β-谷甾醇通过抑制内质网应激改善大鼠脑缺血再灌注损伤

Beta-sitosterol improves cerebral ischemia-reperfusion injury in rats by inhibiting endoplasmic reticulum stress

目的探讨β-谷甾醇对大鼠脑缺血再灌注损伤(CIRI)的影响及其机制是否与内质网应激(ERS)有关。方法将应用改良Longa法制备CIRI模型成功的53只大鼠按随机数字表法分为模型组(n=14)、β-谷甾醇低剂量组(n=13)、β-谷甾醇中剂量组(n=13)、β-谷甾醇高剂量组(n=13),并取进行相同手术操作但不阻断大脑中动脉的12只大鼠作为假手术组。假手术组和模型组大鼠每天灌胃1 mL 5 g/L羧甲基纤维素钠,β-谷甾醇低剂量组、β-谷甾醇中剂量组、β-谷甾醇高剂量组大鼠每天分别灌胃1 mL 10、20、40 mg/kgβ-谷甾醇(溶于5 g/L羧甲基纤维素钠中),连续给药14 d。给药结束后,采用Zea Longa 5分法进行大鼠神经功能评分,然后取脑组织,采用2,3,5-三苯基氯化四氮唑(TTC)染色法评估脑梗死体积,采用HE染色、Nissl染色、TUNEL染色评估脑损伤及神经元凋亡情况,采用水溶性四氮唑-1(WST-1)法检测脑组织中超氧化物歧化酶(SOD)含量,采用比色法检测脑组织中谷胱甘肽过氧化物酶(GSH-Px)含量,采用硫代巴比妥酸(TBA)法检测脑组织中丙二醛(MDA)含量,采用实时荧光定量PCR(qRT-PCR)法及Western blotting法检测脑组织中RNA依赖的蛋白激酶样内质网激酶(PERK)、肌醇需求酶-1(IRE-1)、激活转录因子-6(ATF-6)、葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)和含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-12的mRNA及蛋白表达。结果与假手术组比较,模型组大鼠的神经功能评分明显升高,脑梗死体积比明显升高,TUNEL阳性细胞率明显升高,SOD、GSH-Px含量明显降低,MDA含量明显升高,差异均有统计学意义(P<0.05);模型组大鼠脑组织中PERK、IRE-1、ATF-6、GRP78、CHOP和Caspase-12的mRNA和蛋白水平亦较假手术组明显升高,差异均有统计学意义(P<0.05)。与模型组比较,β-谷甾醇低剂量组、β-谷甾醇中剂量组和β-谷甾醇高剂量组大鼠的神经功能评分明显降低,脑梗死体积比明显降低,TUNEL阳性细胞率明显降低,SOD、GSH-Px含量明显升高,MDA含量明显降低,差异均有统计学意义(P<0.05);β-谷甾醇低剂量组、β-谷甾醇中剂量组和β-谷甾醇高剂量组大鼠脑组织中PERK(mRNA:2.17±0.17、1.79±0.07、1.33±0.07,蛋白:5.11±0.52、2.91±0.26、1.98±0.17)、IRE-1(mRNA:1.75±0.18、1.65±0.08、1.32±0.08,蛋白:5.00±0.31、4.05±0.27、1.98±0.14)、ATF-6(mRNA:2.24±0.12、1.77±0.14、1.37±0.13,蛋白:4.93±0.45、4.04±0.30、3.10±0.20)、GRP78(mRNA:2.67±0.16、2.11±0.16、1.69±0.11,蛋白:5.02±0.38、2.97±0.26、2.05±0.22)、CHOP(mRNA:2.01±0.16、1.70±0.19、1.40±0.10,蛋白:4.92±0.39、4.02±0.27、3.08±0.22)和Caspase-12(mRNA:1.85±0.09、1.61±0.09、1.30±0.09,蛋白:3.03±0.20、2.19±0.11、1.82±0.11)的mRNA和蛋白水平亦较模型组(mRNA:2.99±0.28、2.27±0.12、2.57±0.21、3.46±0.20、2.50±0.23、2.35±0.16,蛋白:6.98±0.48、6.03±0.58、5.98±0.63、7.10±0.45、6.00±0.53、5.02±0.43)明显降低,差异均有统计学意义(P<0.05)。结论β-谷甾醇可减轻大鼠CIRI,其机制可能与抑制ERS有关。

