Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells

In the present study, we investigated the effects of tetramethylpyrazine (TMP) on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease in Bcl-2/Bax protein ratio, release of cytochrome c to cytosol and the activation of caspase-3. TMP not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation and eventually protected against H2O2-induced apoptosis. These results indicated that TMP blocked H2O2-induced apoptosis by the regulation of Bcl-2 family members, suppression of cytochrome c release, and caspase cascade activation in PC12 cells.     

Interaction of (-)-epigallocatechin-3-gallate with human serum albumin: fluorescence, fourier transform infrared, circular dichroism, and docking studies

(-)-Epigallocatechin-3-gallate (EGCG), the major constituent of green tea has been reported to prevent many diseases by virtue of its antioxidant properties. The binding of EGCG with human serum albumin (HSA) has been investigated for the first time by using fluorescence, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and protein-ligand docking. We observed a quenching of fluorescence of HSA in the presence of EGCG. The binding parameters were determined by a Scatchard plot and the results were found to be consistent with those obtained from a modified Stern-Volmer equation. From the thermodynamic parameters calculated according to the van't Hoff equation, the enthalpy change deltaH degrees and entropy change deltaS degrees were found to be -22.59 and 16.23 J/mol K, respectively. These values suggest that apart from an initial hydrophobic association, the complex is held together by van der Waals interactions and hydrogen bonding. Data obtained by fluorescence spectroscopy, CD, and FTIR experiments along with the docking studies suggest that EGCG binds to residues located in subdomains IIa and IIIa of HSA. Specific interactions are observed with residues Trp 214, Arg 218, Gln 221, Asn 295 and Asp 451. We have also looked at changes in the accessible surface area of the interacting residues on binding EGCG for a better understanding of the interaction.     

Anti-apoptotic activity of Bak Foong Pills and its ingredients on 6-hydroxydopamine-induced neurotoxicity in PC12 cells

Bak Foong Pills (BFP), a traditional Chinese medicine used for centuries for the enhancement of women's health, was shown to display neuro-protective activity in the 1-methyl-4-phenyl-1,2,4,6,-tetrahydro-pyridine (MPTP)-induced mouse model in a previous study. In order to elucidate its mechanism of action, we investigated the anti-apoptotic properties of Bak Foong Pills and its main ingredients, including Panax ginseng, Angelica sinensis, Glycyrrhiza uralensis, and Ligusticum chuanxiong, in the 6-hydroxydopamine (6-OHDA)-treated PC12 cell model. The addition of the neurotoxin could cause significant cell death and reduction of cell proliferation, as shown in the results determined by MTT assay, nitric oxide (NO) measurement and flow cytometric propidium iodine (PI) staining analysis, while pre-treatment of PC12 cell with either BFP or its main ingredients prevented the toxicity to some degree. In addition, the neurotoxin caused an elevated activation of caspase-3, the key enzyme for activation of the cellular apoptotic cascade, whereas BFP or its main ingredients inhibited the activation of caspase-3. These results strongly indicate that BFP and its main ingredients may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases, such as Parkinson's disease.     

Puerarin protects PC12 cells against beta-amyloid-induced cell injury

beta-Amyloid protein (Abeta), a major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. In this study, the protective effect of puerarin, an isoflavone purified from the radix of the Chinese herb Pueraria lobata, on Abeta-induced rat pheochromocytoma (PC12) cultures was investigated. Although exposure of PC12 cells to 50 microM Abeta25-35 caused significant viability loss and apoptotic rate increase, pretreatment of the cells with puerarin for 24h reduced the viability loss and apoptotic rate. Puerarin (1 microM) significantly inhibited Abeta25-35-induced apoptosis of PC12 cells. Preincubation of the cell with puerarin also restored the ROS and mitochondrial membrane potential levels that had been altered as a result of Abeta25-35 treatment. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce caspase-3 activation. These results suggest that puerarin could attenuate Abeta25-35-induced PC12 cell injure and apoptosis and could also promote the survival of PC12 cells. Therefore, puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta25-35-induced cell injury.     

