Anti-inflammatory and DNA Repair Effects of Astragaloside IV on PC12 Cells Damaged by Lipopolysaccharide

Objective This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide(LPS)and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses.Methods PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25,0.5,0.75,1,and 1.25 mg/mL for 24 h.Cell morphology was evaluated,and cell survival rates were calculated.A neurocyte inflammatory model was established with LPS treatment,which reached a 50%cell survival rate.PC12 cells were treated with 0.01,0.1,1,10,or 100µmol/L astragaloside IV for 24 h.The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments.NOS activity was detected by colorimetry;the expression levels of ERCC2,XRCC4,XRCC2,TNF-α,IL-1β,TLR4,NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting.The differentially expressed genes(DEGs)between the groups were screened using a second-generation sequence(fold change>2,P<0.05)with the following KEGG enrichment analysis,RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells.Results The viability of PC12 cells was not altered by treatment with 0.01,0.1,or 1µmol/L astragaloside IV for 24 h(P>0.05).However,after treatment with 0.5,0.75,1,or 1.25 mg/mL LPS for 24 h,the viability steadily decreased(P<0.01).The mRNA and protein expression levels of ERCC2,XRCC4,XRCC2,TNF-α,IL-1β,TLR4,NOS,and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h(P<0.01);however,these changes were reversed when PC12 cells were pretreated with 0.01,0.1,or 1µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h(P<0.05).Second-generation sequencing revealed that 1026 genes were upregulated,while 1287 genes were downregulated.The DEGs were associated with autophagy,TNF-α,interleukin-17,MAPK,P53,Toll-like receptor,and NOD-like receptor signaling pathways.Furthermore,PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2,CCL11,CCL7,MMP3,and MMP10,which are associated with the IL-17 pathway.RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results.Conclusion LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage.astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.

Neuroprotective Effects of Bushen Decoction Against Glutamate-Induced Neurotoxicity in PC12 Cells

Aim The enhanced effect of Bushen (Kidney-tonifying) decoction (BS) oncultured PC12 cell proliferation and its antagonistic action on neurotoxicity induced by glutamatewere investigated by serum pharmacological method of the Chinese material medica (CMM) in vitro.Methods The effect of BS on cultured PC12 cell activity and its antagonistic action on neurotoxicityinduced by glutamate was observed by MTT method. Flow cytometry and fluorescence microscopetechniques were employed to observe the antagonistic effect of BS on early period apoptosis of PC12cells induced by glutamate. Results The serum with BS was able to enhance activity of PC12 cells andexert antagonistic effect on glutamate-induced neurotoxicity. Meanwhile, these beneficial effectsproduced by BS were found to be the strongest in 20% concentration of in serum BS. Moreover, it caninhibit apoptosis of PC12 cells induced by glutamate , which occurs in the early period. ConclusionBS may exert a potential neuroprotective effect.

Protective effect of erythropoietin against 1-methyl-4-phenylpyridinium-induced neurodegenaration in PC12 cells

Objective The neuroprotective effect of erythropoietin （EPO） against 1-methyl-4-phenylpyridinium （MPP^＋）- induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated. Methods PC12 ceils impaired by MPP^＋ were used as the cell model of Parkinson＇s disease. Methyl thiazolyl tetrazolium （MTT） was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, and the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） assay was used to analyze the apoptosis ratio of PC 12 cells. The expression of Bcl-2 and Bax in PC 12 cells were examined by Western blot, and the reactive oxygen species （ROS）, the mitochondrial transmembrane potential and the activity of caspase-3 in each group were detected by spectrofluorometer. Results Treatment of PC12 cells with MPP^＋ caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. It was also shown that MPP＋ significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, EPO significantly reversed these responses and had the maximum protective effect at 1 U/mL. Conclusion The inhibitive effect of EPO on the MPP^＋ -induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity, and EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson＇s disease.

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells

Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

Dose Analysis of Methanol, Ethanol, Acetone and Glycerol to PC-12 Tumor Cells' Morphology and Growth

The morphologic changes and growth status of PC12 cells were observed after intervened by different concentrations of methanol, ethanol, acetone, glycerol and the toxic concentrations were ascertained. Four kinds of organic solvents al showed certain cytotoxicity to PC12 cells. Compared with other three kinds of or-ganic solvents, ethanol showed the most obvious cytotoxicity to PC12 cells and the cellviability would be reduced to 60% if the concentration of ethanol was 20 ml/L and the intervention lasted for 24 h. Under the same condition, the reduced per-centages of cellviability for acetone and ethanol were 20% and 15% respectively. Glycerol also showed cytotoxicity to PC12 cells, especial y as the concentration was raised gradual y, but the toxicity was relatively mild. This study would provide refer-ence material for subsequent pharmacological studies.

