Biological functions of connexins in the development of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a group of chronic intestinal inflammatory diseases with unknown etiology. Gap junctions composed of connexins (Cxs) have been recently validated as an important factor in the development of IBD. Under IBD-induced inflammatory response in the gut, gap junctions connect multiple signaling pathways involved in the interaction between inflammatory cells with other intestinal cells, which altogether mediate the development of IBD. This paper is a narrative review aiming to comprehensively elucidate the biological function of connexins, especially the ubiquitously and predominantly expressed Cx43, in the pathogenesis of IBD.

Development of Dietary Thiol Antioxidant via Reductive Modification of Whey Protein and Its Application in the Treatment of Ischemic Kidney Injury.

Thiol antioxidants play important roles in cell and body defense against oxidative stress. In body fluid, albumin is the richest source of thiol antioxidants. One recent study showed that the reductive modification of thiol residues in albumin potentiated its antioxidative activity. Given that whey protein (WP) contains albumin and other thiol-active proteins, this property of WP could be exploited to develop novel thiol antioxidants. The aim of this study was to address this possibility. WP was reductively modified with dithiothreitol (DTT). The modified protein exhibited significantly elevated free sulfhydryl groups (-SH) and thiol antioxidative activity. It detoxified H2O2 and prevented H2O2-initiated protein oxidation and cell death in a -SH group-dependent way in vitro. In addition, it reacted with GSH/GSSG and altered the GSH/GSSG ratio via thiol-disulfide exchange. In vivo, oral administration of the reductively modified WP prevented oxidative stress and renal damage in a mouse model of renal injury caused by ischemia reperfusion. It significantly improved renal function, oxidation, inflammation, and cell injury. These protective effects were not observed in the WP control and were lost after blocking the -SH groups with maleimide. Furthermore, albumin, one of the ingredients of WP, also exhibited similar protective effects when reductively modified. In conclusion, the reductive modification of thiol residues in WP transformed it into a potent thiol antioxidant that protected kidneys from ischemia reperfusion injury. Given that oxidative stress underlies many life-threatening diseases, the reductively modified dietary protein could be used for the prevention and treatment of many oxidative-stress-related conditions, such as cardiovascular diseases, cancer, and aging.

The Combined Administration of Vitamin C and Copper Induces a Systemic Oxidative Stress and Kidney Injury.

Vitamin C (ascorbic acid; AA) and copper (Cu2+) are well used supplements with many health-promoting actions. However, when they are used in combination, the Fenton reaction occurs, leading to the formation of highly reactive hydroxyl radicals. Given that kidney is vulnerable to many toxicants including free radicals, we speculated that the in vivo administration of AA plus Cu2+ may cause oxidative kidney injury. The purpose of this study was to address this possibility. Mice were administered with AA and Cu2+, alone or in combination, via oral gavage once a day for various periods. Changes in the systemic oxidative status, as well renal structure and functions, were examined. The administration of AA plus Cu2+ elevated protein oxidation in serum, intestine, bladder, and kidney, as evidenced by the increased sulfenic acid formation and decreased level of free sulfhydryl groups (-SH). The systemic oxidative stress induced by AA plus Cu2+ was associated with a significant loss of renal function and structure, as indicated by the increased blood urea nitrogen (BUN), creatinine and urinary proteins, as well as glomerular and tubular cell injury. These effects of AA and Cu2+ were only observed when used in combination, and could be entirely prevented by thiol antioxidant NAC. Further analysis using cultured renal tubular epithelial cells revealed that AA plus Cu2+ caused cellular protein oxidation and cell death, which could be abolished by NAC and catalase. Moreover, coincubation of AA and Cu2+ led to H2O2 production. Collectively, our study revealed that a combined administration of AA and Cu2+ resulted in systemic oxidative stress and renal cell injury. As health-promoting supplements, AA and Cu2+ should not be used together.

Tanshinone IIA Stimulates Cystathionine γ-Lyase Expression and Protects Endothelial Cells from Oxidative Injury.

