野黄芩苷通过CCN1介导抑制核转录因子-κB信号通路改善脂多糖诱导的肾损伤

Scutellarin alleviates lipopolysaccharide-induced renal injury via mediating cysteine-rich protein 61-connective tissue growth factor-nephroblastoma overexpressed gene 1 expression to inhibit nuclear factor-κB signaling pathway

目的探讨野黄芩苷(Scu)对脓毒症相关性急性肾损伤(SA-AKI)的保护作用及其机制。方法①体内实验:将36只雄性C57BL/6小鼠按随机数字表法分为生理盐水(NS)对照组、脂多糖(LPS)致SA-AKI模型组(LPS组)、20 mg/kg Scu对照组(Scu 20对照组)及5、10、20 mg/kg Scu预处理组,每组6只。腹腔注射10 mg/kg LPS制备SA-AKI小鼠模型;NS对照组给予等量NS;Scu预处理组于注射LPS前腹腔注射不同剂量Scu,每日1次、持续1周;Scu 20对照组仅给予20 mg/kg Scu,持续1周。各组于LPS处理24 h后处死小鼠取肾脏,观察肾组织损伤情况;采用蛋白质免疫印迹试验(Western blotting)检测核转录因子-κB(NF-κB)信号通路相关分子、凋亡相关蛋白及富含半胱氨酸蛋白61-结缔组织生长因子-肾母细胞瘤过表达基因1(CCN1)的蛋白表达。②体外实验:体外培养人肾小管上皮细胞株HK-2,待细胞融合至80%时用于实验。在未经LPS处理和经过100 g/L的LPS处理后的细胞中,分别转染pcDNA3.1-CCN1过表达CCN1或小干扰RNA(siRNA)抑制CCN1表达,观察CCN1是否参与了NF-κB信号通路激活和细胞凋亡;同时在转染和未转染CCN1 siRNA的HK-2细胞中加入100 g/L的LPS及20μmol/L的Scu,观察Scu对LPS诱导HK-2细胞损伤保护作用的机制。结果①体内实验结果:LPS诱导SA-AKI小鼠肾功能显著下降,肾小管严重受损;Scu可呈剂量依赖性缓解肾功能及组织学损伤。Western blotting条带分析进一步证实,Scu预处理可呈剂量依赖性降低AKI小鼠肾组织中NF-κB信号通路相关分子及CCN1的蛋白表达,并对细胞凋亡具有显著的缓解作用,说明CCN1参与了NF-κB信号通路激活和细胞凋亡。②体外实验结果:在未经LPS处理的HK-2细胞中,过表达CCN1对凋亡相关蛋白及NF-κB信号通路促炎因子表达无显著影响。而在经过LPS处理的HK-2细胞中,过表达CCN1可显著抑制促炎因子白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和单核细胞趋化蛋白-1(MCP-1)的mRNA表达,与单纯LPS刺激细胞比较差异有统计学意义〔IL-1βmRNA(2^(-ΔΔCT)):3.20±0.57比4.88±0.69,TNF-αmRNA(2^(-ΔΔCT)):2.99±0.44比5.00±0.81,MCP-1 mRNA(2^(-ΔΔCT)):2.81±0.50比5.41±0.75,均P<0.05〕,并使凋亡相关蛋白显著下调;当用siRNA抑制CCN1表达时,促炎因子的mRNA表达则较单纯LPS刺激细胞上调〔IL-1βmRNA(2^(-ΔΔCT)):6.01±1.13比4.88±0.69,TNF-αmRNA(2^(-ΔΔCT)):5.15±0.86比5.00±0.81,均P<0.05〕,同时凋亡相关蛋白显著上调。在LPS诱导的细胞模型中,经过Scu处理后HK-2细胞中促炎因子的mRNA表达显著下调,与单纯LPS刺激细胞比较差异有统计学意义〔IL-1βmRNA(2^(-ΔΔCT)):2.55±0.50比6.15±1.04,TNF-αmRNA(2^(-ΔΔCT)):2.58±0.40比3.95±0.52,MCP-1 mRNA(2^(-ΔΔCT)):2.64±0.44比6.21±0.96,均P<0.05〕,而且凋亡相关蛋白显著下调;当用siRNA抑制CCN1表达时,则削弱了Scu对细胞的保护作用,表现为细胞中促炎因子的mRNA表达较未抑制CCN1表达的细胞显著上调〔IL-1βmRNA(2^(-ΔΔCT)):5.34±0.76比2.55±0.50,TNF-αmRNA(2^(-ΔΔCT)):3.66±0.54比2.58±0.40,MCP-1 mRNA(2^(-ΔΔCT)):5.15±0.79比2.64±0.44,均P<0.05〕,且凋亡相关蛋白表达亦显著上调。结论Scu能保护SA-AKI小鼠的肾功能,该保护作用与NF-κB信号通路和CCN1有关;Scu可能通过CCN1调节NF-κB信号通路以减轻LPS诱导的肾损伤。

