金莲花总黄酮腹腔注射对大鼠脑缺血再灌注损伤的治疗作用及其机制

Therapeutic effect of intraperitoneal injection of total flavonoids from trollius chinensis on cerebral ischemia-reperfusion injury in rats and its mechanism

目的观察金莲花总黄酮腹腔注射对大鼠脑缺血再灌注损伤的治疗作用并探讨其机制。方法雄性SD大鼠随机分为假手术组、模型组、金莲花总黄酮组、铁死亡抑制剂组,每组18只。除假手术组外,各组均采用线栓法制作大鼠大脑中动脉阻塞模型;假手术组手术操作与模型组基本相同但未插线栓。金莲花总黄酮组、铁死亡抑制剂组在造模完成后分别腹腔注射金莲花总黄酮及铁死亡抑制剂Lip-1,假手术组、模型组腹腔注射等量生理盐水,连续给药3 d,每天1次。采用Longa评分法对大鼠的神经功能缺损进行评分,TTC染色测算脑组织梗死体积百分比,比色法检测脑组织氧化应激指标活性氧(ROS)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px),Western blotting法检测脑组织铁死亡相关蛋白谷胱甘肽过氧化物酶4(GPX4)、长链脂酰辅酶A合成酶(ACSL4)、转铁蛋白受体1(TFR1)、膜铁转运蛋白1(Ferroportin-1)。结果神经功能缺损评分、脑梗死体积百分比模型组>金莲花总黄酮组、铁死亡抑制剂组>假手术组(P均<0.05)。脑组织ROS、MDA含量模型组>金莲花总黄酮组、铁死亡抑制剂组>假手术组,GSH-Px含量假手术组>金莲花总黄酮组、铁死亡抑制剂组>模型组(P均<0.05)。脑组织GPX4、TFR1、Ferroportin-1蛋白表达模型组<金莲花总黄酮组、铁死亡抑制剂组、假手术组,ACSL4蛋白表达模型组>金莲花总黄酮组、铁死亡抑制剂组、假手术组(P均<0.05)。结论金莲花总黄酮腹腔注射对大鼠脑缺血再灌注损伤具有一定的治疗作用,其机制可能与抑制脑组织铁死亡有关。

Objective To observe the therapeutic effect of intraperitoneal injection of total flavonoids from trollius chinensis on cerebral ischemia-reperfusion injury in rats and to explore its mechanism.Methods Male SD rats were randomly divided into the sham operation group,middle cerebral artery occlusion(MCAO)group,total flavonoids from trollius chinensis group,and ferroptosis inhibitor group,with 18 rats in each group.Except for the sham operation group,we used the thread-embolization method to make the rat MCAO models in other groups;the surgical procedures of the sham operation group was basically the same as that of the model group but the wire-bolt was not inserted.Rats in the total flavonoids from trollius chinensis group and the ferroptosis inhibitor group were intraperitoneally injected with total flavonoids from trollius chinensis and the ferroptosis inhibitor Lip-1,respectively;rats in the sham operation group and the model group were injected with the equal volume of normal saline intraperitoneally,and the drugs were administered once a day,and for 3 consecutive days.The Longa scoring method was used to score the neurological deficits in rats.TTC staining was used to calculate the percentage of infarcted brain tissue.Colorimetric analysis was used to detect oxidative stress indicators such as reactive oxygen species(ROS),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)in the brain tissues.Western blotting was used to detect the ferroptosis-related proteins glutathione peroxidase 4(GPX4),long-chain acyl CoA synthase(ACSL4),transferrin receptor 1(TFR1),and membrane iron transporter-1(Ferroportin-1)in the brain tissues.Results Neurological deficit score and the percentage of cerebral infarction volume were as follows:MCAO group>total flavonoids from trollius chinensis group,ferroptosis inhibitor group>sham operation group(all P<0.05).The content of ROS and MDA in the brain tissues was as follows:MCAO group>total flavonoids from trollius chinensis group,ferroptosis inhibitor group>sham operation group,and the GSH-Px content was in the following order:sham operation group>total flavonoids from trollius chinensis group,ferroptosis inhibitor group group>MCAO group(all P<0.05).The protein expression of GPX4,TFR1,and ferroportin-1 was as follows:MCAO group<total flavonoids from trollius chinensis group,ferroptosis inhibitor group,and sham operation group,and the ACSL4 protein expression was as follows:MCAO group>total flavonoids from trollius chinensis group,ferroptosis inhibitor group,and sham operation group(all P<0.05).Conclusion Intraperitoneal injection of total flavonoids from trollius chinensis has certain therapeutic effect on cerebral ischemia-reperfusion injury in rats,and the mechanism may be related to the inhibition of ferroptosis.