BACKGROUNDMesangial hypercellularity is a critical early histopathological finding in human and experimental glomerular diseases. Hyperlipidemia and the glomerular deposition of lipoproteins are commonly associated with mesangial hypercellularity and play an important pathobiological role in the development of glomerular diseases. The activated cytoplasmic mitogen-activated protein kinase (MAPK), including mainly extracellular-signal regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, has been thought to translocate into the nucleus and activate various transcription factors and protooncogenes associated with cell growth and proliferation. Lipoprotein (a) (Lp(a)) has been shown to stimulate proliferation of mesangial cells, but the events of Lp(a) signaling have not yet been characterized. The purpose of this study was to investigate the signal transduction pathways involved in Lp(a)-induced cell proliferation and provide an evidence for the participation of Lp(a) in intracellular signaling pathways for mesangial cell proliferation.

METHODSLp(a) was isolated from a patient who was being treated with low density lipoprotein (LDL)-apheresis by density gradient ultracentrifugation and then chromatography. Human mesangial cells (HMCs) were isolated by the sequential sieving technique and stimulated with Lp(a) in different concentration and time course. The DNA synthesis of the cells was measured by [3H] thymidine incorporation for detecting the proliferation. The expression of all the three members of MAPK family, including ERK1/ERK2, JNK, and p38, and their phosphorylation were detected by Western blotting.

RESULTSLp(a) could induce a significant dose-dependent proliferation of HMCs. The 3H-TdR incorporation was 1.64+/-0.31, 1.69+/-0.48, 3.59+/-0.68 (P<0.01), 4.14+/-0.78 (P<0.01), and 4.05+/-0.55 (P<0.01) (10(3) cpm) at the Lp(a) concentration of 0, 5, 10, 25, and 50 microg/ml, respectively. Lp(a) induced an increase in ERK1/ERK2 phosphorylation between 5 and 60 minutes, and in JNK phosphorylation between 15 and 30 minutes after incubating with HMCs, whereas the level of p38 and its phosphorylation was not changed.

CONCLUSIONSLp(a) could stimulate the proliferation of HMCs by activiating the phosphorylation of ERK1/ERK2 and JNK MAPK signaling pathway, whereas p38 pathway had no effect on the Lp(a)-induced HMC proliferation, which indicated that three MAPKs seem to be distinctly involved in the effect. In particular, it also provides the evidence that Lp(a) may act as one of the major endogenous modulators for mitogenic signaling response and cell proliferation within the glomerulus.

Blotting, Western ; Cells, Cultured ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lipoprotein(a) ; pharmacology ; Mesangial Cells ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

The cell counting kit-8 (CCK-8) proliferation assay and diameter measure of neurospheres were used to investigate the effect of high-mobility group box 1 (HMGB1) on proliferation of primary rat neural stem cells (NSCs) in vitro, and c-Jun N-terminal protein kinase (JNK) potent inhibitor SP600125 was used to study the mechanism. The results demonstrated that HMGB1 significantly increased CCK-8 absorbance values and neurosphere diameters at concentrations of 1 and 10 ng/mL at 48 h and 72 h (P < 0.05), and the other HMGB1 concentration groups (0.01, 0.1, 100 ng/mL) showed no significant difference, compared with control group (P > 0.05). HMGB1 at 10 ng/mL significantly increased the NSCs proliferation accompanied by the rising of phosphorylated JNK levels (P < 0.01), and 10 μmol/L SP600125 prevented these effects in HMGB1-cultured NSCs (P < 0.01). In conclusion, low concentration of HMGB1 (1-10 ng/mL) can increase NSCs proliferation, which may play a role by promoting JNK phosphorylation.
Animals ; Cell Proliferation ; Cells, Cultured ; HMGB1 Protein ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Neural Stem Cells ; cytology ; Phosphorylation ; Rats

