Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry / 药物分析学报

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 Ca2+,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 Ca2+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.

Berberine inhibits erastin-induced ferroptosis of mouse hippocampal neuronal cells possibly by activating the Nrf2-HO-1/GPX4 pathway / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22).@*METHODS@#Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe2+ fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells.@*RESULTS@#Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P < 0.05) and lowered the levels of intracellular ROS and ferric iron content (P < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P < 0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P < 0.05).@*CONCLUSION@#Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.

Mitophagy mediated by ligustilide relieves OGD/R-induced injury in HT22 cells / 中国中药杂志

Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer's disease by the high-content fluorescence imaging system / 中国天然药物

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 high-throughput 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.

Study on antidepressant effect of total alkaloids of Fibraurea recisa / 中国中药杂志

This study aimed to investigate the antidepressant effects of total alkaloids of Fibraurea recisa in HT22 cells damaged by corticosterone (CORT) in vitro and in a mouse model of chronic unpredictable mild stress (CUMS) as well as the underlying mechanisms.In cellular experiments,the viability of CORT-damaged HT22 cells was detected using cell counting kit-8 (CCK-8),and the cell apoptosis was detected by Hoechst 33258 staining.In animal experiments,C57BL/6N mice were randomly divided into the control group,model group,low (100 mg·kg~(-1)),medium (200 mg·kg~(-1)) and high (400 mg·kg~(-1))-dose of total alkaloids of F.recisa groups,and positive control group.After 21 days of CUMS exposure,their depressive behaviors were observed in behavioral and Morris water maze tests.The serum levels of 5-hydroxytryptamine (5-HT),dopamine (DA),and norepinephrine (NE) were assessed by ELISA.The expression levels of apoptosis-related proteins Bcl-2,Bax and cleaved caspase-3 in HT22 cells and mouse hippocampus were detected by Western blot.The results suggested that total alkaloids of F.recisa alleviated the damage of HT22 cells induced by CORT in a dose-dependent manner.The Hoechst 33258 staining uncovered that total alkaloids of F.recisa better reduced the blue spots and inhibited cell apoptosis.The results of animal experiments showed that total alkaloids of F.recisa significantly improved the depression-like behaviors of mice and increased the serum levels of 5-HT,DA and NE as compared with those in the model group.The Western blot assays revealed a significant up-regulation of Bcl-2 protein expression,but an obvious reduction in Bax and cleaved caspase-3protein expression in the total alkaloids of F.recisa group.In conclusion,total alkaloids of F.recisa inhibited depression possibly by regulating the apoptosis-related protein expression or elevating the monoamine neurotransmitter levels in the brain.

Increased glycogen synthase kinase 3β activity involves in the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 in HT22 cells / 中华神经科杂志

Objective@#To investigate the effect of glycogen synthase kinase 3β (GSK3β) on the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 (Aβ31-35) in HT22 cells.@*Methods@#HT22 mouse hippocampal cells were divided into control group, Aβ31-35 group and LiCl+Aβ31-35 group by random number table method in the present study. Cells were synchronized to G0/G1 phase by 1% serum starvation for 1 hour (circadian time 0 (CT0)). Cell viability was detected by the cell counting kit-8 assay. The mRNA expression of clock gene Bmal1 was examined by real-time PCR at different CT times. The expression of GSK3β and BMAL1 protein was detected by Western blotting.@*Results@#Compared with the control group, Aβ31-35 induced the decreased expression of Bmal1 mRNA; The expression of both Bmal1 mRNA and BMAL1 protein was decreased significantly at CT20 (Bmal1 mRNA: 0.38±0.06 vs 0.83±0.08, t=4.549, P=0.001; BMAL1 protein: 0.67±0.04 vs 1.00±0.04, t=5.943, P<0.001). In the Aβ31-35 group, GSK3β activity was increased and the ratio of phosphorylated GSK3βS9 to GSK3β was decreased compared to the control group (0.66±0.08 vs 1.02±0.14, t=2.217, P=0.025). Aβ31-35 decreased the viability of HT22 cells (71.85%±6.20% in the Aβ31-35 group vs 98.14%±2.68% in the control group, t=3.891, P=0.006), and the GSK3β inhibitor LiCl pretreatment effectively reversed the decline of the viability induced by Aβ31-35 (90.74%±5.74% in the LiCl+Aβ31-35 group vs 71.85%±6.20% in the Aβ31-35 group, t=3.412, P=0.010). LiCl (in the LiCl+Aβ31-35 group) increased the expression of Bmal1 mRNA and BMAL1 protein significantly at CT20 compared with the Aβ31-35 group (Bmal1 mRNA: 0.72±0.05 vs 0.38±0.06, t=4.378, P=0.001; BMAL1 protein: 0.90±0.04 vs 0.67±0.04, t=4.052, P=0.002).@*Conclusion@#Increased GSK3β activity involved in the decreased expression of Bmal1 induced by Aβ31-35 in HT22 cells.

