Adipose-derived mesenchymal stem cell-derived exosomes alleviate hydrogen peroxide-induced PC12 cell apoptosis / 中国组织工程研究

BACKGROUND:Mesenchymal stem cell-derived exosomes may play a crucial role in tissue damage repair,and miRNA is an important component of exosomes for therapeutic effects.Among them,miR-29b-3p has the effect of reducing cell apoptosis,promoting axonal regeneration,and angiogenesis. OBJECTIVE:To study the protective effect of adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p on a neural cell injury model simulated by H2O2-treated PC12 cells,and explore the relevant mechanisms. METHODS:(1)First,the collagenase digestion method was used to extract rat adipose-derived mesenchymal stem cells.Adipose-derived mesenchymal stem cells were transfected with miR-29b-3p mimics and inhibitors.Exosomes were extracted from the culture supernatant by ultracentrifugation and identified so as to construct adipose-derived mesenchymal stem cell-derived exosomes with high expression and knockdown miR-29b-3p.(2)By constructing a neural cell injury model simulated by PC12 cells treated with H2O2,the relevant mechanisms of the protective effect of adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p on the simulated neuronal cell injury model were studied. RESULTS AND CONCLUSION:(1)Adipose-derived mesenchymal stem cell-derived exosome had a typical cup-shaped shape and a diameter distribution in the range of 50-140 nm,expressed membrane proteins Alix,CD63,and TSG101,which were specific markers on the surface of exosomes,and could be successfully ingested by PC12 cells.(2)Adipose-derived mesenchymal stem cell-derived exosome pretreatment could reduce cell apoptosis induced by H2O2 treatment in PC12 cells,and this protective effect was enhanced with the increase of miR-29b-3p expression in the exosomes and weakened with the decrease of miR-29b-3p expression in the exosomes.The mechanism of its effect was related to adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p promoting the expression of anti-apoptotic protein Bcl-2 and inhibiting the expression of apoptotic protein Bax.

Effects of Harmine Inducing Apoptosis on Mitochondrial Fusion and Division in PC 12 Cells / 医药导报

Objective To investigate the effects of harmine(HM)on the expression level of mitochondrion fusion related proteins and mitochondrial function injury in PC 12 cells.Methods PC 12 cells were divided into cell control group,HM group,mitochondrion mitosis inhibitor Mdivi-1 group,HM+Mdivi-1 group,mitochondrion fission agonist WY14643 group,HM+WY14643 group,with drug concentrations of 1,10,25,50,100 μmol·L-1.After 24 h treatment,the MTT method was used to detect the cell survival rate,and a microscope was used to observe the cell morphology,MitoTracker Red probe staining was used to observe the mitochondrial morphology and the length ratio of vertical and horizontal axes,JC-1 staining was used to detect the mitochondrial membrane potential,and a kit was used to detect ATP level and lactate dehydrogenase(LDH)activity.Immunofluorescence staining and Western blotting were used to assess the expression levels of caspase-3,apoptosis-promoting protein(Bax)cytochrome C(cyt-c),mitochondrial fusion protein(Mfn2)and mitochondrial mitotic protein(Drp-1).The interference sequence of Drp1 was transfected by electroporation,and the siRNA sequence with good transfection effect was screened.The related indicators were detected by fluorescence method,MTT method,and immunoblotting method in cooperation with drug intervention.Results MTT results showed that compared with the cell control group,the survival rate of HM group,Mdivi-1 group,HM+Mdivi-1 group,WY14643 group and HM+WY14643 group decreased significantly(P＜0.01),and the EC50 were(11.48±2.32),(12.35±1.67),(14.88±2.07),(39.14±3.25),(20.09±1.97),respectively.According to this,subsequent experiments selected 20 μmol·L-1for HM,WY 14643 and HM+WY14643 as working concentrations to construct PC 12 cell model.Microscopic observation and MitoTracker Red probe staining showed that the cell density in the drug group decreased in varying degrees,and a transition from branched to round morphology in the drug-treated groups was observed.The morphology of mitochondria tended to be round,and the ratio of the length of the longitudinal axis to transverse axis was(3.33±0.72)in the cell control group,(2.19±0.58)in the HM group,(2.45±0.44)in Mdivi-1 group,and(1.43±0.62)in HM+Mdivi-1 group,respectively.The results of JC-1 staining showed that compared with the cell control group,the mitochondrial mode potential of the HM group significantly decreased(P＜0.01).ROS significantly increased(P＜0.01)and ATP levels decreased(P＜0.01),and LDH enzyme activity increased(P＜0.01).Immunofluorescence staining and Western blotting results showed that compared with the cell control group,the expression levels of proapoptotic proteins Bax,cytochrome C,and caspase-3 in the HM group were significantly increased(all P＜0.01).Compared with the cell control group,the expression level of mitochondrial fission related protein Drp1 in HM group was significantly higher(P＜0.01).The expression level of mitochondrial fusion related protein Mfn2 significantly decreased(P＜0.01).After specific interference with Drp1 and synergistic intervention with HM,the survival rate of PC 12 cells in each interference group decreased compared to each drug intervention group.The expression of Drp1 and Mfn2 was downregulated,and the differences were statistically significant(P＜0.05 or P＜0.01).Conclusion HM can reduce the mitochoudrial membrane potential and ATP levels by accumulating ROS,there by activating the caspase-3 apoptosis pathway and promoting cell apoptosis.Mitochondrial fusion division may be involved in the damage of PC12 cells caused by HM,initiating apoptosis through the mitochondrial pathway.

