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 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.     

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.     

Role of p38 MAPK in cyclic mechanical stretch induced HMGB1 expression in alveolar macrophages

AIM: To investigate the role of p38 mitogen-activated protein kinase (MAPK) in cyclic mechanical stretch induced the expression of high mobility group box 1 protein (HMGB1) in alveolar macrophages (AMs). METHODS: AMs were cultured and seeded at 1×108 cells/L in 6-well Bioflex cell culture plates. Subsequently, the cells were exposed to cyclic mechanical stretch at 20% (group B) elongation using Flexercell 4000T cell stretching unit. In group C, cells were pre-treated with SB203580 (40 μmol/L) for 2 h before mechanical stretch. Group A served as control. The expression of HMGB1 mRNA in alveolar macrophages was detected by RT-PCR. p38 MAPK activity and the expression of HMGB1 protein were measured by Western blotting analysis. RESULTS: The expression of HMGB1 mRNA and protein, and the activity of p38 MAPK in AMs were significantly increased in group B than those in group A (P<0.05). SB203580, an inhibitor of p38 MAPK, significantly inhibited the inducing effect of mechanical stretch (P<0.05). CONCLUSION: Mechanical stretch regulates the expression of HMGB1 mRNA and protein in alveolar macrophages by activating p38 MAPK signal pathway.     

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.     

CREG prevents human umbilical vein endothelial cells from apoptosis by inhibiting p38 MAPK activation

AIM: To study the effect of cellular repressor of E1A-stimulated genes(CREG) and its mechanism on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by etoposide (VP-16).METHODS: Primary HUVECs were cultured. RetroviraI eukaryotic expression vectors pLNCX-CREG and pLXSN-shRNA-CREG were transfected into HUVECs. The stable cell clone was selected and obtained by screening with G418 (800 mg/L) and the puromycin (2.5 mg/L), respectively. CREG expression was detected by Western blotting. The cells with overexpression of CREG (H-C) and those with CREG down-regulation (H-S) were pretreated with apoptotic inducer VP-16 at 100 μmol/L for 6 h. The apoptotic rates of the 3 kinds of cells were analyzed by TUNEL and flow cytometry with annexin V/PI dualstaining. Furthermore, the protein levels of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in the 3 kinds of cells were analyzed by Western blotting. The p38-specific inhibitor SB203580(20 μmol/L)was used to investigate the effects of p-p38 expression on apoptosis. RESULTS: Western blotting showed that CREG expression was obviously increased up to 160% in H-C compared to HUVECs. However, CREG expression in H-S cells was identified to be down-regulated to 70% compared with HUVECs. TUNEL assay and annexin V/PI dual-color FACS showed that the apoptotic rate was dramatically increased in H-S cells,but decreased in H-C cells. Subsequently, Western blotting exhibited that p-p38 expression was increased in H-S cells compared to HUVECs and H-C cells. When the H-S was pretreated with SB203580, the apoptotic rate was decreased. CONCLUSION: CREG overexpression might prevent HUVECs from apoptosis by inhibiting p38 MAPK activition.     

Role of p38 MAPK signaling pathway in inflammatory effects on cerulein-induced pancreatic cells

AIM: To investigate the effect of p38 MAPK signal pathway on cerulein-treated pancreatic acinar AR42J cells.METHODS: AR42J cells were divided into control group, cerulein group (treated with 10^-8 mol/L of cerulein), and SB203580 group (treated with 10 μmol/L of SB203580 and 10^-8mol/L of cerulein).The cells were harvested 3 h after treatment.Secretion rate of amylase was measured.The translocation of p-p38 MAPK to nuclei was imaged by immunofluorescence.The protein expression levels of p-p38 MAPK and TNF-α were detected by Western blotting.The activation of NF-κB was measured by electrophoretic mobility assay.RESULTS: Compared with control group, cerulein resulted in increases in the secretion rate of amylase and protein level of TNF-α (P<0.01), as well as the expression levels of p-p38 MAPK and NF-κB (P<0.01).Cerulein induced nuclear translocation of p-p38 MAPK.Compared with cerulein group, the secretion rate of amylase and protein level of TNF-α in SB203580 group decreased significantly (P<0.01).The expression of p-p38 MAPK and NF-κB also decreased greatly (P<0.05).Nuclear translocation of p-p38 MAPK was inhibited by SB203580.CONCLUSION: The p38 MAPK pathway involves in cerulein-induced pancreatic inflammatory response via regulating NF-κB.     

