Role of thioredoxin nitration in doxorubicin-induced apoptosis of neonatal rat cardiomyocytes

AIM：To investigate the role of thioredoxin nitration in the apoptosis of neonatal rat cardiomyocytes (NRCMs) induced by doxorubicin (DOX). METHODS：Cardiomyocytes treated with DOX were isolated from newborn Sprague-Dawley rats and cultured in vitro. NRCMs were treated with DOX alone (DOX group), pretreated with Mn (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), a peroxynitrite (ONOO-) scavenger, and then treated with DOX (MnTMPyP+DOX group), or treated with MnTMPyP alone (MnTMPyP group). NRCMs without any treatment served as a normal control (control group). The viability of the cells was examined by MTT assay, and the apoptosis was measured by Hoechst 33258 nuclear staining kit. The activity of caspase-3 was detected by spectrophotometry. The expression of cleaved poly(ADP-ribose) polymerase 1 (PARP-1), apoptosis signal-regulating kinase 1 (ASK1), phosphorylated ASK1 (p-ASK1), p38 mitogen-activated protein kinase (p38 MAPK) and phosphorylated p38 MAPK (p-p38 MAPK) was measured by Western blotting. Immunoprecipitation and immunoblotting were performed to detect the formation of Trx-ASK1 and Trx-nitrotyrosine. RESULTS：DOX induced significant apoptosis of NRCMs. MnTMPyP could significantly attenuate the apoptosis induced by DOX. Compared with control group, Trx nitration in DOX group increased obviously. The increases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were also observed, besides the expression of Trx-ASK1 compound and p-ASK1 decreased significantly (P<005). MnTMPyP could decrease the nitration of Trx. The decreases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were detected in MnTMPyP+DOX group, while the expression of Trx-ASK1 compound and p-ASK1 increased significantly (P<005). CONCLUSION: DOX could induce significant apoptosis of NRCMs and increase Trx nitration. The process was significantly attenuated by pretreatment with MnTMPyP. Therefore, Trx nitration may play an important role in doxorubicin-induced apoptosis of cardiomyocytes.     

p38 MAPK-Pim-3 signal pathway may be involved in cardiomyocyte anoxia preconditioning against anoxia/reoxygenation injury

AIM: To investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 protects cardiomyocyte against anoxia/reoxygenation (A/R) injury. METHODS: The primarily cultured neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; APC+A/R group; SB203850, U0126 or SP600125+APC+A/R group. The cells were pre-incubated with U0126 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor), or SB203850 (p38 MAPK inhibitor) at concentration of 10 μmol/L for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was detected by TUNEL. RESULTS: U0126, SB203850, and SP600125 abolished the increased expression of ERK1/2, p38-MAPK, and JNK proteins induced by APC+A/R or A/R, respectively. The expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited. Meanwhile, the activity of LDH and the apoptosis index increased, and the viability of cardiomyocytes decreased. CONCLUSION: Pim-3 expression through a p38 MAPK signaling pathway may protect cardiomyocytes from A/R injury.     

Role of thioredoxin-ASK1 in doxorubicin-induced apoptosis of neonatal rat cardiac myocytes

