (qRT-PCR) and Western blot were used to detect the expression of apoptosis-related genes and proteins as well as the expression of autophagy-related genes and proteins. Immunohistochemistry and immunofluorescence were used to detect the expression and distribution of cytochrome c (Cytc) and microtubule-associated protein 1A/1B-light chain 3 (LC3) in kidney tissue. The TUNEL staining was used to detect the apoptosis rate of the kidneys. The results showed that AA could alleviate LPS-induced kidney pathological damage of broilers, and significantly reduce the kidney coefficient and malondialdehyde (MDA) content as well as significantly increase the activities of glutathione peroxidase (GSH-Px), superoxide enzyme (SOD) and catalase (CAT) (P<0.05). AA significantly reduced the mRNA expression of P53, BAX and Caspase3 in LPS-induced AKI of broilers (P<0.05), and significantly promoted the mRNA expression of BCl2, Beclin1, ATG5, LC3-I and LC3-II in LPS-induced AKI of broilers (P<0.05). Moreover, AA pretreatment significantly reduced the protein expression of P53, Bak1, BAX and Cleaved-Caspase3 in LPS-induced AKI of broilers (P<0.05), and significantly promoted the protein expression of Beclin1, ATG5, LC3 II/I in LPS-induced AKI of broilers (P<0.05). Immunohistochemical and immunofluorescence results also showed that AA pretreatment significantly reduced the protein expression and distribution of Cytc in LPS-induced AKI (P<0.001), and significantly promoted the protein expression and distribution of LC3 in LPS-induced AKI (P<0.001). The TUNEL results showed that AA pretreatment significantly reduced the LPS-induced apoptosis rate in the kidney of broilers (P<0.001). In summary, AA alleviates LPS-induced AKI of broilers by inhibiting oxidative stress, promoting renal cell autophagy and reducing apoptosis. This result provides a theoretical basis for AA becoming a potential feed additive and preventing LPS-induced AKI of broilers.

Key words: apoptosis, autophagy, Asiatica acid, lipopolysaccharide, acute renal injury, broiler

中图分类号:

S852.35

邱文粤, 苏依曼, 叶嘉莉, 章心婷, 庞晓玥, 王荣梅, 谢子茂, 张辉, 唐兆新, 苏荣胜. 积雪草酸通过调控细胞凋亡和自噬缓解脂多糖诱导肉鸡急性肾损伤的研究[J]. 畜牧兽医学报, 2024, 55(2): 809-821.

QIU Wenyue, SU Yiman, YE Jiali, ZHANG Xinting, PANG Xiaoyue, WANG Rongmei, XIE Zimao, ZHANG Hui, TANG Zhaoxin, SU Rongsheng. Study on Asiatic Acid Alleviates LPS-induced Acute Kidney Injury by Regulating Apoptosis and Autophagy of Broilers[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 809-821.

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