二苯乙烯苷调控PI3K/AKT信号通路干预SH-SY5Y细胞凋亡的机制研究

目的:探讨二苯乙烯苷(TSG)经PI3K/AKT通路对冈田酸诱导的人神经母细胞瘤细胞(SH-SY5Y)凋亡的影响及调控机制。方法:CCK-8法筛选OA最佳浓度,将SH-SY5Y细胞分为对照组、模型组、TSG组、LY294002组和LY294002+TSG组。通过CCK-8法和TUNEL法检测各组细胞增殖和凋亡情况;Western blotting法和qRT-PCR法检测PI3K、P-PI3K(Y607)、AKT、P-AKT(Ser473)、Bcl-2、Bax蛋白及mRNA表达情况,通路及凋亡蛋白相对表达量以P-PI3K(Y607)/PI3K、P-AKT(Ser473)/AKT、Bcl-2/Bax灰度比值表示。结果:OA最佳处理浓度为40 nmol/L。与对照组相比,模型组细胞活力降低,细胞凋亡率升高,通路及凋亡蛋白、PI3K、AKT、Bcl-2 mRNA表达水平降低,Bax mRNA表达水平升高(均P<0.05);与模型组相比,TSG组细胞活力升高,细胞凋亡率降低,通路及凋亡蛋白、PI3K、AKT、Bcl-2 mRNA相对表达水平升高,Bax mRNA表达水平降低(均P<0.05),LY294002组细胞活力降低,细胞凋亡率升高,P-PI3K(Y607)/PI3K蛋白表达水平显著降低(P<0.05)、P-AKT(Ser473)/AKT、Bcl-2/Bax蛋白表达水平较明显降低,但未有统计学意义,PI3K、AKT、Bcl-2 mRNA表达水平降低,Bax mRNA表达水平升高(均P<0.05);与LY294002组相比,LY294002+TSG组细胞活力升高,细胞凋亡率降低,通路及凋亡蛋白相对表达水平、PI3K、AKT、Bcl-2 mRNA升高,Bax mRNA表达水平降低(均P<0.05)。结论:二苯乙烯苷可能通过干预PI3K/AKT信号通路,进而调节Bcl-2、Bax等凋亡因子的表达,从而缓解冈田酸诱导的SH-SY5Y细胞凋亡。

Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

二苯乙烯苷调控MKK7/JNK信号通路改善阿尔茨海默病大鼠神经元损伤作用及机制研究

目的:探讨二苯乙烯苷(TSG)通过调控MKK7/JNK通路改善阿尔茨海默病大鼠神经元损伤中的作用机制。方法:采用24月龄的雄性老年大鼠42只,随机分为正常组,假手术组,模型组,阳性药组(0.15mg/kg石杉碱甲),以及TSG低剂量组(0.033 g/kg)、TSG中剂量组(0.1 g/kg)、TSG高剂量组(0.3 g/kg),每组6只。模型组、TSG各剂量组采用立体定位Aβ_(25-35)溶液构建老年痴呆模型,于造模28 d后经被动回避实验筛选造模成功的大鼠。筛选后阳性药物组、TSG各剂量组按照对应剂量灌胃给药,给药28 d后进行实验。采HE染色镜下观察每组大鼠大脑内海马区神经元细胞形态学变化;IHC检测各组大鼠脑组织MKK7、JNK蛋白的表达情况;qRT-PCR检测各大鼠脑组织内MKK7、JNK mRNA的表达情况。结果:(1)HE染色观察,与正常组,假手术组相比,模型组神经细胞数量减少且排列紊乱无规则,细胞膜发生皱缩,细胞核溶解变形。与模型组比较,阳性药组以及TSG各组神经细胞数量增多且排列相对整齐。(2)TUNEL染色阳性细胞比值结果:与正常组、假手术组相比,模型组凋亡细胞数量明显上调;与模型组相比,阳性药物组、TSG各组染色凋亡细胞减少(P<0.05)。(3)免疫组化测定结果表明:与正常组和假手术组相比,模型中MKK7、JNK在大鼠脑中的蛋白表达含量显著增加(P<0.05);与模型组比较,阳性药及TSG各剂量组蛋白表达量均有不同程度降低(P<0.05)。(4)qRT-PCR结果显示,与正常组、假手术组相比,模型组大鼠脑组织中MKK7、JNK mRNA水平明显上升(P<0.05);与模型组相比,各组给药大鼠脑组织中MKK7、JNK mRNA的表达含量均明显下调(P<0.05)。结论:二苯乙烯苷对神经元的损伤修复有一定的作用效果,其作用机制可能与下调MKK7、JNK激酶的mRNA转录及蛋白的表达有关。