AIM: To investigate the role of thioredoxin(Trx)-apoptosis signal-regulating kinase 1(ASK1) in doxorubicin-induced apoptosis of neonatal rat cardiac myocytes (NRCMs). METHODS: Primary cardiomyocytes were isolated from newborn Sprague-Dawley rats with the purity of NRCMs >95%. NRCMs were pretreated with the indicated concentrations of ebselen 2 h prior to the addition of doxorubicin, then treated with doxorubicin at concentration of 1 μmol/L for another 24 h. The viability of the cells was examined by MTT assay.Reactive oxygen species(ROS) levels were measured by a ROS-specific probe 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). Apoptotic cardiomyocytes were determined by Hoechst 33258 nuclear staining. The activity of caspase-3 was detected with a caspase-3 colorimetric assay kit. The protein levels of poly(ADP-ribose) polymerase 1(PARP1), ASK1, p-ASK1, p38 and p-p38 were determined by Western blotting. Immunoprecipitation and immunoblotting were performed to detect whether the Trx-ASK1 was dissociated. RESULTS: Doxorubicin induced significant apoptosis of NRCMs. The levels of ROS were significantly increased. Ebselen significantly decreased the apoptosis. Compared with control group, increased activity of caspase-3 was showed in doxorubicin group (P<0.01). Increased protein levels of PARP1, ASK1 and p38 were observed (P<0.01). The increase in the dissociated Trx-ASK1 was also found. Compared with doxorubicin group, ebselen decreased the activity of caspase-3 (P<0.01), the levels of PARP1,ASK1 and p38 proteins (P<0.05), and the dissociated Trx-ASK1. CONCLUSION: Doxorubicin induces significant apoptosis of NRCMs. ASK1 is partly dissociated from Trx, and starts the ASK1-mediated apoptotic signaling. The process is significantly attenuated by pretreatment with ebselen. Trx-ASK1 plays an important role in doxorubicin-induced apoptosis of cardiomyocytes.     

Effect of p38 MAPK on cisplatin-induced renal tubular cell apoptosis

AIM:To investigate the role of p38 MAPK in cisplatin-induced rat renal proximal tubular cell (RPTC) apoptosis. METHODS:To determine the optimal concentration of cisplatin to induce RPTC apoptosis, the cells were treated with 0, 5, 10 and 20 μmol/L cisplatin for 24 h, and then the cell lysates were collected for Western blot analysis of cleaved PARP, p38 and phosphor ylated p38 (p-p38). To determine the role of p38 MAPK in cisplatin-induced RPTC apoptosis, the cells were divided into control group, cisplatin group (the cells were treated with cisplatin for 24 h) and cisplatin+p38 MAPK inhibitor group (the cells were treated with p38 MAPK inhibitor SB203580 for 1 h, and then treated with cisplatin for another 24 h). The morphological changes of apoptotic cells were observed under phase-contrast fluorescence microscope. The apoptotic rate of the cells were analyzed by flow cytometry. The caspase activity of RPTC lysates was examined using Ac-DEVD-AFC kit. The protein levels of p-p38, p38, cleaved PARP and cleaved caspase-3 were determined by Western blot. The pH value of extracellular environment of the cells was measured by pH meter. RESULTS:Cisplatin at 20 μmol/L obviously induced apoptosis of RPTC. The p38 MAPK was phosphorylated and its phosphorylation peaked at 15 min after cisplatin treatment. The apoptotic rate of RPTC was 12.08% after cisplatin induction. Cisplatin treatment also enhanced caspase activity, and increased cleavage of PARP and caspase-3 proteins (P<0.05). The p38 MAPK inhibitor SB203580 effectively inhibited the phosphorylation of p38 MAPK, down-regulated the RPTC apoptosis rate and caspase activity, and reduced the cleavage of PARP and caspase-3 proteins. The pH value change in RPTC culture medium was also inverted by SB203580. CONCLUSION:The phosphorylation of p38 MAPK is involved in cisplatin-induced apoptosis of RPTC. The apoptosis induced by cisplatin results in the change of acidic extracellular environment, which is inhibited by p38 MAPK inhibitor SB203580.     

Involvement of caspase-3 and p38MAPK in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells

AIM: To investigate the function of caspase-3 and mitogen-activated protein kinases (MAPKs) in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells. METHODS: Allogeneic CD8+T cells were isolated from PBMC by positive selection using magnetic beads coated with anti-CD8 antibody. After cocultured with allogeneic CD8+T cells, apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) were detected by AnnexinV-FITC labeling. Western blotting was used to examine the change of MAPK and caspase-3 expression in the vascular endothelial cells. The influence of SB203580 (inhibitor of p38MAPK), SP600125 (inhibitor of JNK), PD98059 (inhibitor of ERK), Z-DEVD-FMK (a caspase-3-specific peptide inhibitor) on apoptosis was also examined. RESULTS: At 24 h and 48 h time-point, the apoptosis rates of HUVECs were 41.7%±10.1% and 29.4%±8.3%, respectively (P<0.01, vs untreated HUVECs); the apoptosis rates of HDMECs were 28.9%±7.2% and 15.2%±4.8%, respectively (P<0.01, vs untreated HDMECs). These effects were largely prevented by Z-DEVD-FMK and SB203580 (P<0.05). Allogeneic CD8+T cells enhanced cleavage of caspase-3 and led to p38MAPK phospholation. CONCLUSION: Caspase-3 and p38MAPK mediate allogeneic CD8+T cells-induced apoptosis of vascular endothelial cells.     