Objective To reveal the effect ofβ-sitosterol on cerebral ischemia-reperfusion injury(CIRI)in rats and whether its mechanism is related to endoplasmic reticulum stress(ERS).Methods Fifty-three CIRI rats(CIRI models established by modified Longa method)were randomly divided into model group(n=14),β-sitosterol low-dose group(n=13),β-sitosterol medium-dose group(n=13)andβ-sitosterol high-dose group(n=13);12 rats underwent the same operation without blocking the middle cerebral artery were selected as sham-operated group.Rats in the sham-operated group and model group were given intragastric administration of 1 mL 5 g/L sodium carboxymethyl cellulose daily.Rats in theβ-sitosterol low-dose group,β-sitosterol medium-dose group andβ-sitosterol high-dose group were given intragastric administration of 1 mLβ-sitosterol at 10,20 and 40 mg/kg/d(dissolved in 5 g/L sodium carboxymethyl cellulose),respectively,for 14 consecutive d.Neurological function was evaluated according to Zea Longa 5 method.Rats were sacrificed and brain tissues were collected.Volume of cerebral infarction was measured by 2,3,5-triphenyl tetrazolium chloride(TTC)staining.Brain injury and neuronal apoptosis were evaluated by HE staining,Nissl staining and TUNEL.Superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)contents were detected by water-soluble tetrazolium 1(WST-1)method,colorimetric method or thiobarbituric acid(TBA)method,respectively.The mRNA and protein expression levels of protein kinase R-like endoplasmic reticulum kinase(PERK),inositol-requiring enzyme-1(IRE-1),activated transcription factor-6(ATF-6),glucose-regulated protein 78(GRP78),C/EBP homologous protein(CHOP)and Caspase-12 in the brain tissues were detected by qRT-PCR or Western blotting.Results Compared with the sham-operated group,the model group had significantly increased neurological function score,cerebral infarction volume and TUNEL positive rate,decreased SOD and GSH-Px content,increased MDA content,and increased mRNA and protein expressions of PERK,IRE-1,ATF-6,GRP78,CHOP and Caspase-12(P<0.05).Compared with the model group,theβ-sitosterol low-dose group,β-sitosterol medium-dose group andβ-sitosterol high-dose group had significantly decreased neurological function score,cerebral infarction volume,and TUNEL positive rate,increased SOD and GSH-Px content,and decreased MDA content(P<0.05);theβ-sitosterol low-dose group,β-sitosterol medium-dose group andβ-sitosterol high-dose group had significantly decreased mRNA and protein PERK expressions(mRNA:2.17±0.17,1.79±0.07 and 1.33±0.07;protein:5.11±0.52,2.91±0.26 and 1.98±0.17),IRE-1 expressions(mRNA:1.75±0.18,1.65±0.08 and 1.32±0.08;protein:5.00±0.31,4.05±0.27 and 1.98±0.14),ATF-6 expressions(mRNA:2.24±0.12,1.77±0.14 and 1.37±0.13;protein:4.93±0.45,4.04±0.30 and 3.10±0.20),GRP78 expressions(mRNA:2.67±0.16,2.11±0.16 and 1.69±0.11;protein:5.02±0.38,2.97±0.26 and 2.05±0.22),CHOP expressions(mRNA:2.01±0.16,1.70±0.19 and 1.40±0.10;protein:4.92±0.39,4.02±0.27 and 3.08±0.22)and Caspase-12 expressions(mRNA:1.85±0.09,1.61±0.09 and 1.30±0.09;protein:3.03±0.20,2.19±0.11 and 1.82±0.11)compared with the model group(mRNA:2.99±0.28,2.27±0.12,2.57±0.21,3.46±0.20,2.50±0.23 and 2.35±0.16;protein:6.98±0.48,6.03±0.58,5.98±0.63,7.10±0.45,6.00±0.53 and 5.02±0.43,P<0.05).Conclusion β-sitosterol attenuates CIRI in rats,whose mechanism may be related to inhibition of ERS signal pathway.