Green tea polyphenol (-)-epigallocatechin-3-gallate induces neurorescue of long-term serum-deprived PC12 cells and promotes neurite outgrowth

Our previous studies have shown that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) prevents neuronal cell death caused by several neurotoxins. The present study sought to determine the neuroprotective effect of EGCG when it is administered after the induction of cell damage ('neurorescue'). In an attempt to imitate a progressive mode of death, PC12 cells were initially subjected to serum-starvation conditions for a period of 1 or 3 days before administration of EGCG (0.1-10 microM) for up to 3 days. In spite of the high percentage of cell death, single or repetitive administration of EGCG (1 microM) significantly attenuated cell death. The neurorescue effect of EGCG was abolished by pre-treatment with the protein kinase C inhibitor GF109203X (2.5 microM), suggesting the involvement of the protein kinase C pathway in neurorescue by the drug. This is consistent with the rapid (15 min) translocation of the protein kinase C alpha isoform to the cell membrane in response to EGCG. The correlative neurite outgrowth activity of EGCG on PC12 cells may also contribute to its neurorescue effect. The present findings suggest that EGCG may have a positive impact on aging and neurodegenerative diseases to retard or perhaps even reverse the accelerated rate of neuronal degeneration.     

Proposed mechanisms of (-)-epigallocatechin-3-gallate for anti-obesity

Green tea catechins (GTCs) are polyphenolic flavonoids formerly called vitamin P. GTCs, especially (-)-epigallocatechin-3-gallate (EGCG), lower the incidence of cancers, collagen-induced arthritis, oxidative stress-induced neurodegenerative diseases, and streptozotocin-induced diabetes. Also, inhibition of adipogenesis by green tea and green tea extract has been demonstrated in cell lines, animal models, and humans. The obesity-preventive effects of green tea and its main constituent EGCG are widely supported by results from epidemiological, cell culture, animal, and clinical studies in the last decade. Studies with adipocyte cell lines and animal models have demonstrated that EGCG inhibits extracellular signal-related kinases (ERK), activates AMP-activated protein kinase (AMPK), modulates adipocyte marker proteins, and down-regulates lipogenic enzymes as well as other potential targets. Also, the catechin components of green tea have been shown to possess anti-carcinogenic properties possibly related to their anti-oxidant activity. In addition, it was shown that dietary supplementation with EGCG could potentially contribute to nutritional strategies for the prevention and treatment of type 2 diabetes mellitus. In this review, the biological activities and multiple mechanisms of EGCG in cell lines, animal models, and clinical observations are explained.     

Neuroprotective Effects of Salidroside in the PC12 Cell Model Exposed to Hypoglycemia and Serum Limitation

The hypoglycemia and serum limitation-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., a traditional Chinese medicinal plant, and has displayed a broad spectrum of pharmacological properties. In this study, MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed that pretreatment with salidroside attenuated, in a dose-dependent manner, cell viability loss, and apoptotic cell death in cultured PC12 cells induced by hypoglycemia and serum limitation. RT-PCR, Western blot analysis, and enzymatic colorimetric assay indicated the changes in expression levels of Bcl-2, Bax, and caspase3 in PC12 cells on exposure to hypoglycemia and serum limitation with and without salidroside pretreatment, respectively. Rhodamine 123 staining and flow cytometry with 2′,7′-Dichlorofluorescin diacetate staining revealed the changes in the mitochondrial membrane potential and radical oxygen species (ROS) production in PC12 cells on exposure to hypoglycemia and serum limitation with and without salidroside pretreatment, respectively. The experimental results suggest that salidroside protects the PC12 cells against hypoglycemia and serum limitation-induced cytotoxicity possibly by the way of the modulation of apoptosis-related gene expression, the restoration of the mitochondrial membrane potential, and the inhibition of the intracellular ROS production. Our findings might raise a possibility of potential therapeutic applications of salidroside for preventing and treating cerebral ischemic and neurodegenerative diseases.     

(-)-Epigallocatechin-3-gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti-tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblast's integrin alpha2beta1, indicating EGCG may affect the expression and affinity of integrin alpha2beta1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen-induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three-dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non-contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti-tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor-stroma interaction.     