TPX2 induces apoptosis of rectal cancer HR-8348 cells through p38 MAPK signaling pathway

AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells.METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA (siRNA) served as TPX2 siRNA group. The non-transfected cells were used as control group. The cells transfected with siRNA negative control (siRNA-NC) were used as siRNA-NC group. The TPX2 siRNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 siRNA+SB203580 group. The expression of TPX2 at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK, p-p38 MAPK, cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot.RESULTS: After transfection, the expression of TPX2 at mRNA and protein levels was decreased in TPX2 siRNA-transfected cells (P<0.05). Transfection with siRNA-NC had no effect on TPX2 mRNA and protein levels in the cells. After knockdown of TPX2 expression, the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased, while the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK were increased significantly reduced (P<0.05). Compared with TPX2 siRNA group, the apopto-tic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 siRNA+SB203580 group were significantly decreased, while the viability was significantly increased (P<0.05).CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.     

Effect of serum amyloid A on invasion,migration and MAPK signaling pathway in osteosarcoma cells

AIM: To investigate the effect of silencing of serum amyloid A (SAA) on the viability, apoptosis, migration and mitogen-activated protein kinase (MAPK) signaling pathway in osteosarcoma U2OS cells. METHODS: Small interfering RNA (siRNA) targeting SAA was transfected into U2OS cells to silence the expression of SAA gene. The U2OS cells were divided into blank control group, negative control group, and experimental group. The cells in negative control group and experimental group were transfected into negative control siRNA and SAA-siRNA, respectively. The cells in blank control group were without any treatment. The viability of the cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry with Annexin V-FITC/PI double staining. The migration and invasion abilities of the cells were detected by Transwell chamber assay. The protein levels of SAA, phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated c-Jun N-terminal kinase (p-JNK) in the cells were determined by Western blot. RESULTS: The protein expression of SAA in SAA-siRNA group was significantly lower than that in blank control group (P<0.05). Compared with blank control group, the cell viability in SAA-siRNA group was significantly decreased (P<0.05), the apoptotic rate was significantly increased (P<0.05), and the invasion and migration abilities were significantly decreased (P<0.05). The protein levels of p-p38 MAPK and p-JNK in SAA-siRNA group were significantly lower than those in blank control group (P<0.05), and no significant difference of total JNK and p38 protein levels was observed. CONCLUSION: Silencing of SAA expression inhibits the viability of osteosarcoma cells, induces apoptosis and decreases the migration of osteosarcoma cells, which may be related to the activation of MAPK signaling pathway.     

Role of TGF-β1-activated p38 MAPK in up-regulation of PAI-1 expression by TGF-β1 in human ovarian cancer cells

AIM: To investigate the relationship between up-regulation of plasminogen activator inhibitor-1(PAI-1) expression and activation of p38 mitogen-activated protein kinase(p38 MAPK) and extracellular signal-regulated kinase(ERK) pathways by TGF-β1 in human ovarian cancer cells. METHODS: PAI-1 expression in human ovarian cancer cells treated with TGF-β1(10 μg/L)was assayed by real-time PCR and Western blotting. The activation of p38 MAPK and ERK was determined by Western blotting using phosphorylated p38 MAPK and phosphorylated ERK antibodies. Specific p38 MAPK inhibitor(SB203580) or ERK inhibitor(PD98059) was used to inhibit their activation. RESULTS: TGF-β1 up-regulated the expression of PAI-1, and activated p38 MAPK and ERK pathways in the ovarian cancer cells. Inhibition of p38 MAPK activation by SB203580 resulted in significant inhibition of the mRNA expression of PAI-1 induced by TGF-β1. However, inhibition of ERK activation did not significantly alter TGF-β1-induced increase in PAI-1 mRNA level. CONCLUSION: TGF-β1-activated p38 MAPK pathway contributes to the up-regulation of PAI-1 expression by TGF-β1 in ovarian cancer cells.     

