Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

PSMA activates JNK/SAPK pathway and regulates apoptosis of prostate cancer cells

AIM：To investigate the effects of prostate-specific membrane antigen (PSMA) on the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway and the apoptosis of prostate cancer cells. METHODS：The shRNA lentiviral vector with high efficiency was constructed in the previous study to block the PSMA expression in the prostate cancer cells as experimental interference group, while the constructed vector of PSMA was transfected into the prostate cancer cells to promote PSMA expression as positive experimental group. The control group was the cell line without any treatment. JNK/SAPK inhibitor SP600125 was used as a negative control. Western blotting and immunocytochemistry were used to observe the p-JNK/SAPK expression. The cell growth curve was determined by CCK-8 assay. The cell cycle and apoptosis were analyzed by flow cytometry. RESULTS：Inhibition of PSMA expression resulted in the decrease of p-JNK/SAPK expression levels, while enhancement of the PSMA expression made the increase in the expression of p-JNK/SAPK. SP600125 decreased the level of p-JNK/SAPK, and no significant difference among the 3 groups was observed. The cell proliferation and S-phase percentage decreased after the inhibition of PSMA, while the cells in the 3 groups with SP600125 treatment only had low levels of cell proliferation and percentage of S phase. The inhibition of PSMA promoted apoptosis, while in the enhanced PSMA expression group, apoptotic rate was significantly reduced. After adding SP600125, the cell apoptotic rate was lower than that in normal culture group. CONCLUSION：PSMA has an impact on the proliferation, cell cycle and apoptosis of prostate cancer cells by up-regulating JNK/SAPK signaling pathway, but the JNK/SAPK signaling is not the only path.     

Harmine induces apoptosis of HepG2 cells via JNK signaling pathway and enhances their chemosensitivity to 5-fluorouracil and cisplatin

AIM: To investigate the proliferation-inhibitory and apoptosis-inducing effects of harmine on human hepatocarcinoma HepG2 cells. METHODS: The proliferation of HepG2 cells was determined in the absence or presence of a JNK inhibitor SP600125 by Cell Counting Kit-8 (CCK-8) assay and colony formation test. The morphology of HepG2 cells was observed by Hoechst 33258 staining under fluorescence microscope. The cell apoptosis was analyzed by Annexin V-PI staining. The expression of apoptosis-regulated proteins,poly(ADP-ribose) polymerase (PARP),c-Jun N-terminal kinase (JNK) and p-JNK, was detected by Western blotting. The sensitizing effects of harmine on HepG2 cells to chemotherapeutic drugs 5-fluorouracil (5-FU) and cisplatin were determined by CCK-8 assay. RESULTS: Harmine inhibited the proliferation of HepG2 cells and induced apoptosis in a dose-dependent manner. After the JNK signaling pathway was blocked by SP600125, the cell apoptotic rate decreased significantly. Hoechst 33258 staining revealed that the nuclear fragmentation, chromosomal condensation, cell shrinkage and attachment loss appeared in the HepG2 cells treated with harmine. The percentage of the sub-G1 fraction was increased and the population of early apoptotic cell death was observed. Apoptosis of HepG2 cells with harmine treatment was associated with the activation of JNK. Combined with harmine, the IC50 values of 5-FU and cisplatin for the tumor cells were 1.47 and 5.78 times sensitized as compared with the correspon-ding single drug treatment groups, respectively. CONCLUSION: Harmine exhibits an anti-proliferative effect on HepG2 cells by inducing apoptosis. The JNK signaling pathway is involved in the apoptosis of HepG2 cells. Harmine enhances the chemosensitivity of the cells to 5-FU and cisplatin.     

Bone marrow-derived mesenchymal stem cells protect against hypoxia-induced injury and apoptosis of PC12 cells via up-regulation of erythropoietin expression

