Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

WNT‐3A and WNT‐5A counteract lipopolysaccharide‐induced pro‐inflammatory changes in mouse primary microglia

Surveying microglia, the resident macrophage‐like cells in the central nervous system, continuously screen their surroundings to sense imbalance in tissue homeostasis. Their activity is tightly regulated in both a pro‐ and anti‐inflammatory manner. We have previously shown that the lipoglycoproteins WNT‐3A and WNT‐5A drive pro‐inflammatory transformation in primary mouse microglia cells, arguing that WNTs have a role in the modulation of the central nervous system immune response. In this study, we address the effects of recombinant WNT‐3A and WNT‐5A on lipopolysaccharide (LPS)‐activated mouse primary microglia to investigate the putative anti‐inflammatory modulation of microglia by WNTs. While both WNT‐3A and WNT‐5A alone induce an up‐regulation of cyclooxygenase 2 (COX2), a generic pro‐inflammatory microglia marker, LPS exceeds these effects dramatically. However, combination of LPS and WNTs results in a dose‐dependent decrease in LPS‐induced cyclooxygenase 2 protein and mRNA expression. In conclusion, our data suggest that WNTs have a dual and context‐dependent effect on microglia acting in a homeostatic pro‐ and anti‐inflammatory manner.     

TLR3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea

Toll‐like receptor‐3 (TLR3) and RNA helicase retinoic‐acid‐inducible protein‐1 (RIG‐I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG‐I signalling pathway was stimulated by viral infection to produce interleukin (IL)‐32‐mediated pro‐inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)‐infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG‐I that are responded to EBV‐encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL‐32‐mediated pro‐inflammatory cytokines and IFN‐β through up‐regulation of TRIF/TRAF family proteins or RIP‐1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF‐κB and IRFs to produce pro‐inflammatory cytokines and IFN‐β than RIG‐I‐siRNA transfection in HCECs/EBV. Blockade of RIP‐1, which connects the TLR3 and RIG‐I pathways, significantly blocked the TLR3/TRIF‐mediated and RIG‐I‐mediated pro‐inflammatory cytokines and IFN‐β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF‐dependent signalling pathway against viral RNA might be a main target to control inflammation and anti‐viral responses in the ocular surface.     

Anti‐inflammatory function of Withangulatin A by targeted inhibiting COX‐2 expression via MAPK and NF‐κB pathways

Withangulatin A (WA), an active component isolated from Physalis angulata L., has been reported to possess anti‐tumor and trypanocidal activities in model systems via multiple biochemical mechanisms. The aim of this study is to investigate its anti‐inflammatory potential and the possible underlying mechanisms. In the current study, WA significantly suppressed mice T lymphocytes proliferation stimulated with LPS in a dose‐ and time‐dependent manner and inhibited pro‐inflammation cytokines (IL‐2, IFN‐γ, and IL‐6) dramatically. Moreover, WA targeted inhibited COX‐2 expression mediated by MAPKs and NF‐κB nuclear translocation pathways in mice T lymphocytes, and this result was further confirmed by the COX‐1/2 luciferase reporter assay. Intriguingly, administration of WA inhibited the extent of mice ear swelling and decreased pro‐inflammatory cytokines production in mice blood serum. Based on these evidences, WA influences the mice T lymphocytes function through targeted inhibiting COX‐2 expression via MAPKs and NF‐κB nuclear translocation signaling pathways, and this would make WA a strong candidate for further study as an anti‐inflammatory agent. J. Cell. Biochem. 109: 532–541, 2010. © 2009 Wiley‐Liss, Inc.     

Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

MiR‐643 inhibits lipopolysaccharide‐induced endometritis progression by targeting TRAF6

Endometritis is a prevalent disease with inflammation of uterus endangering women reproductive health. MicroRNAs (miRNAs) play important roles in inflammatory disorders, including endometritis. However, the role and mechanism of miR‐643 in endometritis development remain unclear. This study aimed to investigate the effect of miR‐643 on lipopolysaccharide (LPS)‐induced inflammatory response and clarify the potential mechanism. LPS‐treated human endometrial epithelial cells (HEECs) were cultured to investigate the role of miR‐643 in vitro. The expression levels of miR‐643 and tumor necrosis factor receptor‐associated factor 6 (TRAF6) were measured via quantitative real‐time polymerase chain reaction and western blot, respectively. LPS‐induced inflammatory response was assessed by inflammatory cytokines secretion via enzyme‐linked immunosorbent assay. The activation of nuclear factor‐κB (NF‐κB) pathway was investigated by western blot. The interaction between miR‐643 and TRAF6 was validated by bioinformatics analysis, luciferase reporter assay, and RNA immunoprecipitation. The expression of miR‐643 was decreased and TRAF6 protein level was enhanced in LPS‐treated HEECs. The overexpression of miR‐643 suppressed LPS‐induced secretion of inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β [IL‐1β], and IL‐6) and activation of NF‐κB pathway. The knockdown of TRAF6 inhibited LPS‐induced inflammatory response in HEECs. TRAF6 was validated as a target of miR‐643 and TRAF6 restoration reversed the effect of miR‐643 on inflammation response in LPS‐treated HEECs. Collectively, miR‐643 attenuated LPS‐induced inflammatory response by targeting TRAF6, indicating a novel avenue for the treatment of endometritis.     