Tanshinone IIA (Tan IIA), an active ingredient of Danshen, is a well-used drug to treat cardiovascular diseases. Currently, the mechanisms involved remain poorly understood. Given that many actions of Tan IIA could be similarly achieved by hydrogen sulfide (H2S), we speculated that Tan IIA might work through the induction of endogenous H2S. This study was to test this hypothesis. Exposure to endothelial cells to Tan IIA elevated H2S-synthesizing enzyme cystathionine γ-Lyase (CSE), associated with an increased level of endogenous H2S and free thiol activity. Further analysis revealed that this effect of Tan IIA was mediated by an estrogen receptor (ER) and cAMP signaling pathway. It stimulated VASP and CREB phosphorylation. Inhibition of ER or PKA abolished the CSE-elevating effect, whereas activation of ER or PKA mimicked the effect of Tan IIA. In an oxidative endothelial cell injury model, Tan IIA potently attenuated oxidative stress and inhibited cell death. In support of a role of endogenous H2S, inhibition of CSE aggerated oxidative cell injury. On the contrary, supplement of H2S attenuated cell injury. Collectively, our study characterized endogenous H2S as a novel mediator underlying the pharmacological actions of Tan IIA. Given the multifaceted functions of H2S, the H2S-stimulating property of Tan IIA could be exploited for treating many diseases.

Reductively modified albumin attenuates DSS-Induced mouse colitis through rebalancing systemic redox state.

Albumin (Alb) is the most abundant plasma protein with multiple biological functions, including antioxidative property through its thiol activity. Given that inflammatory bowel disease is associated with a decreased level of Alb and an increased level of Alb oxidation, we asked whether Alb could have a therapeutic effect on colitis. Here we tested this possibility. Bovine serum albumin (BSA) was reductively modified with dithiothreitol (DTT) and administrated via gavage or intraperitoneal injection. Dextran sulfate sodium (DSS)-induced mice colitis was associated with massive oxidative stress, as indicated by the elevated sulfenic acid formation in blood, colon tissues, and feces. Treatment of mice with the reductively modified albumin (r-Alb) attenuated the oxidative stress and reduced local inflammation and tissue injury. These effects of r-Alb were only partially achieved by unmodified Alb and wholly lost after blocking the -SH groups with maleimide. In cultured colon epithelial cells, r-Alb prevented DSS- and H2O2-induced ROS elevation and barrier dysfunction, preceded by inhibition of sulfenic acid formation and P38 activation. Further analysis revealed that Alb was susceptible to H2O2-induced oxidation, and it detoxified H2O2 in a -SH group-dependent way. Moreover, Alb reacted with GSH/GSSG via thiol-disulfide exchange and reciprocally regulated the availability of -SH groups. Collectively, our study shows that r-Alb effectively attenuates DSS colitis via -SH group-mediated antioxidative action. Given that the oxidative stress underlies many life-threatening diseases, r-Alb, functioning as a potent antioxidant, could have a wide range of applications.

Robust polarization-insensitive strip-slot waveguide mode converter based on symmetric multimode interference.

Strip-slot waveguide mode converters for TE0 have been widely investigated. Here we demonstrate a polarization-insensitive converter numerically and experimentally. The polarization-insensitive performance is achieved by matching the optical field distribution of the 2-fold image of the Multimode Interference (MMI) and the TE0 (TM0) mode of a slot waveguide. The working principle for this MMI-based mode converter is thoroughly analyzed with the quantitatively evaluated optical field overlap ratio that is theoretically derived from the orthonormal relation of eigenmodes. Based on the analysis, the MMI-based polarization-insensitive converters are then simulated and fabricated. The simulation and measurement results indicate that the proposed scheme is a robust design since it is not only polarization-insensitive but also wavelength-insensitive and fabrication-tolerant. Moreover, the mode converter is as small as 1.22 µm × 4 µm while the measured conversion efficiencies are 95.9% for TE0 and 96.6% for TM0. All these excellent properties make the proposed mode converter an ideal solution for coupling light between strip and slot waveguides when both TE and TM polarizations are considered.