The effect and mechanism of stilbene glycosides on improving neuronal injury in Alzheimer's disease rats by regulating ASK/MKK7/JNK pathway

Objective:To investigate the mechanism of action of tetrahydroxy stilbene glycosides(TSG)in ameliorating neuronal damage in Alzheimer's disease rats by regulating MKK7 and JNK kinases.Methods:A total of 24-month-old 42 SD rats were randomly selected for the experiment in 7 groups:normal group,sham-operated group,model group,positive drug group,low,medium and high dose TSG group at 0.033 g/kg,0.1 g/kg,0.3 g/kg.The Model Group and the TSG groups were established by stereotaxic Aβ25-35 solution.After 28 days,the model rats were selected by passive avoidance test.After screening,each dosage group of TSG and positive drug group was given intragastrically according to the corresponding dosage,and the experiment was carried out after 28 days.The pathological changes of hippocampal CA1 region were observed by tissue staining,and the amount of MKK7 and JNK proteins and the expression content of MKK7 and JNK mRNA by histochemical method of protein,and qRTPCR assay.Results:(1)He staining observation:Compared with the normal group and the sham-operated group,the number of nerve cells in the model group decreased and arranged irregularly,the cell membrane shrank,and the nucleus deformed and dissolved.The number of neurons in the positive drug group and TSG Group also increased significantly,the order is also relatively well.(2)From the results of the Tunel staining experiments:the positive apoptotic cells in the model group were higher than control group and sham-operated group,positive drug group and TSG drugs group was significantly smaller than that in the model group(P<0.05).(3)Compared with the control group and the Virtual Operation Group,the MKK7 and JNK protein concentrations in the brain of the model group were increased(P<0.05)by data analysis of immunohistochemistry:Compared with the model group,the protein expression of positive drug and TSG each dose group were reduced(P<0.05).(4)The results of QRTPCR data showed that the levels of MKK7 and JNK mRNA in the brain tissue of the model group were increased compared with the normal group and sham-operated groups(P<0.05).Conclusion:Stilbene glycoside has a certain effect on neuronal injury and repair which may be related to the changes of mRNA transcription and protein expression of MKK7 and JNK kinases.

二苯乙烯苷对拟阿尔茨海默病大鼠Ser199位点磷酸化的影响

目的研究二苯乙烯苷(TSG)对阿尔茨海默病(AD)大鼠Tau蛋白丝氨酸199位点(Ser199位点)磷酸化的影响。方法将SD大鼠随机分为正常组、假手术组、模型组、阳性对照组和低、中、高剂量实验组,每组6只。除正常组和假手术组外,其余5组脑内注射Aβ25-35制备AD模型。低、中、高剂量实验组分别按3.30×10^(-2)、0.10和0.30 g·kg^(-1)的剂量灌胃给予TSG;阳性对照组按0.15 mg·kg^(-1)的剂量灌胃给予石杉碱甲;正常组、假手术组和模型组均灌胃给予等体积0.9%NaCl。用免疫组化法和蛋白质印迹法检测脑组织Tau蛋白和磷酸化Tau蛋白(Ser199位点)分布与表达水平。结果中、高剂量实验组和阳性对照组、模型组、假手术组、正常组的海马区Tau蛋白平均光密度值分别为0.59±0.02、0.54±0.01、0.53±0.07、0.78±0.08、0.54±0.00和0.51±0.04,海马区磷酸化Tau蛋白(Ser199位点)平均光密度值分别为0.73±0.13、0.58±0.04、0.59±0.04、0.91±0.07、0.61±0.12和0.57±0.05,皮质区Tau蛋白平均光密度值分别为0.67±0.03、0.58±0.01、0.57±0.03、0.83±0.02、0.54±0.05和0.54±0.06,皮质区磷酸化Tau蛋白(Ser199位点)平均光密度值分别为0.67±0.02、0.59±0.02、0.56±0.01、0.89±0.10、0.55±0.01和0.53±0.02,脑组织中磷酸化Tau蛋白(Ser199位点)相对表达水平分别为0.64±0.19、0.36±0.06、0.40±0.08、0.96±0.06、0.52±0.12和0.41±0.05。中、高剂量实验组和阳性对照组、正常组、假手术组的上述指标与模型组比较,差异均有统计学意义(P<0.05,P<0.01)。结论TSG可以影响Tau蛋白过度磷酸化进程,可能是通过下调磷酸化Tau蛋白(Ser199位点)的表达。