Low‐intensity pulsed ultrasound attenuates cardiac inflammation of CVB3‐induced viral myocarditis via regulation of caveolin‐1 and MAPK pathways

The aggressive immunological activity elicited by acute viral myocarditis contributes to a large amount of cardiomyocytes loss and poor prognosis of patients in clinic. Low‐intensity pulsed ultrasound (LIPUS), which is an effective treatment modality for osteoarthropathy, has been recently illustrated regulating the overactive inflammatory response in various diseases. Here, we aimed to investigate whether LIPUS could attenuate coxsackievirus B3 (CVB3) infection‐induced injury by coordinating the inflammatory response. Male BALB/c mice were inoculated intraperitoneally with CVB3 to establish the model of acute viral myocarditis. LIPUS treatment was given on Day 1, Day 1, 3 and Day 1, 3, 5 post‐inoculation, respectively. All mice were followed up for 14 days. Day 1, 3, 5 LIPUS treatment significantly improved the survival rate, attenuated the ventricular dysfunction and ameliorated the cardiac histopathological injury of CVB3‐infected mice. Western blotting analysis showed Day 1, 3, 5 LIPUS treatment decreased pro‐inflammatory cytokines, increased the activation of caveolin‐1 and suppressed p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (ERK) signallings in heart tissue. RAW264.7 cells were treated with lipopolysaccharides (LPS) to simulate the augmented inflammatory response in vivo. LIPUS treatment on RAW264.7 inhibited the expression of pro‐inflammatory cytokines, activated caveolin‐1 and suppressed p38 MAPK and ERK signallings. Transfecting RAW264.7 with caveolin‐1 siRNA blunted the suppression of pro‐inflammatory cytokines and MAPK signallings by LIPUS treatment. Taken together, we demonstrated for the first time that LIPUS treatment attenuated the aggressive inflammatory response during acute viral myocarditis. The underlying mechanism may be activating caveolin‐1 and suppressing MAPK signallings.     

Cannabinoids prevent the opposite regulation of astroglial connexin43 hemichannels and gap junction channels induced by pro‐inflammatory treatments

Brain injuries as well as neurodegenerative diseases, are associated with neuro‐inflammation characterized by astroglial and microglial activation and/or proliferation. Recently, we reported that lipopolysaccharide (LPS)‐activation of microglia inhibits junctional channels and promotes hemichannels, two connexin43 functions in astrocytes. This opposite regulation is mediated by two pro‐inflammatory cytokines, interleukin‐1 beta and tumor necrosis factor‐alpha, released from activated microglia. Because cannabinoids (CBs) have anti‐inflammatory properties and their receptors are expressed by glial cells, we investigated on primary cortical cultures the effects of CB agonists, methanandamide and synthetic CBs on (i) cytokines released from LPS‐activated microglia and (ii) connexin43 functions in astrocytes subjected to pro‐inflammatory treatments. We observed that CBs inhibited the LPS‐induced release of interleukin‐1 beta and tumor necrosis factor‐alpha from microglia. Moreover, the connexin43 dual regulation evoked by the pro‐inflammatory treatments, was prevented by CB treatments. Pharmacological characterizations of CB actions on astrocytic connexin43 channels revealed that these effects were mainly mediated through CB1 receptors activation, although non‐CB1/CB2 receptors seemed to mediate the action of the methanandamide. Altogether these data demonstrate that in inflammatory situations CBs exert, through the activation of different sub‐types of glial CB receptors, a regulation on two functions of connexin43 channels in astrocytes known to be involved in neuron survival.     

