miR-6722-3p调控Toll样受体/核因子-κB对脓毒症大鼠肝肾损伤的影响

Influence of miR-6722-3p targeting Toll-like receptor/nuclear factor-kappa B on hepatic and renal injury in septic rats

目的 探讨miR-6722-3p靶向壳多糖酶3样蛋白1(chitinase-3-like protein 1,CHI3L1)调控Toll样受体(Toll-like receptor,TLR)/核因子-κB(nuclear factor-kappa B,NF-κB)信号通路对脓毒症大鼠肝肾损伤的影响及可能机制。方法18只成年雄性SD大鼠随机分为假手术组、模型组和过表达组各6只,模型组和过表达组采用盲肠结扎穿孔术制备脓毒症模型,假手术组仅开腹暴露盲肠后关腹。术后72 h过表达组尾静脉注射miR-6722-3p siRNA慢病毒,假手术组和模型组正常饲养。造膜后第7天测定3组大鼠尿肌酐及血清白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平。采用HE染色观察3组大鼠肝、肾组织形态结构,采用免疫组织化学法检测肝、肾组织CHI3L1表达,采用TUNEL染色检测肝、肾组织细胞凋亡情况,采用实时荧光定量PCR法检测肾组织CHI3L1、TLR4、NF-κB p65 mRNA相对表达量,采用Western blot法检测肾组织CHI3L1、TLR4、NF-κB p65蛋白相对表达量。结果 过表达组、模型组血清IL-1β[(10.07±1.88)、(8.62±1.12)ng/L]、TNF-α[(58.93±10.21)、(56.77±3.62)ng/L]及尿肌酐[(757±210)、(1 189±890)mmol/L]水平均高于假手术组[(6.77±1.03)ng/L、(41.62±3.46)ng/L、(529±102)mmol/L](P<0.05);过表达组血清IL-1β水平高于模型组(P<0.05),血清TNF-α及尿肌酐水平与模型组比较差异均无统计学意义(P>0.05)。假手术组肝细胞及肾小管上皮细胞排列规则,细胞核轮廓清晰,肾脏和肝脏组织CHI3L1均呈阳性;模型组个别肾小球萎缩,肝细胞胞浆空泡样变性,肾脏和肝脏组织CHI3L1均呈阳性;过表达组肾小管上皮细胞肿胀、空泡样变性,肾小管上皮脱落、色素沉着,肝细胞胞浆空泡样变性,色素沉着,肾脏组织CHI3L1呈阴性,肝脏组织CHI3L1呈弱阳性。模型组较假手术组肝脏和肾脏细胞凋亡增多,过表达组较模型组细胞凋亡减少。过表达组肾组织CHI3L1、TLR4 mRNA相对表达量均高于假手术组、模型组(P<0.05),NF-κB p65 mRNA相对表达量与假手术组、模型组比较差异均无统计学意义(P>0.05);模型组肾组织CHI3L1、TLR4、NF-κB p65 mRNA相对表达量与假手术组比较差异均无统计学意义(P>0.05)。模型组肾组织CHI3L1、TLR4、NF-κB p65蛋白相对表达量均高于过表达组、假手术组(P<0.05),过表达组均高于假手术组(P<0.05)。结论 miR-6722-3p靶向CHI3L1调控TLR/NF-κB信号通路延缓脓毒症大鼠肝肾损伤进展,miR-6722-3p过表达可减轻脓毒症大鼠肝肾损伤。

Objective To explore the influence of miR-6722-3p on hepatic and renal injury via regulating Toll-like receptor(TLR)/nuclear factor-kappa B(NF-κB)by targeting chitinase-3-like protein 1(CHI3L1),and its potential mechanism in septic rats.Methods Eighteen adult male SD rats were randomly divided into sham-operation group,model group and overexpression group,with 6 rats in each group.Model group and overexpression group were established the sepsis models by cecal ligation and perforation,and sham-operation group was only opened abdomen to expose the cecum and then was closed the abdomen.Model group and sham-operation group were fed normally,and overexpression group was injected with miR-6722-3p siRNA lentivirus via caudal vein 72 h after modeling.The serum levels of urine creatinine,interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by day 7 after modeling.The morphologies and structures of hepatic and renal tissues were observed by HE staining.The expression of CHI3L1 was detected by immunohistochemistry.The apoptosis of hepatic and renal tissues was detected by TUNEL staining.The relative expressions of CHI3L1,TLR4 and NF-κB p65 mRNAs in renal tissues were detected by real-time fluorescence quantitative PCR.The relative expressions of CHI3L1,TLR4 and NF-κB p65 proteins in renal tissues were detected by Western blot.Results The levels of serum IL-lβ,TNF-αand urine creatinine were higher in overexpression group[(10.07±1.88)ng/L,(58.93±10.21)ng/L,(757±210)mmol/L]and model group[(8.62±1.12)ng/L,(56.77±3.62)ng/L,(1189±890)mmol/L]than those in sham-operation group[(6.77±1.03)ng/L,(41.62±3.46)ng/L,(529±102)mmol/L](P<0.05).The IL-1βlevel was higher in overexpression group than that in model group(P<0.05),and the serum TNF-αand urine creatinine levels showed no significant differences between overexpression group and model group(P>0.05).In sham-operation group,the hepatocytes and renal tubular cells were regularly arranged with clear outline of nuclei,and the expressions of CHI3L1 in both hepatic and renal tissues were positive.In model group,individual renal glomeruli were atrophied,cytoplasmic vacuole-like lesions of hepatocytes were present,and the expressions of CHI3L1 in both hepatic and renal tissues were positive.In overexpression group,the renal tubular epithelial cells were swollen and showed vacuole-like lesions,the renal tubular epithelium was detached and pigmented,the hepatocytes showed cytoplasmic vacuole-like lesions and hyperpigmentation,the renal CHI3L1 was negative and the hepatic CHI3L1 was weakly positive.Compared with sham-operation group,the apoptosis of hepatic and renal tissues increased in model group,and the apoptosis was significantly reduced in overexpression group compared with model group.The relative expressions of CHI3L1 and TLR4 mRNAs in renal tissues were higher in overexpression group than those in sham-operation and model groups(P<0.05),and the relative expression of NF-κB p65 mRNA showed no significant difference compared with sham-operation and model groups(P>0.05).The relative expressions of CHI3L1,TLR4 and NF-κB p65 mRNAs in renal tissues showed no significant differences between model group and sham-operation group(P>0.05).The relative expressions of CHI3L1,TLR4 and NF-κB p65 proteins were higher in model group than those in overexpression group and sham-operation group(P<0.05),and higher in overexpression group than those in sham-operation group(P<0.05).Conclusion miR-6722-3p regulates TLR/NF-κB signaling pathway by targeting CHI3L1 to slow the damage process of hepatic and renal tissues in septic rats,and the overexpression of miR-6722-3p can relieve the hepatic and renal injuries.