To explore the effect of propofol on human cardiac AC16 cells under CoCl2-induced hypoxic injury and the possible mechanisms.
 Methods: Human AC16 cardiomyocytes were treated with cobalt chloride (CoCl2) to mimic hypoxic condition in cultured cardiomyocytes. The AC16 cells were divided into 3 groups: a control group, a CoCl2 hypoxia group (CoCl2 group), and a propofol+CoCl2 group (propofol+ CoCl2 group). The cell viability was assessed by cell counting kit-8 (CCK-8). Cell apoptosis ratio (AR) and the mitochondrial membrane potential (Δψm) were detected by flow cytometry. The reactive oxygen species (ROS) production in AC16 cells were determined with the ROS-sensitive fluorescent probe. Meanwhile, total intracellular levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in AC16 cells were detected with commercially available kits. Western blot was used to evaluate the activation of c-Jun N-terminal kinase (JNK) and p38 signaling pathways.
 Results: 1) Compared with the control group, AC16 cell viability was decreased significantly in the CoCl2 group following the treatment with 500 μmol/L CoCl2 (P<0.01); 2) Compared with the control group, AR value in AC16 cells was increased significantly in the CoCl2 group, while Δψm was decreased significantly (all P<0.01). Compared with the CoCl2 group, AR value in AC16 cells was decreased significantly in the propofol+CoCl2 group, while Δψm was increased significantly (both P<0.05); 3) Compared with the control group, the levels of ROS and MDA were increased significantly, and the level of SOD was significantly decreased in the CoCl2 group (all P<0.01). Compared with the CoCl2 group, the ROS and MDA levels in the propofol+CoCl2 group were increased significantly and the SOD levels were decreased significantly (all P<0.05); 4) Compared with the control group, the phosphorylation levels of JNK and p38 were increased significantly (both P<0.05) in the CoCl2 group. Compared with the CoCl2 group, the phosphorylation levels of JNK and p38 were decreased significantly in the propofol+CoCl2 group (both P<0.05).
 Conclusion: The pretreatment with propofol may protect human cardiac AC16 cells from the chemical hypoxia-induced injury through regulation of JNK and p38 signaling pathways.
Apoptosis ; Cell Hypoxia ; Cell Line ; Cell Survival ; Cobalt ; pharmacology ; Humans ; Hypoxia ; JNK Mitogen-Activated Protein Kinases ; Propofol ; Reactive Oxygen Species

OBJECTIVETo investigate the effects of mitogen activated protein kinase (MAPK) signal transduction pathways on heat shock protein 70 (HSP70) gene expression in endothelial cells exposed to benzo(a)pryene (BaP).

METHODSPorcine aortic endothelial cells were pre-treated or by PD98059 (10 micro mol/L) or SB203580 (20 micro mol/L) for 1 hour, then treated with different concentrations of BaP (0, 0.1, 0.5, 1.0, 5.0 and 10.0 micro mol/L) for 24 hours respectively;Expression levels of three phosphorylated MAPKs [extracellular signal regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38] and HSP70 were determined by Western-blot.

RESULTSThe three phosphorylated MAPKs expressional levels especially p-ERK1 had different extents of changes with dose-response relationship under BaP exposure. BaP inhibited the expression of HSP70, which significantly decreased in medium and high dose group (>or= 1.0 micro mol/L) but did not decrease in control group (P < 0.05). Although the inhibitor of ERK (PD98059) could partly weaken the inhibited effects of BaP on HSP70 expression, HSP70 expression levels of endothelial cells pre-treated with PD98059 were still significantly lower than that of control cells (P < 0.05).

CONCLUSIONERK1 pathway might play some roles in HSP70 gene expression in endothelial cells exposed to BaP, and other unknown signal pathways might also have some effects on this process.