AIM： To investigate the effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on cobalt chloride (CoCl2)-induced injury and apoptosis of PC12 cells. METHODS：PC12 cells were divided into control group, CoCl2 group, BM-MSCs-siCTL+CoCl2 group and BM-MSCs-siEPO+CoCl2 group. The viability of the PC12 cells was measured by MTT assay. Flow cytometry and Hoechst 33258 staining were used to determine the apoptotic rate and the changes of chromatin distribution in PC12 cells. The expression of erythropoietin (EPO) in BM-MSCs was measured by RT-PCR and Western blotting. The mRNA expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR. Caspase-9 and caspase-3 assay kits were used to detect the activity of caspase-9 and caspase-3. RESULTS：The viability of PC12 cells treated with CoCl2 for 24 h and 48 h decreased to （43.0±6.4）% and （33.8±5.7）%, respectively, while 1∶15 ratio of BM-MSCs co-culture increased the cell viability to （77.9±3.8）% and （75.2±9.7）%,respectively. The expression of EPO in BM-MSCs was up-regulated after treated with 0.6 mmol/L CoCl2 for 24 h and 48 h, while EPO siRNA significantly abrogated the EPO expression in BM-MSCs. BM-MSCs-siCTL co-culture significantly inhibited the apoptosis of PC12 cells induced by CoCl2. However, EPO siRNA the protective effect of BM-MSCs. Compared with CoCl2 treatment group, BM-MSCs co-culture induced remarkable increase in the expression of Bcl-2 and decrease in the expression of Bax in PC12 cells, which was reversed by EPO siRNA. BM-MSCs-siCTL co-culture remarkably abrogated the CoCl2 induced up-regulation of caspase-9 and -3, while BM-MSCs-siEPO co-culture significantly reversed the down-regulation of caspase-9 and -3 induced by BM-MSCs-siCTL co-culture. CONCLUSION：BM-MSCs protect PC12 cells from apoptosis induced by CoCl2. The protective effect of BM-MSCs might be executed by up-regulating the expression of EPO.     

EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by mTOR regulation

AIM:Hypoxia (evoked by CoCl2)-induced apoptosis and autophagy are emerging as crucial events in the etiopathology of many neurodegenerative diseases. Epigallocatechin gallate (EGCG) is the active ingredient in tea polyphenols with abilities of anti-apoptosis and anti-autophagy, but the mechanism has not been fully elucidated. In recent years, studies have reported that the mammalian target of rapamycin (mTOR) involved in the regulation of a variety of neurological like differentiation and maturation of nerve cells, anti-oxidative stress, etc. Therefore, we investigate that whether EGCG protects PC12 from hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. METHODS:The expression of mTOR and beclin-1 were detected by Western blotting. The expression of caspase-3 was measured by ELISA. The cell viability was detected by CCK-8 assay. The LC-3 expression in nucelus was observed by immunofluorescence. RESULTS:Hypoxia induced apoptosis and autophagy in PC12 cells. EGCG antagonized hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. Blocking the pathway of mTOR reversed the protective effect of EGCG on PC12 cells. CONCLUSION: EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by controlling mTOR regulation.     

Fasudil hydrochloride prevents cisplatin-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition

AIM: To explore the protective effect of fasudil hydrochloride against cisplatin(CP)-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition. METHODS: Healthy male Sprague-Dawley (SD) rats were randomly divided into control group, CP group and CP+ fasudil group. All animals were sacrificed 96 h after injection of 0.9% saline or CP. Blood samples and kidney tissues were collected to evaluate levels of blood urea nitrogen (BUN), serum creatinine (sCr) and morphological alteration of the kidneys, respectively. The apoptosis of renal tubular epithelium cells was detected by TUNEL. Protein levels of Rho-associated protein kinase 1 (ROCK1), PTEN and Akt were measured by Western blotting and immunohistochemistry. The protein level of p-Akt was analyzed by Western blotting. RESULTS: Compared with control group, the sCr and BUN levels, the expression of ROCK1 and PTEN and TUNEL-positive cells were increased, while the level of p-Akt was decreased in CP group and CP+fasudil group. The histological structure of the kidneys observed by PAS staining was developed marked structural damage in CP group(P<0.05). Compared with CP group, sCr level, the expression of ROCK1 and PTEN and TUNEL-positive cells were decreased, while the level of p-Akt was increased in CP+fasudil group (P<0.05). Very little structural damage was detected in fasudil-treated groups. CONCLUSION: Fasudil hydrochloride has a protective effect on CP-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition 1.     

Effects of Mage-H1 expression on PC12 cell differentiation and cell cycle

AIM: To explore the effects of melanoma-associated antigen H1 (Mage-H1) on cell proliferation and differentiation. METHODS: A phase contrast microscope was used to observe the morphological changes of PC12 cells treated with or without nerve growth factor (NGF). The expression of Mage-H1 in pre-and post-differentiated PC12 cells was detected by RT-PCR and Western blotting, and its potential effects on the cell cycle were analyzed by flow cytometry. RESULTS: After induced by NGF for 8 days, over 92% of PC12 cells were differentiated. The relative levels of Mage-H1 mRNA and protein in the differentiated PC12 cells were 4.6 times and 2.6 times higher than those in control cells,respectively. Moreover, the PC12 cells transiently expressed Mage-H1 were significantly arrested in G₀-G₁ phase as compared to the cells transfected with an empty vector.CONCLUSION: Mage-H1 inhibits the proliferation of PC12 cells and promotes the differentiation of the cells.     