The anti‐seizure drugs vinpocetine and carbamazepine,but not valproic acid,reduce inflammatory IL‐1β and TNF‐α expression in rat hippocampus

In the present study, the effects of the two classical anti‐epileptic drugs, carbamazepine and valproic acid, and the non‐classical anti‐seizure drug vinpocetine were investigated on the expression of the pro‐inflammatory cytokines IL‐1β and TNF‐α in the hippocampus of rats by PCR or western blot after the administration of one or seven doses. Next, the effects of the anti‐seizure drugs were investigated on the rise in cytokine expression induced by lipopolysaccharides (LPS) inoculation in vivo. To validate our methods, the changes induced by the pro‐convulsive agents 4‐aminopyridine, pentylenetetrazole and pilocarpine were also tested. Finally, the effect of the anti‐seizure drugs on seizures and on the concomitant rise in pro‐inflammatory cytokine expression induced by 4‐aminopyridine was explored. Results show that vinpocetine and carbamazepine reduced the expression of IL‐1β and TNF‐α from basal conditions, and the increase in both pro‐inflammatory cytokines induced by LPS. In contrast, valproic acid failed to reduce both the expression of the cytokines from basal conditions and the rise in IL‐1β and TNF‐α expression induced by LPS. Tonic‐clonic seizures induced either by 4‐aminopyridine, pentylenetetrazole or pilocarpine increased the expression of IL‐1β and TNF‐α markedly. 4‐aminopyridine‐induced changes were reduced by all the tested anti‐seizure drugs, although valproic acid was less effective. We conclude that the anti‐seizure drugs, vinpocetine and carbamazepine, whose mechanisms of action involve a decrease in ion channels permeability, also reduce cerebral inflammation.

     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Microparticles stimulate angiogenesis by inducing ELR+ CXC‐chemokines in synovial fibroblasts

Microparticles (MPs) are small membrane‐vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro‐inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co‐cultured with increasing numbers of MPs. The effects of supernatants from co‐cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans‐well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro‐angiogenic ELR⁺ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co‐cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR⁺ chemokines. Consistent with the specific induction of ELR⁺ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio‐chamber assay, supernatants from RASFs co‐cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro‐angiogenic ELR⁺ chemokines. These pro‐angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis.     

Augmented LPS responsiveness in type 1 diabetes‐derived osteoclasts

Bone abnormalities are frequent co‐morbidities of type 1 diabetes (T1D) and are principally mediated by osteoblasts and osteoclasts which in turn are regulated by immunologic mediators. While decreased skeletal health in T1D involves alterations in osteoblast maturation and function, the effect of altered immune function on osteoclasts in T1D‐associated bone and joint pathologies is less understood. Here T1D‐associated osteoclast‐specific differentiation and function in the presence and absence of inflammatory mediators was characterized utilizing bone marrow‐derived osteoclasts (BM‐OCs) isolated from non‐obese diabetic (NOD) mice, a model for spontaneous autoimmune diabetes with pathology similar to individuals with T1D. Differentiation and osteoclast‐mediated bone resorption were evaluated along with cathepsin K, MMP‐9, and immune soluble mediator expression. The effect of lipopolysaccharide (LPS), a pro‐inflammatory cytokine cocktail, and NOD‐derived conditioned supernatants on BM‐OC function was also determined. Although NOD BM‐OCs cultures contained smaller osteoclasts, they resorbed more bone concomitant with increased cathepsin K, MMP‐9, and pro‐osteoclastogenic mediator expression. NOD BM‐OCs also displayed an inhibition of LPS‐induced deactivation that was not a result of soluble mediators produced by NOD BM‐OCs, although a pro‐inflammatory milieu did enhance NOD BM‐OCs bone resorption. Together these data indicate that osteoclasts from a T1D mouse model hyper‐respond to RANK‐L resulting in excessive bone degradation via enhanced cathepsin K and MMP‐9 secretion concomitant with an increased expression of pro‐osteoclastic soluble mediators. Our data also suggest that inhibition of LPS‐induced deactivation in NOD‐derived BM‐OC cultures is most likely due to NOD osteoclast responsiveness rather than LPS‐induced expression of soluble mediators. J. Cell. Physiol. 228: 349–361, 2013. © 2012 Wiley Periodicals, Inc.     