Effects of tricholoma matsutake polysaccharides on 1-methy-4-pehnyl-pyridine ion-induced PC12 cell damage / 解剖学报

Obｊective To investigate the protective mechanism of tricholoma matsutake polysaccharides(TMP) against 1-methy-4-pehnyl-pyridine ion (MPP

Protective effect of saikosaponin b2 on corticosterone induced PC12 cell injury based on cell metabonomics / 中国药理学与毒理学杂志

OBJECTIVE To study the protective effect of saikosaponin b2(SSb2)on corticosterone(CORT)induced PC12 cell injury and its mechanism.METHODS ① PC12 cells were divided into the cell control group(24 h of culture with RPMI-1640 medium),CORT group(24 h of culture with CORT 100-800 μmol·L-1)and SSb2 group(24 h of culture with SSb2 1.5625,3.125,6.25,12.5,25,50 and 100 μmol·L-1).MTT assay was used to detect the cell survival rate.②PC12 cells were divided into the cell control group(24 h of culture with RPMI 1640 medium),model group(24 h of culture with CORT 400 μmol·L-1),and model+SSb2 group(3 h pretreatment with SSb2 1.5625,3.125,6.25,12.5 and 25 μmol·L-1,removal of the supernatant before cells were co-incubated with CORT 400 μmol·L-1 and corresponding concentrations of SSb2 for 24 h).MTT assay was used to detect the cell survival rate while micro-plate assay was used to detect the lactate dehydrogenase(LDH)leakage rate of PC12 cells.③PC12 cells were divided into the cell control group,model group and model+SSb2 12.5 μmol·L-1 group.AnnexinV-FITC/PI flow cytometry assay was used to detect PC12 cell apoptosis,ultra-perfor-mance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)cell metabonomics was used to detect metabolic profile changes and colorimetric assay was employed to detect the glutamic acid content and glutaminase activity in PC12 cells.RESULTS Compared with the cell control group,the cell viability decreased to(55±6)%(P<0.01)when the concentration of CORT was 400 μmol·L-1.When the concentration of SSb2 was higher than 50 μmol·L-1,there was significant toxicity to PC12 cells(P<0.01).②Compared with the cell control group,the cell survival rate was signif-icantly decreased(P<0.01),while the release rate of LDH was significantly increased(P<0.01)in the model group.Compared with the model group,the cell survival rate significantly increased(P<0.05,P<0.01),while the LDH release rate significantly decreased(P<0.01)in the model+SSb2 group.③ Com-pared with the cell control group,cell apoptosis was significantly increased in the model group(P<0.05).Compared with the model group,cell apoptosis was significantly decreased(P<0.05)in the model+ SSb2 group.Metabolomics results show that SSb2 significantly back-regulated nine differential metabo-lites of glutamate,creatine,N-acetylaspartate,L-tyrosine,citric acid,L-isoleucine,lactic acid,glutamine and choline.Further network analysis of the key metabolites regulated by SSb2 yielded five major metabolic pathways:D-glutamine and D-glutamate metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,alanine,aspartate and glutamate metabolism,tyrosine metabolism and arginine biosynthesis.Compared with the cell control group,the content of glutamate and activity of glutaminase were significantly decreased in the model group(P<0.01).Compared with the model group,the content of glutamate(P<0.01)and activity of glutaminase(P<0.05)were significantly increased in the model+SSb2 group.CONCLUSION SSb2 has a neuroprotective effect on CORT-injured PC12 cells,and the mechanism of which is related to inhibition of apoptosis and regulation of metabolic disorders.