p38 MAPK involves in cepharanthine-induced apoptosis in cardiomyocytes

AIM: To investigate the apoptotic effect of cepharanthine (CEP) on neonatal rat cardiomyocytes(NRCMs) and the underlying mechanisms. METHODS: MTT assay was used to detect the viability of the cells. CEP-induced apoptosis in NRCMs was evaluated by Hoechst 33342 staining and the expression of activated caspase-3. The phosphorylation levels of mitogen-activated protein kinases (MAPKs),such as extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38 MAPK,were examined by Western blotting. The specific inhibitors of ERK and p38 MAPK were applied for identifying the roles of the corresponding signal pathways in CEP-induced apoptosis of cardiomyocytes. RESULTS: CEP inhibited the viability of NRCMs in a dose-and time-dependent manners. Positive nuclear fragmentation and activated caspase-3 were found in CEP-treated NRCMs. The phosphorylation levels of ERK and p38 MAPK were significantly elevated in CEP-treated NRCMs, but the change of JNK was not obvious. SB203580, an inhibitor of p38 MAPK, significantly alleviated the apoptotic effect induced by CEP. However, PD98059, an inhibitor of ERK1/2, did not significantly reduce the apoptotic effect.CONCLUSION: p38 MAPK is involved in CEP-induced apoptosis in NRCMs.     

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.     

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.     

Propofol attenuates aquaporin-4 over-expression through p38 pathway on ammonia-induced neocortical astrocyte in rats

AIM: To investigate the effects of propofol on ammonia-induced neocortical astrocyte aquaporin-4 expression in rats and its mechanism. METHODS: Astrocytes were separated from newborn Sprague Dawley rats. Glial fibrillary acidic protein, the specific protein of astrocyte, was labeled by cell immunofluorescence method. The purity of astrocyte achieved to 95% was considered to be used in the study. Grouping: cultured astrocytes were randomly divided into 5 groups (n=3): normal control group (N); ammonia-incubated for 24 h group (NH4Cl-24 h); p38 antagonist SB203580 (10 μmol/L) pretreated group (SB); propofol (10 μmol/L) pretreated group (P); solvent (DMSO) control group (C). SB203580, propofol and DMSO were pretreated for 30 min before astrocytes were exposed to NH4Cl. Cell morphology was assessed by light microscopy. The expression of aquaporin-4; p38 and p-p38 were detected by Western blotting. RESULTS: Astrocytes were found significant swelling when exposed to 5 mmol/L NH4Cl in NH4Cl-24 h group compared to control group. Pretreatment with propofol and SB203580 decreased astrocyte swelling. No significant change in total p38 in all groups was observed (P>0.05) by Western blotting analysis, while the levels of p-p38 and AQP4 in group SB and group P were significantly decreased compared to group C and group NH4Cl-24 h (P<0.05).CONCLUSION: The expression of AQP4 is regulated by p38 pathway. Propofol pretreatment down-regulates aquaporin-4 expression through preventing the ammonia-induced p38 phosphorylation.     

Signal mechanism of endothelin-1-mediated activation of airway fibroblasts induced by injured airway epithelial cells

AIM: To explore the effects of p38 mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinases (PI3K)/Akt on interleukin (IL)-6, the endothelin (ET)-1-mediated process of airway fibroblast activation induced by injured human bronchial epithelial cells (HBE). METHODS: Human primary cultured airway fibroblasts were co-cultured with HBE pre-treated with or without poly-L-arginine (PLA). The procedure was also performed in the presence or absence of p38 MAPK selective inhibitor SB203580, PI3K selective inhibitor LY294002 or ETA receptor blocker BQ123, respectively. Immunostaining, Western blotting or ELISA were used for detecting α-smooth muscle actin (α-SMA) expression, the activities of p38 MAPK and Akt in fibroblasts or IL-6 levels in supernatants of fibroblasts. In addition, fibroblasts were mixed with soluble collagen and cultured with HBE treated as the same mentioned above, the gel contraction was measured by serial area measurements. RESULTS: ET-1 and IL-6 levels ［(13.69±1.36) ng/L, (56.7±10.7) ng/L］ in the supernatants of fibroblasts cultured with injured HBE were significantly higher than those in the supernatants of fibroblasts cultured with HBE ［(3.79±0.64) ng/L, (15.5±3.2) ng/L］. BQ123, SB203580 or LY294002 decreased IL-6 levels ［(27.2±3.1) ng/L, (31.5±3.6) ng/L, (41.3±3.2) ng/L］ differently in the supernatants of fibroblasts induced by injured HBE. Activation of p38 MAPK preceded Akt in fibroblasts cultured with injured HBE. BQ123 reduced the phosphorylation levels of p38 MAPK and Akt. SB203580 concentration-dependently attenuated Akt phosphorylation, while LY294002 had little effect on p38 MAPK phosphorylation. Fibroblasts expressed more α-SMA after cultured with injured HBE and showed significant increase in the gel contraction compared to fibroblasts cultured with HBE ［percentage of gel contraction: (61.2±2.7)% vs (15.4±7.3)%］, all these effects were diminished or inhibited by BQ123, SB203580 or LY294002. Furthermore, the effects of BQ123 and SB203580 on decreased gel contraction were stronger than the effect of LY294002. CONCLUSION: ET-1 exerts a key role in the airway fibroblasts activation induced by injured HBE through activating p38 MAPK, PI3K/Akt signaling and promoting IL-6 expression.     