miR-422a regulates SOX6 to inhibit injury of PC12 cells induced by hydrogen peroxide

AIM: To investigate the effects of microRNA-422a (miR-422a) on the damage of rat adrenal gland pheochromocytoma PC12 cells induced by hydrogen peroxide (H₂O₂). METHODS: The expression of miR-422a in the PC12 cells treated with H₂O₂ was detected by real-time PCR. After miR-422a mimics were transfected into PC12 cells, the cell viability was measured by MTT assay, the lactate dehydrogenase (LDH) leakage rate was detected, and the apoptosis was analyzed by flow cytometry. Target gene prediction software was used to predict that sex-determining region Y box 6 (SOX6) may be the target gene of miR-422a. Luciferase reporter assay was used to identify the targeting relationship. miR-422a mimics and SOX6 over-expression vector were co-transfected into the PC12 cells. The effects of SOX6 over-expression on the viability, LDH leakage rate and apoptosis of PC12 cells treated with H₂O₂ and transfected with miR-422a mimics were evaluated. RESULTS: The expression of miR-422a in the PC12 cells was decreased after treatment with H₂O₂ (P<0.05). The viability of PC12 cells treated with H₂O₂ was decreased, and the LDH leakage rate and apoptotic rate were increased. Transfection with miR-422a mimics enhanced the viability of PC12 cells treated with H₂O₂, and the leakage rate of LDH and apoptotic rate of the PC12 cells were reduced. The expression of SOX6 was negatively regulated by miR-422a. SOX6 over-expression reversed the effects of miR-422a on PC12 cell viability, LDH leakage and apoptosis. CONCLUSION: miR-422a reduces the damage of PC12 cells induced by H₂O₂ via targeting SOX6.     

Phosphorylation PKCδ participates in apoptosis of PC12 cells induced by 6-hydroxydopamine

AIM: To observe the effect of phosphorylation protein kinase C delta (PKCδ) on the procedure of PC12 cells apoptosis induced by 6-hydroxydopamine(6-OHDA) and to investigate the potential molecular pathogenesis of Parkinson disease.METHODS: TUNEL staining and transmission electron microscope were applied to measure apoptosis when dopaminergic PC12 cells exposed to the excitomotors and inhibitors of PKC before 6-OHDA for 18 hours. The expression of phosphorylation of PKCδ was detected by Western blotting. RESULTS: PMA, an activating agent of PKCδ, significantly increased PC12 cell apoptosis induced by 6-OHDA. Rottlerin, an inhibitor of PKCδ, protected PC12 cells apparently. As contrast, bisindolylmaleimide I, an inhibitor of general PKC and G6976, the inhibitor of calcium-dependent PKC, did not show any protective role. CONCLUSION: The phosphorylation PKCδ is one of the important links in the process of PC12 cell apoptosis induced by 6-OHDA. PKCδ may directly participate in neurodegeneration process in parkinsonian.     

Inhibitory effects of tumor associated mitochondrial protein 12 on HepG2 cell apoptosis

AIM: To explore the inhibitory effects of tumor associated mitochondrial protein 12 (TAMP12) on tumor cell apoptosis. METHODS: (1) A retrovirus expression vector was recombinated and transfected into the packaging cell line PA317. The virus particles were obtained to infect the target cell line HepG2 low expressing of TAMP12. The expression of TAMP12 mRNA was detected by RT-PCR. The subcellular localization and quantification of TAMP12 protein labeled with double fluorescein were observed under confocal laser scanning microscope (CLSM). (2) Hoechst33258 staining and flow cytometry (FACS) were used to analysis the apoptosis of HepG2 cells treated with 5-fluorouracil (5-FU). RESULTS: (1) The CLSM observation showed that TAMP12 protein was mainly expressed in mitochondria of HepG2 cells. The expressions of TAMP12 gene and protein were stable and high in transfected HepG2 cells. (2) Upon treatment with 5-FU, the transfected HepG2 cells showed a fairly integrated nucelus while the control HepG2 cells exhibited chromatin condensation, marginalization and karyorhexix. Moreover, the apoptosis rate of transfeced HepG2 cells was significantly lower than that in control HepG2 cells (P<0.05 or P<0.01) by FACS. CONCLUSION: TAMP 12 is an anti-apoptotic element in 5-FU induced HepG2 cell apoptosis.     

