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.

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.

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.

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

Aim To explore the effects of NaAsO

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.

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.

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.

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

Establishment of a hypobaric hypoxia-induced cell injury model in PC12 cells / 浙江大学学报·医学版

To construct a hypobaric hypoxia-induced cell injury model. Rat pheochromocytoma PC12 cells were randomly divided into control group, normobaric hypoxia group and hypobaric hypoxia group. The cells in control group were cultured at normal condition, while cells in other two groups were cultured in normobaric hypoxia and hypobaric hypoxia conditions, respectively. CCK-8 method was used to detect cell viability to determine the optimal modeling conditions like the oxygen concentration, atmospheric pressure and low-pressure hypoxia time. The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by microplate method. The apoptosis ratio and cell cycle were analyzed by flow cytometry. The hypobaric hypoxia-induced cell injury model can be established by culturing for 24 h at 1% oxygen concentration and 41 kPa atmospheric pressure. Compared with the control group and normobaric hypoxia group, the activity of LDH and the content of MDA in hypobaric hypoxia group were significantly increased, the activity of SOD was decreased, the percentage of apoptosis was increased (all <0.05), and the cell cycle was arrested in G0/G1 phase. A stable and reliable cell injury model induced by hypobaric hypoxia has been established with PC12 cells, which provides a suitable cell model for the experimental study on nerve injury induced by hypoxia at high altitude.

Low molecular weight heparin through TLR4/MyD88/NF-κB pathway protects PC12 cells against oxygen glucose deprivation induced inflammation / 中国医师杂志

Objective:To investigate the effect of low molecular weight heparin (LMWH) on the inflammatory response of PC12 cells induced by oxygen glucose deprivation (OGD) and its related mechanism.Methods:The PC12 cells were cultured in vitro were randomly divided into sham(control) group, OGD group, LMWH group and blocking agent group. The latter group was divided into six groups: Eritoran+ OGD group, LMWH+ Eritoran+ OGD group, ST2825+ OGD group, LMWH+ ST2825+ OGD group, pyrrolidinedithiocarbamate (PDTC)+ OGD group and LMWH+ PDTC+ OGD group. OGD cell model was established. Cell counting kit-8 (CCK-8) assay was used to detect cell activity. The expressions of toll-like receptor 4 (TLR4), MyD88 and nuclear factor κB (NF-κB) mRNA and protein were detected by real time polymerase chain reaction (qRT-PCR) and Western blot. The concentration of interleukin (IL)-1β, IL-6, tumor necrosis factor-α(TNF-α) and S100β were determined by enzyme linked immunosorbent assay (ELISA). Results:The cell activity of OGD group was significantly lower than that of control group on the first, second, third day ( P<0.05). Compared with OGD group, the activity of LMWH group was increased on the second, third day ( P<0.05), but lower than that of control group ( P<0.01). The mRNA expression of TLR4, MyD88 and NF-κB was significantly increased in OGD group compared with the control group ( F=144.9, F=710.5, 79.51, P<0.01). Compared with OGD group, the mRNA expression of TLR4, MyD88 and NF-κB were significantly decreased after treatment with LMWH ( P<0.01), and the specific inhibitor of TLR4, MyD88 and NF-κB enhanced the anti-inflammatory effect of LMWH. The protein expression of this pathway was consistent with that of the gene. The concentration of IL-1β, IL-6, TNF-α and S100β in OGD group was significantly higher than control group ( P<0.05). After treatment with LMWH, the concentrations of inflammatory factors and S100β were significantly decreased compared with OGD group ( P<0.01). When hinder TLR4, MyD88 and NF-κB respectively by Eritoran, ST2825 and PDTC, the concentrations of inflammatory factors and S100β were significantly decreased, but it was still higher than control group ( P<0.05). Conclusions:OGD can cause pathological damage of PC12 cells, including high expression level of S100β and aggravation of inflammatory reaction. LMWH can improve cell activity, down-regulate inflammatory reaction degree and protect the cells. Using inhibitors of TLR4/MyD88/NF-κB pathway to inhibit the corresponding target, the up-regulation of inflammatory factors by OGD can be inhibited in varying degrees. These suggested that LMWH may regulate inflammatory reaction of PC12 cells induced by OGD through TLR4/MyD88/NF-κB pathway.