Animals ; Benzo(a)pyrene ; toxicity ; Blotting, Western ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; HSP70 Heat-Shock Proteins ; analysis ; Imidazoles ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 4 ; Mitogen-Activated Protein Kinase Kinases ; analysis ; Mitogen-Activated Protein Kinases ; analysis ; antagonists & inhibitors ; Pyridines ; pharmacology ; Signal Transduction ; physiology ; Swine ; p38 Mitogen-Activated Protein Kinases

The aim of this study was to investigate the effect of proteasome inhibitor bortezomib on the expression of ERK, JNK, and P38 in daunorubicin (DNR)-resistant K562 cells and its mechanism. MTT method was used to determine the drug-resistant K562 cells and the cellular toxicity of bortezomib; Western blot was used to detect the expression of protein ERK, JNK and P38 in K562 cells after treatment with 100 nmol/L DNR alone or combined with 1 nmol/L and 10 nmol/L bortezomib for 36 hours. Flow cytometry assay was used to detect the apoptosis rate in each group cells. The results indicated that the expression of ERK and P38 were significantly suppressed (p < 0.05) and the expression of JNK was significantly enhanced (p < 0.05) in the cells treated by DNR combined with bortezomib. It is concluded that bortezomib can decrease the expressions of protein ERK and P38 and enhance the expression of JNK, the bortezomib reverses the cellular drug-resistance and promote cell apoptosis through MAPK pathway.
Antineoplastic Agents ; administration & dosage ; pharmacology ; Boronic Acids ; administration & dosage ; pharmacology ; Bortezomib ; Drug Resistance, Neoplasm ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; K562 Cells ; Protease Inhibitors ; administration & dosage ; pharmacology ; Pyrazines ; administration & dosage ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective: To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate. Methods: In September 2020, primary hippocampal neuronal cells were isolated and cultured from fetal rats, and identified using cellular immunofluorescent. MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis. MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate. According to the results, different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells, after 24 h, lead acetate was added to the cells, meanwhile, blank and model groups were set up, the content of reactive oxygen species (ROS) , superoxide dismutase (SOD) , lactate dehydrogenase (LDH) , malondialdehyde (MDA) and Caspase-3 were measured. Extracellular signal regulated kinase (ERK) , phosphorylated ERK (p-ERK) , p38 mitogen -activated protein kinases (p38MAPK) , phosphorylated p38MAPK (p-p38MAPK) , c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) protein expression in hippocampal neuronal cells were determined by Western blotting. Results: The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate, MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25 μmol/L. Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80 μmol/L. Compared with the model group, the activity of hippocampal neuronal cells was significantly increased after treating with 20, 40 or 80 μmol/L paeoniflorin (P<0.05) . Compared with the blank group, the ROS activity, LDH release level, MDA content and caspase-3 content were significantly increased (P<0.01) , and the SOD activity was significantly decreased (P< 0.01) in the hippocampal neuronal cells of the model group. Compared with the model group, the ROS activity, LDH release level, MDA content and caspase-3 content were obviously decreased (P<0.05) , SOD activity was significantly increased (P <0.01) after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin. Relative to the model group, the ratio of p-ERK/ERK were significantly up-regulated (P<0.01) , while the ratios of p-p38MAPK/p38MAPK and p-JNK/JNK were significantly down-regulated after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin (P<0.05) . Conclusion: Paeoniflorin may down-regulate the expression of p-p38MAPK and p-JNK protein, up-regulate the expression of p-ERK protein, and inhibit the apoptosis of hippocampal neurons induced by lead acetate through the MAPK signaling pathway.
Acetates/pharmacology* ; Animals ; Apoptosis ; Caspase 3/metabolism* ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; Glucosides ; Hippocampus/metabolism* ; JNK Mitogen-Activated Protein Kinases/pharmacology* ; Lead ; Monoterpenes ; Neurons/metabolism* ; Rats ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/metabolism* ; p38 Mitogen-Activated Protein Kinases/metabolism*

BACKGROUNDAdrenomedullin (ADM), a potent hypotensive small peptide, was recently found to inhibit the proliferation of glomerular mesangial cells (MsC) in vitro and to attenuate glomerular lesions in vivo, however the mechanisms remain poorly understood. In this study, we attempted to elucidate them using molecular signal transduction.

METHODSCultured rat MsC were treated with ADM and several inhibitors of signalling molecules. Methyl thiazoleterazolium (MTT) assay and BrdU incorporation method were employed for examining MsC proliferation. Western blot analysis was used for detecting total mitogen activated protein kinases (t-MAPKs) and phosphorylated MAPKs (p-MAPKs) proteins.