Verbascoside down‐regulates some pro‐inflammatory signal transduction pathways by increasing the activity of tyrosine phosphatase SHP‐1 in the U937 cell line

Polyphenols are the major components of many traditional herbal remedies, which exhibit several beneficial effects including anti‐inflammation and antioxidant properties. Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) is a redox sensitive protein tyrosine phosphatase that negatively influences downstream signalling molecules, such as mitogen‐activated protein kinases, thereby inhibiting inflammatory signalling induced by lipopolysaccharide (LPS). Because a role of transforming growth factor β‐activated kinase‐1 (TAK1) in the upstream regulation of JNK molecule has been well demonstrated, we conjectured that SHP‐1 could mediate the anti‐inflammatory effect of verbascoside through the regulation of TAK‐1/JNK/AP‐1 signalling in the U937 cell line. Our results demonstrate that verbascoside increased the phosphorylation of SHP‐1, by attenuating the activation of TAK‐1/JNK/AP‐1 signalling. This leads to a reduction in the expression and activity of both COX and NOS. Moreover, SHP‐1 depletion deletes verbascoside inhibitory effects on pro‐inflammatory molecules induced by LPS. Our data confirm that SHP‐1 plays a critical role in restoring the physiological mechanisms of inducible proteins such as COX2 and iNOS, and that the down‐regulation of TAK‐1/JNK/AP‐1 signalling by targeting SHP‐1 should be considered as a new therapeutic strategy for the treatment of inflammatory diseases.     

5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

Peptide‐based systems analysis of inflammation induced myeloid‐derived suppressor cells reveals diverse signaling pathways

A better understanding of molecular signaling between myeloid‐derived suppressor cells (MDSC), tumor cells, T‐cells, and inflammatory mediators is expected to contribute to more effective cancer immunotherapies. We focus on plasma membrane associated proteins, which are critical in signaling and intercellular communication, and investigate changes in their abundance in MDSC of tumor‐bearing mice subject to heightened versus basal inflammatory conditions. Using spectral counting, we observed statistically significant differential abundances for 35 proteins associated with the plasma membrane, most notably the pro‐inflammatory proteins S100A8 and S100A9 which induce MDSC and promote their migration. We also tested whether the peptides associated with canonical pathways showed a statistically significant increase or decrease subject to heightened versus basal inflammatory conditions. Collectively, these studies used bottom‐up proteomic analysis to identify plasma membrane associated pro‐inflammatory molecules and pathways that drive MDSC accumulation, migration, and suppressive potency.     

HSPA12B inhibits lipopolysaccharide‐induced inflammatory response in human umbilical vein endothelial cells

Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)‐induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein‐HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1 μg/ml LPS for 4 hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound‐healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT‐qPCR and Western blot, respectively. The release of cytokines interleukin‐6 and tumour necrosis factor‐α was measured by ELISA. HSPA12B suppressed LPS‐induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS‐induced up‐regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS‐induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS‐induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway.     

Hydrogen sulfide reduces cell adhesion and relevant inflammatory triggering by preventing ADAM17‐dependent TNF‐α activation

H₂S is the third endogenous gaseous mediator, after nitric oxide and carbon monoxide, possessing pleiotropic effects, including cytoprotection and anti‐inflammatory action. We analyzed, in an in vitro model entailing monocyte adhesion to an endothelial monolayer, the changes induced by H₂S on various potential targets, including cytokines, chemokines, and proteases, playing a crucial role in inflammation and cell adhesion. Results show that H₂S prevents the increase in monocyte adhesion induced by tumor necrosis factor‐α (TNF‐α). Under these conditions, downregulation of monocyte chemoattractant protein‐1 (MCP‐1), chemokine C‐C motif receptor 2, and increase of cluster of differentiation 36 could be detected in monocytes. In endothelial cells, H₂S treatment reduces the increase in MCP‐1, inter‐cellular adhesion molecule‐1, vascular cell adhesion molecule‐1, and of a disintegrin and metalloproteinase metallopeptidase domain 17 (ADAM17), both at the gene expression and protein levels. Cystathionine γ‐lyase and 3‐mercaptopyruvate sulfurtransferase, the major H₂S forming enzymes, are downregulated in endothelial cells. In addition, H₂S significantly reduces activation of ADAM17 by PMA in endothelial cells, with consequent reduction of both ADAM17‐dependent TNF‐α ectodomain shedding and MCP‐1 release. In conclusion, H₂S is able to prevent endothelial activation by hampering endothelial activation, triggered by TNF‐α. The mechanism of this protective effect is mainly mediated by down‐modulation of ADAM17‐dependent TNF‐converting enzyme (TACE) activity with consequent inhibition of soluble TNF‐α shedding and its relevant MCP‐1 release in the medium. These results are discussed in the light of the potential protective role of H₂S in pro‐inflammatory and pro‐atherogenic processes, such as chronic renal failure. J. Cell. Biochem. 114: 1536–1548, 2013. © 2013 Wiley Periodicals, Inc.