An effective vaccine against colon cancer in mice:Use of recombinant adenovirus interleukin-12 transduced dendritic cells

AIM： To investigate the effect of a vaccine with recombinant adenovirus interleukin-12 （AdVIL-12） transduced dendritic cells （DCs） against colon cancer in mice. METHODS： DCs and AdVIL-12 were incubated together at different time intervals and at different doses. Supernatant was collected and tested for IL-12 by enzyme-linked immunosorbent assay （ELISA）. In order to determine whether tumor cell lysate-pulsed （TP） AdVIL-12/DCs enhance therapeutic potential in the established tumor model, CT26 colon tumor cells were implanted subcutaneously （s.c.） in the midflank of naive BALB/c mice. Tumor-bearing mice were injected with a vaccination of CT26 TP AdVIL-12/DCs on d 3 and 10. As a protective colon tumor model, naive BALB/c mice were immunized s.c. in their abdomens with CT26 TP AdVIL-12/DCs twice at seven day intervals. After the immunization on d 7, the mice were challenged with a lethal dose of CT26 tumor cells and survival times were evaluated. Subsequently, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNy） secretion was evaluated in the immunized mice, and assayed CTL ex vivo. RESULTS： Murine DCs were retrovirally transduced with AdVIL-12 efficiency, and the AdVIL-12 transduced DCs secreted a high level of IL-12 （AdVIL-12/DCs, 615.27 ± 42.3 pg/mL vs DCs, 46.32 ± 7.29 pg/mL, P 〈 0.05）. Vaccination with CT26 TP AdVIL-12/DCs could enhance anti-tumor immunity against CT26 colon tumor in murine therapeutic models （tumor volume on d 19：CT26 TP AdVIL-12/DCs 107 ± 42 mm^3 vs CT26 TP DCs 383± 65 mm^3, P 〈 0.05） and protective models. Moreover, the CT26 TP AdVIL-12/DC vaccination enhances tumor-specific CTL activity, producing high levels of IFN7 in immunized mice. Ex vivo primed T cells with AdVIL-12/DCs were able to induce more effective CTL activity than in primed T cells with CT26 TP/DCs （E：T = 100：1, 69.49% ± 6.11% specific lysis vs 37.44% ＋ 4.32% specific lysis, P 〈 0.05）.CONCLUSION： Vaccination with recombinant AdVIL-12 transduced DC pulsed tumor cell lysate enhance antitumor immunity specific to colon cancer in mice.

High glucose causes apoptosis of rabbit corneal epithelial cells involving activation of PERK-eIF2α-CHOP-caspase-12 signaling pathway

AIM: To investigate the effect of high concentration of glucose(HCG) on double stranded RNA-activated protein kinase-like ER kinase(PERK)-eukaryotic initiation factor-2α(eIF2α)-transcription factor C/EBP homologous protein(CHOP)-cysteine aspartate specific proteinase(caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells(RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48 h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 k Da glucose-regulated protein 78(GRP78), CHOP, B-cell lymphoma 2(Bcl-2), B-cell lymphoma-2-associated X protein(Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eI F2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs(P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12(P<0.05), and the alterations caused by glucose were in concentration-and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups(P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group(P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits(P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eI F2α in the HCG group(P<0.05), while still did not affect the expression of PERK and eIF2α among groups(P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose(P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOPcaspase-12 signaling pathway and promotes apoptosis of RCECs.

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells

Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury.Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology,the mechanisms are not fully understood.To address this issue,we first co-cultured 1.5×10^5 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1,and then intervened with cobalt chloride(CoCl2)for 24 hours.Reactive oxygen species in PC12 cells was measured by Mito-sox.Mitochondrial membrane potential(ΔΨm)in PC12 cells was determined by JC-1 staining.Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining.Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy.Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry.Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria.Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells.CoCl2-induced PC12 cell damage was dose-dependent.Co-culture with mesenchymal stem cells significantly reduced apoptosis and restoredΔΨm in the injured PC12 cells under CoCl2 challenge.Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling,the disappearance of cristae,and chromatin margination in the injured PC12 cells.After direct co-culture,mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells.The transfer efficiency was greatly enhanced in the presence of CoCl2.More importantly,inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury.Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.