Increased glycogen synthase kinase 3β activity involves in the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 in HT22 cells / 中华神经科杂志

Objective@#To investigate the effect of glycogen synthase kinase 3β (GSK3β) on the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 (Aβ31-35) in HT22 cells.@*Methods@#HT22 mouse hippocampal cells were divided into control group, Aβ31-35 group and LiCl+Aβ 31-35 group by random number table method in the present study. Cells were synchronized to G0/G1 phase by 1% serum starvation for 1 hour (circadian time 0 (CT0)). Cell viability was detected by the cell counting kit-8 assay. The mRNA expression of clock gene Bmal1 was examined by real-time PCR at different CT times. The expression of GSK3β and BMAL1 protein was detected by Western blotting.@*Results@#Compared with the control group, Aβ31-35 induced the decreased expression of Bmal1 mRNA; The expression of both Bmal1 mRNA and BMAL1 protein was decreased significantly at CT20 (Bmal1 mRNA: 0.38±0.06 vs 0.83±0.08, t=4.549, P=0.001; BMAL1 protein: 0.67±0.04 vs 1.00±0.04, t=5.943, P<0.001). In the Aβ31-35 group, GSK3β activity was increased and the ratio of phosphorylated GSK3βS9 to GSK3β was decreased compared to the control group (0.66±0.08 vs 1.02±0.14, t=2.217, P=0.025). Aβ31-35 decreased the viability of HT22 cells (71.85%±6.20% in the Aβ31-35 group vs 98.14%±2.68% in the control group, t=3.891, P=0.006), and the GSK3β inhibitor LiCl pretreatment effectively reversed the decline of the viability induced by Aβ31-35 (90.74%±5.74% in the LiCl+Aβ31-35 group vs 71.85%±6.20% in the Aβ31-35 group, t=3.412, P=0.010). LiCl (in the LiCl+Aβ31-35 group) increased the expression of Bmal1 mRNA and BMAL1 protein significantly at CT20 compared with the Aβ31-35 group (Bmal1 mRNA: 0.72±0.05 vs 0.38±0.06, t=4.378, P=0.001; BMAL1 protein: 0.90±0.04 vs 0.67±0.04, t=4.052, P=0.002).@*Conclusion@#Increased GSK3β activity involved in the decreased expression of Bmal1 induced by Aβ31-35 in HT22 cells.

Increased glycogen synthase kinase 3β activity involves in the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 in HT22 cells / 中华神经科杂志

Objective To investigate the effect of glycogen synthase kinase 3β (GSK3β) on the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 (Aβ31-35) in HT22 cells.Methods HT22 mouse hippocampal cells were divided into control group,Aβ31-35 group and LiCl+Aβ 31-35 group by random number table method in the present study.Cells were synchronized to G0/G1 phase by 1％ serum starvation for 1 hour (circadian time 0 (CT0)).Cell viability was detected by the cell counting kit-8 assay.The mRNA expression of clock gene Bmal1 was examined by real-time PCR at different CT times.The expression of GSK3β and BMAL1 protein was detected by Western blotting.Results Compared with the control group,Aβ31-35 induced the decreased expression of Bmal1 mRNA;The expression of both Bmal1 mRNA and BMAL1 protein was decreased significantly at CT20 (Bmal1 mRNA:0.38±0.06 vs 0.83±0.08,t=4.549,P=0.001;BMAL1 protein:0.67±0.04 vs 1.00±0.04,t=5.943,P＜0.001).In the Aβ31-35group,GSK3β activity was increased and the ratio of phosphorylated GSK3βS9 to GSK3β was decreased compared to the control group (0.66±0.08 vs 1.02±0.14,t=2.217,P=0.025).Aβ31-35 decreased the viability of HT22 cells (71.85％±6.20％ in the Aβ31-35 group vs 98.14％±2.68％ in the control group,t=3.891,P=0.006),and the GSK3β inhibitor LiC1 pretreatment effectively reversed the decline of the viability induced by Aβ31-35 (90.74％±5.74％ in the LiCl+Aβ31-35 group vs 71.85％±6.20％ in the Aβ31-35 group,t=3.412,P=0.010).LiCl (in the LiCl+Aβ31-35 group) increased the expression of Bmal1 mRNA and BMAL1 protein significantly at CT20 compared with the Aβ31-35 group (Bmal1 mRNA:0.72±0.05 vs 0.38±0.06,t=4.378,P=0.001;BMAL1 protein:0.90±0.04 vs 0.67±0.04,t=4.052,P=0.002).Conclusion Increased GSK3β activity involved in the decreased expression of Bmal 1 induced by Aβ31-35 in HT22 cells.