Effect of Radix Tetrastigma hemsleyani flavone on apoptosis and MAPK signaling pathway in myeloid leukemia NB-4 cells

AIM:To study the effects of Radix Tetrastigma hemsleyani flavone (RTHF) on the viability, apoptosis and MAPK signaling pathway in human acute promyelocytic leukemia NB-4 cells. METHODS:The inhibitory effects of RTHF on the viability and proliferation of NB-4 cells were measured by CCK-8 assay and BrdU test. Flow cytometry was used to analyze the apoptosis of NB-4 cells induced by RTHF, and the cell cycle distribution after RTHF treatment. The levels of apoptosis- and MAPK pathway-related proteins in the NB4 cells were determined by Western blot. RESULTS:RTHF inhibited the viability and proliferation of NB-4 cells in a time- and dose-dependent manner, and the IC₅₀ at 48 h was 2.26 g/L. RTHF blocked NB-4 cells into the cell proliferation cycle, with stagnation in the G₂ phase. Meanwhile, RTHF induced apoptosis of the cells, down-regulated the expression of anti-apoptotic protein Bcl-2, and up-regulated the expression of pro-apoptotic proteins Bax, caspase-3 and Cyt-C, in a dose-dependent manner (P<0.05). The expression of ERK5 was decreased, and p38 was increased induced after RTHF treatment. However, no obvious change of ERK1/2 and JNK after RTHF treatment was observed. CONCLUSION:RTHF effectively inhibits the viability and proliferation, and induces apoptosis of leukemic NB-4 cells in vitro. Its mechanism may be related to signaling pathways of p38 MAPK and apoptosis proteins.     

Involvement of extracellular signal regulated kinase in the regulation of amyloid precursor protein processing in PC12 cells by TPPB

AIM: To explore the signal transduction pathways involved in the regulation of amyloid precursor protein (APP) processing by protein kinase C (PKC) activator TPPB.METHODS: PC12 cells were treated with TPPB (PKC activator) for 3 h and various signal transduction inhibitors were added to the conditioned medium to investigate their effects on α-secretase form of soluble amyloid precursor protein (sAPPα) secretion after TPPB treatment via Western blotting. Extracellular signal regulated kinase (ERK, p42/44MAPK) and phospho-p42/44MAPK were also measured after TPPB treatment.RESULTS: TPPB (1 μmol/L) significantly increased sAPPα secretion as compared with control group. The increase in sAPPα secretion by TPPB was partially blocked by ERK inhibitor U0126, c-Jun N-terminal kinase (JNK) inhibitor SP600125 and protein tyrosine kinase (PTK) inhibitor genistein, but not by p38MAPK inhibitor SB203580. TPPB (1 μmol/L) increased the expression of phospho-p42/44MAPK without altering total p42/44MAPK levels.CONCLUSION: ERK, JNK and PTK may be involved in the regulation of APP processing by TPPB.     

Manumycin inhibits the activity of breast cancer cell line SK-BR-3 via inducing apoptosis

