丹红注射液对蛛网膜下腔出血大鼠早期脑损伤氧化应激及神经元自噬的影响

Effects of Danhong injection on oxidative stress and neuronal autophagy in early brain injury in rats with subarachnoid hemorrhage

目的 探究丹红注射液对蛛网膜下腔出血(SAH)大鼠早期脑损伤(EBI)氧化应激及神经元自噬的影响。方法 71只大鼠随机分出12只作为假手术组,其余接受血管内穿孔建立SAH模型,建模后存活52只,随机分入模型组、低剂量组、高剂量组和EX-527组,每组13只。假手术组、模型组接受腹腔注射5 ml·kg^(-1)·d^(-1)生理盐水,低剂量组接受腹腔注射5 ml·kg^(-1)·d^(-1)用生理盐水稀释1倍的丹红注射液,高剂量组接受腹腔注射5 ml·kg^(-1)·d^(-1)丹红注射液原液,EX-527组接受腹腔注射EX-52710 mg·kg^(-1)·d^(-1)×3d。使用改良Garcia评分、参考Sugawara等评分标准完成大鼠神经功能评价与SAH评分,检测脑含水量。苏木精-伊红染色观察脑组织病理形态,透射电镜观察脑组织线粒体结构变化,检测脑组织炎性因子与氧化应激指标。免疫组化检测Beclin-1表达情况,qRT-PCR、WB检测SIRT1、AMPK、mTOR mRNA与蛋白、p-AMPK、p-mTOR表达情况。结果 假手术组大脑组织外观无明显出血,锥体细胞层细胞密集,神经元形态正常,细胞核浅染,未见明显变性或坏死,线粒体结构、形态完整;模型组、EX-527组可见明显血凝块,锥体细胞大量坏死或减少,细胞坏死后体积变小,染色加深,胞浆凝集,线粒体肿胀,内部结构遭到破坏。与假手术组相比,模型组、低、高剂量组、EX-527组神经功能评分、MnSOD、CAT、GSH-Px、SIRT1、AMPK mRNA与p-AMPK水平降低(P<0.05),脑含水量、SAH评分、TNF-α、IL-6、IL-8、IL-1β、MDA、ROS、Beclin-1阳性神经元百分比、mTOR mRNA与p-mTOR水平升高(P<0.05);与模型组、EX-527组相比,低、高剂量组出血情况减少,坏死细胞较少,核固缩、染色深,线粒体结构更完整,神经功能评分、MnSOD、CAT、GSH-Px、SIRT1、AMPK mRNA与p-AMPK水平上升(P<0.05),脑含水量、SAH评分、TNF-α、IL-6、IL-8、IL-1β、MDA、ROS、Beclin-1阳性神经元百分比、mTOR mRNA与p-mTOR水平下降(P<0.05);与低剂量组相比,高剂量组出血情况更轻,细胞形态与线粒体结构更完好,神经功能评分、MnSOD、CAT、GSH-Px、SIRT1、AMPK mRNA与p-AMPK水平上升(P<0.05),脑含水量、SAH评分、TNF-α、IL-6、IL-8、IL-1β、MDA、ROS、Beclin-1阳性神经元百分比、mTOR mRNA与p-mTOR水平下降(P<0.05)结论 丹红注射液能够通过降低氧化应激与炎症反应水平、调节神经元自噬、保护线粒体与脑组织细胞,减轻SAH大鼠的EBI,其作用机制可能与SIRT1/AMPK/mTOR信号通路相关。

Objective Effects of Danhong injection on oxidative stress and neuronal autophagy in early brain injury in rats with subarachnoid hemorrhage.Methods 71 rats were randomly divided into sham operation group(n=12)and intravascular perforation(SAH)model.52 rats survived after modeling and were randomly divided into model group(n=13),low dose group(n=13),high dose group(n=13)and EX-527 group(n=13).The sham operation group and model group received intraperiloneal injection of 5 ml·kg^(-1)·d^(-1)normal saline,the low dose group received intraperitoneal injection of 5 ml·kg^(-1)·d^(-1)Danhong injection diluted with normal saline,the high dose group received intraperitoneal injection of 5 ml·kg^(-1)·d^(-1)Danhong injection,and EX-527 group received 10 mg·kg~(-1)EX-527 intraperitoneal injection once a day for 3 days.The neurological function evaluation and SAH score of rats were evaluated by modified Garcia score and referring to the scoring standard of Sugawara et al.Detection of brain water content.Observation of pathological morphology of brain tissue by hematoxylin-eosin(HE)staining.Observation of structural changes of mitochondria in brain tissue by transmission electron microscope.Detection of inflammatory factors and oxidative stress indexes in brain tissue.Immunohistochemical detection of Beclin-1 expression.The expression of SIRT1,AMPK,mTOR mRNA and protein,p-AMPK and p-mTOR was detected by qRT-PCR and Wb.Results In the sham operation group,there was no obvious hemorrhage in the appearance of brain tissue,dense cells in pyramidal layer,normal morphology of neurons,light staining of nucleus,no obvious degeneration or necrosis,and complete structure and morphology of mitochondria.Obvious blood clots,massive necrosis or decrease of pyramidal cells,smaller size,deeper staining,cytoplasmic condensation,swelling of mitochondria and destruction of internal structure were observed in the model group and EX-527 group.Compared with the sham operation group,the neurological function score,MnSOD,CAT,GSH-Px,SIRT1,AMPK mRNA and p-AMPK in the model group,low and high dose groups decreased(P<0.05),while brain water content,SAH score,TNF-α,IL-6,IL-8,IL-1β,MDA,ROS,Beclin-1 positive neurons,mTOR mRNA and p-mTOR levels increased(P<0.05).Compared with the model group,the low and high dose groups had less bleeding,fewer necrotic cells,nuclear pyknosis,deep staining,more intact mitochondrial structure,higher neurological function score,MnSOD,CAT,GSH-Px,SIRT1,AMPK mRNA and p-AMPK levels(P<0.05),while brain water content,SAH score,TNF-α,IL-6,IL-8,IL-1β,MDA,ROS,Beclin-1 positive neuron percentage,mTOR mRNA and p-mTOR levels decreased(P<0.05).Compared with the low dose group,the high dose group had less bleeding,better cell morphology and mitochondrial structure,higher neurological function score,MnSOD,CAT,GSH-Px,SIRT1,AMPK mRNA and p-AMPK levels(P<0.05),and decreased brain water content,SAH score,TNF-α,IL-6,IL-8,IL-1β,MDA,ROS,Beclin-1 positive neurons,mTOR mRNA and p-mTOR levels(P<0.05).Conclusion Danhong injection can reduce EBI in SAH rats by reducing the level of oxidative stress and inflammation,regulating neuronal autophagy,protecting mitochondria and brain tissue cells,and its mechanism may be related to SIRT1/AMPK/mTOR signal pathway.