Protective effect of baicalein on injured PC12 cell induced by Aβ and its mechanism / 国际中医中药杂志

Objective:To investigate the protective effect of baicalein on injured PC12 cell induced by Aβ and explore its mechanism.Methods:The method of MTT was used to detect the cell activity of each group and screened the concentration of baicalein. The PC12 cells were randomly divided into the blank group, the Aβ group, the baicalin group and the estradiol group. 24 hours after inoculation, baicalein group was intervened with 1×10 -6 mol/L baicalein solution, and estradiol group was intervened with 1×10 -5 mol/L estradiol solution. Two hours later, except the blank group, the other groups were added with 1.5×10 -4 mol/L Aβ to make the model. MTT assay was used to detect the cell viability of each group after 24 hours of cultivation. Then used oxidation kit to detect the contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and lactate dehydrogenase (LDH) in each group. And the level of caspase-3 mRNA was detected by Real-time Quantitative PCR (RT-PCR). Then the Western blot method was used to detect the expressions of p-PI3K, p-AKT and caspase-3. Results:Compared with the Aβ group, the PC12 cell viability [(96.348 ± 0.571)%, (97.183 ± 0.714)% vs. (86.922 ± 0.429)%] in the baicalin group and the estradiol group significantly increased( P<0.01). The activities of SOD [(54.31 ± 1.34) U/mgprot, (57.38 ± 2.25) U/mgprot vs. (36.18 ± 2.24) U/mgprot] and GSH-PX [(4.46 ± 0.23) U/mgprot, (4.72 ± 0.31) U/mgprot vs. (2.05 ± 0.37) U/mgprot] significantly increased, and the level of LDH [(85.43 ± 0.92) nmol/ml, (82.46 ± 0.27) nmol/ml vs. (99.17 ± 0.52) nmol/ml] significantly decreased ( P<0.01). The expression of caspase-3 mRNA (2.24 ± 0.64, 2.33 ± 0.75 vs. 3.46 ± 0.46) and p-PI3K (0.46 ± 0.03, 0.44 ± 0.06 vs. 0.66 ± 0.09), p-AKT (0.43 ± 0.05, 0.41 ± 0.02 vs. 0.58 ± 0.03), caspase-3 (0.61 ± 0.03, 0.56 ± 0.53 vs. 0.92 ± 0.07) protein significantly decreased ( P<0.01). Conclusion:Baicalein could slow down cell apoptosis and oxidative reaction, reduce the damage of Aβ to PC12 cells by inhibiting the expression of PI3K/AKT pathway.

Protective effect and mechanism of active peptide GRGDS on PC12 cells damage by oxygen-glucose deprivation / 药学实践杂志

Objective To study the protective effect of active peptide GRGDS on rat nerve cells (PC12 cells) in oxygen glucose deprivation (OGD) injury model and explore its mechanism of action. Methods PC12 cells were divided into control group, ODG group, and active peptide GRGDS treatment group. The injury model was established by simulating in vitro cerebral ischemia by oxygen and sugar deprivation. MTT and flow cytometry were used to detect apoptosis after oxygen-glucose deprivation. ELISA method was used to detect the changes of inflammatory factors TNF-α and IL-1β in PC12 cell supernatant after oxygen-glucose deprivation. Western blot was used to detect the expression of apoptosis pathway-related proteins. Results The results of MTT and flow cytometry showed that the active peptide GRGDS significantly reduced the apoptosis of PC12 cells after oxygen glucose deprivation (P<0.05). ELISA test results showed that the active peptide GRGDS significantly reduced the content of TNF-α and IL-1β in the supernatant of PC12 cells after oxygen-glucose deprivation. (P<0.05). Western blot results showed that the active peptide GRGDS significantly reduced the expression levels of p-JNK, Bax, and cleaved caspase 3 in PC12 cells mediated by oxygen-glucose deprivation injury (P <0.01). Conclusion The active peptide GRGDS has protective effect on PC12 cells damaged by oxygen and glucose deprivation. The mechanism may be related to anti-apoptotic and anti-inflammatory effects.

