当归挥发油通过抑制自噬减轻拟缺血再灌注神经细胞损伤

目的:探讨自噬在拟缺血再灌注损伤神经细胞中的作用以及当归挥发油对自噬的影响。方法:采用"缺糖-复糖"结合"缺氧-复氧"的方法,在体外用连二亚硫酸钠(Na2S2O4)处理高分化PC12细胞,模拟"缺血再灌注"损伤,用CCK-8比色法检测当归挥发油对细胞增殖的影响,AO荧光染色观察自噬细胞形态,Western-blot检测自噬相关蛋白Beclin-1、Atg5、LC3B-Ⅱ的表达。结果:在6.25~25μg/ml的浓度范围内,当归挥发能够显著提高PC12细胞存活率,减少受损细胞酸性自噬泡的数量,25μg/ml的当归挥发油能够抑制自噬相关蛋白Beclin-1、Atg5、LC3B-Ⅱ的表达。结论:当归挥发油可能通过抑制自噬减轻拟缺血再灌注神经细胞的损伤。

三七总苷对大鼠缺血再灌注肾损伤的保护作用

目的观察三七总苷对大鼠肾缺血再灌注损伤的保护作用。方法30只SD大鼠随机等分为假手术对照组、模型对照组、三七总苷组。建立大鼠肾缺血再灌注损伤模型后24h,下腔静脉取血,制备肾组织匀浆,分别测定大鼠血肌酐(Cr)、血尿素氮(BUN)、肾组织匀浆超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果与假手术组比较,模型对照组大鼠血清BUN、Cr含量与肾组织MDA含量升高,肾组织SOD活性降低(P均<0.01);与模型对照组比较,三七总苷组大鼠血清BUN、Cr含量和肾组织MDA含量降低,肾组织SOD活性升高(P均<0.01)。结论三七总苷可通过抗自由基损伤和减轻脂质过氧化实现对肾缺血再灌注损伤的保护作用。

缺血预处理减轻沙土鼠脑缺血再灌注损伤与海马CA1区p-p38MAPK表达的关系

目的:研究脑缺血预处理减轻沙土鼠脑缺血再灌注损伤与海马CA1区磷酸化p38MAPK(p-p38MAPK)含量之间的关系。方法:采用双侧颈总动脉夹闭造成前脑缺血的动物模型。动物随机分为假手术组(SH组)、预缺血组(IA组,前脑缺血3min)、缺血再灌注组(IR组)、预处理组(IP组)。后3组末次缺血后再分为再灌注15min、2h、6h、1天和5天时间点,每时间点8只动物。前4亚组用免疫组化法测定海马CA1区p-p38MAPK含量。再灌注1天、5天2亚组观察行为学变化和海马CA1区形态学存活细胞数目。结果:IA组、IR组、IP组沙土鼠海马CA1区再灌注15min、2h、6h和1天p-p38MAPK表达均多于SH组,差异有统计学意义(P<0.01);IR组沙土鼠海马CA1区再灌注15min、2h、6h和1天p-p38MAPK表达均多于IA组,差异有统计学意义(P<0.01);IP组沙土鼠海马CA1区再灌注15min和2hp-p38MAPK表达多于IA组,差异有统计学意义(P<0.05),再灌注2h、6h和1天p-p38MAPK表达较IR组下降,差异有统计学意义(P﹤0.05)。IA组与SH组沙土鼠行为学改变无统计学意义(P>0.05);IR组沙土鼠再灌注1天和5天探索活动均较IA组增多,差异有统计学意义(P<0.01);IP组沙土鼠再灌注1天、5天探索活动均少于IR组,差异有统计学意义(P<0.01)。IR组沙土鼠海马CA1区再灌注1天和5天存活神经元少于IA组,差异有统计学意义(P<0.01);IP组沙土鼠海马CA1区再灌注5天存活神经元多于IR组,差异有统计学意义(P<0.01)。结论:3min预缺血可引起海马CA1/3区p-p38MAPK水平轻度升高,但能降低24h后5min缺血再灌注引起的海马CA1区p-p38MAPK水平的上升程度。同时缺血预处理减少海马CA1区神经元死亡,并改善沙土鼠行为学变化。

