芹菜素对小鼠肺缺血再灌注损伤影响的实验研究

Apigenin alleviates lung ischemia-reperfusion injury in mice

目的观察并探讨芹菜素对小鼠肺缺血再灌注(I/R)损伤的影响及机制。方法根据随机数表法,将40只雄性C57/B6小鼠平均分为假手术组(Sham组)、I/R组、低剂量芹菜素组和高剂量芹菜素组,每组10只。采用左肺门夹闭1 h,再灌注2 h的方法构建小鼠肺I/R损伤模型。低剂量芹菜素组和高剂量芹菜素组小鼠分别在肺I/R造模前连续7 d给予芹菜素(10 mg·kg-1·d-1和50 mg·kg-1·d-1)灌胃。再灌注2 h结束后,留取小鼠肺组织,通过HE染色评估小鼠肺损伤状况并评分;RT-PCR检测小鼠肺组织白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、高迁移率族蛋白B1(HMGB1)、环加氧酶2(PTGS2)和谷胱甘肽过氧化物酶4(GPX4)的mRNA表达水平;Western blot检测小鼠肺组织Bax、Bcl-2、缺氧诱导因子-1α(HIF-1α)、PTGS2和GPX4的蛋白表达。最后,按处理方式不同,将处理后的A549肺上皮细胞分为PBS组、缺氧/复氧(H/R)组、H/R+芹菜素组和H/R+芹菜素+莫立司他组。用莫立司他激活HIF-1α后,进一步观察芹菜素(50μmol/L)对缺氧/复氧(H/R)诱导的A549肺上皮细胞铁死亡的影响。所有两组间比较采用t检验,多组间比较采用One-way ANOVA分析。结果I/R组与Sham组小鼠肺损伤评分分别为(7.05±0.66)分和(1.25±0.42)分,肺湿/干重比分别为(8.65±1.12)%和(4.17±0.54)%,Tunel阳性细胞比例分别为(58.22±4.92)%和(8.23±1.22)%,促炎症细胞因子IL-1β、TNF-α和HMGB1的mRNA相对表达量分别为7.82±0.16比1.00±0.14、4.24±0.12比1.00±0.19和6.24±0.19比1.00±0.11,两组上述参数间比较,差异均有统计学意义(P均<0.05)。低剂量芹菜素组和高剂量芹菜素组小鼠肺损伤评分分别为(4.88±0.31)分和(2.11±0.29)分,肺湿/干重比分别为(6.42±1.03)%和(4.88±1.62)%,Tunel阳性细胞比例分别为(41.46±6.73)%和(16.02±5.31)%,促炎症细胞因子IL-1β、TNF-α和HMGB1的mRNA相对表达量分别为5.88±0.13和3.21±0.19、3.56±0.11和2.12±0.09及4.12±0.14和3.12±0.09,两组上述参数分别与I/R组比较,差异亦均有统计学意义(P均<0.05)。低剂量芹菜素组、高剂量芹菜素组、I/R组小鼠肺组织中HIF-1α和PTGS2蛋白水平分别为2.00±0.10、0.93±0.11、2.99±0.06和4.12±0.14、2.51±0.18和6.11±0.12,前两组均较I/R组降低,且差异有统计学意义(P<0.05)。低剂量芹菜素组、高剂量芹菜素组、I/R组GPX4蛋白水平分别为0.55±0.02、0.83±0.02和0.38±0.04,前两组较I/R组表达升高,且差异均有统计学意义(P<0.05)。体外实验显示,H/R+芹菜素组和H/R组A549肺上皮细胞PTGS2蛋白表达水平分别为1.11±0.0和4.55±0.12,GPX4蛋白表达水平分别为0.93±0.11比0.32±0.04,组间比较,差异均有统计学意义(P均<0.05);但H/R+芹菜素组+莫立司他组HIF-1α激活后,PTGS2和GPX4蛋白表达水平升至4.01±0.12和降至0.52±0.05,与H/R+芹菜素组比较,差异均有统计学意义(P均<0.05)。结论芹菜素可能通过抑制HIF-1α/铁死亡轴减轻小鼠肺缺血再灌注相关的肺损伤、凋亡及炎症反应。

Objective To explore the effects and mechanism of apigenin on lung ischemia-reperfusion(I/R)injury in mice.Methods A total of 40 male C57/B6 mice were randomized into 4 groups of sham,I/R,low-dose apigenin and high-dose apigenin(n=10 each).Lung I/R injury model was established by clipping left hilum for 1h and reperfusion for 2 h.Low/high-dose apigenin group received a gavage of apigenin(10/50 mg·kg-1·d-1)for 7 days before lung I/R.After 2-hour reperfusion,lung tissue was collected and lung injury status evaluated and scored by hematoxylin-eosin(H&E)stain;mRNA expression levels of interleukin-1β(IL-1β),tumor necrosis factor-alpha(TNF-α),high mobility group box 1(HMGB1),prostaglandin-endoperoxide synthase 2(PTGS2)and glutathione peroxidase 4(GPX4)were detected by reverse transcription-polymerase chain reaction(RT-PCR);Western blot was utilized for detecting the protein expressions of Bax,Bcl-2 and HIF-1α.Finally,after morinostat activated Hif-1α,the effect of apigenin(50μmol/L)on hypoxia-reoxygenation(H/R)-induced ferroptosis was further observed in A549 lung epithelial cells.Results As compared with sham group,lung injury score spiked markedly in I/R group(7.05±0.6 vs.1.25±0.42),pulmonary edema worsened obviously(8.65±1.12 vs.4.17±0.54),the percentage of Tunel positive cells rose significantly(58.22±4.92 vs.8.23±1.22)and mRNA expression levels of IL-1β,TNF-αand HMGB1 increased(7.82±0.16 vs.1.00±0.14,4.24±0.12 vs.1.00±0.19,6.24±0.19 vs.1.00±0.11)(P<0.05);Compared with I/R group,lung injury score declined markedly in low/high-dose apigenin group(4.88±0.31/2.11±0.29 vs.7.05±0.66)(P<0.05),pulmonary edema lessened markedly(6.42±1.03/4.88±1.62 vs.8.65±1.12)(P<0.05)and the percentage of Tunel positive cells(41.46±6.73/16.02±5.31 vs.58.22±4.92)and the mRNA expression levels of IL-1β,TNF-αand HMGB1 became obviously suppressed(5.88±0.13/3.21±0.19 vs.7.82±0.16,3.56±0.11/2.12±0.09 vs.4.24±0.12,4.12±0.14/3.12±0.09 vs.6.24±0.19)(P<0.05);protein expressions of HIF-1αand PTGS2 dropped sharply in low/high-dose apigenin group(2.00±0.10/0.93±0.11 vs.2.99±0.06,4.12±0.14/2.51±0.18 vs.6.11±0.12)while GPX4 protein rose obviously(0.55±0.02/0.83±0.02 vs.0.38±0.04)(P<0.05).In vitro experiments further showed that apigenin could significantly suppress the H/R-induced protein expression of PTGS2 in A549 lung epithelial cells(1.11±0.0 vs.4.55±0.12)(P<0.05)while up-regulating the protein expression of GPX4(0.93±0.11 vs.0.32±0.04)(P<0.05).The inhibition of PTGS2 protein(4.01±0.12 vs.1.11±0.05)and the up-regulation of GPX4 were significantly blocked(0.52±0.05 vs.0.93±0.11)(P<0.05).Conclusions Through an inhibition of HIF-1α/ferroptosis axis,apigenin can alleviate lung injury,apoptosis and inflammatory response associated with lung I/R in mice.