该文研究黑枸杞多酚(Lycium ruthenicum Murr.polyphenols,LRP)对丙烯酰胺(acrylamide,ACR)诱导SH-SY5Y细胞氧化损伤的保护机制。ACR诱导细胞建立氧化损伤模型,LRP预处理。分为对照组,ACR组(2.5 mmol/L)和LRP组(500、1 000、1 500 μg/mL)。CCK-8法测定细胞活力,试剂盒检测活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽(glutathione,GSH)水平,Western blot测定核因子E2相关因子2 (nuclear factor E2-related factor 2,Nrf2)、谷氨酸半胱氨酸连接酶催化亚基(glutamate-cysteine ligase catalytic subunit,GCLC)、谷胺酸半胱氨酸连接酶(glutamate-cysteine ligase,GCLM)、血红素氧合酶1(heme oxygenase-1,HO-1)、c-Jun N-末端激酶(c-Jun N-terminal kinase,JNK)、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase, p38)、半胱氨酸蛋白酶-3(cysteinyl aspartate-specific proteinase-3,Caspase-3)蛋白表达水平。观察小干扰RNA(small interfering RNA,siRNA)干扰Nrf2表达对以上蛋白影响。结果LRP组细胞活力提高,ROS和MDA水平降低、SOD和GSH含量增加,Nrf2、GCLC、GCLM和HO-1蛋白表达提高,JNK、p38和Caspase-3蛋白表达降低,1 500 μg/mL LRP组细胞活性和抗氧化能力最高。siRNA沉默Nrf2后各组Nrf2、GCLC、GCLM和HO-1蛋白表达降低,JNK、p38和Caspase-3蛋白表达增加,LRP组siRNA干扰前差异显著(P<0.05)。研究证明LRP有抗氧化、抗凋亡的生物活性,通过激活Nrf2通路和抑制JNK、p38和Caspase-3蛋白减弱ACR对SH-SY5Y细胞的毒性。
The protective mechanism of Lycium ruthenicum Murr. polyphenols (LRP) on acrylamide (ACR)-induced oxidative stress injury in SH-SY5Y cells were investigated.The oxidative stress injury model of SH-SY5Y cells was established by ACR, cells were pretreated with LRP.SH-SY5Y cells were divided into control group, ACR group (2.5 mmol/L) and LRP groups (500, 1 000, and 1 500 μg/mL).The cell viability was measured by CCK-8, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) activity were evaluated by biochemical kits, the expression levels of the nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase (GCLM), heme oxygenase-1(HO-1), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38), cysteinyl aspartate-specific proteinase-3 (Caspase-3) protein were measured by Western blot.The effects of expression of the above proteins were also observed after small interfering RNA (siRNA) blocking Nrf2 expression.The results showed that after the treatment of LRP, cell viability was increased, the levels of ROS and MDA reduced, SOD and GSH content increased, the expression levels of Nrf2, GCLC, GCLM, and HO-1 protein increased, while the expression levels of JNK, p38, and Caspase-3 protein decreased, the SH-SY5Y cells treated with 1 500 μg/mL LRP had the highest activity and the strongest ability to resist acrylamide-induced oxidative stress injury.After silencing Nrf2 gene expression with siRNA, the expression of Nrf2, GCLC, GCLM, and HO-1 protein decreased, the expression of JNK, p38, and Caspase-3 protein increased.In addition, there were significant differences between LRP group and LRP group with siRNA treated (P<0.05).Our research proved that LRP showed antioxidant and anti-apoptosis biological activities.And LRP can attenuate the toxicity of ACR to SH-SY5Y cells by activating Nrf2 signaling pathway and inhibiting apoptotic proteins JNK, p38, and Caspase-3.
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