p38 MAPK/ATF-2 pathway is involved in C-relative protein-induced endothelial cell activation

AIM: To investigate the role of p38 MAPK/ATF-2 pathway in C-relative protein (CRP)-induced endothelial cell activation. METHODS: Human coronary artery endothelial cells (HCAEC) were cultured and were used between passages 3 and 7. CRP served as a stimulus for endothelial cell activation. Western blotting was performed to determine the expression and phosphorylation of eNOS, p38 and ATF2. ELISA was carried out to detect the levels of ICAM-1, VCAM-1 and MCP-1 released from HCAEC. Pharmacological p38 inhibitors SB203580 and SB202190 were used to determine the effect of p38/ATF-2 pathway. RESULTS: CRP reduced the p-eNOS level in a concentration-dependent manner and induced the release of ICAM-1, VCAM-1 and MCP-1. The p38/ATF-2 pathway was activated by CRP treatment. SB203580 and SB202190 partially rescued p-eNOS level and suppressed the secretion of ICAM-1, VCAM-1 and MCP-1. CONCLUSION: p38MAPK/ATF-2 pathway participates in CRP-induced endothelial activation.     

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.     

Erianin induces apoptosis of human lung cancer A549 cells via ROS/p38 MAPK pathway

AIM:To investigate the effect of erianin on the viability and apoptosis of human lung cancer A549 cells and its possible mechanism. METHODS:A549 cells and BEAS-2B cells were cultured in vitro and treated with erianin at 10, 20, 40, 80 and 160 nmol/L for 48 h. The cell viability was measured by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. The activity of superoxide dismutase (SOD) was detected by WST-8 method, and the content of malondialdehyde (MDA) was detected by barbituric acid method. The protein levels of nuclear factor E2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), p38 MAPK, p-p38 MAPK, caspase-3 and cleaved caspase-3 were determined by Western blot.RESULTS:Erianin remarkably reduced the viability of A549 cells in a dose-dependent manner (P<0.05) with IC₅₀ at 52.64 nmol/L. Erianin also induced apoptosis (P<0.05), increased ROS level and MDA content (P<0.05), diminished SOD activity (P<0.05), and down-regulated the protein levels of Nrf2, NQO1 and HO-1 (P<0.05), in a dose-dependent manner. Meanwhile, erianin up-regulated the levels of p-p38 MAPK and cleaved caspase-3 (P<0.05), and these effects were inhibited by N-acetyl-L-cysteine and SB203580 (P<0.05).CONCLUSION:Erianin may induce apoptosis of human lung cancer A549 cells most likely via inhibiting SOD activity and down-regulating the protein levels of Nrf2, NQO1 and HO-1, thus resulting in an increase in ROS and activation of p38 MAPK.     

p38 MAPK signaling pathway is involved in the regulation of receptor-mediated endocytosis

AIM: To investigate the role of p38 MAPK signaling pathway in the receptor-mediated endocytosis. METHODS: The effects of p38 MAPK on the receptor-mediated endocytosis were observed by using Alexa 594-conjugated Transferrin, in the presence of p38 specific inhibitor SB203580 or ERK pathway specific inhibitor PD98059, or using p38 knockout techniques. RESULTS: In the process of receptor-mediated endocytosis, p38 was activated by phosphorylation. Furthermore, the receptor-mediated endocytosis was inhibited by pretreatment with SB203580 or p38 knockout, while pretreatment with PD98059 had no effect on this process.CONCLUSION: p38 MAPK signaling pathway plays a role in the regulation of receptor-mediated endocytosis.     

