VEGF对缺氧复氧小鼠海马神经元细胞线粒体凋亡的抑制作用

目的研究血管内皮生长因子(VEGF)对缺氧复氧小鼠海马神经元细胞系HT22细胞凋亡与线粒体动力相关蛋白1(Drp1)表达情况的影响。方法取对数生长期的HT22细胞,随机分3组:正常对照组(Control)、缺氧复氧组(Model)、Model+VEGF组(Model+VEGF)。除Control组外,其余各组细胞在缺氧缺糖处理6h后,进行复氧复糖继续培养18h,Model+VEGF组在缺氧复氧结束后加入VEGF 164处理24h。倒置显微镜观察各组细胞形态,Hoechst33258染色及流式细胞术检测细胞凋亡情况,Western Blot检测细胞中Drp1的表达情况。结果Control组细胞呈两级或者多级,胞体明显,细胞更为短小与饱满,细胞交织成片连接在一起;Model组细胞皱缩,胞体变圆,细胞显微镜下观察细胞突触变长,细胞间连接减少;Model+VGEF组细胞形态与Model组相比有所缓解;与Control组相比,Model组细胞凋亡与Drp1蛋白表达量显著增加(P<0.05);与Model组相比,Model+VEGF组细胞凋亡率与Drp1蛋白表达量明显下降(P<0.05)。结论VEGF可能通过抑制细胞线粒体凋亡对缺氧复氧的HT22细胞起到保护作用。

Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry

Aconitine,a common and main toxic component of Aconitum,is toxic to the central nervous system.However,the mechanism of aconitine neurotoxicity is not yet clear.In this work,we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine.HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system.Meanwhile,to confirm the metabolic mechanism of aconitine toxicity on HT22 cells,the levels of lactate dehydrogenase,intracellular Ca^(2+),reactive oxygen species,glutathione and superoxide dismutase,and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology.Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid,which was followed by the accumulation of lactic acid and reduction of glucose.The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca^(2+)overload and oxidative stress,and eventually result in cell apoptosis.In general,we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer’s disease by the high-content fluorescence imaging system

Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important.In this study,we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD.At first,stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells,together with G418 screening.The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1.By employing the high-content fluorescence imaging system,together with stable YFP-CL1 HT22 cells,the UPS activators were successfully screened from our established TCM library.The representative images were captured and analyzed,and quantification of the YFP fluorescence intensity was performed by flow cytometry.Then,the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP(APP),pRK5-EGFP-Tau P301L(Tau P301L),or pRK5-EGFP-Tau(Tau)transiently transfected HT22 cells using fluorescence imaging,flow cytometry,and Western blot.In conclusion,our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the highthroughput screening of the UPS activators.Three compounds,namely salvianolic acid A(SAA),salvianolic acid B(SAB),and ellagic acid(EA),were identified to significantly decrease YFP fluorescence intensity,which suggested that these three compounds are UPS activators.The identified UPS activators were demonstrated to clear AD-related proteins,including APP,Tau,and Tau P301L.Therefore,these findings provide a novel insight into the discovery and development of anti-AD drugs.

BHBA对HT22细胞中ACE表达的影响

生酮饮食(KD)已被证实具有神经保护作用,但其机制尚不明确。本试验以小鼠海马神经元HT22为细胞模型,利用2 mmol/Lβ-羟基丁酸酯(BHBA)对HT22细胞进行处理,通过荧光定量PCR,免疫蛋白印迹和信号通路阻断试验,观察BHBA对HT22细胞ACE表达的影响,并探讨参与调控的信号通路。结果表明,2 mmol/L BHBA可通过激活HT22细胞内GPR109A受体和ERK1/2信号通路进而增加HT22细胞中ACE的表达。