虎杖苷通过调控HMGB1/TLR4/NF-κB信号通路对脓毒症急性肺损伤的保护作用

The Protective Effect of Polydatin on Sepsis-induced Acute Lung Injury in Rats by Regulating HMGB1/TLR4/NF-κB Signaling Pathway

[目的]观察虎杖苷(polydatin,PD)对脓毒症急性肺损伤大鼠的保护作用,并探究其作用机制。[方法]将50只无特定病原体(specific pathogen free,SPF)级SD大鼠随机分为假手术组、模型组和PD高、中、低剂量组,每组10只。模型组和PD高、中、低剂量组大鼠采用盲肠结扎穿孔术制备脓毒症急性肺损伤模型,假手术组打开腹腔翻动盲肠后缝合。PD高、中、低剂量组大鼠分别予100、50、25mg·kg-1 PD灌胃,假手术组和模型组大鼠予等量0.5%羧甲基纤维素钠(carboxymethyl cellulose sodium,CMC-Na)灌胃,1次/d,连续7d。实验结束检测各组大鼠肺组织湿干重比(wet-to-dry weight ratio,W/D)、丙二醛(malondialdehyde,MDA)水平和超氧化物歧化酶(superoxide dismutase,SOD)活力;苏木精-伊红(hematoxylin-eosin,HE)染色观察肺组织病理变化;酶联免疫吸附检测(enzyme linked immunosorbent assay,ELISA)法检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)水平;实时定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Realtime qPCR)法和Western blot法检测肺组织高迁移率族蛋白B1(high mobility group protein B1,HMGB1)、Toll样受体4(Toll like receptor 4,TLR4)和核因子-κB(nuclear factor-κB,NF-κB)p65 mRNA和蛋白表达水平。[结果]与假手术组比较,模型组TNF-α、IL-1β、IL-6、MDA水平升高,SOD活力降低,W/D值显著升高(P<0.05);与模型组比较,PD高、中剂量组TNF-α、IL-1β、IL-6、MDA水平降低,SOD活力升高,W/D值显著降低(P<0.05),而PD低剂量组TNF-α、IL-1β、IL-6、MDA水平,SOD活力及W/D值差异无统计学意义(P>0.05);PD中剂量组TNF-α、IL-1β、IL-6、MDA水平和W/D值高于PD高剂量组,低于PD低剂量组,SOD活力低于PD高剂量组,高于PD低剂量组(P<0.05)。HE染色显示,模型组大鼠肺泡壁增厚水肿,有大量炎性细胞浸润;与模型组比较,PD高、中剂量可显著改善大鼠肺损伤,减轻肺泡壁增厚、水肿及炎性细胞浸润。Real-time qPCR和Western blot检测显示,与假手术组比较,模型组HMGB1、TLR4、NF-κB p65 mRNA和蛋白表达均增加(P<0.05);与模型组比较,PD高、中剂量组HMGB1、TLR4、NF-κB p65 mRNA和蛋白表达降低(P<0.05),PD低剂量组HMGB1、TLR4和NF-κB p65 mRNA和蛋白表达差异无统计学意义(P>0.05);与PD高剂量组比较,PD中、低剂量组HMGB1、TLR4和NF-κB p65 mRNA和蛋白表达增高,其中PD中剂量组HMGB1、TLR4和NF-κB p65 mRNA和蛋白表达低于PD低剂量组,差异有统计学意义(P<0.05)。[结论]PD对大鼠脓毒症相关急性肺损伤具有保护作用,其作用机制可能与抑制HMGB1/TLR4/NF-κB信号通路的活化有关。

[Objective]To observe the protective effect of polydatin(PD)on sepsis-induced acute lung injury in rats,and to explore its mechanism.[Methods]Fifty specific pathogen free(SPF)SD rats were randomly divided into sham operation group,model group,PD high-dose group,PD mediumdose group and PD low-dose group,with 10 rats in each group.Sepsis-induced acute lung injury model was established by cecal ligation and perforation in model group,PD high-dose group,PD medium-dose group and PD low-dose group.Rats in sham operation group were only opened the abdominal cavity and turned the cecum and sutured.Rats in PD high-dose group,PD medium-dose group and PD low-dose group were given 100,50 and 25mg·kg-1 PD by gavage separately.Rats in sham operation group and model group were given 0.5%carboxymethyl cellulose sodium(CMC-Na)by gavage,once a day,for 7 consecutive days.At the end of the experiment,the wet-to-dry weight ratio(W/D)value,the level of malondialdehyde(MDA)and superoxide dismutase(SOD)activity in lung tissue of each group were detected.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissue of the rats.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of serum inflammatory factors tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6).Real-time quantitative polymerase chain reaction(Real-time qPCR)and Western blot were used to detect the expression levels of high mobility group protein B1(HMGB1),Toll like receptor 4(TLR4)and nuclear factor-κB(NF-κB)p65 mRNA and protein in lung tissues.[Results]Compared with sham operation group,the levels of TNF-α,IL-1β,IL-6 and MDA in model group increased,SOD activity decreased,and the W/D value increased significantly(P<0.05).Compared with model group,the levels of TNF-α,IL-1β,IL-6 and MDA in PD high-dose and medium-dose groups were decreased,SOD activity increased,and the W/D value was significantly reduced(P<0.05);while the levels of TNF-α,IL-1β,IL-6,MDA,SOD activity and W/D values in PD low-dose group were not significantly different from those in model group(P>0.05).Among them,the levels of TNF-α,IL-1β,IL-6,MDA and W/D values of PD medium-dose group were higher than those of PD high-dose group and lower than those of PD low-dose group,and SOD activity was lower than that of PD high-dose group,higher than PD low-dose group(P<0.05).HE staining showed that the alveolar wall thickened and edema in model group,and there was a large number of inflammatory cell infiltration.Compared with model group,high and medium doses of PD can significantly relieve lung injury;reduce alveolar wall thickening,edema and inflammatory cells infiltration.The results of Real-time qPCR and Western blot showed that compared with sham operation group,the expressions of HMGB1,TLR4,NF-κB p65 mRNA and protein in model group increased(P<0.05).Compared with model group,the expressions of HMGB1,TLR4,NF-κB p65 mRNA and protein in PD high-dose and medium-dose groups were significantly reduced(P<0.05),while the expression levels of HMGB1,TLR4,NF-κB p65 mRNA and protein in PD low-dose group were not significantly different from those in model group(P>0.05).Compared with PD high-dose group,the expression of HMGB1,TLR4,NF-κB p65 mRNA and protein in PD medium-dose and low-dose groups increased;among them,the expression levels of HMGB1,TLR4,NF-κB p65 mRNA and protein in PD medium-dose group were lower than those of PD low-dose group,and the differences were statistically significant(P<0.05).[Conclusion]PD has a protective effect on sepsis-induced acute lung injury in rats,and its mechanism may be related to inhibition of the activation of HMGB1/TLR4/NF-κB signaling pathway.