Ligustilide protects PC12 cells from oxygen-glucose deprivation/reoxygenation-induced apoptosis via the LKB1-AMPK-mTOR signaling pathway

Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;however,it is unclear whether this neuroprotective effect involves autophagy.In this study,PC12 cells were treated with 1×10^-5–1×10^-9 M LIG for 0,3,12 or 24 hours,and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Treatment with 1×10^-6 M LIG for 3 hours had the greatest effect on cell proliferation,and was therefore used for subsequent experiments.PC12 cells were pre-treated with 1×10^-6 M LIG for 3 hours,cultured in 95%N2/5%CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours,and then cultured normally for 16 hours,to simulate oxygen-glucose deprivation/reoxygenation(OGD/R).Cell proliferation was assessed with the MTS assay.Apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2 and Bax,autophagy-related proteins,Beclin 1 and microtubule-associated protein l light chain 3B(LC3-II),and liver kinase B1(LKB1)-5′-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway-related proteins were assessed by western blot assay.Immunofluorescence staining was used to detect LC3-II expression.Autophagosome formation was observed by electron microscopy.LIG significantly decreased apoptosis,increased Bcl-2,Beclin 1 and LC3-II expression,decreased Bax expression,increased LC3-II immunoreactivity and the number of autophagosomes,and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R.The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG.Taken together,our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by OGD/R via the LKB1-AMPK-mTOR signaling pathway.

Protection of PC12 Cells against Superoxide-induced Damage by Isoflavonoids from Astragalus mongholicus

Objective To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine （XA）/xanthine oxidase （XO）-induced injury to PC12 cells. Methods PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isofavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry. Results Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-β-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/XO-induced injury to PC12 cells. Their ECs0values of formononetin and calycosin were 0.05 μg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px）, while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 μg/mL and 50 μg/mL. Formononetin and calycosin had no significant infuence on Bcl-2 or Bax protein contents. Conclusion Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.

Synthesis of cystine C_(60) derivative and its protective effects on hydrogen peroxide-induced apoptosis in PC12 cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the usable ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene is a novel compound that behaves as a free radical scavenger with excellent biology consistent. In the present study, we have synthesized and characterized a novel cystine C60 derivative for the first time, and investigated the effects on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by MTT, PI/Hoechst 33342 staining and flow cytometry analysis. These results suggested that cystine C60 derivative has the potential to prevent oxidative stress-induced cell death and has no evident toxicity.

Edaravone protects PC12 cells from ischemic-like injury via attenuating the damage to mitochondria

Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.

Long non-coding RNA GAS5 promotes PC12 cells differentiation into Tuj1-positive neuron-like cells and induces cell cycle arrest

Growth arrest-specific 5 (GAS5) is an anti-oncogene that has been extensively studied in tumors. However, research on GAS5 in the context of nervous system disease is rare at present. This study aimed to investigate the role of the long non-coding RNA GAS5 in rat pheochromocytoma cells (PC12 cells). GAS5-overexpressing lentivirus was transfected into PC12 cells, and expression levels of GAS5 and C-myc were detected by real-time PCR. Ratios of cells in S phase were detected by 5-ethynyl-2′-deoxyuridine. Immunohistochemical staining was used to detect the immunoreactivity of neuron microtubule markers Tuj1, doublecortin, and microtubule-associated protein 2. Apoptosis was detected by flow cytometry, while expression of acetylcholine in cells was detected by western blot assay. We found that GAS5 can promote PC12 cells to differentiate into Tuj1-positive neuron-like cells with longer processes. In addition, cell proliferation and cell cycle were significantly suppressed by GAS5, whereas it had no effect on apoptosis of PC12 cells. Our results indicate that GAS5 could increase the expression of choline acetyltransferase and acetylcholine release. Thus, we speculate that GAS5 is beneficial to the recovery of neurons and the cholinergic nervous system.

Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

The killing effects of lymphocytes on Hela cells expressing interleukin-12 （IL-12） in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL- 12 between 24 h and 72 h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express 1L-12 and were more easily killed by lymphocytes.

Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson＇s Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal a-synuclein accumulation in cells Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

Neuroprotective Effects of Dexmedetomidine Preconditioning on Oxygen-glucose Deprivation-reoxygenation Injury in PC12 Cells via Regulation of Ca^2+-STIM1/Orail Signaling

