积雪草酸通过抑制HMGB1/TLR4/NF-κB通路减轻脂多糖诱导肉鸡肾细胞焦亡

doi:10.11843/j.issn.0366-6964.2024.04.039

摘要/Abstract

摘要： 旨在研究积雪草酸(asiatic acid，AA)通过抑制HMGB1/TLR4/NF-κB通路减轻脂多糖(lipopolysaccharide, LPS)诱导肉鸡肾细胞焦亡的作用机制。将40只1日龄健康黄羽肉鸡适应性饲养至7日龄，随机分成4组：空白对照组(CON)、LPS诱导模型组(LPS)、AA低剂量组(LPS+AA 15 mg·kg^-1)和AA高剂量组(LPS+AA 30 mg·kg^-1)。AA处理组的肉鸡用对应剂量的AA预处理14 d。除空白对照组外，其余肉鸡在16、18和20日龄时腹腔注射0.5 mg·kg^-1的LPS构建急性肾损伤模型，在20日龄，腹腔注射LPS 12 h后处死肉鸡，并采集肾组织样品。采用苏木精-伊红染色(HE)观察肾组织病理变化；RT-qPCR法检测肾组织中HMGB1、TLR4、NF-κB、IκB、NLRP3、Caspase-1、IL-18、IL-1β和TNF-α的mRNA表达水平；Western blot法检测肾组织中HMGB1、TLR4、P-NF-κB、NLRP3、GSDMD、IL-1β和Cleaved caspase-1/Caspase-1的蛋白表达水平；免疫组化技术检测P-IκB蛋白在肾组织中的表达水平；免疫荧光技术检测HMGB1、NLRP3和ASC蛋白在肾组织中的表达水平。结果显示，LPS会引发肾小管上皮细胞产生空泡变性以及肾小球发育不良，而经AA处理可以改善由LPS诱导引起的病理损伤。同时，LPS使肉鸡肾组织中HMGB1/TLR4/NF-κB通路和焦亡相关基因蛋白表达升高(P < 0.05)，AA可以显著下调由LPS诱导所导致的肾组织中HMGB1、TLR4、NF-κB、IκB、NLRP3、Caspase-1、TNF-α和IL-18的mRNA水平升高(P < 0.05)；TLR4、P-NF-κB、NLRP3、IL-1β和Cleaved caspase-1/Caspase-1的蛋白水平显著下降(P < 0.05)；免疫组化和免疫荧光结果表明，AA可以显著降低LPS所诱导的肾组织中P-IκB、HMGB1、NLRP3和ASC蛋白的表达与分布。综上所述，AA能够通过抑制HMGB1/TLR4/NF-κB通路减轻LPS诱导肉鸡肾细胞焦亡。

关键词: 积雪草酸, 脂多糖, HMGB1/TLR4/NF-κB通路, 肾, 细胞焦亡

Abstract: The aim of this study was to explore the mechanism of asiatic acid (AA) on LPS-induced pyroptosis in kidney cells of yellow-feather broilers through HMGB1/TLR4/NF-κB pathway. Forty one-day-old healthy broilers were reared to 7 days of age and randomly divided into four groups: control group (CON), LPS-induced model group (LPS), AA low-dose group (LPS+AA 15 mg·kg^-1) and AA high-dose group (LPS+AA 30 mg·kg^-1). Broilers in the AA treatment groups were given the corresponding dose of AA by intragastric administration for 14 days. Except the control group, all broilers were injected intraperitoneally 0.5 mg·kg^-1 LPS to establish the model of acute kidney injury at 16, 18 and 20 days of age. Broilers were killed on the 20th day after LPS was intraperitoneally injected for 12 h and renal samples were collected. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue. The mRNA expression levels of HMGB1, TLR4, NF-κB, IκB, NLRP3, Caspase-1, IL-18, IL-1β and TNF-α in renal tissue were detected by RT-qPCR. Protein expression levels of HMGB1, TLR4, P-NF-κB, NLRP3, GSDMD, IL-1β and Cleaved caspase-1/Caspase-1 were detected by Western blot. The expression level of P-IκB was detected by immunohistochemical method. The protein expression levels of HMGB1, NLRP3 and ASC were detected by immunofluorescence. The results showed that LPS induced vesicular degeneration of renal tubular epithelial cells and glomerular dysplasia, which could be ameliorated by treatment with AA. At the same time, the mRNA levels of HMGB1, TLR4, NF-κB, IκB, NLRP3, Caspase-1, TNF-α and IL-18 in renal tissue were significantly downregulated by AA(P < 0.05). Protein levels of HMGB1, TLR4, P-NF-κB, NLRP3, GSDMD, IL-1β, and Cleaved caspase-1/Caspase-1 decreased. The results of immunohistochemistry and immunofluorescence showed that AA could significantly reduce the expression and distribution of P-IκB, HMGB1, NLRP3 and ASC in renal tissue by LPS-induced. In conclusion, AA can alleviate LPS-induced pyroptosis in kidney cells of broilers by inhibiting the HMGB1/TLR4/NF-κB pathway.

Key words: asiatic acid, lipopolysaccharide, HMGB1/TLR4/NF-κB pathway, kidney, pyroptosis

中图分类号:

S859.7

苏依曼, 叶嘉莉, 邱文粤, 章心婷, 庞晓玥, 王荣梅, 唐兆新, 苏荣胜. 积雪草酸通过抑制HMGB1/TLR4/NF-κB通路减轻脂多糖诱导肉鸡肾细胞焦亡[J]. 畜牧兽医学报, 2024, 55(4): 1777-1786.

SU Yiman, YE Jiali, QIU Wenyue, ZHANG Xinting, PANG Xiaoyue, WANG Rongmei, TANG Zhaoxin, SU Rongsheng. Asiatic Acid Alleviates LPS-induced Pyroptosis in Renal Cell by Inhibiting HMGB1/TLR4/NF-κB Pathway in Broilers[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1777-1786.

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