Effects of AT1 receptor autoantibody on renal cell apoptosis and JNK expression in diabetic nephropathy rats

AIM:To study the effects of angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) on the apoptosis of renal cell and the expression of c-Jun N-terminal kinase (JNK) in diabetic nephropathy (DN) rats. METHODS:High-sucrose and high-fat diet and intraperitoneal injection of streptozotocin were utilized to induce DN rat model. We employed enzyme-linked immunosorbent assay (ELISA) for serum AT1-AA and TUNEL staining for renal cell apoptosis. Furthermore, Western blotting was performed to measure the levels of endoplasmic reticulum stress (ERS) chaperone glucose-regulated protein 78 (GRP78) and ERS-associated apoptosis protein p-JNK. RESULTS:The renal cell apoptotic rate in DN group was significantly increased compared with NC group, and the apoptotic renal cells in AT1-AA positive DN rats were much greater than those in AT1-AA negative DN rats (P<0.05). The protein levels of GRP78 and p-JNK were significantly increased compared with NC group. GRP78 and p-JNK protein levels also significantly increased in AT1-AA positive DN rats compared with AT1-AA negative DN rats. CONCLUSION: AT1-AA activates ERS response and induces renal cell apoptosis via the JNK apoptotic pathway in the renal tissues of DN rats.     

Relationship between morphological changes of autophagy and apoptosis in PC12 cells induced by oxygen-glucose deprivation and reoxygenation

AIM: To investigate the relationship between morphological changes of autophagy and apoptosis in the PC12 cells induced by oxygen-glucose deprivation and reoxygenation. METHODS: The PC12 cells were randomly divided into normal control group, oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group. The cells in oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group were exposed to reoxygenation (12 h) after 3 h of oxygen-glucose deprivation, and autophagy inhibitor 3-methyladenine and autophagy activator rapamycin were added into the cells at the same time. Using transmission electron microscope and monodansylcadaverine fluorescence staining, the morphological changes of autophagosome were observed. The apoptosis of the PC12 cells were analyzed by flow cytometry with Annexin V-FITC/PI staining and TUNEL method. RESULTS: Compared with normal control group, the numbers of autophagosomes and the apoptotic rates increased in oxygen-glucose deprivation and reoxygenation group (P<0.05). Compared with oxygen-glucose deprivation and reoxygenation group, the numbers of autophagosomes decreased obviously (P<0.05) and the apoptotic rates increased markedly in autophagy inhibitor group (P<0.05). The numbers of autophagosomes increased obviously (P<0.05), the apoptotic rates decreased markedly (P<0.05), the autophagosomes became bigger in size, and autolysosomes was also found in autophagy activator group. CONCLUSION: Oxygen-glucose deprivation and reoxygenation induce autophagy in PC12 cells, and autophagy inhibits cell apoptosis to play a protective role.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

Effects of metoprolol on cardiomyocyte apoptosis and caspase-12 activation after coronary microembolization in rats

AIM: To investigate the effects of metoprolol on cardiomyocyte apoptosis and caspase-12 activation after coronary microembolization in rats. METHODS: 30 rats were randomized to sham-operated group (S group), coronary microembolization group (CME group) and metoprolol group. Coronary microembolization models were produced by injection of 42 μm microspheres (3000/0.1mL) into the left ventricle during 10 seconds ascending aorta occlusion in rats. The S groups were injected saline instead. Intravenous metoprolol was infused into the rats assigned to the metoprolol groups.Cardiomyocyte apoptosis was detected with in TUNEL staining. The activation of caspase-12 was measured by Western blotting analysis. Left ventricular ejection fraction (LVEF) was assessed by transthoracic two-dimensional echocardiography. RESULTS: ① LVEF was significantly decreased in CME group compared to S group (P<0.05). No statistical difference between the metoprolol group and CME group was observed. ②Compared with S group, the apoptosis rate of cardiomyocytes and the levels of activated caspase-12 proteins in CME group were significantly increased (all P<0.05). Compared with CME group, the apoptosis rate of cardiomyocyte and the levels of activated caspase-12 proteins in metoprolol group were significantly decreased (all P<0.05). CONCLUSION: Metoprolol inhibits the apoptosis of cardiomyocytes and the activation of caspase-12 after coronary microembolization.     

Dual immunoregulatory effects of CD69 on cell activation and apoptosis

CD69, a member of C type lectin superfamily and NK cell gene family, plays important roles in signal transduction in the regulation of cellular functions. Previous studies demonstrate that CD69 has a vital role in cell activation and CD69 is used as a marker of cell activation because of its early expression in activated cells. CD69 is also the costimulatory signal for cell activation, proliferation and differentiation. Furthermore, recent studies show the close association between CD69 and apoptosis. CD69 mediates apoptosis selectively in activated cells, mainly in activated eosinophils and monocytes. Using CD69 as an apoptotic inducer in certain cells may have good result and less side effect. This article tries provide a brief summary of the dual immunoregulatory effects of CD69 on cell activation and apoptosis.