Bile acid modulates transepithelial permeability via the generation of reactive oxygen species in the Caco-2 cell line

The barrier functions in epithelial and endothelial cells seem to be very important for maintaining normal biological homeostasis. However, it is unclear whether or how bile acids affect the epithelial barrier. We examined the bile acid-induced disruption of the epithelial barrier. We measured the transepithelial electrical resistance (TEER) of Caco-2 cells as a marker of disruption of the epithelial barrier. Reactive oxygen species (ROS) generation was also measured. Cholic acid (CA) decreased the TEER and increased intracellular ROS generation. PLA2 (phospholipase A2), COX (cyclooxygenase), PKC (protein kinase), ERK 1/2 (extracellular signal-regulated kinase 1/2), PI 3 K (phosphatidylinositol 3-kinase), p38 MAPK (p38 mitogen-activated protein kinase), MLCK (myosin light-chain kinase), NADH dehydrogenase, and XO (xanthine oxidase) inhibitors or ROS scavengers prevented the CA-induced TEER decrease. PLA2, COX, PKC, NADH dehydrogenase, and XO inhibitors prevented the CA-induced ROS generation but not ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors. If the cells were treated with ROS generators such as superoxide dismutase, the TEER decreased. ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors prevent these ROS generators from inducing the TEER decrease. These results suggest that ROS play an important role. In addition, PLA2, COX, PKC, NADH dehydrogenase, and XO are located upstream of the ROS generation, but ERK 1/2, PI 3 K, p38 MAPK, and MLCK are downstream during the signaling of CA-induced TEER alterations.     

Involvement of p38 MAPK in haemozoin‐dependent MMP‐9 enhancement in human monocytes

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase‐9 (MMP‐9), and a major role for 15‐(S,R)‐hydroxy‐6,8,11,13‐eicosatetraenoic acid (15‐HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen‐activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal‐regulated kinase‐1/2 and c‐jun N‐terminal kinase‐1/2. 15‐HETE mimicked nHZ effects on p38 MAPK, whereas lipid‐free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15‐HETE also promoted phosphorylation of MAPK‐activated protein kinase‐2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ‐dependent and 15‐HETE‐dependent enhancement of MMP‐9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15‐HETE upregulate MMP‐9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP‐9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria. Copyright © 2013 John Wiley & Sons, Ltd.     

Acute hyperglycaemia enhances oxidative stress and aggravates myocardial ischaemia/reperfusion injury: role of thioredoxin‐interacting protein

Hyperglycaemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin‐interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether or not hyperglycaemia enhances Txnip expression in myocardial ischaemia/reperfusion (MI/R) and consequently exacerbates MI/R injury. Rats were subjected to 30 min. of left coronary artery ligation followed by 4 hrs of reperfusion and treated with saline or high glucose (HG, 500 g/l, 4 ml/kg/h intravenously). In vitro study was performed on cultured rat cardiomyocytes subjected to simulated ischaemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion during MI/R significantly impaired cardiac function, aggravated myocardial injury and increased cardiac oxidative stress. Meanwhile, Txnip expression was enhanced whereas thioredoxin activity was inhibited following HG treatment in ischaemia/reperfusion (I/R) hearts. In addition, HG activated p38 MAPK and inhibited Akt in I/R hearts. In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity. Overexpression of Txnip enhanced HG‐induced superoxide generation and aggravated cardiomyocyte apoptosis, whereas Txnip RNAi significantly blunted the deleterious effects of HG. Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG‐induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury in HG‐treated rats. Hyperglycaemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequently, amplification of cardiac injury following MI/R.     