AIM: To investigate the anticancer effect of manumycin on abdominal metastatic breast cancer cell line - SK-BR-3 and its relationship with p38 MAPK. METHODS: The test of anticancer effect was performed by the method of MTT, apoptosis induced by manumycin and affected by SB203580, a specific p38 MAPK inhibitor, were examined by caspase-3 activity assay kit, and the protein expression was detected by immunoblotting assay. RESULTS: The inhibition rates at 24 h after treatment with manumycin of 6 μmol/L, 18 μmol/L, 54 μmol/L were (7.4±3.9)%, (21.0±4.4)% and (64.7±4.1)%, respectively and showed dosage-effect relationship. Compared with the control group, the survival rates of the last two treatment groups were decreased significantly (P<0.01). The value of IC₅₀ 24 h after treatment with manumycin was 42.5 μmol/L. Manumycin simultaneously activated caspase-3 protein, which was partly blocked by p38 MAPK inhibitor, SB203580. The results of immunoblotting showed that manumycin increased p38 MAPK protein phosphorylation. CONCLUSION: Manumycin exerts anticancer effect on SK-BR-3 cell line via inducing cell apoptosis, which is partly regulated by p38 MAPK.     

Effect of normal mesenteric lymph on multiple organ injury in mice with endotoxic shock

AIM: To observe the effects of normal mesenteric lymph (NML) on the lung, heart and liver injuries and the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) in the mice with endotoxic shock (ES). METHODS: The NML was drained form health male BALB/c mice for the intervention of ES after the removal of cellular constituent. Lipopolysaccharide (LPS, 35 mg/kg) was intraperitoneally injected into the mice for the establishment of ES model. After 60 min of LPS injection, the administration of NML (1/15 of whole blood volume) was performed through the femoral artery in NML+ES group. Meanwhile, the mean arterial pressure (MAP) was monitored during the experiment. At 6 h after intraperitoneal injection of LPS or the corresponding time point, blood samples were harvested from the heart through apical centesis for determination of the biochemical indexes to reflect myocardial and hepatocyte injuries. Simultaneously, the lung, heart and liver tissue specimens from a fixed location were harvested for the observation of histomorphology and the measurement of phosphorylation levels of p38 MAPK, ERK1/2 and JNK. RESULTS: Compared with sham shock (SS) group, MAP in ES group and NML+ES group remarkably decreased at multiple time points after intraperitoneal injection of LPS. However, MAP in NML+ES group at 80 min, 90 min, 190 min, 210 min, 240 min, 250 min, 340 min, 350 min, and 360 min were significantly increased compared with ES group. There were normal structures in the lung, liver and myocardium of the mice in SS group, while the morphological damages of these tissues appeared in ES group. Meanwhile, the damages were attenuated in the mice of NML+ES group. The activities of AST, ALT and CK-MB in the plasma in ES group were remarkably higher than those in SS group. The CK-MB activity in NML+ES group was also increased compared with SS group, and the activities of AST and LDH-1 were lower than those in ES group. At 6 h after LPS injection, the phosphorylation levels of p38 MAPK, ERK1/2 and JNK in the lung tissues were remarkably increased. Meanwhile, no statistical difference of these indexes between the myocardial and hepatic tissues was observed. NML intervention decreased the phosphorylation levels of p38 MAPK in the lung tissues, and p38 MAPK, ERK1/2 and JNK in the myocardial tissues. CONCLUSION: The NML administration alleviates multi-organ injuries and reduces the phosphorylation level of p38 MAPK in the lung tissues in the mice subjected to ES.     

Oxidative stress plays an important role in acetaldehyde-induced cardiomyocytes apoptosis

AIM: To elucidate the mechanism of alcoholic myocardiopathy (AHMD) by exploring the role of ROS mediated oxidative stress in acetaldehyde-induced cardiomyocytes apoptosis. METHODS: Cultured rat cardiomyocytes were stimulated with acetaldehyde (100 μmol/L) for 24 h, then the cell apoptotic index were examined and compared to that with alcohol (100 μmol/L) stimulation. The changes of ROS levels and SOD activities were explored by a time-dependent model in acetaldehyde-induced cardiomyocytes. Meanwhile, the phosphorylation changes of ROS mediated MAPK signaling pathway correlated proteins were also detected, with which compared that changes both in pre-adding NAC acetaldehyde-induced cardiomyocytes, and in H2O2 (100 μmol/L) induced cardiomyocytes, respectively. RESULTS: Acetaldehyde had more obvious apoptotic effect than alcohol through caspase 3 activating (P<0.05, vs control), both cellular ROS level and SOD activity increased in a time-dependent way, and reached the peak value of (78.84%±4.33%) for ROS and (0.55±0.02) for SOD among 18 to 24 h, respectively. Meanwhile, JNK and ERK protein phosphorylation upregulated in acetaldehyde-induced cardiomyocytes, and was reversed by NAC anti-oxidative effect. However all the phosphorylation levels were weaker than that in H2O2-induced group. CONCLUSION: Acetaldehyde causes oxidative damage in cardiomyocytes through enhancing cellular ROS level, and induces cardiocytes apoptosis by activating ROS mediated JNK pathway. The novel way of depressing cellular ROS level or blocking the special apoptotic pathway may have effects on AHMD preservation and therapy.     