Effects of latroeggtoxin-VI on dopamine and α-synuclein in PC12 cells and the implications for Parkinson's disease

Background Parkinson's disease (PD) is characterized by death of dopaminergic neurons leading to dopamine deficiency, excessive α-synuclein facilitating Lewy body formation, etc. Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin discovered from the eggs of spider L. tredecimguttatus, was previously found to promote the synthesis and release of PC12 cells, showing a great potential as a drug candidate for PD. However, the relevant mechanisms have not been understood completely. The present study explored the mechanism underlying the effects of LETX-VI on dopamine and α-synuclein of PC12 cells and the implications for PD. Results After PC12 cells were treated with LETX-VI, the level of dopamine was significantly increased in a dose-dependent way within a certain range of concentrations. Further mechanism analysis showed that LETX-VI upregulated the expression of tyrosine hydroxylase (TH) and L-dopa decarboxylase to enhance the biosynthesis of dopamine, and downregulated that of monoamine oxidase B to reduce the degradation of dopamine. At the same time, LETX-VI promoted the transport and release of dopamine through modulating the abundance and/or posttranslational modification of vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT). While the level of dopamine was increased by LETX-VI treatment, α-synuclein content was reduced by the spider toxin. α-Synuclein overexpression significantly decreased the dopamine level and LETX-VI efficiently alleviated the inhibitory action of excessive α-synuclein on dopamine. In the MPTP-induced mouse model of PD, application of LETX-VI ameliorated parkinsonian behaviors of the mice, and reduced the magnitude of MPTP-induced α-synuclein upregulation and TH downregulation. In addition, LETX-VI displayed neuroprotective effects by inhibiting MPTP-induced decrease in the numbers of TH-positive and Nissl-stained neurons in mouse brain tissues. Conclusions All the results demonstrate that LETX-VI promotes the synthesis and release of dopamine in PC12 cells via multiple mechanisms including preventing abnormal α-synuclein accumulation, showing implications in the prevention and treatment of PD.

Inhibitory Effect of Sophora alopecuroides Total Alkaloids and Two Monomer Alkaloids on PC12 Cell Activity / 中国实验方剂学杂志

ObjectiveTo compare the effects of total alkaloids， matrine， and sophoridine extracted from Sophora alopecuroides on the activity of pheochromocytoma cells （PC12 cells）. MethodThe effect of S. alopecuroides total alkaloids， matrine， and sophoridine at concentrations of 2， 1， 0.5， 0.25， 0.125， and 0.062 5 g·L-1 on the proliferation of PC12 cells was evaluated using the 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyl tetrazolium bromide （MTT） assay. The lactate dehydrogenase （LDH） leakage rate was measured by enzyme-linked immunosorbent assay （ELISA）. Flow cytometry was used to assess cell apoptosis rate， cell cycle distribution， and intracellular Ca2+ levels. Real-time quantitative polymerase chain reaction （Real-time PCR） was performed to determine the mRNA transcription levels of B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. Protein expression levels of apoptosis-related proteins Caspase-3， Caspase-8， Bcl-2， and Bax were detected by Western blot. ResultCompared to the control group， S. alopecuroides total alkaloids， matrine， and sophoridine inhibited the proliferation of PC12 cells， increased LDH leakage rate， enhanced intracellular Ca2+ fluorescence intensity， and induced cell apoptosis in concentration-dependent manner （P<0.05， P<0.01）. Among them， S. alopecuroides total alkaloids had the strongest inhibitory effect on cell proliferation and induction of apoptosis in PC12 cells （P<0.01）. After treatment with S. alopecuroides total alkaloids， matrine， and sophoridine， the cell cycle progression of PC12 cells was arrested at G1/G0 in the S. alopecuroides total alkaloids group， and at G1/S in the matrine and sophoridine groups. The expression levels of Bax mRNA were significantly increased （P<0.05， P<0.01）， while the expression levels of Bcl-2 mRNA were significantly decreased （P<0.05， P<0.01）. All treatments significantly downregulated the expression of the anti-apoptotic protein Bcl-2 （P<0.05， P<0.01） and upregulated the expression of the pro-apoptotic proteins Bax， Caspase-3， and Caspase-8 （P<0.05， P<0.01）， with the most significant protein expression changes observed in the S. alopecuroides total alkaloids group. ConclusionAmong the S. alopecuroides total alkaloids， matrine， and sophoridine， S. alopecuroides total alkaloids exhibit the strongest inhibitory effect on the activity of PC12 cells， and its mechanism of action may be related to the inhibition of anti-apoptotic protein expression， upregulation of pro-apoptotic protein expression， and activation of the mitochondrial Caspase-8 apoptotic pathway.

