姜黄素对心脏停搏/心肺复苏大鼠肠黏膜损伤的抑制作用

Effect of curcumin on intestinal mucosal injury in a rat model of cardiac arrest and cardiopulmonary resuscitation

目的·观察姜黄素对心肺复苏(cardiopulmonary resuscitation,CPR)后大鼠肠缺血/再灌注(ischemia/reperfusion,I/R)损伤的影响,并初步探讨其作用机制。方法·24只健康雄性SD大鼠随机分为4组,每组6只,即对照组(Sham组)、姜黄素组(Cur组)、CPR后肠I/R损伤组(I/R组)和姜黄素+CPR后肠I/R损伤组(Cur+I/R组)。4个组均行麻醉后气管插管,股动、静脉置管;I/R组和Cur+I/R组通过窒息法建立心脏停搏/CPR模型,Sham组和Cur组不进行该操作;Cur+I/R组于CPR开始前1 min腹腔注射姜黄素100 mg/kg,同一时间Cur组腹腔注射等量姜黄素,Sham组、I/R组注射同等体积二甲基亚砜。自主循环恢复后6、12、24 h静脉采血检测肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白介素6(interleukin-6,IL-6)的浓度;自主循环恢复后24 h处死大鼠,检测肠组织中丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(glutathione,GSH)浓度和超氧化物歧化酶(superoxide dismutase,SOD)活力,苏木精-伊红染色观察肠组织病理学变化,TUNEL法检测肠黏膜上皮细胞凋亡情况,Western blotting法检测肠组织中缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)、胱天蛋白酶3(caspase-3)的表达情况,透射电子显微镜下观察肠组织超微结构改变。结果·Cur+I/R组3个时间点的TNF-α、IL-6的浓度均低于I/R组(均P<0.05),高于Sham组(均P<0.05)。Cur+I/R组的MDA含量低于I/R组(P<0.01),高于Sham组(P<0.01);Cur+I/R组的SOD活力和GSH浓度高于I/R组(均P<0.05),低于Sham组(均P<0.01)。Cur+I/R组的HIF-1α蛋白表达水平显著高于I/R组(P<0.01),低于Sham组(P<0.01);而Cur+I/R组caspase-3蛋白表达水平显著低于I/R组(P<0.01),高于Sham组(P<0.01)。病理学观察发现,I/R组可见回肠黏膜水肿,炎症细胞浸润,绒毛顶端上皮脱落、粘连,固有层裸露、破溃,而Cur+I/R组肠黏膜损伤较I/R组明显减轻。TUNEL法检测显示,Cur+I/R组凋亡指数较I/R组显著降低(P<0.01),但较Sham组升高(P<0.01)。电子显微镜下Sham组、Cur组肠黏膜形态正常,I/R组肠黏膜上皮细胞微绒毛排列稀疏、倒伏,内质网脱颗粒,线粒体空化、嵴断裂;Cur+I/R组肠黏膜上皮细胞微绒毛排列密集、有序,结构清晰,细胞间的连接复合体完整,细胞器形态大致正常。结论·姜黄素对CPR后大鼠肠黏膜I/R损伤具有抑制作用,其机制与抑制炎症、降低氧化应激、抗凋亡及上调HIF-1α蛋白表达有关。

Objective·To observe the effect of curcumin on intestinal ischemia/reperfusion(I/R)injury in the rats after cardiopulmonary resuscitation(CPR),and explore the mechanism.Methods·Twenty-four healthy male SD rats were randomly divided into 4 groups,namely control group(Sham group,n=6),curcumin group(Cur group,n=6),intestinal I/R injury after CPR group(I/R group,n=6),and curcumin+intestinal I/R injury after CPR group(Cur+I/R group,n=6).Tracheal and femoral artery and vein catheterization were carried out after anesthesia in the 4 groups.The cardiac arrest/CPR model was established by asphyxiation in I/R group and Cur+I/R group,while not in the other two groups.Curcumin(100 mg/kg)was injected intraperitoneally 1 min before CPR in Cur+I/R group.Meanwhile,equivalent curcumin was injected intraperitoneally in Cur group,and equivalent dimethylsulfoxide was injected in Sham group and I/R group.Venous blood samples were collected 6,12 and 24 h after recovery of spontaneous circulation(ROSC)to detect the concentrations of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6).The rats were sacrificed 24 h after ROSC,and the concentrations of malondialdehyde(MDA)and glutathione(GSH)and the activity of superoxide dismutase(SOD)in the intestinal tissues were measured.The pathological changes of intestinal tissues were observed by hematoxylin-eosin staining.The apoptosis of intestinal mucosal epithelial cells was detected by TUNEL assay.Hypoxia-inducible factor-1α(HIF-1α)and caspase-3 expressions in the intestinal tissues were detected by Western blotting.Ultrastructural changes of the intestinal tissues were observed by using transmission electron microscopy.Results·The concentrations of TNF-αand IL-6 in Cur+I/R group at 3 time points were lower than those in I/R group(all P<0.05),while higher than those in Sham group(all P<0.05).The MDA content in Cur+I/R group was lower than that in I/R group(P<0.01),while higher than that in Sham group(P<0.01).The SOD activity and GSH concentration in Cur+I/R group were higher than those in I/R group(both P<0.05),while lower than those in Sham group(both P<0.01).The expression of HIF-1αprotein in Cur+I/R group was significantly higher than that in I/R group(P<0.01),but lower than that in Sham group(P<0.01).The expression of caspase-3 protein in Cur+I/R group was significantly lower than that in I/R group(P<0.01),while higher than that in Sham group(P<0.01).Pathological observation showed that ileal mucosal edema,inflammatory cell infiltration,epithelial exfoliation and adhesion at the top of villi,and exposed and ruptured lamina propria were found in I/R group.Intestinal mucosal damage in Cur+I/R group was significantly alleviated,compared with that in I/R group.TUNEL assay revealed that the apoptotic index in Cur+I/R group was significantly lower than that in I/R group(P<0.01),but higher than that in Sham group(P<0.01).Under electron microscopy,the intestinal mucosae of Sham group and Cur group were normal.In I/R group,intestinal mucosal epithelial cells presented sparsely arranged and lodged microvilli,degranulation of endoplasmic reticulum,and vacuolated mitochondria with broken ridges.Intestinal mucosal epithelial cells in Cur+I/R group showed densely and orderly arranged microvilli with clear structure,normal organelles,and complete junctional complexes between cells.Conclusion·Curcumin has a protective effect on intestinal mucosa I/R injury in rats after CPR.Its mechanism is related to inhibiting inflammation,reducing oxidative stress,anti-apoptosis and up-regulating HIF-1αprotein expression.