Regulation of MAP kinase-mediated endothelial dysfunction in hyperglycemia via arginase I and eNOS dysregulation

Emerging evidence suggests that arginase contributes to endothelial dysfunction in diabetes. Intracellular signaling pathways, which interplay between arginase and eNOS enzyme activity leading to the development of endothelial dysfunction in hyperglycemia are not fully understood. Here, we analyzed the possible involvement of hyperglycemia (HG) induced arginase expression in eNOS protein regulation and activity and also the impact of arginase inhibition on eNOS activity. Furthermore, the roles of p38 MAPK and Erk1/2 phosphorylation in upregulation of arginase expression and eNOS dysregulation in endothelial cells (ECs) under hyperglycemia were evaluated. Protein analysis showed a concurrent increase in arginase I expression and decrease in eNOS expression and phosphorylation at Ser¹¹⁷⁷ under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr⁴⁹⁵ in HG. Arginase inhibition prevented increased arginase activity, restored impaired NO bioavailability and reduced superoxide anion generation. Inhibition of MAP-kinases demonstrated that, unlike Erk1/2, p38 MAPK is an upstream activator in a signaling cascade leading to increased arginase I in HG conditions. P38 MAPK protein expression and phosphorylation were increased in response to HG. In the presence of a p38 MAPK inhibitor, HG-induced arginase expression was blunted. Although Erk1/2 was activated in HG, increased arginase expression was not blocked by co-treatment with an Erk1/2 inhibitor. Activation of both, p38 MAPK and Erk1/2 in HG, induced a downregulation in eNOS activity. Hence, applying MAPK inhibitors increased eNOS phosphorylation in HG.In conclusion, these findings demonstrate contributions of arginase I in the development of endothelial cell dysfunction under HG conditions via impaired eNOS regulation, which maybe mediated by p38 MAPK.     

Candesartan and epigallocatechin‐3‐gallate ameliorate gentamicin‐induced renal damage in rats through p38‐MAPK and NF‐κB pathways

This study pointed to estimate the possible protective impacts of candesartan and/or epigallocatechin‐3‐gallate (EGCG) against gentamicin‐induced nephrotoxicity. The current work revealed that gentamicin significantly elevated relative kidney weight and the serum level of creatinine and urea. Also, renal level of malondialdehyde was significantly increased with a concurrent decrease in renal glutathione‐S‐transferase and superoxide dismutase activities. Moreover, renal levels of nuclear factor‐kappa B (NF‐κB) and p38 mitogen‐activated protein kinase (p38‐MAPK) were increased together with the elevation of tumor necrosis factor‐alpha and interleukin‐1 beta levels after gentamicin treatment. In addition, caspase‐3 expression was elevated, and histological examination revealed extreme alterations enlightening inflammation, degeneration, and necrosis. Pretreatments with candesartan and/or EGCG attenuated gentamicin‐induced nephrotoxicity. Importantly, the altered expression of p38‐MAPK and NF‐κB may play a significant role in the protective mechanisms exerted by candesartan and EGCG. Coadministration of candesartan and EGCG exhibited more profound response compared with the monotherapy.     

Amyloid‐β‐induced occludin down‐regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation

Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer’s disease (AD). A leaky blood–brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid β (Aβ) peptides of 1–40 and 1–42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Aβ 1–40, the Aβ variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Aβ 1–40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC‐dextran when compared with cells incubated with the scrambled Aβ 1–40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin‐5 and ZO‐1 were unaffected. JNK and p38MAPK inhibition prevented both Aβ 1–40‐mediated down‐regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.     

Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti‐tumour properties. The underlying mechanisms by which statins‐induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti‐tumour mechanisms of a lipophilic statin, lovastatin, in MCF‐7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF‐7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1‐AMPK signalling blockade abrogated lovastatin‐induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1‐AMPK‐p38MAPK‐p53‐survivin cascade to cause MCF‐7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.     

Peroxisome proliferator‐activated receptor delta protects against obesity‐related glomerulopathy through the P38 MAPK pathway

Objective:

Obesity is a prominent component of metabolic syndrome and a major risk factor for renal disease. The aim of this study was to explore the effect of cross‐talk between peroxisome proliferator‐activated receptor (PPAR)δ and p38 mitogen‐activated protein kinase (p38 MAPK) on obesity‐related glomerulopathy.