Objective To explore the protective effect and mechanism of scutellarin(Scu)on sepsis associated-acute kidney injury(SA-AKI).Methods①In vivo experiment:36 male C57BL/6 mice were divided into normal saline(NS)control group,lipopolysaccharide(LPS)induced SA-AKI model group(LPS group),20 mg/kg Scu control group(Scu 20 control group),and 5,10,20 mg/kg Scu pretreatment groups by random number table with 6 mice in each group.The SA-AKI model was reproduced by intraperitoneal injection of 10 mg/kg LPS.The NS control group was injected with NS intraperitoneally.The Scu pretreatment groups were intraperitoneally injected with different doses of Scu every day before LPS injection for 1 week.Scu 20 control group was injected with 20 mg/kg Scu for 1 week.After 24 hours of LPS treatment,mice in each group were sacrificed,kidney tissues were collected,and kidney injury was detected by hematoxylin-eosin(HE)staining.Western blotting was used to detect the protein expression levels of nuclear factor-κB(NF-κB)signaling pathway related molecules,apoptosis-related proteins and cysteine-rich protein 61-connective tissue growth factor-nephroblastoma overexpressed gene 1(CCN1).②In vitro experiment:human renal tubular epithelial cell line HK-2 was cultured in vitro and used for experiment when the cells fused to 80%.In the cells without LPS treatment and after 100 g/L LPS treatment,pcDNA3.1-CCN1 and small interfering RNA(siRNA)CCN1 sequence were transfected to overexpress and inhibit CCN1 expression,respectively,to observe whether CCN1 was involved in NF-κB signaling pathway activation and apoptosis.In addition,100g/L LPS and 20μmol/L Scu were added into HK-2 cells transfected with and without CCN1 siRNA to investigate the mechanism of protective effect of Scu on LPS-induced HK-2 cells injury.Results①The results of in vivo experiment:the renal function of SA-AKI mice induced by LPS was significantly decreased,and had kidney histological damage and severely damaged renal tubules.Scu could alleviate renal function and histological damage in a dose-dependent manner.Western blotting results showed Scu could reduce the protein expression of NF-κB signaling pathway related molecules and CCN1 in the renal tissue,and had a significant alleviating effect on apoptosis,indicating that CCN1 was involved in NF-κB signaling pathway activation and apoptosis.②The results of in vitro experiment:in HK-2 cells not treated with LPS,CCN1 overexpression had no effect on apoptosis related protein and pro-inflammatory factors of NF-κB signaling pathway.In HK-2 cells treated with LPS,overexpression of CCN1 significantly inhibited the mRNA expressions of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and monocyte chemoattractant protein-1(MCP-1),with significant differences as compared with cells stimulated only by LPS[IL-1βmRNA(2^(-ΔΔCT)):3.20±0.57 vs.4.88±0.69,TNF-αmRNA(2^(-ΔΔCT)):2.99±0.44 vs.5.00±0.81,MCP-1 mRNA(2^(-ΔΔCT)):2.81±0.50 vs.5.41±0.75,all P<0.05],and the apoptosis-related protein was significantly down-regulated.However,when siRNA was used to inhibit the expression of CCN1,the mRNA expressions of pro-inflammatory factors were significantly increased as compared with cells stimulated only by LPS[IL-1βmRNA(2^(-ΔΔCT)):6.01±1.13 vs.4.88±0.69,TNF-αmRNA(2^(-ΔΔCT)):5.15±0.86 vs.5.00±0.81,all P<0.05],and apoptosis-related protein was significantly up-regulated.In the LPS-induced HK-2 cells,the mRNA expressions of pro-inflammatory factors were significantly down-regulated after Scu treatment as compared with cells stimulated only by LPS[IL-1βmRNA(2^(-ΔΔCT)):2.55±0.50 vs.6.15±1.04,TNF-αmRNA(2^(-ΔΔCT)):2.58±0.40 vs.3.95±0.52,MCP-1 mRNA(2^(-ΔΔCT)):2.64±0.44 vs.6.21±0.96,all P<0.05],and apoptosis-related protein was also significantly reduced.When the expression of CCN1 was inhibited by siRNA,the protective effect of Scu on cells was weakened,which showed that the mRNA expressions of pro-inflammatory factors in cells was significantly up-regulated compared with the cells without inhibition of CCN1 expression[IL-1βmRNA(2^(-ΔΔCT)):5.34±0.76 vs.2.55±0.50,TNF-αmRNA(2^(-ΔΔCT)):3.66±0.54 vs.2.58±0.40,MCP-1 mRNA(2^(-ΔΔCT)):5.15±0.79 vs.2.64±0.44,all P<0.05],and the expression of apoptosis related protein was also significantly up-regulated.Conclusions Scu could protect the renal function in SA-AKI mice,and the protective effect is associated with NF-κB signaling pathway and CCN1.Thus,Scu could alleviate LPS-induced kidney injury by regulating the NF-κB signaling pathway.