Molecular mechanism of ligustilide attenuating OGD/R injury in PC12 cells by inhibiting ferroptosis / 中国中药杂志

The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.

Study on Anti-Depression Active Ingredients of Bupleuri Radix Based on UPLC-Q-TOF/MS Integrated with CORT-Induced Poorly Differentiated PC12 Depression Cell Model / 世界科学技术-中医药现代化

Objective To analyze the chemical constituents in the water extract of Bupleuri Radix and investigate the active ingredients of Bupleuri Radix for the treatment of depression.Methods The chemical constituents in the water extract of Bupleuri Radix were identified by Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS).CORT-induced poorly differentiated PC12 depression cell model was launched,and PC12 cells were pretreated with monomeric compounds from Bupleuri Radix for 24 h.The cell viability and LDH release rate were measured by CCK-8 assy kit and LDH assay kit,respectively.Results A total of 53 compounds were identified in the water extract of Bupleuri Radix,mainly including type Ⅰ,type Ⅱ and type Ⅲsaikosaponins.Among them,saikosaponin A,saikosaponin B2,saikosaponin C,saikosaponin E,saikosaponin F and 6″-acetyl saikosaponin A contributed the most to the metabolite profile of Bupleuri Radix,and could improve the viability of CORT-induced PC12 cells(P<0.05,P<0.01).Furthermore,saikosaponin A,saikosaponin B2,saikosaponin C,saikosaponin E and saikosaponin F could decrease the LDH release rate of CORT-induced PC12 cells(P<0.05,P<0.01).Conclusion The major anti-depression active ingredients in Bupleuri Radix may be Saikosaponin A,saikosaponin B2,saikosaponin C,saikosaponin E and saikosaponin F,which lays a foundation for the research of the quality control and pharmacodynamic material basis of Bupleuri Radix.

Role of extracellular signal-regulated kinase 1/2 in glutamate-induced ferroptosis in PC12 cells / 中华麻醉学杂志

Objective:To evaluate the role of extracellular signal-regulated kinase (ERK)1/2 in glutamate-induced ferroptosis in PC12 cells.Methods:PC12 cells were divided into 6 groups ( n=21 each) using a random number table method: control group (C group), glutamategroup (Glu group), glutamate+ ERK1/2 over-expression group (Glu+ ERK1/2-OE group), glutamate+ ERK1/2 plasmid empty vector group (Glu+ Vec group), glutamate+ ERK1/2 knockdown group (Glu+ si-ERK1/2 group)and glutamate+ ERK1/2 SiRNA negative control group (Glu+ si-NC group). Cells were treated with glutamate at a final concentration of 6 mmol/L for 72 h in Glu group and with the equal volume of PBS buffer for 72 h in C group. Glu+ ERK1/2-OE group was transfected with ERK1/2 overexpression plasmid, Glu+ Vec group was transfected with plasmid empty vector, and Glu+ si-ERK1/2 group was transfected with ERK1/2 siRNA, Glu+ si-NC group was transfected with siRNA negative control for 48 h, and then glutamate at a final concentration of 6 mmol/L was added and cells were treated for 72 h. The cell viability, lactic dehydrogenase (LDH)activity and contents of glutathione (GSH), ferrous ions and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Mitochondrial membrane potential (MMP) and lipid reactive oxygen species (Lip-ROS) were measured by flow cytometry. Results:Compared with C group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, ferrous ions content, MDA content and Lip-ROS levels were increased in Glu group ( P<0.05). Compared with Glu+ Vec group, the cell viability, GSH content and MMP were significantly increased, and the activity of LDH, contents of ferrous ions and MDA, and Lip-ROS levels were decreased in Glu+ ERK1/2-OE group( P<0.05). Compared with Glu+ si-NC group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, contents of ferrous ions and MDA, and Lip-ROS level were increased in Glu+ si-ERK1/2 group ( P<0.05). Conclusions:ERK1/2 is involved in glutamate-induced ferroptosis in PC12 cells.