Design and Methods:

Male Wistar rats were randomly assigned to standard laboratory chow or a high‐fat diet for 32 weeks. Glomerular mesangial cells HBZY‐1 and mature differentiation 3T3‐L1 cells were cocultured and were transfected with PPARδ‐expressing vectors or treated with agonist or inhibitor of PPARδ or p38 MAPK.

Results:

Rats on a high‐fat diet showed typical characteristics of metabolic syndrome including obesity, dyslipidemia, insulin resistance, and hypertension. Rats on a high‐fat diet also had significant glomerular hypertrophy and extracellular matrix accumulation, which were accompanied by increased p38 MAPK phosphorylation and decreased PPARδ expression in the kidney tissue. The roles of p38 MAPK and PPARδ in a coculture system of mesangial cells and mature differentiation 3T3‐L1 cells were further explored. PPARδ suppression promoted laminin and type IV collagen secretion through p38 MAPK phosphorylation in mesangial cells, whereas PPARδ overexpression or PPARδ agonist attenuated phosphorylation of p38 MAPK and laminin and type IV collagen secretion.

Conclusions:

The characteristics of obesity‐related glomerulopathy, which might be partly caused by PPARδ suppression‐induced p38 MAPK activation and laminin and type IV collagen secretion was demonstrated.     

Role of the p38 mitogen‐activated protein kinase signaling pathway in estrogen‐mediated protection following flap ischemia‐reperfusion injury

Ischemia‐reperfusion (I/R) injury often occurs during skin flap transplantation and results in tissue damage. Although estrogen treatment significantly alleviates this I/R injury‐induced damage, the detailed molecular mechanism is not clear. In this study, a superficial epigastric artery flap I/R injury model was created in adult Wistar rats. Severe necrosis was observed in skin tissue after I/R injury. Histological examination of skin tissue revealed that I/R injury damages skin structure and results in neutrophil infiltration. Inflammation‐related parameters, including neutrophil count, tumor necrosis factor‐α, and interleukin‐10 levels, were increased due to I/R injury. These pathological phenomena were reduced by estradiol treatment. Further investigation found that I/R injury triggers the p38 mitogen‐activated protein kinase (p38‐MAPK) pathway. The expression levels of p38‐MAPK and phosphorylated p38‐MAPK were increased after I/R injury. Estradiol increased the expression level of MAPK phosphatase‐2, a putative phosphatase of p38, and reduced the levels of p38‐MAPK and phosphorylated p38‐MAPK. These results suggest that estradiol can improve skin flap survival, possibly by inhibiting neutrophil infiltration and the expression of p38‐MAPK. This study provides an explanation for how estrogen alleviates I/R injury‐induced damage that occurs during skin flap transplantation. In a rat pathological model, I/R injury leads to skin necrosis, skin structure damage, neutrophil infiltration, and inflammatory cytokine secretion, which are probably downstream effects of activation of the p38‐MAPK pathway. On the other hand, estradiol treatment triggers the expression of MAPK phosphatase‐2, a putative phosphatase of p38‐MAPK, and reduced all examined pathological phenomena. Therefore, estrogen may reduce the deleterious effect of I/R injury on skin flap transplantation through modulating the p38‐MAPK pathway.     

Role of mitogen‐activated protein kinase in Zn‐BC‐AM PDT‐induced apoptosis in nasopharyngeal carcinoma cells