Connexin43 hemichannel-mediated regulation of connexin43.

BACKGROUND: Many signaling molecules and pathways that regulate gap junctions (GJs) protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43) hemichannels were activated by cadmium (Cd(2+)), we tested this hypothesis. PRINCIPAL FINDINGS: Incubation of Cx43-transfected LLC-PK1 cells with Cd(2+) led to an increased expression of Cx43. This effect of Cd(2+) was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd(2+) on JNK activation and Cx43 expression. Indeed, Cd(2+) induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd(2+). CONCLUSIONS: Collectively, our results thus indicate that Cd(2+)-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.

NADPH oxidase-mediated upregulation of connexin43 contributes to podocyte injury.

The gap junction protein connexin43 (Cx43) was markedly increased in podocytes in a rat model of nephrosis induced by puromycin. However, the mechanisms and roles of the altered Cx43 in podocytes are still unclear. Given that oxidative stress mediates podocyte injury under a variety of pathological situations, we examined the possible involvement of an oxidative stress-related mechanism in the regulation of Cx43. Incubation of podocytes with puromycin led to a time- and concentration-dependent loss of cell viability, which was preceded by an elevation in Cx43 levels. Concomitantly, puromycin also induced NOX4 expression and promoted superoxide (O(2)(·-)) generation. Inhibition of NADPH oxidase with apocynin and diphenyleneiodonium chloride or addition of the superoxide dismutase mimetic tempol completely abrogated, whereas the O(2)(·-) donors menadione and 2,3-dimethoxy-1,4-naphthoquinone reproduced, the effects of puromycin on Cx43 expression and cell injury. Further analysis demonstrated that treatment of podocytes with several structurally different gap-junction inhibitors significantly attenuated the cytotoxicity of puromycin. Our results thus indicate that NADPH oxidase-mediated upregulation of Cx43 contributes to podocyte injury.

Reciprocal regulation between proinflammatory cytokine-induced inducible NO synthase (iNOS) and connexin43 in bladder smooth muscle cells.

Gap junctions (GJs) play an important role in the control of bladder contractile response and in the regulation of various immune inflammatory processes. Here, we investigated the possible interaction between inflammation and GJs in bladder smooth muscle cells (BSMCs). Stimulation of BSMCs with IL1ß and TNFα increased connexin43 (Cx43) expression and function, which was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. Inhibition of PKA with H89 or down-regulation of CREB with specific siRNAs largely abolished the Cx43-elevating effect. Further analysis revealed that IL1ß/TNFα induced NFκB-dependent inducible NO synthase (iNOS) expression. Inhibition of iNOS with G-nitro-l-arginine methyl ester abrogated and an exogenous NO donor mimicked the effect of the cytokines on Cx43. Intraperitoneal injection of LPS into mice also induced bladder Cx43 expression, which was largely blocked by an iNOS inhibitor. Finally, the elevated Cx43 was found to negatively regulate iNOS expression. Dysfunction of GJs with various blockers or down-regulation of Cx43 with siRNA significantly potentiated the expression of iNOS. Fibroblasts from Cx43 knock-out (Cx43(-/-)) mice also displayed a significantly higher response to the cytokine-induced iNOS expression than cells from Cx43 wild-type (Cx43(+/+)) littermates. Collectively, our study revealed a previously unrecognized reciprocal regulation loop between cytokine-induced NO and GJs. Our findings may provide an important molecular mechanism for the symptoms of bladder infection. In addition, it may further our understanding of the roles of GJs in inflammatory diseases.

Nonsteroidal anti-inflammatory drug flufenamic acid is a potent activator of AMP-activated protein kinase.