Effects of naringin on MAPK signal pathway in rat bone marrow mesenchymal stem cells during osteogenic differentiation

AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β₁), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10^-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β₁ did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β₁, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.     

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in the vascular smooth muscle cells (VSMCs). METHODS: mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RT-PCR and ELISA, respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies (a specific TLR4 inhibitor), PD98059 (ERK1/2 specific inhibitor), SB23015 (p38MAPK specific inhibitor) and SP600125 (JNK specific inhibitor) were used to investigate the underlying mechanisms. RESULTS: The mRNA and protein expressions of MCP-1 in VSMCs were up-regulated by ox-LDL (P<0.05), while those were inhibited by TLR4 neutralizing antibodies, PD98059 or SB23015 (P<0.05), but not by SP600125 (P>0.05). TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION: ox-LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4/ERK1/2 and TLR4/p38MAPKs pathways.     

Low concentration MNNG inhibition of JNK/SAPK is independent of a nuclear signal while MNNG activation of p38MAPK may depend on a nuclear signal

AIM:To identify the effect of alkylating agent N-methyl-N'- nitro-N-nitrosoguanidine (MNNG) with low concentration on JNK/SAPK and p38MAPK and the origins of JNK/SAPK and p38MAPK cascade.METHODS:p38 and JNK kinase activity were detected by immunoprecipitation and Western immunoblotting in intact and enucleated Vero cells.RESULTS:With the same experimental conditions, low concentration of MNNG inhibited JNK kinase in both intact cell and enucleated Vero cell. MNNG activated p38 kinase in intact cell while no effect on p38 kinase in enucleated cell was observed.CONCLUSION:Inhibition of JNK/SAPK by low concentration of MNNG was independ of a nuclear signal while MNNG activation of p38MAPK may depend on a nuclear signal.     

Leptin protects rat cardiomyocytes from H2O2-induced apoptosis

AIM: To investigate the effect of leptin on H₂O₂-induced apoptosis in rat cardiomyocytes (H9c2 cells) and the underlying mechanisms.METHODS: Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to determine H₂O₂-induced apoptosis in H9c2 cells in the absence or presence of leptin. The activities of caspase-3 and extracellular signal-regulated kinase (ERK) were examined by Western blotting.RESULTS: (1)Leptin significantly inhibited H₂O₂-induced apoptosis in H9c2 cells (P<0.01). This effect of leptin was opposed by MEK inhibitor PD98059. (2)H₂O₂ inhibited basal ERK activity. Leptin activated ERK and partially inhibited H₂O₂-induced caspase-3 activation.CONCLUSION: Leptin protects H9c2 cells from H₂O₂-induced apoptosis possibly by activating ERK.     

p38MAPK expression and apoptosis in the rat unilateral ureteral obstruction model

AIM: To investigate the expression of p38 mitogen-activated protein kinase (p38MAPK) in the kidney after unilateral ureteral obstruction (UUO) in rats and the functional role of it on apoptosis and fibrosis.METHODS: Eighteen Wistar rats underwent UUO were killed at 3, 7, 14 days. Additional 7 rats were sham operated. Histological changes were observed by HE and Masson staining. Immunohistochemistry study was performed on renal tissue for proliferating cell nuclear antigen (PCNA). Apoptotic cells were determined by terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) and the electrophoresis analysis of genomic DNA. Western blotting of cysteinyl aspartate specific proteinase-3 (caspase-3), p38MAPK and p-p38MAPK were measured.RESULTS: UUO induced a significant increase in renal tubular and interstitial cell apoptosis, immunohistochemistry of PCNA and Western blotting of caspase-3, p-p38MAPK as well as severe morphology changes. However, there was no significant difference between UUO and the control in Western blotting of p38MAPK.CONCLUSION: An in vivo model of renal fibrosis after UUO demonstrates that activated or phosphorylated p38MAPK plays a role in apoptosis of renal tubulointerstitial cells.     