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn‐BC‐AM was found to effectively induce apoptosis in a well‐differentiated nasopharyngeal carcinoma (NPC) HK‐1 cell line. Sustained activation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal kinase (JNK) as well as a transient increase in activation of extracellular signal‐regulated kinase (ERK) were observed immediately after Zn‐BC‐AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDT‐induced apoptosis of HK‐1 cells. PD169316 also prevented the loss of Bcl‐2 and Bcl‐xL in PDT‐treated HK‐1 cells. However, inhibition of JNK with SP600125 had no effect on Zn‐BC‐AM PDT‐induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn‐BC‐AM PDT‐induced apoptosis. Further study showed that knockdown of the p38β isoform with siRNA also increased Zn‐BC‐AM PDT‐induced apoptosis, indicating that the anti‐apoptotic effect of PD169316 in PDT‐treated HK‐1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38β and ERK may enhance the therapeutic efficacy of Zn‐BC‐AM PDT on NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution. Copyright © 2010 John Wiley & Sons, Ltd.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Caffeine induces matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 down‐regulation in human leukemia U937 cells via Ca2+/ROS‐mediated suppression of ERK/c‐fos pathway and activation of p38 MAPK/c‐jun pathway

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Down‐regulation of MMP‐2 and MMP‐9 in U937 cells was abrogated by abolishment of caffeine‐elicited increase in intracellular Ca²⁺ concentration and ROS generation. Pretreatment with BAPTA‐AM (Ca²⁺ chelator) and N‐acetylcysteine (ROS scavenger) abolished caffeine‐induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase‐1 (MKP‐1) down‐regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up‐regulation, which were involved in cross‐talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP‐2 and MMP‐9 protein expression in caffeine‐treated cells. Caffeine treatment repressed ERK‐mediated c‐Fos phosphorylation but evoked p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun by siRNA reflected that c‐Fos counteracted the effect of c‐Jun on MMP‐2/MMP‐9 down‐regulation. Taken together, our data indicate that MMP‐2/MMP‐9 down‐regulation in caffeine‐treated U937 cells is elicited by Ca²⁺/ROS‐mediated suppression of ERK/c‐Fos pathway and activation of p38 MAPK/c‐Jun pathway. J. Cell. Physiol. 224: 775–785, 2010. © 2010 Wiley‐Liss, Inc.     

Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.     

Zinc rescues obesity‐induced cardiac hypertrophy via stimulating metallothionein to suppress oxidative stress‐activated BCL10/CARD9/p38 MAPK pathway

Obesity often leads to obesity‐related cardiac hypertrophy (ORCH), which is suppressed by zinc‐induced inactivation of p38 mitogen‐activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4‐week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B‐cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate‐treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate‐induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate‐induced up‐regulation of BCL10 and phospho‐p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress‐mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress‐activated BCL10 expression and p38 MAPK activation.     

ECM‐dependent mRNA expression profiles and phosphorylation patterns of p130Cas,FAK, ERK and p38 MAPK of osteoblast‐like cells

Osteoblast cells synthesize collagen‐rich ECM (extracellular matrix) in response to various environmental cues, but little is known about ECM‐dependent variations in phosphorylation patterns. Using MC3T3 E1 osteoblast‐like cells and mouse whole‐genome microarrays, we investigated molecular signalling affected by collagen‐based ECMs. A genome‐wide expression analysis revealed that cells grown in the 3D collagen matrix partially suppressed the genes associated with cell adhesion and cell cycling. Western analysis demonstrated that the expression of the active (phosphorylated) form of p130Cas, FAK (focal adhesion kinase) and ERK1/2 (extracellular‐signal‐regulated protein kinase 1/2) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK (p38 mitogen‐activated protein kinase) was elevated in the 3D matrix, and its up‐regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. Although multiple characteristics such as surface topography, chemical composition and mechanical properties differ in the preparations of our collagen‐rich milieu, our observations support the notion that geometrical alterations in ECM environments can alter the phosphorylation pattern of p130Cas, FAK, ERK1/2 and p38 MAPK and lead to a differential developmental fate.     

Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKCζ‐mediated phosphorylation of occludin

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however, during respiratory bacterial infection TJs become disrupted compromising barrier function. This promotes glucose flux/accumulation into the lumen which acts as a nutrient source for bacterial growth. Metformin used for the treatment of diabetes increases transepithelial resistance (TEER) and partially prevents the effect of bacteria but the mechanisms of action are unclear. We investigated the effect of metformin and Staphylococcus aureus on TJ proteins, zonula occludins (ZO)‐1 and occludin in human airway epithelial cells (H441). We also explored the role of AMP‐activated protein kinase (AMPK) and PKCζ in metformin‐induced effects. Pretreatment with metformin prevented the S. aureus‐induced changes in ZO‐1 and occludin. Metformin also promoted increased abundance of full length over smaller cleaved occludin proteins. The nonspecific PKC inhibitor staurosporine reduced TEER but did not prevent the effect of metformin indicating that the pathway may involve atypical PKC isoforms. Investigation of TJ reassembly after calcium depletion showed that metformin increased TEER more rapidly and promoted the abundance and localization of occludin at the TJ. These effects were inhibited by the AMPK inhibitor, compound C and the PKCζ pseudosubstrate inhibitor (PSI). Metformin increased phosphorylation of occludin and acetyl‐coA‐carboxylase but only the former was prevented by PSI. This study demonstrates that metformin improves TJ barrier function by promoting the abundance and assembly of full length occludin at the TJ and that this process involves phosphorylation of the protein via an AMPK‐PKCζ pathway.     

Effect of heme oxygenase‐1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase‐1 (HO‐1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO‐1 recombinant adenovirus (HO‐MSCs) for stable expression of HO‐1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor‐α (TNF‐α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad‐MSCs, Ad‐HO + MSCs or HO‐MSCs. mRNA and protein expression of Zona occludens‐1 (ZO‐1) and human HO‐1 and the release of cytokines were measured. ZO‐1 and human HO‐1 in Caco2 were significantly decreased after treatment with TNF‐α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO‐1 was not significantly affected by Caco2 treatment with TNF‐α, Ad‐HO, and MSCs. In contrast, ZO‐1 and human HO‐1 increased significantly when the damaged Caco2 was treated with HO‐MSCs. HO‐MSCs showed the strongest effect on the expression of ZO‐1 in colon epithelial cells. Coculture with HO‐MSCs showed the most significant effects on reducing the expression of IL‐2, IL‐6, IFN‐γ and increasing the expression of IL‐10. HO‐MSCs protected the intestinal epithelial barrier, in which endogenous HO‐1 was involved. HO‐MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti‐inflammatory factors. These results suggested that HO‐MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO‐1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases.     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

The role of β‐adrenergic receptors and p38MAPK signaling pathways in physiological processes of cardiosphere‐derived cells

The effects of β adrenergic receptors (β‐ARs) and p38 mitogen‐activated protein kinases (MAPK) pathways on cardiosphere‐derived cells (CDCs) are largely unknown. This study aimed to investigate the roles of β‐ARs and p38MAPK pathways on the proliferation, apoptosis, and differentiation capacity of CDCs. The CDCs were treated with β1‐AR blocker (Met group), β2‐AR antagonist (ICI group), and p38MAPK inhibitor (SB group), non‐selective β‐AR blocker (PRO group), and β‐AR agonist (ISO group). The viability, apoptotic rate and differentiation status of CDCs were determined by MST‐1 assay, flow cytometery, and Western blot, respectively. The CDCs viability significantly reduced in ICI group (all P < 0.05), and SB group had a significant high viability after 48 h treatment (P < 0.05). Compared with control group, all treated groups had a low apoptotic rate. After treatment for 72 h, ISO treatment elevated the expression of Nkx2.5, and could partially or fully attenuate the inhibitory effects of β‐AR antagonists and/or p38MAPK inhibitor. A similar overall trend of protein expression levels among all groups could be observed between protein pairs of cTnT and β1‐AR as well as c‐Kit and β2‐AR, respectively. These results suggested that β‐ARs and p38MAPK signaling pathways play crucial roles in the proliferation and differentiation of CDCs. Our findings should be helpful for better understanding the molecular mechanism underlying the physiological processes of CDCs.