当归挥发油通过激活ERK信号通路以减轻缺血再灌注神经细胞凋亡的作用

目的研究当归挥发油(EOAS)对缺血再灌注损伤神经细胞凋亡的影响。方法将高分化PC12细胞分为空白组、模型组、对照组及当大中小3个剂量实验组。除空白组外,其余各组用含10 mmol·L^(-1)连二亚硫酸钠(Na2S2O4)的无糖培养基缺氧缺糖损伤1 h,大中小3个剂量实验组加入终浓度分别为25.00,12.50,6.25μg·m L^(-1)EOAS,对照组加入10μmol·L^(-1)依达拉奉,复氧48 h。用MTT比色法检测当归挥发油对细胞增殖的影响,以试剂盒检测乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,用流式细胞仪检测细胞凋亡,以AO/PI免疫荧光染色观察凋亡细胞形态,以比色法检测细胞caspase-3的活性,以免疫印迹法检测p-ERK1/2蛋白的表达。结果与空白组相比,模型组细胞存活率降至(67.4±0.10)%,差异有统计学意义(P<0.01)。与模型组相比,对照组和大剂量实验组的细胞存活率分别升至(86.2±0.10)%,(94.5±0.05)%,差异均有统计学意义(P<0.05,P<0.01)。与空白组比较,模型组细胞LDH活性、MDA含量分别升高至(912.53±16.71)U·L^(-1)及(9.05±0.25)μmol·L^(-1);而SOD水平降至(12.53±0.29)U·m L^(-1),差异均有统计学意义(均P<0.01)。与模型组相比,大剂量实验组的LDH活性降低至(565.61±11.72)U·L^(-1),而SOD水平升高至(12.53±0.29)U·m L^(-1),差异均有统计学意义(P<0.05,P<0.01)。与模型组相比,大中2个剂量实验组MDA含量分别降至(3.32±0.68),(5.79±0.68)μmol·L^(-1),差异均有统计学意义(均P<0.01)。模型组与大中小3个剂量实验组细胞的光密度(A)分别为0.75±0.06,0.10±0.02,0.16±0.03及0.49±0.04,与模型组相比,差异均有统计学意义(P<0.05,P<0.01)。与空白组相比,模型组细胞凋亡率为(31.17±2.44)%,差异有统计学意义(P<0.01);与模型组相比,大中2个剂量组细胞凋亡率分别为(4.57±0.32)%,(5.93±0.81)%,差异均有统计学意义(均P<0.01)。与模型组相比,大剂量实验组能够明显促进细胞p-ERK1/2蛋白的表达(P<0.01)。结论 EOAS通过激活ERK信号通路抑制拟缺血再灌注神经细胞凋亡。

Effects of Chinese Herbal Medicine Serum on the Apoptosis of Sinoatrial Node Cells Induced by Simulated Ischemia-reperfusion

Objective:To study the effect of Chinese herbal medicine Kangxin Fumai Granule(康心复脉颗粒 Granule for heart diseases) serum on the primary cultured sinoatrial node(SAN) cell apoptosis induced by simulated ischemia-reperfusion(IR).Methods:The SAN cells removed from SAN tissue of neonatal Wistar rats were cultured and purified with differential attachment and 5'-bromodeoxyuridine(BrdU) treatment.Simulated IR model was adopted.The obtained cells were morphologically observed with inverted microscopy.By using the method of serum pharmacology,the cell apoptosis was measured with TUNEL staining qualitatively and with flow cytometry quantitatively.Results:Three kinds of cells were observed in the cultured SAN cells:spindle,triangle and irregular.The spindle cells comprised the greatest proportion.The SAN cells in the model group showed moderate positive brown staining in the nucleus,and the apoptosis rate increased significantly compared to that in the control group(P<0.01).While the SAN cells in the Kangxin Fumai Granule high-dose group did not demonstrated positive staining in the nucleus,and the apoptosis rate decreased significantly compared to that in the model group(P<0.05).Conclusion:Of the cells cultured from SAN,the spindle cells were pacemaker cells of SAN in rats.Blockade and/or inhibition of the SAN cell apoptosis might be one of the important mechanisms of Kangxin Fumai Granule in preventing and treating sinoatrial injury induced by simulated IR.