KLHL21基因在小鼠心肌梗死中的作用及机制

The role and mechanism of KLHL21 gene in mouse myocardial infarction

摘要:

目的]探讨KLHL21基因在小鼠心肌梗死(MI)中的作用及机制。 [方法]使用CRISPR/Cas技术构建KLHL21基因敲除小鼠(KO小鼠),并选取C57BL/6野生型小鼠作为对照。60只KLHL21基因KO小鼠与60只野生型小鼠随机分为四组:野生型假手术组(WT+Sham)、野生型心肌梗死组(WT+MI)、KO假手术组(KO+Sham)和KO心肌梗死组(KO+MI)。术后通过TTC及Evens Blue双染法计算缺血区和梗死区面积,ELISA法测量心肌损伤标志物,心脏超声检测心功能,HE染色和Masson染色观察病理变化,Western blot检测核因子κB(NF-κB)信号通路相关蛋白。 [结果]KO小鼠心肌组织KLHL21蛋白表达显著低于WT小鼠。KO+MI组小鼠梗死区面积显著增加,心功能较WT+MI组明显降低。HE染色显示KO+MI组心肌细胞减少,出现液化性坏死、核碎裂及大量炎症细胞浸润,Masson染色提示纤维化加重。KO+MI组血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、肌酸激酶同工酶(CK-MB)和心肌肌钙蛋白I(cTnI)水平显著升高。Western blot结果表明KO+MI组磷酸化核因子κB抑制蛋白α(p-IKBα)、P65和P50蛋白水平增高,IKBα蛋白水平下降。 [结论]KLHL21基因对小鼠心肌梗死具有预防作用,可能通过抑制NF-κB信号通路的激活来发挥作用。

Abstract:

Aim To investigate the role and mechanism of KLHL21 gene in myocardial infarction (MI) of mice. Methods KLHL21 gene knockout (KO) mice were generated using CRISPR/Cas9 technology, and C57BL/6 wild-type mice were used as controls. Sixty KLHL21 KO mice and 60 wild-type mice were randomly divided into four groups:WT+Sham group (n＝30), WT+MI group (n＝30), KO+Sham group (n＝30) and KO+MI group (n＝30). Postoperative ischemic and infarct areas were assessed using TTC and Evans Blue staining, myocardial injury markers were measured by ELISA, cardiac function was evaluated by ultrasound, and histological changes were examined using HE and Masson staining. Western blot was used to detect proteins related to the nuclear factor-κB (NF-κB) signaling pathway. ResultsKLHL21 protein expression in the myocardial tissue of KO mice was significantly lower than that in WT mice. The infarct area in KO+MI mice was significantly larger than that in WT+MI group. KO+MI mice showed reduced cardiac function compared with WT+MI mice. HE staining revealed myocardial cell loss, liquefactive necrosis, nuclear fragmentation, and significant neutrophil infiltration, while Masson staining showed aggravated fibrosis in KO+MI group. Serum tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), creatine kinase-MB (CK-MB), and cardiac troponin I (cTnI) levels were significantly increased in KO+MI mice compared with WT+MI mice. Western blot analysis showed increased levels of phosphorylated inhibitor of nuclear factor-κB alpha (p-IKBα), P65, and P50, and decreased nuclear factor-κB alpha (IKBα) in KO+MI mice. Conclusion KLHL21 gene plays a preventive role in myocardial infarction in mice, possibly through inhibition of NF-κB signaling pathway activation.