荜茇酰胺通过miR-132-3p调控FOXO1改善脓毒症肾损伤机制研究

The mechanism of piperlongumine in improving sepsis-induced renal injury via regulating miR-132-3p targeting FOXO1

目的探究荜茇酰胺(PPL)对脓毒症肾损伤的作用及其可能机制。方法24只雄性C57BL/6小鼠随机数字表法分为四组:Sham组、盲肠结扎穿刺(CLP)组、CLP+PPL-L组和CLP+PPL-H组,每组6只。使用CLP法构建脓毒症小鼠模型,CLP+PPL-L组和CLP+PPL-H组小鼠分别按5、10 mg/kg剂量灌胃PPL溶液,CLP组和Sham组小鼠灌胃等量生理盐水。随后使用酶联免疫吸附试验(ELISA)检测小鼠外周血肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、单核细胞趋化因子1(MCP-1)表达情况以评估模型构建,苏木素-伊红(HE)染色观察小鼠肾组织形态。使用脂多糖(LPS)刺激人近端肾小管上皮细胞(HK-2)构建体外脓毒症细胞模型,随后使用CCK-8和流式细胞术分别检测细胞活性和凋亡水平,qRT-PCR检测miR-132-3p表达量,蛋白免疫印迹法检测叉头框蛋白O1(FOXO1)蛋白的表达情况。使用双荧光素酶实验检测miR-132-3p与FOXO1靶向结合关系。结果小鼠体内实验中,与Sham组比较,CLP组小鼠TNF-α[(13.26±2.15)ng/mL比(2.61±0.33)ng/mL,P<0.01]、IL-6[(6.56±0.84)ng/mL比(1.20±0.13)ng/mL,P<0.01]、MCP-1[(55.35±5.13)ng/mL比(20.85±2.09)ng/mL,P<0.01]表达上升,病理显示肾损伤显著,miR-132-3p表达显著上升[(2.84±0.24)比(1.00±0.03),P<0.01],FOXO1蛋白表达下降;与CLP组比较,CLP+PPL-L组和CLP+PPL-H组小鼠TNF-α[(9.55±0.57)ng/mL、(6.69±1.13)ng/mL比(13.26±2.15)ng/mL,P<0.01]、IL-6[(4.79±0.35)ng/mL、(3.24±0.35)ng/mL比(6.56±0.84)ng/mL,P<0.01]、MCP-1[(44.68±3.80)ng/mL、(29.79±4.10)ng/mL比(55.35±5.13)ng/mL,P<0.01]表达下降,病理显示肾损伤有所缓解,miR-132-3p表达显著下降[(2.36±0.28)、(1.44±0.18)比(2.84±0.24),P<0.05或P<0.01],FOXO1蛋白表达上升。在体外细胞模型中,PPL能显著提高LPS刺激下肾小管细胞活性[(67.11±3.48)%、(88.53±4.42)%比(50.23±4.44)%,P<0.05或P<0.01],抑制凋亡水平,并且过表达miR-132-3p会抑制PPL对LPS刺激下细胞的影响。同时,在体外细胞中证实了miR-132-3p对FOXO1的靶向调控作用。结论PPL可能通过miR-132-3p调控FOXO1的表达从而缓解脓毒症肾损伤。

Objective To evaluate the effect of piperlongumine(PPL)on sepsis-induced renal injury and its underlying mechanism.Methods Twenty-four male C57BL/6 mice were divided into 4 groups using the random number table method,Sham,cecal ligation puncture(CLP),CLP+PPL-L,and CLP+PPL-H groups,with 6 mice in each group.A mouse sepsis model was constructed using CLP.Mice in CLP+PPL-L and CLP+PPL-H groups were administrated with PPL solution at 5 mg/kg and 10 mg/kg by gavage,respectively,while mice in CLP and Sham groups were administrated with an equal volume of normal saline by gavage.Then the expression levels of tumor necrosis factorα(TNF-α),interleukin 6(IL-6),and monocyte chemotactic protein-1(MCP-1)in the peripheral blood of mice were detected using enzyme-linked immunosorbent assay to evaluate model.Hematoxylin-eosin staining was used to observe the morphology of kidney tissue in mice.Human proximal renal tubular epithelial cells were stimulated by lipopolysaccharide(LPS)to construct in vitro sepsis cell model,and cell activity and apoptosis were detected using CCK8 assay and flow cytometry,respectively.The expression levels of miR-132-3p were detected using qRT-PCR,while the expression of Forkhead box protein(FOXO1)was detected using Western blotting.In addition,a dual luciferase assay was used to detect the targeting binding relationship between miR-132-3p and FOXO1.Results In the in vivo experiments,compared with the Sham group,the expression levels of TNF-α[(13.26±2.15)vs.(2.61±0.33)ng/mL,P<0.01],IL-6[(6.56±0.84)vs.(1.20±0.13)ng/mL,P<0.01],and MCP-1[(55.35±5.13)vs.(20.85±2.09)ng/mL,P<0.01]were increased in CLP group.Pathological examination showed significant kidney damage.The expression of miR-132-3p was significantly increased(2.84±0.24 vs.1.00±0.03,P<0.01),while FOXO1 protein expression was decreased.Compared with the CLP group,mice in the CLP+PPL-L and CLP+PPL-H groups showed a significant decrease in the expression of TNF-α[(9.55±0.57),(6.69±1.13)vs.(13.26±2.15)ng/mL,P<0.01],IL-6[(4.79±0.35),(3.24±0.35)vs.(6.56±0.84)ng/mL,P<0.01],MCP-1[(44.68±3.80),(29.79±4.10)vs.(55.35±5.13)ng/mL,P<0.01].Pathological examination showed a partial relief of renal damage.The expression of miR-132-3p was significantly decreased(2.36±0.28,1.44±0.18 vs.2.84±0.24,P<0.05,P<0.01),while FOXO1 protein expression was increased.In the in vitro cell models,PPL significantly increased the activity of LPS-stimulated renal tubule cells[(67.11±3.48),(88.53±4.42)vs.(50.23±4.44)%,P<0.05,P<0.01]and inhibited apoptosis levels.Moreover,overexpression of miR-132-3p inhibited the effect of PPL on LPS-stimulated cells.Additionally,the targeted regulatory effect of miR-132-3p on FOXO1 was confirmed in vitro.Conclusion PPL may alleviate sepsis-induced renal injury by regulating the expression of FOXO1 through miR-132-3p.