Resveratrol inhibits advanced glycation end products induced PC12 cell apoptosis through ROS-JNK pathway / 中国药理学通报

Aim To investigate the effect of resveratrol (RSV)on apoptosis of PC 12 cells induced by advanced glycation end products(AGEs) and its possible molecular mechanism. Methods MTS assay was used to detect the effects of AGEs (0, 50, 100, 200, 400 g • L"1) and RSV (0, 5, 10, 20, 40 jxmol • L"1) on cell viability. PC12 cells were treated with AGEs (400 g • L-1) and RSV (10 |xmol • L"1). TUNEL staining was used to detect apoptosis; flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) ; Western blot was used to detect protein expression of p-JNK, JNK, PUMA, Bcl-2, Bax and Caspase-3. Apoptosis was observed by flow cytometry after pretreat- ment with JNK specific phosphorylation inhibitor (sp600125). Results Compared with normal control group, the cell viability of AGEs group decreased, the apoptotic rate and ROS levels increased, the expressions of p-JNK, PUMA, Bax and Caspase-3 protein in-creased, the expression of Bcl-2 protein decreased; Compared with AGEs group, the cell viability of the AGEs + RSV group increased, the levels of apoptotic rate and ROS were reduced, the expressions of p-JNK, PUMA, Bax and caspase-3 protein decreased, the expression of Bcl-2 protein increased. Sp600125 could partially reverse the effect of AGEs on PC)2 cell apopto-sis. Conclusions RSV can significantly inhibit the apoptosis of PC 12 cells induced by AGEs, which may be related to the activation of ROS-JNK pathway.

ZNRF2 attenuates PC12 cell injury caused by oxygen-glucose deprivation and reoxygenation through inhibiting autophagy / 中国药理学通报

Aim To study the protective effect of ZN-RF2 on OGD/R-induced injury and the autophagy-related mechanism in PC12 cells. Methods PC12 cells were cultured in vitro and divided into normal group and OGD/R group. qRT-PCR and Western blot were used to measure the mRNA and protein expressions of ZNRF2. To explore the effect of ZNRF2 on OGD/R-induced injury in PC12 cells, cells were grouped into normal group, OGD/R group, LV-ZNRF2 group, LV-NC group, siR-ZNRF2 group and siNC group. Cell viability was detected by MTT assay, cell apoptosis was measured by flow cytometry and the expressions of autophagy-related proteins LC3II, p62, Beclin-l were accessed by Western blot. Results Compared with normal group, the cell viability decreased in OGD/R group, the cell apoptosis increased markedly, and the expressions of ZNRF2 mRNA and protein were downregulated significantly. Simultaneously, the proteins expressions of LC3II and Beclin-1 increased, and the expression of p62 protein decreased in OGD/R group. Compared with OGD/R group, the cell viability was enhanced, the cell apoptosis and autophagy were decreased in LV-ZNRF2 group. In contrast, the cell viability decreased and the cell apoptosis and autophagy were aggravated after transfecting siR-ZNRF2. Conclusions ZNRF2 protects PCI2 cells from the injury caused by OGD/R and its mechanism may be related to the inhibition of autophagy.

Study of astragaloside IV alleviating oxidatIVe stress injury of pc12 cells induced by oxygen-glucose deprIVation/reoxygenation through regulating autophagy / 中国药理学通报

