丹参酮ⅡA通过调控HMGB1表达抑制TLR4/My D88/NF-κB通路减轻大鼠脓毒症肺损伤机制研究

Tanshinone Ⅱ A protects lung injury in a rat model of sepsis by downregulation of the expression of HMGB1 via inhibiting TLR4/My D88/NF-κB pathway

目的 探讨丹参酮ⅡA对脓毒症大鼠肺损伤中高迁移率族蛋白B1(HMGB1)及Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核转录因子-κB(NF-κB)通路的影响。方法 20只清洁级健康成年雄性Sprague-Dawley(SD)大鼠采用盲肠结扎穿孔法(CLP)造模,造模成功后随机分为四组,即模型组,低、中、高剂量治疗组,每组5只。模型组腹腔注射0.9%氯化钠注射液,低、中、高剂量治疗组腹腔注射不同剂量的丹参酮ⅡA(5、10、20 mg/kg)。另取5只SD大鼠作为假手术组,开腹翻动肠道后关闭腹腔并腹腔注射0.9%氯化钠注射液。各组大鼠干预完成后等待24 h处死。测定各组肺湿/干重比(W/D),观察光镜下各组肺组织结构变化,实时荧光定量PCR(RT-PCR)检测肺组织Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子-κB(NF-κB)、高迁移率族蛋白B1(HMGB1)mRNA的表达。结果 与模型组比较,中、高剂量治疗组的肺组织W/D显著减小[(4.46±0.29)、(4.27±0.21)比(5.09±0.42),P<0.05或P<0.01];并且中、高剂量治疗组的肺组织TLR4、MyD88、NF-κB、HMGB1 mRNA表达下调[TLR4:(1.54±0.12)、(1.29±0.18)比(2.25±0.35);MyD88:(1.78±0.28)、(1.52±0.29)比(2.59±0.28);NF-κB:(1.35±0.09)、(1.09±0.30)比(1.87±0.29);HMGB1:(1.45±0.10)、(1.26±0.27)比(2.06±0.22);P<0.01],而低剂量治疗组肺组织TLR4、MyD88、HMGB1mRNA的表达下调[TLR4:(1.85±0.20)比(2.25±0.35);MyD88:(2.14±0.26)比(2.59±0.28);HMGB1:(1.72±0.10)比(2.06±0.22);P<0.05或P<0.01],而NF-κB表达下调不明显,差异无统计学意义[(1.63±0.13)比(1.87±0.29),P>0.05]。光镜下低、中剂量治疗组大鼠肺组织较模型组损伤明显减轻,但较假手术组损伤严重,高剂量治疗组肺组织受损最轻。结论 丹参酮ⅡA可能通过下调HMGB1表达,抑制TLR4/MyD88/NF-κB通路,减少炎症因子释放来保护脓毒症大鼠肺组织。

Objective To assess the protective effect of tanshinone ⅡA on lung injury in a rat model of sepsis and the underlying molecular events.Methods Twenty-five male specific pathogen-free(SPF) grade Sprague-Dawley(SD) rats were subjected to cecal ligation and puncture method(CLP) to establish sepsis with lung injury or a sham operation as a control.The model group of rats was then randomly divided into model control and low-,medium-,and high-dose treatment groups(n=5 per group).Treatment groups of rats were intraperitoneally injected with tanshinone ⅡA(5,10,and 20 mg/kg for low-,medium-,and high-dose groups,respectively).Control and model rats were injected intraperitoneally with normal saline.These rats were executed 24 h after completion of the treatment and their lungs were resected and measured for the wet/dry weight ratio(W/D).Changes in lung tissue structure were assessed under a light microscope after HE staining,while expression of Toll-like receptor 4(TLR4),Myeloiddifferentiation-factor88(MyD88),nuclear factor-κB(NF-κB),and high mobility group protein B1(HMGB1) mRNA was detected using qRT-PCR.Results Compared with the model group,the medium-and high-dose tanshinone ⅡA treatment reduced the lung tissue W/D(4.46±0.29 and 4.27±0.21 vs.5.09±0.42;P0.05or P0.01) and mRNA levels of TLR4(1.54±0.12 and 1.29±0.18 vs.2.25±0.35),MyD88(1.78±0.28 and 1.52±0.29vs.2.59±0.28),NF-κB(1.35±0.09 and 1.09±0.30 vs.1.87±0.29),and HMGB1(1.45±0.10 and 1.26±0.27 vs.2.06±0.22,respectively;P0.01).Although the low-dose tanshinone ⅡA treatment reduced downregulated level of TLR4(1.85±0.20 vs.2.25±0.35),MyD88(2.14±0.26 vs.2.59±0.28),and HMGB1(1.72±0.10 vs.2.06±0.22)mRNA in lung tissues(P0.05 or P0.01),NF-κB mRNA level was not significantly changed(1.63±0.13 vs.1.87±0.29;P0.05).Furthermore,the low-and medium-dose tanshinone Ⅱ A treatment also significantly reduced lung tissue injury compared with the model group of rats.However,the high-dose tanshinone Ⅱ A showed worse lung tissue injury vs.that of the model and low-and medium-dose treatment groups.Conclusion Tanshinone ⅡA was able to reduce the level of HMGB1,TLR4,MyD88,and NF-κB mRNA to protect the lung tissues in a rat model of sepsis.