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黄连解毒汤抑制NLRP3炎症小体活化改善脓毒血症小鼠急性肝损伤的机制
作者:
作者单位:

1.长春中医药大学 药学院,长春 130117;2.长春中医药大学 临床医学院,长春 130117

作者简介:

赵芮竹,在读硕士,从事中药防治脓毒血症作用机制研究,E-mail:564111319@qq.com

通讯作者:

孙聪,教授,硕士生导师,从事中药抗常见多发病作用机制研究,E-mail:373673266@qq.com

中图分类号:

R2-0;R33;R289;R364.5

基金项目:

吉林省科技厅重点研发项目(20210204090YY);吉林省教育厅科学研究项目(JJKH20230949KJ);吉林省大学生创新训练项目(S202210199043)


Mechanism of Huanglian Jiedutang in Inhibiting Activation of NLRP3 Inflammasomes and Ameliorating Acute Liver Injury in Septic Mice
Author:
Affiliation:

1.School of Pharmacy,Changchun University of Chinese Medicine,Changchun 130117,China;2.School of Clinical Medicine,Changchun University of Chinese Medicine,Changchun 130117,China

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    摘要:

    目的 探讨黄连解毒汤通过抑制炎症小体NOD样受体蛋白3(NLRP3)活化抑制细胞焦亡来缓解脂多糖(LPS)诱导的脓毒血症小鼠急性肝损伤(ALI)的机制研究。方法 将54只雄性C57BL/6小鼠随机分为空白组、模型组、黄连解毒汤低、中、高剂量组(3.08、6.15、12.30 g·kg-1)、地塞米松组,每组9只,治疗组灌胃对应剂量的黄连解毒汤7 d,治疗结束后腹腔注射LPS(15 mg·kg-1)建立小鼠脓毒血症模型,地塞米松组在造模后1.5 h腹腔注射地塞米松(0.05 g·kg-1)进行治疗,造模12 h后记录小鼠脓毒血症评分(MSS);处死小鼠并取血液与肝脏组织;生化分析仪检测丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平;酶联免疫吸附测定法(ELISA)检测血清中肿瘤坏死因子(TNF-α)、白细胞介素(IL)-6、IL-1β、IL-18含量;苏木素-伊红(HE)染色观察小鼠肝脏的病理变化;免疫荧光法检测NLRP3蛋白含量;免疫组化法检测适配蛋白(ASC)蛋白含量;蛋白免疫印迹法(Western blot)检测肝脏组织中NLRP3、ASC、胱天蛋白酶-1(Caspase-1)、消皮素D(GSDMD)蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测肝脏组织中GSDMD、Caspase-1、IL-1β、IL-18 mRNA表达。结果 与空白组比较,模型组小鼠ALT和AST水平显著上升(P<0.01);炎症因子在血清中含量显著上升(P<0.01);小鼠肝脏有水肿、坏死等病理学损伤现象;NLRP3、ASC、Caspase-1、GSDMD蛋白表达显著上升(P<0.01);GSDMD、Caspase-1、IL1β、IL-18 mRNA表达显著上升(P<0.01)。与模型组比较,各治疗组小鼠炎症因子含量显著下降(P<0.01);肝脏组织病理损伤减轻;肝组织NLRP3、ASC、Caspase-1、GSDMD蛋白表达明显下降(P<0.05,P<0.01);GSDMD、Caspase-1、IL-1β、IL-18 mRNA表达水平明显下降(P<0.05,P<0.01)。结论 黄连解毒汤可通过抑制NLRP3炎症小体的活化抑制细胞焦亡,减轻炎症反应,进而对LPS诱导的脓毒血症小鼠急性肝损伤起到治疗作用。

    Abstract:

    Objective To explore the mechanism of Huanglian Jiedutang in inhibiting the pyroptosis mediated by NOD-like receptor protein 3 (NLRP3) inflammasomes and alleviating the acute liver injury (ALI) induced by lipopolysaccharide (LPS) in the mouse model of sepsis.Method Fifty-four male C57BL/6 mice were randomized into blank, model, low- (3.08 g·kg-1), medium- (6.15 g·kg-1), and high-dose (12.30 g·kg-1) Huanglian Jiedutang, and positive control (dexamethasone) groups (n=9). The mice were administrated with Huanglian Jiedutang at different doses by gavage for 7 days, and then LPS (15 mg·kg-1) was injected intraperitoneally for the modeling of sepsis. In the positive control group, dexamethasone (0.05 g·kg-1) was injected intraperitoneally 1.5 h after modeling, and the mouse sepsis score (MSS) was recorded 12 h after modeling. The mice were sacrificed for the collection of blood and liver tissue samples. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) were measured by a biochemical analyzer. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18 in the serum were measured by enzyme-linked immunosorbent assay kits. Hematoxylin-eosin staining was used to observe the pathological changes in the liver tissue. The content of NLRP3 was observed by the immunofluorescence assay. The expression of apoptosis-associated speck-like protein containing CARD (ASC) was detected by immunohistochemistry. The protein levels of NLRP3, ASC, Caspase-1, and gasdermin D (GSDMD) in the liver tissue were determined by Western blot. Real-time quantitative polymerase chain reaction(Real-time PCR) was employed to determine the mRNA levels of GSDMD, Caspase-1, IL-1β, and IL-18.Result Compared with the blank group, the model group showed elevated levels of ALT and AST (P<0.01) and risen levels of inflammatory cytokines in the serum (P<0.01). In addition, the modeling resulted in edema and necrosis in the liver, and up-regulated the protein levels of GSDMD, NLRP3, ASC, and Caspase-1 (P<0.01) and the mRNA levels of GSDMD, Caspase-1, IL-1β, and IL-18 (P<0.01). Compared with the model group, the drug intervention groups showed reduced content of inflammatory cytokines (P<0.01), alleviated pathological damage in the liver tissue, and down-regulated protein levels of GSDMD, NLRP3, ASC, and Caspase-1 (P<0.05,P<0.01) and mRNA levels of GSDMD, Caspase-1, IL-1β and IL-18 (P<0.05,P<0.01) in the liver tissue.Conclusion Huanglian Jiedutang can inhibit pyroptosis and reduce inflammation by inhibiting the activation of NLRP3 inflammasomes, thus demonstrating a therapeutic effect on acute liver injury in the mouse model of sepsis induced by LPS.

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赵芮竹,滑正阳,王宇航,任鑫悦,范丁兴,娄石磊,阎慧,孙聪.黄连解毒汤抑制NLRP3炎症小体活化改善脓毒血症小鼠急性肝损伤的机制[J].中国实验方剂学杂志,2024,30(22):27~34

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  • 收稿日期:2024-03-22
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  • 在线发布日期: 2024-10-17
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