Protective effects of green tea polyphenols and their major component, (-)-epigallocatechin-3-gallate (EGCG), on 6-hydroxydopamine-induced apoptosis in PC12 cells

Green tea polyphenols exert a wide range of biochemical and pharmacological effects, and have been shown to possess antimutagenic and anticarcinogenic properties. Oxidative stress is involved in the pathogenesis of Parkinson's disease. However, although green tea polyphenols may be expected to inhibit the progression of Parkinson's disease on the basis of their known antioxidant activity, this has not previously been established. In the present study, we evaluated the neuroprotective effects of green tea polyphenols in the Parkinson's disease pathological cell model. The results show that the natural antioxidants have significant inhibitory effects against apoptosis induced by oxidative stress. 6-Hydroxydopamine (6-OHDA)-induced apoptosis in catecholaminergic PC12 cells was chosen as the in vitro model of Parkinson's disease in our study. Apoptotic characteristics of PC12 cells were assessed by MTT assay, flow cytometry, fluorescence microscopy and DNA fragmentation. Green tea polyphenols and their major component, EGCG at a concentration of 200 microM, exert significant protective effects against 6-OHDA-induced PC12 cell apoptosis. EGCG is more effective than the mixture of green tea polyphenols. The antioxidant function of green tea polyphenols may account for this neuroprotective effect. The present study supports the notion that green tea polyphenols have the potential to be effective as neuropreventive agents for the treatment of neurodegenerative diseases.     

Phospholipase D2 activity suppresses hydrogen peroxide-induced apoptosis in PC12 cells

Phospholipase D (PLD) plays an important role as an effector in the membrane lipid-mediated signal transduction. However, the precise physiological functions of PLD are not yet well understood. In this study, we examined the role of PLD activity in hydrogen peroxide (H(2)O(2))-induced apoptosis in rat pheochromocytoma (PC12) cells. Treatment of PC12 cells with H(2)O(2) resulted in induction of apoptosis in these cells, which is accompanied by the activation of PLD. This H(2)O(2)-induced apoptosis was enhanced remarkably when phosphatidic acid production by PLD was selectively inhibited by pretreating the PC12 cells with 1-butanol. Expression of PLD2, but not of PLD1, correlated with increased H(2)O(2)-induced PLD activity in a concentration- and time-dependent manner. Concomitant with PLD activation, the PLD2 activity suppressed H(2)O(2)-induced apoptosis in PC12 cells. Expression of PLD2 lipase-inactive mutant (K758R) had no effect on either PLD activity or apoptosis. PLD2 activity also suppressed H(2)O(2)-induced cleavage and activation of caspase-3. Taken together, the results suggest that PLD2 activity is specifically up-regulated by H(2)O(2) in PC12 cells and that it plays a suppressive role in H(2)O(2)-induced apoptosis.     

Neuroprotective effect of Korea Red Ginseng extract on 1-methyl-4-phenylpyridinium-induced apoptosis in PC12 Cells

Background: Korean Red Ginseng (KRG) is a valuable herb in Asian countries that is used as a crude substance to inhibit inflammation and to enhance vitality, longevity and immunity. The protective effects of KRG against the toxicity of 1-methyl-4-phenylpyridinium (MPP+) were investigated in vitro in the present study.

Methods: PC12 cells were pretreated with the water extract of KRG for 24?h, then incubated with MPP+ for 24?h. The growth of the cells was assessed using a live cell viability assay, the ratio of apoptotic cells was measured using flow cytometry and morphology of the apoptotic cells was detected using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The expressions of caspase-3 and -9 were measured by quantitative real-time PCR.

Results: Pretreatment of the KRG extract increased cell viability significantly when compared with that of only MPP+-treated cells (p?p?p?p?Conclusion: These results indicate that KRG treatment suppresses MPP+-induced apoptosis in PC12 cells by regulating caspase cascades, suggesting a possible role for KRG in the prevention and treatment of Parkinson’s disease.     

黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。     

Distinct effects of tea catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (-)-epigallocatechins gallate (EGCG) and (-)-epicatechin gallate (ECG) in 50-200 microM had obvious concentration-dependent protective effects on cell viability, while (-)-epicatechin (EC), (+)-catechin ((+)-C), and (-)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 microM EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECG> or = EGCG>EC> or = (+)-C>EGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD.     

Chloride channels involve in hydrogen peroxide-induced apoptosis of PC12 cells

Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H₂O₂)-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H₂O₂ activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H₂O₂ elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1 h and induced apoptosis of most PC12 cells tested in 24 h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H₂O₂-induced high membrane permeability and cell shrinkage, suppressed H₂O₂-activated chloride currents and protected PC12 cells from apoptosis induced by H₂O₂. The results suggest that chloride channels may contribute to H₂O₂-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.     