Hippo-YAP pathway is involved in the effect of NaAsO / 中国药理学通报

Aim To explore the effects of NaAsO

Effect of bergapten on glucose and oxygen deprivation induced PC12 cell injury by regulating long non-coding RNA opioid receptor gene expression / 解剖学报

Objective To study whether bergapten (BG) protects PC12 cells from oxygen-glucose deprivation (OGD) induced cell injury by regulating long non-coding RNA (lncRNA) opioid receptor gene (Oprm1) expression. Methods PC12 cells were divided into control (Con) group, OGD group, OGD+ low concentration BG (BG-L) group, OGD+medium concentration BG (BG-M) group, OGD + high concentration BG (BG-H) group, OGD + pcDNA group, OGD+pcDNA-Oprm1 group, OGD+BG+si-NC group, OGD+BG+si-Oprm1 group. The malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were measured by the kits. Cell apoptosis rate was analysed by flow cytometry. The expression level of Oprm1 was analysed by Real-time PCR. Results Compared with the Con group, the apoptosis rate and MDA content of PC12 cells in OGD group increased significantly, whereas Oprm1 expression, SOD and GSH-Px activity decreased significantly (P < 0. 05). Compared with the OGD group, the apoptosis rate and MDA content of PC12 cells in the OGD + BG-L group, OGD + BG-M group, OGD + BG-H group were significantly reduced, whereas the Oprm1 expression, SOD and GSH-Px activities increased significantly (P < 0. 05). Compared with the OGD+pcDNA group, the apoptosis rate and MDA content of the PC12 cells in the OGD+pcDNA-Oprm1 group reduced significantly, whereas the SOD and GSH-Px activities increased significantly (P<0. 05). Compared with the OGD+BG+si-NC group, the apoptosis rate and MDA content of PC12 cells in the OGD+BG+si-Oprm1 group increased significantly, whereas the SOD and GSH-Px activities decreased significantly (P < 0. 05). Conclusion Bergapten may alleviate OGD-induced PC12 cell injury, which is correlated to the up-regulation of lncRNA Oprm1 expression.

Investigation on improvement effects and mechanism of 7-hydroxyethyl chrysin on PC12 cell injury induced by hypobaric hypoxia / 中国药房

OBJECTIVE To investigate the improvement effects and possible mechanism of 7-hydroxyethyl chrysin （7-HEC） on PC12 cell injury induced by hypobaric hypoxia. METHODS The rat adrenal pheochromocytoma cell line PC12 was cultured under low-pressure hypoxia （5%CO2， 94%N2， 1%O2， 54 004 Pa） to investigate the different concentrations of 7-HEC （100， 10， 1， 0.1， 0.01 μmol/L） on the survival rate of hypoxic cells； the effects of 7-HEC（1 μmol/L） on the contents of lactate dehydrogenase （LDH） and malondialdehyde （MDA）， superoxide dismutase （SOD） activity， apoptotic rate， cell cycle， and the expressions of cleaved caspase-3， Bcl-2 and Bax were detected. RESULTS Compared with control group， the survival rate of cells in hypobaric hypoxia group was decreased significantly （P＜0.01）； 10， 1， 0.1 μmol/L 7-HEC could reverse the decrease of cell survival rate caused by hypobaric hypoxia （P＜0.05 or P＜0.01）. Compared with control group， LDH content in supernatant， MDA content in cells， apoptotic rate， the proportion of cells at G1 stage and the protein expression of cleaved caspase-3 were increased significantly in hypobaric hypoxia group， while SOD activity in cells， the proportion of cells at S stage and G2 stage and Bcl-2/Bax ratio were decreased significantly （P＜0.05 or P＜0.01）. Compared with hypobaric hypoxia group， the contents of LDH and MDA， apoptotic rate， the proportion of cells at G1 stage and the expression of cleaved caspase-3 in 7-HEC group were decreased significantly， while SOD activity， the proportion of cells at G2 stage and Bcl-2/Bax ratio were increased significantly （P＜ 0.01）. CONCLUSIONS 7-HEC can significantly increase the survival rate of hypobaric hypoxia cells， reduce the LDH content in supernatant， improve cell cycle arrest， and reduce the rate of apoptosis. Its improvement effects on hypobaric hypoxia cell injury may be related to the inhibition of caspase-3/Bax/Bcl-2 pathway activation.