RESULTSADM suppressed MsC proliferation in a concentration- and time-dependent fashion. This response was inhibited by ADM receptor antagonist CGRP8-37 and a potent protein kinase-A (PKA) inhibitor, H89. Forskolin, a direct adenylate cyclase activator, also significantly inhibited MsC proliferation. SB203580, a P38MAPK inhibitor, and U0126, a MEK inhibitor, both completely blocked ADM mediated responses in MsC. However, curcumin, a SAPK/JNK inhibitor, and GF109203X, a potent protein kinase-C (PKC) inhibitor, had no effect on MsC growth. Western blot analysis showed that ADM did not change the expression of t-MAPKs but increased p-SAPK/JNK and p-P38MAPK levels and decreased p-ERK level. These responses were inhibited by CGRP8-37. All these kinase phosphorylations, except for the increase in p-SAPK/JNK, could be stimulated using forskolin. In addition, only ADM mediated changes in ERK and P38MAPK phosphorylations were inhibited by H89. GF109203X did not affect ADM induced changes in three p-MAPKs expressions.

CONCLUSIONSADM inhibits MsC proliferation possibly through cAMP-PKA pathway. Both phosphorylations of ERK and P38MAPK pathways were necessary in mediating the antiproliferative response of ADM. It does not preclude the involvement of cAMP independent pathways in the ADM mediated responses.

Adrenomedullin ; Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Glomerular Mesangium ; cytology ; drug effects ; JNK Mitogen-Activated Protein Kinases ; physiology ; Peptides ; pharmacology ; Rats ; Signal Transduction ; physiology ; p38 Mitogen-Activated Protein Kinases ; physiology

Sauchinone has been known to have anti-inflammatory and antioxidant effects. We determined whether sauchinone is beneficial in regional myocardial ischemia/reperfusion (I/R) injury. Rats were subjected to 20 min occlusion of the left anterior descending coronary artery, followed by 2 hr reperfusion. Sauchinone (10 mg/kg) was administered intraperitoneally 30 min before the onset of ischemia. The infarct size was measured 2 hr after resuming the perfusion. The expression of cell death kinases (p38 and JNK) and reperfusion injury salvage kinases (phosphatidylinositol-3-OH kinases-Akt, extra-cellular signal-regulated kinases [ERK1/2])/glycogen synthase kinase (GSK)-3beta was determined 5 min after resuming the perfusion. Sauchinone significantly reduced the infarct size (29.0% +/- 5.3% in the sauchinone group vs 44.4% +/- 6.1% in the control, P < 0.05). Accordingly, the phosphorylation of JNK and p38 was significantly attenuated, while that of ERK1/2, Akt and GSK-3beta was not affected. It is suggested that sauchinone protects against regional myocardial I/R injury through inhibition of phosphorylation of p38 and JNK death signaling pathways.
Animals ; Benzopyrans/*pharmacology ; Dioxoles/*pharmacology ; Glycogen Synthase Kinase 3/metabolism ; JNK Mitogen-Activated Protein Kinases/*metabolism ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Myocardial Reperfusion Injury/*metabolism/pathology/prevention & control ; Phosphorylation ; Protective Agents/*pharmacology ; Rats ; Signal Transduction/*drug effects ; p38 Mitogen-Activated Protein Kinases/*metabolism

OBJECTIVETo study the possible anti-apoptotic mechanism of ginsenoside Rg1 on the apoptosis of hippocampal neuron after cerebral ischemia/reperfusion (I/R) injury rats.

METHODSTotally 120 healthy male adult SD rats were randomly divided into the cerebral I/R model group (the model group), the low dose ginsenoside Rg1 group (10 mg/kg), the middle dose ginsenoside Rg1 group (20 mg/kg), the high dose ginsenoside Rg1 group (40 mg/kg), and the sham-operation group, 18 in each group. Rats received medication by peritoneal injection. Equal volume of normal saline was peritoneally injected to rats in the sham-operation group and the model group, once daily, for 7 successive days. The cerebral I/R injury model was prepared by 2-h middle cerebral artery occlusion (MCAO) followed by 24-h reperfusion. Rats in the sham-operation group received the same surgical procedure without the carotid arteries occluded. The neurofunction was assessed using Longa EZ method. The injury of hippocampal pyramidal cells was observed by Nissel staining and TUNEL assay. The nerve cell apoptosis rate was calculated. The protein expression levels of extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), c-Jun N-terminal kinases (JNK), and phosphorylated c-Jun N-terminal kinase (p-JNK) were detected using Western blot.

