广州医科大学附属广州市妇女儿童医疗中心儿童重症监护室,广东 广州 510120
张春敏,硕士,主治医师,研究方向:脓毒症相关急性肾损伤,E-mail: min_135@163.com
纸质出版日期:2022-11-20,
收稿日期:2022-07-21,
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张春敏,陈飞燕,杨文敏等.抑制miR-4321减轻脓毒症相关急性肾脏损伤的作用和机制研究[J].中山大学学报(医学科学版),2022,43(06):928-937.
ZHANG Chun-min,CHEN Fei-yan,YANG Wen-min,et al.Effect and Mechanism of miR-4321 Inhibition Alleviates Sepsis Associated Acute Kidney Injury[J].Journal of Sun Yat-sen University(Medical Sciences),2022,43(06):928-937.
张春敏,陈飞燕,杨文敏等.抑制miR-4321减轻脓毒症相关急性肾脏损伤的作用和机制研究[J].中山大学学报(医学科学版),2022,43(06):928-937. DOI: 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608.
ZHANG Chun-min,CHEN Fei-yan,YANG Wen-min,et al.Effect and Mechanism of miR-4321 Inhibition Alleviates Sepsis Associated Acute Kidney Injury[J].Journal of Sun Yat-sen University(Medical Sciences),2022,43(06):928-937. DOI: 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608.
目的
2
本研究旨在探讨抑制
miR-4321
在改善脓毒症相关急性肾损伤(Sepsis associated acute kidney injury, SA-AKI)中的作用和机制。
方法
2
采用RT-qPCR方法检测LPS诱导的HK2细胞模型和CLP模型小鼠肾脏组织中
miR-3165
、
miR-4270
和
miR-4321
的表达水平。利用
miR-4321
-Inhibitor抑制LPS诱导的HK2细胞损伤模型中
miR-4321
的表达,分别通过CCK-8分析、EdU染色分析和western blotting评估
miR-4321
对细胞增殖、细胞活力、细胞因子和凋亡相关蛋白水平的影响。利用
miR-4321
-Antago干预CLP模型小鼠肾组织中
miR-4321
的表达,通过H
&
E染色检测肾组织结构变化,比色法检测血清中肌酐和血尿氮水平,ELISA检测血清中白细胞介素6(IL-6)、白细胞介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达,IHC染色检测组织中mTOR水平,western blotting检测肾组织中凋亡相关蛋白表达水平。机制上,使用Targetscan软件预测
miR-4321
的靶基因,并通过双荧光素酶报告基因实验对靶基因进行验证。
结果
2
与对照组相比,LPS诱导的HK2细胞中
miR-4321
(
n
=3,
t
=7.154,
P
=0.001 3)表达升高;
miR-4321
-Inhibitor可促进HK2细胞增殖和细胞活力,并降低LPS诱导的IL-1β,IL-6和TNF-α水平的表达。体内实验证明
miR-4321
-Antago能够抑制CLP小鼠血清中肌酐和血尿氮水平,改善肾组织损伤,降低血清中IL-1β,IL-6和TNF-α水平,促进肾组织
mTOR
表达,抑制凋亡相关蛋白的表达。此外,
mTOR
是
miR-4321
的靶基因之一。
结论
2
抑制
miR-4321
可明显减轻脓毒症相关急性肾损伤,这一作用可能是通过增加
mTOR
的表达实现的。
Objective
2
Sepsis associated acute kidney injury (SA-AKI) is a critical clinical disease. The purpose of this study was to investigate the role and molecular mechanism of
miR-4321
in the HK2 cellular damage induced by lipopolysaccharides (LPS).
Methods
2
RT-qPCR was conducted to detect the expression of
miR-3165
,
miR-4270
and
miR-4321
in LPS-induced HK2 cell model and cecal ligation and puncture (CLP)-induced renal injury model. In LPS-induced HK2 cell model,
miR-4321
-inhibitor was used to inhibit the
miR-4321
expression.
The effects of
miR-4321
on cell proliferation, cell viability, cytokines and apoptosis-related protein levels were evaluated by CCK-8 analysis, EdU staining analysis and western blotting analysis, respectively. In CLP-induced renal injury model,
miR-4321
-Antago was used to intervene the
miR-4321
expression. The changes of renal tissue structure were examined by H
&
E staining. The levels of serum creatinine and blood urea nitrogen (BUN) were measured by colorimetric method. ELISA was employed to assess the expression of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum. IHC staining and western blotting were performed to determine the mTOR level and apoptosis-related protein expression in kidney tissues. Target genes of
miR-4321
were predicted by Targetscan software and verified by dual-luciferase reporter assay.
Results
2
Compared with the control group, the
miR-4321
expression was increased in LPS-induced HK2 cell model (
n
=3,
t
=7.154,
P
=0.001 3).
miR-4321
inhibitor promoted the proliferation and viability of HK2 cells, decreased the expression of LPS-induced IL-6, IL-1β and TNF-α and apoptosis-related proteins. In vivo experiments showed that
miR-4321
-Antago inhibited serum creatinine and BUN levels in CLP mice, improved renal injury, reduced levels of IL-1β, IL-6 and TNF-α, promoted the mTOR expression in renal tissues and inhibited the apoptosis-related protein expression. mTOR signaling pathway was believed the target gene of
miR-4321
.
Conclusion
2
Inhibition of
miR-4321
significantly alleviates SA-AKI, which may be achieved by increasing the expression of mTOR.
脓毒症相关急性肾损伤脂多糖miR-4321mTOR
sepsis associated acute kidney injurylipopolysaccharidemiR-4321mTOR
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