Flufenamic acid (FFA) is a nonsteroidal anti-inflammatory drug (NSAID). It has anti-inflammatory and antipyretic properties. In addition, it modulates multiple channel activities. The mechanisms underlying the pharmacological actions of FFA are presently unclear. Given that AMP-activated protein kinase (AMPK) has both anti-inflammatory and channel-regulating functions, we examined whether FFA induces AMPK activation. 1) Exposure of several different types of cells to FFA resulted in an elevation of AMPKα phosphorylation at Thr172. This effect of FFA was reproduced by functionally and structurally similar mefenamic acid, tolfenamic acid, niflumic acid, and meclofenamic acid. 2) FFA-induced activation of AMPK was largely abolished by the treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (an intracellular Ca(2+) chelator) or depletion of extracellular Ca(2+), whereas it was mimicked by stimulation of cells with the Ca(2+) ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3-benzoxazole-4-carboxylic acid (A23187) or ionomycin. 3) FFA triggered a rise in intracellular Ca(2+), which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca(2+)/calmodulin-dependent kinase kinase ß (CaMKKß) with 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609) or down-regulation of CaMKKß with short interfering RNA largely abrogated FFA-induced activation of AMPK. 5) FFA significantly suppressed nuclear factor-κB activity and inducible nitric-oxide synthase expression triggered by interleukin-1ß and tumor necrosis factor α. This suppression was also largely abrogated by STO-609. Taken together, we conclude that FFA induces AMPK activation through the Ca(2+)-CaMKKß pathway. Activation of AMPK is a presently unrecognized important mechanism underlying the pharmacological effects of FFA.

Multi-glycoside of Tripterygium wilfordii Hook. f. reduces proteinuria through improving podocyte slit diaphragm dysfunction in anti-Thy1.1 glomerulonephritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) has been proved clinically effective in reducing proteinuria in chronic kidney disease in China. However, the mechanisms involved are still unclear. In this study we examined the effects of GTW at the different dosages on proteinuria and podocyte slit diaphragm (SD) dysfunction in anti-Thy1.1 glomerulonephritis (GN). MATERIALS AND METHODS: Rats with anti-Thy1.1 GN were divided into 2 groups, a GTW group and a vehicle group, and sacrificed at 30 min, on day 7, and on day 14 in Experiments 1, 2 and 3, respectively. The administration of GTW at the moderate and high doses was started 3 days before or at the same time of antibody injection till sacrifice. Proteinuria was determined in Experiments 1, 2, and 3. After sacrifice, the staining intensity of SD-associated key functional molecules including nephrin and podocin, podocyte structure, mesangial change, macrophage infiltration, and blood biochemical parameters were examined, respectively. Protein and mRNA expressions of nephrin and podocin in glomeruli were also investigated. Besides, liver histological characteristics were analyzed. RESULTS: In Experiment 1, GTW pretreatment at the medium dose (75 mg/kg body weight) caused no influence on the induction of anti-Thy1.1 GN and the basal nephrin expression. In Experiment 2, the high dosage (100mg/kg body weight) of GTW ameliorated proteinuria, the distribution of nephrin and podocin, mesangial proliferation, and the activated macrophage accumulation, as compared with vehicle group (P<0.05). Additionally, it increased mRNA and protein expressions of nephrin and podocin in glomeruli on day 7, but had no influence on podocyte structure. In Experiment 3, the medium dosage (75 mg/kg body weight) of GTW improved proteinuria, the partial matrix expansion, and the distribution of nephrin and podocin on day 14, as compared with anti-Thy1.1 GN rats (P<0.05). GTW at the high or moderate dose did not affect hepatic function on day 7 and on day 14. CONCLUSIONS: Podocyte SD dysfunction, such as the disordered distribution and down-regulation of nephrin and podocin expression, is critically involved in the pathogenesis of anti-Thy1.1 GN induced by mAb 1-22-3. The restoration of the distribution and expression of nephrin and podocin by GTW could be an important mechanism by which GTW ameliorates proteinuria and podocyte SD dysfunction.

[Regulative mechanism of Chinese herbal medicine on cell signaling pathway in kidney].