Neuroprotective effect of methanol extract of Phellodendri Cortex against 1-methyl-4-phenylpyridinium (MPP)-induced apoptosis in PC-12 cells

Phellodendri Cortex (PC) is a traditional herbal medicine, widely used in Korea and China. The effects of the methanol extract of Phellodendri Cortex (PC extract) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced neuronal apoptosis in PC-12 cells have been investigated. MPP⁺-induced apoptosis in PC-12 cells was accompanied by an increased bax/bcl-2 ratio, release of cytochrome c to the cytosol and activation of caspase-3. PC extract inhibited the downregulation of bcl-2 and the upregulation of bax, as well as the release of mitochondrial cytochrome c into the cytosol. In addition, PC extract attenuated caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP). These results suggest that the PC extract has protective effects against MPP⁺-induced neuronal apoptosis in PC-12 cells.     

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl⁻ channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl⁻ channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl⁻ channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively. These authors contributed equally to this work.     

Cytoprotective effect of two synthetic enhancer substances, (-)-BPAP and (-)-deprenyl, on human brain capillary endothelial cells and rat PC12 cells

Enhancer regulation is a new control mechanism in the brain [Knoll, J., 2003. Enhancer regulation/endogenous and synthetic enhancer compounds: a neurochemical concept of the innate and acquired drives. Neurochemical Research 28(8), 1275-1297]. Enhancer substances exert their effect in bi-modal form with a highly characteristic dose-dependency. Two bell-shaped concentration curves have been published. The one in ultra low concentration range (fM) specific form of enhancer regulation and the other at high concentration (100 microM) non-specific form of enhancer regulation. Catecholaminergic neurons proved to be enhancer-sensitive cells. Since rat PC12 cells and human brain endothelial cells (HBEC) work under catecholaminergic influence, it was reasonable to expect that both the specific and non-specific form of the enhancer regulation might be detectable in these cells. We tested this possibility on these cultured cells under normoxia and hypoxia-reoxygenation. After 1 h hypoxia produced by Argon gas and 0, 2, 4, and 20 h reoxygenation the cell loss was calculated by propidiumiodide assay and the cell activity was investigated by Alamar Blue assay colorimetric measurement. The percentages of living and necrotic cells were expressed after propidiumiodide staining. Broad scale concentrations of the two compounds (1 fM-100 microM) were added to the culture strait after the oxygen deprivation. (-)-BPAP and (-)-deprenyl, due to their enhancer effect, exerted a significant cytoprotective effect on both HBECs and PC12 cells. In harmony with Knoll's publications we were able to demonstrate by the aid of (-)-BPAP and (-)-deprenyl that both HBEC and PC12 are enhancer-sensitive cells. We detected the specific form of the enhancer regulation in the ultra low concentration range (fM-pM) and also the non-specific form of the enhancer regulation was visible (mM-microM).     

BDNF RNA干扰在多巴胺能PC12细胞凋亡中的作用研究

目的：探讨脑源性神经营养因子（brain derived neurotrophic factor,BDNF）在PC12细胞凋亡中的作用。方法：设计并合成针对BDNF mRNA序列的小片段干扰RNA（siRNA）,利用lipofectamine 2000将siRNA转染入PC12细胞中或给与6-OHDA损伤,给与/不给予BDNF蛋白保护,采用定量PCR和免疫荧光法检测BDNF mRNA和蛋白表达水平;采用上清液乳酸脱氢酶（LDH）释放量测定和流式细胞仪法检测siRNA对细胞凋亡的影响。结果：转染siRNA的细胞的BDNF mRNA的表达量比正常组细胞减少73%,而转染作为对照的scrambled siRNA的细胞的BDNF mRNA的表达没有明显变化。BDNF RNA干扰与6-OHDA神经毒性一样可诱导PC12细胞的LDH释放和细胞凋亡。给予BDNF蛋白保护后细胞毒性减轻。结论：BDNF基因下调可以导致PC12细胞的凋亡,BDNF蛋白对PC12细胞有保护作用,为进一步进行动物体内研究奠定了基础。