Influence of high mobility group box-1 protein inhibition on protein expression of rage/toll-like receptor 4 signal passway in ht22 cells induced with aβ25-35 / 中国药理学与毒理学杂志

OBJECTIVE To investigate the influence of high mobility group box-1 protein (HMGB1) inhibition on the expression of receptors for advanced glycation end products (RAGE)/Toll-like receptor 4 (TLR4) signal passway related proteins, RAGE, TLR4, NF-kB, interieukin-1β (IL-1βand tumor necrosis factor-α(TNF-α) in HT22 cells stimulated with amyloid-β25-35 (Aβ25-35). METHODS HT22 cells were stimulated with Aβ25-350, 2.5, 5, 10, 20 and 40 ymol • L"1 for 24 h, and the vitality of HT22 cells was determined using MTT assay. Half maximal inhibitory concentration (IC50) was calculated. HT22 cells were divided into 5 groups: Normal cell control group, Apas-K 40 Mmol-L"' group, siRNA 50 Mmol-L"' group, siRNA or scramble siRNA+APas-as group (HT22 cells were transfected with siRNA or scramble siRNA at 50 pmol • L"' for 24 h, then treated with synthetic Apa-as at a final concentration of 40 pmol • L"1 for 24 h). The morphology of HT22 cells was observed under a microscope. The location of HMGB1 was observed by immunofluorescence. The protein expressions of HMGB1, RAGE, TLR4 and NF-kB P65 were detected by Western blotting. The levels of IL-IP and TNF-α in the culture supernatants were examined by ELISA. RESULTS The ICso of HT22 cells treated with Apas-as for 24 h was 41.17 Mmol-L-1, and A25-35 40 Mmol-L"1 was selected in subsequent experiments. After 24 h treatment with A25-35 40 nmol-L"', a large number of cells died, or aggregated into clusters, processes decreased, cell gaps increased, and HMGB1 was released from the nucleus to the outside. The protein expressions of HMGB1, RAGE, TLR4 and NF-kB P65 in HT22 cells were significantly increased (P<0.05) after treatment with Apas-as, and the levels of IL-1p and TNF-α in the culture supernatants were significantly increased (P<0.05). The protein expressions of HMGB1, RAGE, TLR4 and NF-kB P65 in HT22 cells were significantly decreased (P<0.05) after 24 h treatment with siRNA 50 pmol-L"' and the levels of IL-1p and TNF-α in the culture supernatants were significantly decreased (P<0.05). CONCLUSION RNA interference with HMGB1 reduces the expression of HMGB1, RAGE, TLR4 and NF-kB P65 in HT22 cells stimulated with A25-35

Focal Cerebral Ischemia Induces Decrease of Astrocytic Phosphoprotein PEA-15 in Brain Tissue and HT22 Cells / 한국실험동물학회지

PEA-15 is a small phosphoprotein (15 kDa) that is enriched in brain astrocytes. PEA-15 acts as an important modulator of cellular function including apoptosis and signal integration. This study investigated the expression of PEA-15 in focal cerebral ischemic injury. Cerebral ischemia was surgically induced in adult male rats by middle cerebral artery occlusion (MCAO), and brains were collected 24 hr after MCAO. A proteomic approach demonstrated decreases of PEA-15 protein spots in MCAO-operated animals in comparison to sham-operated animals. Western blot analysis clearly demonstrated that MCAO induces decreases in PEA-15 levels. We previously showed that glutamate toxicity induces cell death in a hippocampus-derived cell line (HT22). Glutamate exposure induces decreases of PEA-15 levels in HT22 cells. The results of this study suggest that focal cerebral ischemia induces cell death through downregulation of PEA-15 protein.

Focal Cerebral Ischemia Induces Decrease of Astrocytic Phosphoprotein PEA-15 in Brain Tissue and HT22 Cells / 한국실험동물학회지

PEA-15 is a small phosphoprotein (15 kDa) that is enriched in brain astrocytes. PEA-15 acts as an important modulator of cellular function including apoptosis and signal integration. This study investigated the expression of PEA-15 in focal cerebral ischemic injury. Cerebral ischemia was surgically induced in adult male rats by middle cerebral artery occlusion (MCAO), and brains were collected 24 hr after MCAO. A proteomic approach demonstrated decreases of PEA-15 protein spots in MCAO-operated animals in comparison to sham-operated animals. Western blot analysis clearly demonstrated that MCAO induces decreases in PEA-15 levels. We previously showed that glutamate toxicity induces cell death in a hippocampus-derived cell line (HT22). Glutamate exposure induces decreases of PEA-15 levels in HT22 cells. The results of this study suggest that focal cerebral ischemia induces cell death through downregulation of PEA-15 protein.

Cytoprotective Effects of Serum Hormone Deprivation against Glutamate Toxicity in HT22 Mouse Hippocampal Cells / 대한해부학회지

Reactive oxygen species (ROS) are important signaling molecules or mediators in many cellular responses, including the oxidative-burst defense response. Certain hormones are neuroprotective because they are modulators of neuronal activity or ROS scavengers. We have examined the effect of a hormone-free condition on ROS levels following glutamate-induced excitotoxicity in the mouse hippocampal HT22 cell line. We show that hormone starvation slightly elevates ROS and that continuous low concentrations of ROS induce expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1). In addition, N-acetyl-L-cysteine (NAC) restores the expression of ERK1/2 protein in hormone-starved HT22 cells. These findings suggest that whereas high-dose ROS are cytotoxic and lead to tissue damage in the brain low-dose ROS may act in neuroprotective signaling.