Summary:Dexmedetomidine(DEX),a potent and highly selective agonist for a2-adrenergic receptors(a2AR),exerts neuroprotective effects by reducing apoptosis through decreased neuronal Ca^2+influx.However,the exact action mechanism of DEX and its effects on oxygen-glucose deprivation-reoxygenation(OGD/R)injury in vitro are unknown.We demonstrate that DEX pretreatment reduced OGD/R injury in PC12 cells,as evidenced by decreased oxidative stress,autophagy,and neuronal apoptosis.Specifically,DEX pretreatment decreased the expression levels of stromal interaction molecule 1(STIM1)and calcium release-activated calcium channel protein 1(Orail),and reduced the concentration of intracellular calcium pools.In addition,variations in cytosolic calcium concentration altered apoptosis rate of PC12 cells after exposure to hypoxic conditions,which were modulated through STIM 1/Orail signaling.Moreover,DEX pretreatment decreased the expression levels of Beclin-1 and microtubule-associated protein 1A/1B-light chain 3(LC3),hallmark markers of autophagy,and the formation of autophagosomes.In conclusion,these results suggested that DEX exerts neuroprotective effects against oxidative stress,autophagy,and neuronal apoptosis afier OGD/R injury via modulation of Caf-STIM1/Orai1 signaling.Our results offer insights into the molecular mechanisms of DEX in protecting against neuronal ischemia-reperfusion injury.

Mechanisms of rotenone-induced neurotoxicity in PC 12 cells

BACKGROUND： Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson＇s disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson＇s disease remains unclear. OBJECTIVE： To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING： A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS： PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS： PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco＇s modified eagle＇s medium （DMEM）, was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES： Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry （MALDI-TOF-MS） was used. RESULTS： In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition （P 〈 0.01）. Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition （P 〈 0.01）, as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition （P 〈 0.01）. By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION： γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.

Enzyme-digested Colla Corii Asini(E’jiao) prevents hydrogen peroxide-induced cell death and accelerates amyloid beta clearance in neuronal-like PC12 cells

As an aging-associated degenerative disease,Alzheimer’s disease is characterized by the deposition of amyloid beta(Aβ),oxidative stress,inflammation,dysfunction and loss of cholinergic neurons.Colla Corii Asini(CCA)is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging.CCA might delay aging-induced degenerative changes in neurons.In the present study,we evaluated antioxidant activity,cytoprotective effects,and Aβremovability of enzyme-digested Colla Corii Asini(CCAD).Oxygen radical absorbance capacity(ORAC)activity assay showed that,as compared to gelatins from the skin of porcine,bovine and cold water fish,CCA exhibited the highest ORAC activity.The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage.Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008,2013,2017 and gelatin from cold water fish skin,CCA manufactured in 2008 presented the smoothest surface structure.We further tested the protective effects of CCAD(manufactured in 2008)and enzyme-digested gelatin from cold water fish skin(FGD)on hydrogen peroxide(H2O2)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells.Presto blue assay showed that both FGD and CCAD at 0.5 mg/m L increased cell viability in H2O2-treated neuronal-like PC12 cells.The protection of CCAD was significantly superior to that of FGD.Acetylcholinesterase(Ach E)assay showed that both FGD and CCAD inhibited Ach E activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1%and 74.5%of that in non-treated cells,respectively.The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine.Although CCAD inhibited Ach E activity in neuronal-like PC12 cells,CCAD prevented H2O2-induced abnormal deterioration of Ach E.ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aβ1–42-treated neuronal-like PC12 cells,suggesting that CCAD can enhance Aβclearance.Our results suggest that CCA might be useful for preventing and treating Alzheimer’s disease.

IMMUNOTHERAPY OF SPONTANEOUS METASTATIC LUNG CANCER WITH TUMOR ANTIGEN-PULSED, INTERLEUKIN-12 GENE-MODIFIED DENDRITIC CELLS

Objective: To investigate the treatment of spontaneous metastatic lung cancer by tumor antigen-pulsed, interleukin-12 (IL-12) gene-modified dendritic cells (DC). Methods:The spontaneous metastatic lung cancer model, prepared by injection of the 3LL Lewis lung cancer cells into the footpads of C57BL/6 mice, was treated by subcutaneous vaccination with tumor antigen peptide mut1-pulsed, IL-12 gene-modified dendritic cells (DC-IL-12/mut1) derived from the normal bone morrow. After treatment, the lung weight, the number of lung metastatic nodes and the survival time of the tumor-bearing mice were observed, and the NK and CTL activity were determined respectively. The mice were divided into 8 groups with 12 mice in each group. Results: Compared with mice treated with mut1-pulsed, control LacZ gene modified DC and untreated DC, tumor-bearing mice treated with DC-IL-12/mut1 had the lightest lung weights (P<0.01), the least lung metastatic node number (P<0.01), the longest survival time (P<0.01), also with the induction of potent CTL activity (P<0.01) and NK activity (P<0.01). Conclusion: Tumor antigen-pulsed, IL-12 gene-modified dendritic cells have significant therapeutic effects on the spontaneous metastatic lung cancer, providing a new approach to treatment of lung tumors.

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells(DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium(DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor(NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2(MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction(qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors(NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCsCM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls;however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.