Huangdi Anxiao Capsules-containing serum protects cell model from cognitive dysfunction in diabetes via inhibiting NLRP3-mediated pyroptosis / 中国中药杂志

This study aims to investigate the effects and the molecular mechanism of Huangdi Anxiao Capsules(HDAX)-containing serum in protecting the rat adrenal pheochromocytoma(PC12) cells from diabetes-associated cognitive dysfunction induced by high glucose and whether the mechanism is related to the regulation of NOD-like receptor thermal protein domain associated protein 3(NLRP3)-mediated pyroptosis. The PC12 cell model of diabetes-associated cognitive dysfunction induced by high glucose was established and mcc950 was used to inhibit NLRP3. PC12 cells were randomized into control, model, HDAX-containing serum, mcc950, and HDAX-containing serum+mcc950 groups. Methyl thiazolyl tetrazolium(MTT) assay was employed to determine the viability, and Hoechst 33258/PI staining to detect pyroptosis of PC12 cells. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1 beta(IL-1β) and IL-18. Western blot was employed to determine the protein levels of postsynaptic density protein 95(PSD-95), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), GSDMD-N, and cleaved cysteinyl aspartate specific proteinase-1(caspase-1), and RT-PCR to determine the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1. The immunofluorescence assay was adopted to measure the levels and distribution of NLRP3 and GSDMD-N in PC12 cells. Compared with the control group, the model group showed decreased cell proliferation, increased PI positive rate, down-regulated protein level of PSD-95, up-regulated protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1, up-regulated mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and elevated levels of IL-1β and IL-18. Compared with the model group, HDAX-containing serum, mcc950, and the combination of them improved cell survival rate and morphology, decreased the PI positive rate, down-regulated the protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1 and the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and promoted the secretion of IL-1β and IL-18. The findings demonstrated that HDAX-containing serum can inhibit the pyroptosis-mediated by NLRP3 and protect PC12 cells from the cognitive dysfunction induced by high glucose.

Study on molecular mechanism of etomidate by promoting expression of miR-142-3p to reduce hypoxia-induced neuro-inflammatory response and cell apoptosis in PC12 cells / 中国免疫学杂志

Objective:To investigate whether etomidate affects inflammatory response and apoptosis of PC12 cells induced by hypoxia by regulating miR-142-3p.Methods:PC12 cells were pretreated with different doses(2,6,12 μmol/L)of etomidate to establish hypoxia model;PC12 cells that transfected with miR-142-3p mimics or inhibitors were pretreated with 0 or 12 μmol/L of etomidate to establish hypoxia model.Cell viability,apoptosis and protein(CyclinD1,Cleaved-caspase-3)expressions were detected by CCK-8 method,flow cytometry and Western blot,respectively.ELISA was used to detect levels of inflammatory factors TNF-α,IL-1β,IL-6.Expression of miR-142-3p was detected by RT-qPCR.Results:Etomidate increased hypoxia-induced PC12 cells activity and expres-sion of CyclinD1 protein and miR-142-3p,while decreased cell apoptosis rate,Cleaved-caspase-3 protein expression and levels of inflammatory factors TNF-α,IL-1β,IL-6(P<0.05).Up-regulation of miR-142-3p increased activity and expression of CyclinD1 pro-tein of hypoxia-induced PC12 cells,while decreased cell apoptosis rate,Cleaved-caspase-3 protein expression and levels of inflamma-tory factors TNF-α,IL-1β,IL-6(P<0.05).Down-regulation of miR-142-3p reversed effects of etomidate on hypoxia-induced PC12 cell activity,apoptosis and expressions of inflammatory factors(P<0.05).Conclusion:Etomidate can reduce inflammatory response and apoptosis of PC12 cells induced by hypoxia,and its mechanism may be related to the up-regulation of miR-142-3p expression in cells.