Role of p38MAPK activation in high glucose-induced collagen Ⅲ synthesis in normal rat kidney tubular epithelial cells NRK52E

AIM: To study the role of p38 mitogen-activated protein kinase (p38MAPK) activation in high glucose-induced collagen Ⅲ synthesis in NRK52E cells. METHODS: Normal rat tubular epithelial cell line NRK52E was cultured in D-glucose of different concentrations, pretreated with SB203580 and collected at different time points. The levels of phospho-p38MAPK and extracellular matrix collagen Ⅲ were examined by Western blotting. RESULTS: The activation of p38MAPK was shown to be dependent upon D-glucose concentration and the time-course. Pretreatment with SB203580 blocked p38MAPK activation induced by high concentration of D-glucose in NRK52E cells. CONCLUSIONS: The activation of p38MAPK induced by high concentration of glucose may play a role in diabetic interstital renal fibrosis. SB203580 has a potential value of clinical applications in the prevention and treatment of diabetic nephropathy.     

Establishment of human lung infection model in vitro and mechanisms of NTHi-induced inflammatory responses

AIM: To investigate the key signal pathways of inflammatory responses in lung tissues induced by the infection of nontypeable Haemophilus influenzae(NTHi). METHODS: Human lung tissues were co-incubated with NTHi (10¹⁰ CFU/L) for 4 h and 24 h, respectively. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected by Western blotting. The nuclear translocation of nuclear factor (NF)-κB was examined by electrophoretic mobility shift assay (EMSA). The expression of Toll-like receptor (TLR) 2 was measured by real-time quantitative RT-PCR, and the level of interleukin (IL)-8 was detected by enzyme-linked immunosorbent assay (ELISA). Furthermore, lung tissues were incubated with anti-TLR2 monoclonal antibody (5 mg/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or NF-κB inhibitor PDTC (25 μmol/L) for 2 h, then stimulated with NTHi (10¹⁰ CFU/L) for another 24 h. The supernatants were collected for IL-8 detection. RESULTS: The TLR2-p38 MAPK-NF-κB signaling pathway in lung tissues was rapidly activated 4 h after NTHi stimulation. IL-8 secreted from lung tissues infected with NTHi was significantly increased compared with uninfected lungs (P<0.05). The pre-incubation with anti-TLR2 antibody, p38 MAPK inhibitor or NF-κB inhibitor markedly decreased IL-8 production induced by NTHi. CONCLUSION: NTHi induces inflammatory responses in lung tissues by activation of TLR2-p38 MAPK-NF-κB signaling pathway. Human lung infection model provides a new research tool for the study of interaction between pathogens and hosts.     

Akebia saponin D promotes differentiation of bone marrow mesenchymal stem cells into osteoblasts through MAPK signaling pathways

AIM:To explore the role of Akebia saponin D(ASD) in the differentiation of rat bone marrow-derived mesenchymal stem cells(BMSCs) into osteoblasts. METHODS:The rat BMSCs were cultured using routine methods. The effects of ASD on the differentiation of MSCs into osteoblasts were observed. The p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 and extracellular signal-regulated kinase(ERK) inhibitor PD098059 were used to evaluate the mechanisms. The activity of alkaline phosphate(ALP) and content of osteocalcin(OC) were assayed during differentiation. The mRNA expression of osteoprotegerin(OPG) and receptor activator of nuclear factor-κB ligand(RANKL) was determined by real-time fluorescence quantitative PCR. The activity of p38 MAPK and ERK was measured by Western blotting. RESULTS:Six days after treatment with ASD, the mRNA expression of OPG significantly increased, while the mRNA level of RANKL significantly decreased in induced cells. ASD increased the activity of ALP and the content of OC. Moreover, ASD enhanced the activity of both p38 MAPK and ERK, which was inhibited by SB203580 and PD098059. SB203580 and PD098059 also inhibited the positive role of ASD in the differentiation of MSCs into osteoblasts. CONCLUSION:Akebia saponin D significantly enhances differentiation of rat BMSCs into osteoblasts in vitro, which may be mediated by the p38 MAPK and ERK signaling pathways.