Aim To observe the effects of astragaloside IV on autophagy and oxidative stress induced by oxy gen-glucose deprivation/reoxygenation in PCI2 cells. Methods PCI2 cells were divided into normal group, model group (ox-ygen-glucose deprivation/reoxygenation group) , astragaloside IV group, and autophagy inhibitor + astragaloside IV group. CCK-8 was used to detect cell viability, and ELISA to detect MDA, SOD and GSH-Px. Transmission electron microscope and MDC fluorescent staining were employed to observe the changes of au-tophagosome, and Western blot to detect the protein expression of Beclinl. Results Compared with normal group, the cell activity in model group decreased (P <0.05), MDA content increased, SOD and CSH-Px activity decreased (P < 0.05), and autophagosomes could be seen and the protein expression of Beclinl increased ( P < 0.05). Compared with model group, the cell activity in astragaloside IV group increased ( P < 0. 05), MDA content decreased, SOD and GSH-Px activity increased (P < 0.05), the number of autophagosomes and the protein expression of Beclinl increased (P<0.05). When autophagy inhibitor was given at the same time, the autophagy inhibitor could obviously antagonize the antioxidant effect of astragaloside IV while alleviating autophagy. Conclusions Astragaloside IV can protect PC 12 cells from oxidative stress injury induced by oxygen-glucose deprivation/reoxygenation by up-regulating autophagy.

Neuroprotective effects of kukoamine A against rotenone-induced neurotoxicity in PC12 cells / 中草药

Objective: To investigate the neuroprotective effects of kukoamine A (KuA) on rotenone-induced PC12 cells damage and to preliminary verify its potential action mechanisms. The present study may lay the foundation for finding leading compounds with anti-Parkinson's disease (PD) effects. Methods: A PD model induced by rotenone was established in vitro, and MTT, LDH, and Hoechst33342 staining were used for preliminary confirmation of KuA resistance to rotenone-induced PC12 cell injury in vitro. The effects of KuA on superoxide dismutase (SOD) activity, malondialdehyde (MDA) and reactive oxygen species (ROS) content, and mitochondrial membrane potential (MMP) were investigated by colorimetric method and fluorescence staining, respectively. Western blotting was applied to explore the underlying mechanisms of protective effects of KuA against rotenone-induced PC12 cells damage. Results: The PC12 cell viability was significantly decreased after exposure to 0.5 μmol/L rotenone, whereas pretreatment with different concentrations of KuA could attenuate the cell injury induced by rotenone. Compared with the rotenone-treated group, KuA could decrease the ROS production and MDA level, while increase the SOD activity. In addition, KuA could effectively increase the MMP, decrease the cytochrome c release and the Bax/Bcl-2 ratio as well as inhibit caspase-3, caspase-9, and α-synuclein protein expressions. Conclusion: KuA showed neuroprotective ability on rotenone-induced PC12 cells PD model and the potential protective mechanisms of KuA can be related with inhibition of ROS generation, protection of MMP, regulation of protein expressions involved in the mitochondrial apoptosis pathway and reduction of α-synuclein expression.

Study on Improvement Effects of Total Ginsenosides on D-galactose Induced PC 12 Cell Senescence and Its Me- chanism / 中国药房