Biocompatible chitosan/polyethylene glycol/multi-walled carbon nanotube composite scaffolds for neural tissue engineering / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Carbon nanotube (CNT) composite materials are very attractive for use in neural tissue engineering and biosensor coatings. CNT scaffolds are excellent mimics of extracellular matrix due to their hydrophilicity, viscosity, and biocompatibility. CNTs can also impart conductivity to other insulating materials, improve mechanical stability, guide neuronal cell behavior, and trigger axon regeneration. The performance of chitosan (CS)/polyethylene glycol (PEG) composite scaffolds could be optimized by introducing multi-walled CNTs (MWCNTs). CS/PEG/CNT composite scaffolds with CNT content of 1%, 3%, and 5% (1%=0.01 g/mL) were prepared by freeze-drying. Their physical and chemical properties and biocompatibility were evaluated. Scanning electron microscopy (SEM) showed that the composite scaffolds had a highly connected porous structure. Transmission electron microscope (TEM) and Raman spectroscopy proved that the CNTs were well dispersed in the CS/PEG matrix and combined with the CS/PEG nanofiber bundles. MWCNTs enhanced the elastic modulus of the scaffold. The porosity of the scaffolds ranged from 83% to 96%. They reached a stable water swelling state within 24 h, and swelling decreased with increasing MWCNT concentration. The electrical conductivity and cell adhesion rate of the scaffolds increased with increasing MWCNT content. Immunofluorescence showed that rat pheochromocytoma (PC12) cells grown in the scaffolds had characteristics similar to nerve cells. We measured changes in the expression of nerve cell markers by quantitative real-time polymerase chain reaction (qRT-PCR), and found that PC12 cells cultured in the scaffolds expressed growth-associated protein 43 (GAP43), nerve growth factor receptor (NGFR), and class III β‍-tubulin (TUBB3) proteins. Preliminary research showed that the prepared CS/PEG/CNT scaffold has good biocompatibility and can be further applied to neural tissue engineering research.

Effect of baicalein on the expression of glutamate receptor related protein in PC12 cells damaged by Aβ 25-35 / 国际中医中药杂志

Objective:To study the effect of baicalein on the expression of glutamate receptor related protein in PC12 cells injured by Aβ 25-35. Methods:PC12 cells were divided into control group, model group, estradiol group and baicalein group with different concentrations. The survival condition of PC12 cells in each group were detected by thiazole blue (MTT). PC12 cells were divided into control group, model group, estradiol group and baicalein group. The control group and model group were cultured with DMEM medium, and the estradiol group was added with 1×10 -3 μmol/L estradiol DMEM medium, baicalein group was added with 1 μmol/L baicalein DMEM medium. After 2 hours of intervention, 20 μmol/L Aβ 25-35 was added to the model group, estradiol group and baicalein group with induced PC12 cell injury. After 22 hours of intervention, flow cytometry was used to detect the apoptosis of PC12 cells. The expression of estrogen receptor β (ER β), phosphorylated c-Jun N-terminal kinase (p-JNK/JNK) and ionic receptor N-methyl-D-aspartate receptor 1 (NMDAR1), glutamate receptor 2 (GluR2) and calcium/calmodulin dependent protein kinase Ⅱ (CaMK Ⅱ) were detected by Western blot. Results:Compared with model group, 1 μmol/L baicalein significantly increased the proliferation rate [(95.80±2.47)% vs. (64.34±3.84)%]. The apoptosis rate of PC12 cells[(7.83±0.67)% vs. (12.84±0.91)%] was significantly decreased in baicalein group ( P<0.01). The expression of NMDAR1 (0.582±0.012 vs. 0.352±0.012), GluR2(0.538±0.017 vs. 0.355±0.006), ER β (0.362±0.015 vs. 0.262±0.018) in baicalein group were significantly increased ( P<0.01), the expression of p-JNK/JNK (0.476±0.013 vs. 0.752±0.014) and CaMK Ⅱ(0.499±0.019 vs. 0.670±0.016) in baicalein group were significantly decreased ( P<0.01). Conclusions:Baicalein has a protective effect on PC12 cells injured by Aβ 25-35. Its mechanism may be related to the inhibition of p-JNK/JNK activity by activating ERβ and regulating the expression of glutamate receptor related protein.