Effect of PARP-1 on cisplatin resistance in breast cancer MCF-7 cells

AIM: To study the effect of poly(ADP-ribose) polymerase-1 (PARP-1) on cisplatin resistance of human breast cancer MCF-7 cells and its possible mechanisms.METHODS: The expression of PARP-1 at mRNA and protein levels in MCF-7 cells and MCF-7/DDP cells was determined by real-time PCR and Western blot. The expression of PARP-1 in the MCF-7/DDP cells was blocked by PARP-1 siRNA. The cell viability and apoptosis were detected by the CCK-8 assay and flow cytometry analysis, respectively. Furthermore, the protein levels of PARP-1, Bcl-2, Bax, cleaved caspase-3, caspase-3, cytochrome C (Cyto-C), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) were detected by Western blot.RESULTS: The expression of PARP-1 at both mRNA and protein levels was significantly up-regulated in the MCF-7/DDP cells. The expression of PARP-1 was increased in the MCF-7 cells treated with cisplatin. Knockdown of PARP-1 induced the apoptosis of MCF-7/DDP cells with an increased sensitivity to cisplatin. Meanwhile, knockdown of PARP-1 down-regulated the protein levels of Bcl-2/Bax and p-ERK, but up-regulated the protein levels of cleaved caspase-3 and Cyto-C. After incubated with a specific ERK inhibitor U0126, the cell viability in PARP-1 siRNA group was down-regulated significantly.CONCLUSION: Knockdown of PARP-1 increases the sensitivity of MCF-7/DDP cells to cisplatin, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of ERK signaling pathway by inhibiting phosphorylation of ERK.     

Dexmedetomidine reduced isoflurane-induced neuroapoptosis by inhibiting activation of p38 and JNK proteins in hippocampus of neonatal rats

AIM：To investigate the effect of dexmedetomidine (Dex) on neuronal apoptosis induced by isoflurane (Iso) and its relationship with the expression of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) proteins in the hippocampus of neonatal rats. METHODS：Forty-eight neonatal SD rats at postnatal day 7 were randomly divided into control group (Con), Dex group, Iso group and Iso combined with Dex (Iso+Dex) group. Rats in Iso and Iso+Dex groups were exposed to 0.75% Iso for 6 h, while rats in Con and Dex groups were exposed to air for 6 h. Rats were intraperitoneally injected with 25 μg·kg-1 Dex (Dex and Iso+Dex groups) or 150 μL saline (Con and Iso groups) 20 min before exposure and 2 and 4 h after exposure. After the termination of anesthesia, the neuronal apoptosis in hippocampal CA1 region was detected by TUNEL staining, and the protein expression of cleaved caspase-3, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK) and JNK in hippocampal tissues was detected by Western blotting. RESULTS：The number of TUNEL positive cells in hippocampal CA1 region of the rats in Iso group was increased by 447.57% (P<0.01) compared with Con group, while Dex significantly inhibited the increased TUNEL positive cells in Iso group by 75.18% (P<0.01). The expression of cleaved caspase-3 protein in Iso group was increased by 126.29% (P<0.01) compared with Con group, while Dex reversed the increased cleaved caspase-3 protein expression (P<0.01). Iso significantly increased the phosphorylation of p38 and JNK proteins (P<0.01), while Dex reversed the increased p-p38 and p-JNK proteins (P<0.01). CONCLUSION：Dex attenuates Iso-induced neuroapoptosis in the hippocampus of neonatal rats through inhibiting the phosphorylation of p38 and JNK proteins.