In kidney, the role of cell proliferation, differentiation, apoptosis, inflammatory mediators and cytokines expression is closely related with cell signaling pathways, including tyrosine kinase pathway, transforming growth factor-beta/Smad pathway, Rho/Rho-associated coiled-coil forming protein kinase pathway, phosphoinositol pathway, cyclic nucleotide pathway, nuclear factor kappaB pathway and so on. Some Chinese herbs and their extracts, such as rhubarb and triptolide, as well as some Chinese herbal prescriptions, such as astragalus-angelica mixture and Chailing decoction, not only could ameliorate proliferation, differentiation and apoptosis of renal cell by regulating cell signaling pathways, but also could control target gene transcription, expression and its biological effects through inhibiting the phosphorylation of key signaling molecules.

Connexin43 hemichannels contribute to cadmium-induced oxidative stress and cell injury.

We investigated the potential involvement of connexin hemichannels in cadmium ions (Cd(2+))-elicited cell injury. Transfection of LLC-PK1 cells with a wild-type connexin43 (Cx43) sensitized them to Cd(2+)-elicited cell injury. The cell susceptibility to Cd(2+) was increased by depletion of glutathione (GSH) with DL-buthionine-[S,R]-sulfoximine, and decreased by N-acetyl-cysteine or glutathione reduced ethyl ester. Fibroblasts derived from Cx43 wild-type (Cx43+/+) and knockout (Cx43-/-) fetal littermates displayed different susceptibility to Cd(2+). Cd(2+) induced a higher concentration of reactive oxygen species, a stronger activation c-Jun N-terminal kinase, and significantly more severe cell injury in Cx43+/+ fibroblasts, as compared with Cx43-/- fibroblasts. Cd(2+) caused a reduction in intracellular GSH, whereas it elevated extracellular GSH. This effect of Cd(2+) was more dramatic in Cx43+/+ than Cx43-/- fibroblasts. Treatment of Cx43+/+ fibroblasts with Cd(2+) caused a Cx43 hemichannel-dependent influx of Lucifer Yellow and efflux of ATP. Collectively, our study demonstrates that Cx43 sensitizes cells to Cd(2+)-initiated cytotoxicity, possibly through hemichannel-mediated effects on intracellular oxidative status.

Multi-glycoside of Tripterygium wilfordii Hook f. ameliorates prolonged mesangial lesions in experimental progressive glomerulonephritis.

BACKGROUND/AIMS: Multi-glycoside from Tripterygium wilfordii Hook f. (GTW) is used for treatment of progressive glomerulonephritis (GN) in China. We have previously reported the beneficial effects of GTW on acute GN induced by an anti-Thy-1.1 monoclonal antibody (mAb). In the present study, the effect and potential mechanisms of GTW on the chronic irreversible model of GN were investigated. METHODS: Progressive GN was induced in rats by two intravenous injections of anti-Thy-1.1 mAb 1-22-3. Daily oral administration of GTW was started before the second injection of mAb until the day of sacrifice. Ten rats were randomly divided into a control (vehicle-treated) and a GTW-treated group, and sacrificed on day 45 after the first injection of mAb 1-22-3. Proteinuria was determined on days 0, 1, 3, 5, 7, 10, 14, 20, 25, 30, 35, 40, and 45. Blood biochemical parameters, morphological changes of mesangium, glomerular infiltration of macrophage and T lymphocyte, and glomerular mRNA expression of cytokines (TGF-beta, IL-2, and IFN-gamma) were examined from the samples taken at terminal sacrifice. RESULTS: GTW treatment significantly ameliorated proteinuria, renal function, prolonged mesangial lesions and inflammatory cell accumulation in glomerulus. In addition, it significantly reduced the glomerular mRNA expression for TGF-beta, IL-2, and IFN-gamma. CONCLUSION: GTW ameliorates prolonged glomerular lesions presumably through suppression of cytokine production (TGF-beta, IL-2, and IFN-gamma). GTW could be an effective therapeutic agent for treatment of chronic renal diseases.