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微小RNA-544对脓毒症肝损伤小鼠模型的调控作用及其机制
DOI: 10.12449/JCH250922
伦理学声明：本研究方案于2021年3月20日经由西南大学实验动物伦理委员会审批，批号：IACUC-20210320-02，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：段世刚负责研究构思与设计；黄培悟、龚小保负责实验操作和数据收集；林康强负责对结果分析与解释；段世刚、官松美负责文章撰写。
Regulatory effect of microRNA-544 on liver injury in mice with sepsis and its mechanism
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摘要:
目的 本研究旨在探讨微小RNA（miRNA）-544在脂多糖（LPS）诱导的脓毒症小鼠肝损伤中的作用及其潜在机制，为脓毒症肝损伤的治疗提供新靶点。 方法 选取40只C57BL/6J小鼠，随机分为对照组（腹腔注射生理盐水）、模型组（腹腔注射LPS，5 mg/kg）、激动剂组（腹腔注射LPS+miR-544抑制剂，5 mg/kg）和miR-544抑制剂组（腹腔注射LPS+miR-544激动剂，5 mg/kg），每组10只。采用全自动生化仪检测血清和肝组织中ALT、AST和TBil水平；Western Blot检测肝脏中单核细胞趋化蛋白1（MCP-1）、CD16/32以及NF-κB信号通路相关蛋白表达，实时定量聚合酶链式反应（qRT-PCR）和酶联免疫吸附测定法（ELISA）检测血清中TNF-α、IL-6、IL-1β的表达。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 与对照组相比，LPS诱导的脓毒症模型组小鼠血清和肝组织中miR-544表达水平显著降低（P值均<0.01），肝脏出现明显炎性细胞浸润、中央静脉充血等病理改变，肝损伤指标（ALT、AST、TBil）在血清和肝组织中均显著升高（P值均<0.001），炎性因子（MCP-1、CD16/32、TNF-α、IL-6、IL-1β）表达水平均显著升高（P值均<0.01），NF-κB通路关键蛋白（p-IKK、p-I-κB-α、p-p65）磷酸化水平均显著升高（P值均<0.01）。与模型组相比，miR-544抑制剂组血清和肝组织中miR-544表达水平均显著降低（P值均<0.01），肝脏病理学进一步加重，同时，肝损伤指标和炎性因子（ALT、AST、TBil、MCP-1、CD16/32、TNF-α、IL-6、IL-1β）水平均显著升高（P值均<0.05），NF-κB通路关键蛋白（p-IKK、p-IκB-α、p-p65）磷酸化水平均显著升高（P值均<0.01），而miR-544 激动剂组血清和肝组织中miR-544水平均显著升高（P值均<0.01），肝脏病理学显著减轻，肝损伤指标和炎性因子（ALT、AST、TBil、MCP-1、CD16/32、TNF-α、IL-6、IL-1β）水平均显著降低（P值均<0.05），NF-κB通路关键蛋白（p-IKK、p-I-κB-α、p-p65）磷酸化水平均显著降低（P值均<0.05）。 结论 miR-544可通过抑制炎症相关蛋白表达及NF-κB信号通路激活，减轻LPS诱导的脓毒症小鼠肝损伤。 -
关键词:
- 脓毒症 /
- 脂多糖类 /
- 微RNAs /
- 小鼠， 近交C57BL
Abstract:Objective To investigate the role and potential mechanism of microRNA-544 （miRNA-544） in lipopolysaccharide （LPS）-induced liver injury in mice with sepsis， and to provide a new target for the treatment of liver injury in sepsis. Methods A total of 40 C57BL/6J mice were randomly divided into control group （intraperitoneal injection of normal saline）， model group （intraperitoneal injection of LPS at a dose of 5 mg/kg）， agonist group （intraperitoneal injection of LPS and miR-544 agonist at a dose of 5 mg/kg）， and miR-544 inhibitor group （intraperitoneal injection of LPS and miR-544 inhibitor at a dose of 5 mg/kg）， with 10 mice in each group. An automatic biochemical analyzer was used to measure the levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， and total bilirubin （TBil） in serum and the liver； Western blot was used to measure the expression levels of monocyte chemotactic protein-1 （MCP-1）， CD16/32， and proteins associated with the NF-κB signaling pathway in the liver； q-PCR and ELISA were used to measure the expression levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β） in serum. A one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the control group， the model group of LPS-induced sepsis had a significant reduction in the expression level of miR-544 in serum and liver tissue （P<0.01）， significant pathological changes of the liver （such as