芍药醇对脓毒症模型大鼠急性肺损伤保护作用及其机制研究

Protective Effect of Paeonol on Acute Lung Injury in Septic Rats and Its Mechanism

目的探究芍药醇对脓毒症模型大鼠急性肺损伤的保护作用及其机制。方法将18只SD大鼠按照随机数字表法分成对照组、模型组和芍药醇组,每组6只。模型组大鼠腹腔注射10mg/kg脂多糖制备脓毒症模型;芍药醇组大鼠腹腔注射10mg/kg脂多糖2h后,尾静脉单次注射50mg/kg芍药醇;对照组大鼠腹腔注射等量生理盐水。造模24h后,处死大鼠,取肺组织。采用氧化应激试剂盒检测肺组织丙二醛(MDA)、总抗氧化能力(T-AOC)水平和过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性,酶联免疫吸附实验(ELISA)检测肺组织肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、转化生长因子-β(TGF-β)水平,免疫印迹法(Western blot)检测肺组织炎症、氧化应激及凋亡相关蛋白表达,实时定量PCR(qRT-PCR)检测相关非编码小RNA(miRNA)表达。结果与对照组比较,模型组大鼠肺组织MDA水平显著增高[(1.74±0.13)nmol/mg比(0.72±0.05)nmol/mg,P<0.05],T-AOC水平显著下降[(1.34±0.08)U/mg比(1.89±0.07)U/mg,P<0.05],CAT[(1.23±0.05)U/mg比(2.73±0.10)U/mg]和SOD[(54.73±5.11)U/mg比(91.61±4.21)U/mg]活性减低(P均<0.01);TNF-α[(138.24±8.99)pg/mL比(28.56±1.73)pg/mL]、IL-6[(193.21±11.23)pg/mL比(70.31±5.19)pg/mL]、TGF-β[(132.43±8.21)ng/mL比(31.92±3.24)ng/mL]水平升高(P<0.05或P<0.01);高迁移率蛋白1(HMGB1)、半胱天冬氨酸酶9剪切体(cleaved-Caspase9)表达增多,B淋巴细胞瘤-2(Bcl-2)、核转录因子2(Nrf2)表达减少;miR-126[(0.79±0.02)比(1.01±0.03)]、miR-223[(0.61±0.04)比(1.02±0.02)]相对水平降低(P均<0.05),miR-21[(3.23±0.05)比(0.99±0.01)]相对水平升高(P<0.01)。与模型组比较,芍药醇组大鼠肺组织MDA[(0.97±0.07)nmol/mg]水平降低,T-AOC[(1.68±0.06)U/mg]水平升高,CAT[(2.25±0.07)U/mg]和SOD[(79.99±5.21)U/mg]活性增强均P<0.05;TNF-α[(81.89±5.32)pg/mL]、IL-6[(100.31±9.43)pg/mL]、TGF-β[(50.21±4.13)ng/mL]水平降低(P<0.05或P<0.01);HMGB1、cleaved-Caspase9蛋白表达减少,Bcl-2、Nrf2蛋白表达增多;miR-126(1.78±0.03)、miR-223(0.89±0.03)相对水平升高,miR-21(1.45±0.03)相对水平降低(P<0.05或P<0.01)。结论芍药醇抑制脓毒症大鼠炎症反应、氧化应激及细胞凋亡,对急性肺损伤具有保护作用。

Objective To explore the protective effect of paeonol on acute lung injury in septic rats and its mechanism.Methods Eighteen SD rats were randomly divided into control group,model group and paeonol group,with 6 animals in each group.Model group rats were intraperitoneally injected with 10 mg/kg lipopolysaccharide to prepare the sepsis model;Paeonol group rats were intraperitoneally injected with 10 mg/kg lipopolysaccharide first,2 hours later,50 mg/kg paeonol was injected by the tail vein of rat;control group rats were intraperitoneally injected with the same amount of normal saline.After 24 hours,rats were sacrificed and lung tissues were collected.The levels of malondialdehyde(MDA),total antioxidant capacity(T-AOC),catalase(CAT)and superoxide dismutase(SOD)in lung tissues were detected by oxidative stress kit.Enzyme-linked immunosorbent assay(ELISA)was used to test the levels of necrosis factor-α(TNF-α),interleukin-6(IL-6),transforming growth factor-β(TGF-β).Western blot was performed to check the expression of inflammation,oxidative stress and apoptosis-related proteins,real-time quantitative PCR(qRT-PCR)was applied to detect the expression of related non-coding micro RNA(miRNA).Results Compared to the control rats,in the model group rats,MDA level increased significantly[(1.74±0.13)vs(0.72±0.05)nmol/mg,P<0.05],T-AOC decreased[(1.34±0.08)vs(1.89±0.07)U/mg,P<0.05],CAT[(1.23±0.05)vs(2.73±0.10)U/mg]and SOD[(54.73±5.11)vs(91.61±4.21)U/mg]decreased(P<0.01,respectively);TNF-α[(138.24±8.99)vs(28.56±1.73)pg/mL],IL-6[(193.21±11.23)vs(70.31±5.19)pg/mL]and TGF-β[(132.43±8.21)vs(31.92±3.24)ng/mL]increased(P<0.05 or P<0.01,respectively);expressions of high mobility protein 1(HMGB1)and cleaved-Caspase 9 increased,B-cell CLL/lymphoma 2(Bcl-2)and nuclear transcription factor 2(Nrf2)decreased;The relative levels of miR-126[(0.79±0.02)vs(1.01±0.03)]and miR-223[(0.61±0.04)vs(1.02±0.02)]decreased(P<0.05,respectively),miR-21[(3.23±0.05)vs(0.99±0.01)]increased(P<0.01).Compared to the model rats,in the paeonol group rats,MDA level[(0.97±0.07)nmol/mg]decreased,T-AOC[(1.68±0.06)U/mg]increased,CAT[(2.25±0.07)U/mg]and SOD[(79.99±5.21)U/mg]activity increased(P<0.05,respectively);The expression of TNF-α[(81.89±5.32)pg/mL],IL-6[(100.31±9.43)pg/mL]and TGF-β[(50.21±4.13)ng/mL]decreased(P<0.05 or P<0.01,respectively);The expression of HMGB1 and cleaved-Caspase9 decreased,and Bcl-2 and Nrf2 increased;The relative levels of miR-126[(1.78±0.03)]and miR-223[(0.89±0.03)]increased,and miR-21[(1.45±0.03)]decreased(P<0.05 or P<0.01,respectively).Conclusion Paeonol inhibits inflammatory response,oxidative stress and apoptosis in septic rats,and it has protective effects on acute lung injury.