大黄素对重症急性胰腺炎大鼠肺损伤的保护作用及对MAPK通路的影响

The Protective Effect of Emodin on Lung Injury in Severe Acute Pancreatitis Rats and Its Effect on MAPK Pathway

[目的]观察大黄素对重症急性胰腺炎(severe acute pancreatitis,SAP)大鼠肺损伤的保护作用,并初步探究其作用机制。[方法]将80只无特定病原体(specific pathogen free,SPF)级SD大鼠随机分为对照组、模型组、大黄素高剂量组和大黄素低剂量组,每组20只。除对照组外,其余组大鼠均使用牛磺脱氧胆酸钠建立SAP大鼠模型,动物苏醒后1h及6h,大黄素高、低剂量组大鼠分别给予60mg·kg^(-1)和30mg·kg^(-1)的大黄素灌胃,对照组和模型组大鼠以等量0.5%羧甲基纤维素钠(carboxymethylcellulose sodium,CMC-Na)灌胃。造模12h后检测大鼠肺组织湿干重比(wet-to-dry weight ratio,W/D)、动脉血氧分压(partial pressure of oxygen,PaO2)、二氧化碳分压(partial pressure of carbon dioxide,PaCO2)、氧合指数(oxygenation index,OI);苏木精-伊红(hematoxylin-eosin,HE)染色检测肺组织病理变化;酶联免疫吸附检测(enzyme linked immunosorbent assay,ELISA)试剂盒检测血清脂肪酶、淀粉酶、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)含量、肺组织超氧化物歧化酶(superoxide dismutase,SOD)活力和丙二醛(malondialdehyde,MDA)含量;实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,RT-qPCR)检测肺组织B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein gene,Bax)mRNA表达水平;Western blot检测肺组织Bcl-2、Bax蛋白表达水平和p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)、细胞外信号调节激酶(extracellular signalregulated kinase 1/2,ERK1/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)蛋白磷酸化水平。[结果]与对照组比较,模型组W/D和PaCO2升高,PaO2和OI降低,血清脂肪酶、淀粉酶、TNF-α、IL-6、MDA含量升高,SOD活力降低,肺组织充血、水肿,有大量炎症细胞浸润,Bcl-2 mRNA和蛋白表达降低,BaxmRNA和蛋白表达增加,p38 MAPK、ERK1/2、JNK蛋白磷酸化水平增加。与模型组比较,大黄素可降低W/D和PaCO2,增加PaO2和OI,降低血清脂肪酶、淀粉酶、TNF-α、IL-6、MDA含量,提高SOD活力,改善肺组织肺损伤,提高Bcl-2 mRNA和蛋白表达,减少Bax mRNA和蛋白表达,降低p38 MAPK、ERK1/2、JNK蛋白磷酸化水平,其中高剂量大黄素的作用优于低剂量。上述指标差异均有统计学意义(P<0.05)。[结论]大黄素对SAP大鼠肺损伤具有保护作用,其作用机制可能与抑制MAPK信号通路的关键蛋白活化有关。

[Objective]To observe the protective effect of emodin on lung injury in severe acute pancreatitis(SAP)rats,and to explore its mechanism of action.[Methods]Eighty specific pathogen free(SPF)SD rats were randomly divided into control group,model group,emodin high-dose group and emodin low-dose group,20 rats in each group.Except for control group,the rats in the other groups used sodium taurodeoxycholate to establish the SAP rat model.One hour and six hours after awakening,the rats in emodin high-dose group and low-dose group were given 60mg·kg^(-1) and 30mg·kg^(-1) emodin by gavage,the rats in control group and model group were gavaged with the same amount of 0.5%carboxy methylcellulose sodium(CMC-Na).After 12 hours of modeling,the wet-to-dry weight ratio(W/D),partial pressure of oxygen(PaO2),partial pressure of carbon dioxide(PaCO2),and oxygenation index(OI)were detected.Hematoxylin-eosin(HE)staining was used to detect pathological changes of lung tissues.Enzyme linked immunosorbent assay(ELISA)kits were used to detect serum lipase,amylase,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)content,lung tissue superoxide dismutase(SOD)activity and malondialdehyde(MDA)content.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein gene(Bax)mRNA levels of lung tissues.Western blot was used to detect the expression of Bcl-2 and Bax protein in lung tissues and the protein phosphorylation levels of p38 mitogen-activated protein kinase(p38 MAPK),extracellular signal-regulated kinase 1/2(ERK1/2),c-Jun Nterminal kinase(JNK).[Results]Compared with control group,W/D and PaCO2 of model group increased,PaO2 and OI decreased,serum lipase,amylase,TNF-α,IL-6,MDA content increased,and SOD activity decreased.The lung tissues of mice in model group were congested with edema,and infiltrated with a large number of inflammatory cells.In model group,the expression of Bcl-2 mRNA and protein decreased,the expression of Bax mRNA and protein increased,and the protein phosphorylation level of p38 MAPK,ERK1/2,and JNK protein increased.Compared with model group,emodin reduced W/D and PaCO2,increased PaO2 and OI,reduced serum lipase,amylase,TNF-α,IL-6,MDA content,increased SOD activity,improved lung damage,and also increased Bcl-2 mRNA and protein expression,reduced Bax mRNA and protein expression,and reduced p38 MAPK,ERK1/2,and JNK protein phosphorylation levels,and the effect of high-dose emodin was better than that of low-dose emodin.The differences in the above indicators were statistically significant(P<0.05).[Conclusion]Emodin has a protective effect on lung injury in SAP rats,and its mechanism may be related to the inhibition of the activation of key proteins in the MAPK signaling pathway.