inflammatory cell infiltration and central vein congestion）， and significant increases in the levels of liver injury markers （ALT， AST， and TBil） in serum and the liver （all P<0.001）， the expression levels of inflammatory factors （MCP-1， CD16/32， TNF-α， IL-6， and IL-1β） （all P<0.01）， and the phosphorylation levels of key proteins of the NF-κB pathway （p-IKK， p-I-NF-κ， and p-p65） （all P<0.01）. Compared with the model group， the miR-544 inhibitor group had a significant reduction in the expression level of miR-544 in serum and liver tissue （P<0.01）， aggravated pathological changes of the liver， and significant increases in the levels of liver injury markers and inflammatory factors （ALT， AST， TBil， MCP-1， CD16/32， TNF-α， IL-6， and IL-1） （all P<0.05）， as well as significant increases in the phosphorylation levels of key proteins of the NF-κB pathway （p-IKK， p-I-κB-α， and p-p65） （all P<0.01）. On the contrary， the miR-544 agonist group had a significant increase in the expression level of miR-544 in serum and liver tissue （P<0.01）， significant alleviation of liver pathological changes， and significant reductions in the levels of liver injury markers and inflammatory factors （ALT， AST， TBil， MCP-1， CD16/32， TNF-α， IL-6， and IL-1β） （all P<0.05）， as well as significant reductions in the phosphorylation levels of key proteins of the NF-κB pathway （p-IKK， p-I-κB-α， and p-p65） （all P<0.05）. Conclusion This study shows that miR-544 can alleviate LPS-induced liver injury in mice with sepsis by inhibiting the expression of inflammatory-related proteins and the activation of the NF-κB signaling pathway. -
Key words:
- Sepsis /
- Lipopolysaccharides /
- MicroRNAs /
- Mice， Inbred C57BL
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注： a，LPS诱导脓毒症小鼠的血清中miR-544的表达；b，LPS诱导脓毒症小鼠的肝脏中miR-544的表达；c，LPS处理的脓毒症小鼠存活率情况；d，LPS处理的脓毒症小鼠肝组织HE染色（×200）。
图 1 LPS诱导的脓毒症小鼠血清和肝组织中的miR-544表达水平
Figure 1. Underexpression of miR-544 in LPS-induced sepsis
注： a，血清肝功能指标；b，肝组织肝功能指标。
图 2 miR-544对LPS诱导的脓毒症小鼠肝功能的影响
Figure 2. Effects of miR-544 on liver function in mice with LPS-induced sepsis
注： a，MCP-1和CD16/32蛋白免疫印迹分析；b，MCP-1蛋白免疫印迹分析的量化分析；c，CD16/32蛋白免疫印迹量化分析；d，MCP-1免疫组化分析（ECL，×100）；e，CD16/32免疫组化分析（ECL，×100）。
图 3 miR-544对LPS诱导的脓毒症小鼠肝脏炎症的影响
Figure 3. Effect of miR-544 on hepatic inflammation in LPS-induced sepsis
注： a，血清促炎因子；b，肝组织促炎因子。
图 4 miR-544对LPS诱导的脓毒症小鼠血清和肝组织中炎症因子的影响
Figure 4. Effect of miR-544 on inflammatory factors in LPS-induced sepsis
图 5 miR-544抑制脓毒症小鼠肝组织中NF-κB信号的激活
Figure 5. miR-544 inhibits activation of NF-κB signaling in uremic liver
表 1 用于qRT-PCR分析的引物
Table 1. Primers for qRT-PCR analysis
底物 正向引物（5′-3′） 反向引物（5′-3′） miR-544 ACACTCCAGCTGGGATTCTG TGGTGTCGTGTGAGTCG IL-1β ATGGCAACTGTTCCTGAACTCAACT CAGGACAGGTATAGATTCTTTCCTTT IL-6 GCTATGAAGTTCCTCTCTGC CTAGGTTTGCCGAGTAGATC TNF-α CCAGACCCTCACACTCAGAT AACACCCATTCCCTTCACAG GAPDH ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGTA 
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