OBJECTIVE：To study the improvement effects of total ginsenosides on the senescence of PC 12 cells induced by D-galactose and its mechanism. METHODS ：Rat pheochromocytoma （PC12）cells were treated with D-galactose to establish cell senescence model. CCK- 8 method was used to screen the D-galactose modeling concentration and total ginsenosides concentration. Normal control group ，model group ，total ginsenosides low and high concentration groups were set up. Cell senescence ，cell apoptosis rate ，apoptotic cycle and mitochondrial membrane potential （MMP），cell adenosine triphosphate （ATP）and reactive oxygen species （ROS）levels in each group were detected. The expression of apoptosis related proteins [B lymphoma 2（Bcl-2）and its related egg X protein （Bax），cytochrome C （Cyt-C）] and oxidative damage related proteins [nuclear factor 2 related factor 2 （Nrf2），heme oxygenase 1（HO-1）] were detected. In addition ，positive drug group [ 5 mmol/L N-acetyl-L-cysteine（NAC）] and positive control group [ D-galactose+5 mmol/L NAC] were set up to compare the levels of oxidative damage related proteins. RESULTS：D-galactose could significantly inhibit the survival rate of PC 12 cells，with a critical concentration of 20 mg/mL. The total ginsenosides could significantly increase the survival rate of D-galactose induced senescent cells with a median effective concentration（EC50）of 65 μg/mL，and then the low and high concentrations of total ginsenosides were set at 55 and 65 μg/mL. Compared with normal control group ，the number of aging cells increased ，the apoptotic rate and percentage of G 1 phase were significantly increased i n model group. the percentage of S phase ，MMP and ATP contents ，the protein expression of Bcl- 2 and Cyt-C in mitochondria were decreased significantly ，whileROS content ，the protein expression of Bax ，Nrf2 and Cyt-C protein in endochylema were increased significantly （P＜0.05 or P＜0.01）. Compared with model group ，the number of E-mail：sunqiao150509@163.com aging cells reduced ，the apoptosis rates and percentage of G 1 phase were significantly decreased in total ginsenosides low and high concentration groups ，the percentage of S phase ，the contents of MMP and ATP （except for low concentration group ），protein expression of Bcl- 2，Nrf2 and HO- 1 as well as protein expression of Cyt-C in mitochondria were increased significantly ；ROS level （except for low concentration group ）and Bax protein as well as protein expression of Cyt-C were decreased significantly. The protein expression of Nrf 2 and HO- 1 were increased significantly in positive control group （P＜0.05 or P＜0.01）， but it was lower than that of total ginsenosides groups . CONCLUSIONS：Total ginsenosides can improve D-galactose induced senescence of P 12 cells，the mechanism of which may be related to activating Nrf 2 antioxidant signal pathway to antagonize D-galactose induced oxidative stress and alleviating mitochondrial dysfunction.

Effects of stachyine on apoptosis in an Aβ-induced PC12 cell model of Alzheimer's disease / 南方医科大学学报

OBJECTIVE@#To investigate the effects of stachydrine (STA) on apoptosis of Aβ-induced PC12 cells mimicking Alzheimer's disease and explore the mechanisms.@*METHODS@#The differential genes of STA were analyzed based on GSE85871 data, and the target genes of STA were identified using STITCH database. PC12 cells were treated with Aβ to establish a cell model of Alzheimer's disease, and the changes in cell viability and cell cycle in response to STA treatment were assessed using MTT assay and flow cytometry, respectively. RT-PCR and Western blotting were used to detect the relevant gene or protein expressions in the treated cells.@*RESULTS@#GSE85871 data showed 37 up-regulated genes and 48 down-regulated genes in cells following treatment with STA. Analysis of the data from the STITCH database indicated that RPS8 and EED were the target genes of STA. Treatment of PC12 cells with Aβ significantly lowered the cell viability ( < 0.05) and the expressions of RPS8 and EED at both the mRNA and protein levels ( < 0.05), and obviously inhibited the expression of apoptosis-related proteins Bcl-2 and p53 ( < 0.05). STA treatment of the cells significantly reversed the effect of Aβ and induced cell cycle arrest in G2/M phase, causing also significantly increases in the expression levels of RPS8, EED, Bcl-2 and p53 ( < 0.05).@*CONCLUSIONS@#STA plays an important role in inhibiting the apoptosis of PC12 cells induced by Aβ possibly by regulating RPS8 and EED expression to promote the expressions of Bcl-2 and p53.

The effect of four compounds of astragalus isoflavones on the differentiation of PC 12 cells / 药学实践杂志

Objective To investigate the effects of calycosin, formononetin, calycosin-7-glucoside and ononin on PC 12 cells differentiation. Methods PC 12 cells were cultured and treated with different concentrations of nerve growth factor (NGF), calycosin, formononetin, calycosin-7-glucoside and ononin for 5 days, once a day, 3 times in a row. The neurite outgrowth of PC 12 cells was observed and the expression of β III-tubulin were measured by immunofluorescence. Results Compared with the vehicle group, neurite outgrowth and the expression of β III tubulin in PC 12 cells had not promoted by calycosin, formononetin, calycosin-7-glucoside and ononin (0.01-10.00 μmol/L). Conclusion PC 12 cells differentiation could not be induced by calycosin, formononetin, calycosin-7-glucoside and ononin.