Humulus japonicus extract alleviates oxidative stress and apoptosis in 6-hydroxydopamine-induced PC12 cells

@# Objective: To explore the possible neuroprotective activities of Humulus japonicus extract against Parkinson's disease (PD) in a cellular model. Methods: PD was modeled in PC12 cells using 6-hydroxydopamine (6-OHDA). The cell activity, intracellular levels of reactive oxygen species (ROS), anti-oxidative and anti-apoptotic effects, and other related indicators and related signaling pathways were evaluated to elucidate the neuroprotective effects of Humulus japonicus extract. Results: Humulus japonicus extract exhibited anti-oxidative and anti-apoptotic effects in 6-OHDA-stimulated PC12 cells. It also reduced oxidative stress-induced ROS accumulation; upregulated antioxidant enzymes, such as glutathione, catalase, heme oxidase-1, and 8-oxguanine glycosylase 1; promoted cell survival by decreasing BAX and increasing Bcl-2 and sirtuin 1 expression via the MAPK and/or Nrf2 signaling pathways. Conclusions: Humulus japonicus extract has antioxidative and anti-apoptotic effects and could be developed as a promising candidate for preventing and treating oxidative stress-related neurodegenerative diseases.

Effects of sodium arsenite on proliferation, apoptosis and Hippo-YAP pathway of PC12 cells / 中国药理学通报

To investigate the effects of sodium arsenite(NaAsO

MicroRNA-486 targeting TRIM10 to inhibit Parkinson’s disease cell model damage / 解剖学报

[Abstract] Objective To study the effect and mechanism of microRNA-486 (miR-486) on 1-methyl-4-phenylpyridine (MPP

Comparison of PC-12 cells’ apoptosis induced by triazine herbicides atrazine, simazine and cyanazine / 西安交通大学学报(医学版)

【Objective】 To compare PC-12 cells’ apoptosis caused by the serotonin herbicides atrazine （ATR）， simazine （SIM） and cyanazine （CYA）. 【Methods】 The rat adrenal medullary pheochromoma PC-12 cell line was selected for routine culture. At the cell logarithmic growth phase， ATR， SIM and CYA were used at a concentration of 200 μmol/L for 24 h， respectively， and the same solvent was added in the control group. The CCK-8 method was used to detect the cell survival rate； the content of reactive oxygen species （ROS） in PC-12 cells was detected； the Real-time PCR and Western blotting methods were used to detect the mRNA and protein expressions of Bax， p53， Bcl-2 and Caspase-3. 【Results】 Compared with that in the control group， the survival rate of the cells in ATR group， SIM group and CYA group was significantly decreased. The intracellular ROS activity of the three groups was significantly increased， and the mRNA and protein expressions of Bax， p53 and Caspase-3 were increased. mRNA and protein expressions of Bcl-2 were significantly reduced （P<0.01）. Compared with that in ATR group， the cell survival rate of SIM group and CYA group was significantly increased， the intracellular ROS activity of the two groups was significantly decreased， the expressions of Bax， p53 and Caspase-3 mRNA and protein were significantly reduced， and the expressions of Bcl-2 mRNA and protein were significantly increased （P<0.01）. Compared with that of SIM group， the cell survival rate of CYA group was significantly increased， while the intracellular ROS activity was significantly decreased； the Bax， p53 and Caspase-3 mRNA and protein expressions were significantly reduced， and Bcl-2 mRNA and protein expressions were significantly increased （P<0.01）. 【Conclusion】 ATR， SIM and CYA can all promote PC-12 cells’ apoptosis； ATR has the strongest effect while CYA has the weakest effect.