RESULTSCompared with the sham-operation group, the score of neurofunction, the apoptosis rate, the expression levels of p-JNK and p-ERK1/2 increased, the survived number of pyramidal cells decreased in the model group (P < 0.05, P < 0.01). Compared with the model group, the score of neurofunction and the apoptosis rate decreased in each ginsenoside Rg1 group (P < 0.05, P < 0.01). The survived number of pyramidal cells increased in the high and middle dose ginsenoside Rg1 groups, the expression of p-JNK in the hippocampal CA1 region decreased, and the expression level of p-ERK1/2 increased (P < 0.05, P < 0.01). Compared with the low dose ginsenoside Rg1 group, the score of neurofunction, the apoptosis rate, the p-JNK protein expression decreased, the survived number of pyramidal cells increased, the expression of p-ERK1/2 increased in the high and middle dose ginsenoside Rg1 groups (P < 0.05, P < 0.01). Three to four layers of pyramidal cells were arranged tightly and compactly in the hippocampal CA1 region of the sham - operation group. The nucleus was big and round under high power lens, with 1 -2 kernel. After cerebral I/R injury, the hippocampal nerve cells were severely injured. Normal structure was lost in the CA1 region, with disarranged cell line and reduced cell amount. Partial neurons were shrunken, and the kernel was condensed and darkenedly stained. They were in triangular, long strip, fusiform, or irregular shape. The staining of nucleus was clustered and the kernel was not clear. Ginsenoside Rg1 (20 and 40 mg/kg) could improve the morphology of ischemic nerve cells, reduce their loss. Of them, stronger effects were shown in the high dose ginsenoside Rg1 group than in the middle dose ginsenoside Rg1 group. The JNK protein band was divided into two subzones, JNK1 (46 kD) and JNK2 (54 kD). ERK protein band was also divided into two subzones, ERK1 (44 kD) and ERK2 (42 kD).

CONCLUSIONThe protective effect of ginsenoside Rg1 on cerebral I/R injury was correlated with inhibiting the apoptosis of hippocampal neurons, regulating the expression levels of p-ERK1/2 and p-JNK.

Animals ; Brain Ischemia ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Ginsenosides ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism

The aim of this study was to investigate the apoptosis-inducing effect of gambogic acid (GA) on Jurkat cells and its underlying signaling pathway. Apoptosis induced by GA and some inhibitors was assayed by Annexin V/PI doubling staining. The levels of caspase 3, caspase 8 and caspase 9 activated in living Jurkat cells were measured by flow cytometry. The expressions of caspase 3, caspase 9, p-JNK and P38 were detected by Western blot. The results showed that GA induced apoptosis of Jurkat cells in a dose-dependent manner. The positive cell number of activated caspase 3, caspase 8, caspase 9 and the levels of activated caspase 3, caspase 9, p-JNK, P38 increased after Jurkat cells were treated with GA. ROS, CaMKII, caspase 3, caspase 9, MAPKK, JNK1/2 and P38 inhibitors had some significant effect on GA-induced apoptosis. ROS, CaMKII, MAPKK, JNK1/2 and P38 inhibitors decreased the levels of activated caspase 3, caspase 9 by GA.ROS, CaMKII, MAPKK, JNK1/2 inhibitors decreased the levels of p-JNK by GA. ROS, CaMKII, MAPKK, P38 inhibitors decreased the levels of P38 by GA. It is concluded that GA induced apoptosis of Jurkat cells by activated caspases through activating of ROS-CaMKII-MAPKK-JNK/P38 pathway.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Jurkat Cells ; MAP Kinase Signaling System ; drug effects ; Xanthones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism