PKA regulates HMGB1 through activation of IGFBP-3 and SIRT1 in human retinal endothelial cells cultured in high glucose

Objective and Design

Inflammation is a key component of a number of diseases, including diabetic retinopathy. We investigated the cellular pathway by which protein kinase A (PKA) inhibited high mobility group box 1 (HMGB1).

Methods

Primary human retinal endothelial cells (REC) were grown in normal glucose (5 mM) or high glucose (25 mM). Cells in high glucose were treated with exchange protein for cAMP 1 (Epac1) and IGFBP-3 siRNA. Additional cells in high glucose were treated with forskolin, a PKA agonist, and Epac1 siRNA. Some cells were treated with a plasmid for insulin-like growth factor binding protein 3 (IGFBP-3) that does not bind IGF-1. Finally, some REC received Ex527, a sirtuin 1 (SIRT1) antagonist, prior to forskolin treatment. Protein analyses were done for HMGB1, Epac1, IGFBP-3, SIRT1, and PKA.

Results

PKA inhibited cytoplasmic HMGB1, independent of Epac1 actions. PKA activated IGFBP-3 and SIRT1 to inhibit cytoplasmic HMGB1. High glucose inhibited SIRT1 levels and increased cytoplasmic HMGB1 in REC.

Conclusions

PKA requires active IGFBP-3 and SIRT1 to inhibit HMGB1 inflammatory actions in the retina vasculature. Activation of these pathways may offer new targets for therapy development.

     

HMGB1 inhibits insulin signalling through TLR4 and RAGE in human retinal endothelial cells

In the past decade, the role of inflammation has been shown in diabetes and its complications. Little is available on high mobility group box 1 (HMGB1) actions on the proteins involved in insulin signal transduction, which may be altered to result in insulin resistance in the retina. Retinal endothelial cells (REC) were grown in normal or high glucose and treated with recombinant human HMGB1, an Epac1 agonist, or both. Additional cells were treated with advanced glycation end-products (RAGE) or toll-like receptor 4 (TLR4) siRNA prior to rhHMGB1. Proteins lysates were processed for Western blotting for TLR4, RAGE, insulin receptor, Akt, and IRS-1 phosphorylation. We found that rhHMGB1 blocked insulin and Akt phosphorylation through either RAGE or TLR4 actions. Epac1 overcame both endogenous and exogenous HMGB1 to maintain normal insulin signalling. Taken together, these data offer upstream targets to maintain proper insulin signal transduction in the retinal vasculature.     

Salsalate ameliorates the atherosclerotic response through HO-1- and SIRT1-mediated suppression of ER stress and inflammation

Objective and design

Inflammation plays a causative role in atherosclerosis development. Salsalate is an anti-inflammatory drug used to treat atherosclerosis, but the mechanisms by which it affects atherosclerotic progression remain unclear.

Methods

Human umbilical vascular endothelial cells (HUVECs) and THP-1 human monocytes were treated with salsalate. Heme oxygenase 1 (HO-1) and sirtuin 1 (SIRT1) small interfering RNAs (siRNAs) were used to suppress each gene expression. Protein analyses were performed for measuring the expression of HO-1, SIRT1, nuclear factor kappa B (NFκB), cell adhesion molecules, and endoplasmic reticulum (ER) stress markers. Furthermore, cell adhesion assay, caspase 3 activity assay, and ELISA were also performed.

Results

In this study, we show that salsalate increases the expression of HO-1 and SIRT1 in HUVEC and suppresses lipopolysaccharide (LPS)-induced atherosclerotic responses via HO-1- and SIRT1-mediated pathways. Salsalate treatment of HUVEC and THP-1 cells reduced LPS-induced phosphorylation of NFκB and secretion of the proinflammatory cytokines TNFα and MCP-1. Salsalate treatment of HUVEC reduced the expression of the adhesion molecules ICAM, VCAM, and E-selectin and the LPS-induced adhesion of THP-1 cells to HUVEC. Salsalate treatment also attenuated LPS-induced ER stress and cell apoptosis. These anti-atherosclerotic effects were reversed by treating cells with siRNA for HO-1 and SIRT1.

Conclusions

Salsalate ameliorates LPS-induced atherosclerotic reactions via HO-1 and SIRT1-dependent reduction of inflammation and ER stress. Activation of these pathways by salsalate may provide therapeutic strategies for treating atherosclerosis.

     

SIRT7 affects autophagy and activation of hepatic stellate cells by regulating the acetylation level of high mobility group protein 1

ObjectivePreventing the progression of hepatic fibrosis is an important strategy to improve the prognosis of liver disease. The purpose of this study was to investigate the role of sirtuin7 (SIRT7) and high mobility group box 1 (HMGB1) acetylation in the occurrence and development of hepatic fibrosis.Materials and methodsHepatic fibrosis mice model was induced by CCl₄. TGF-β1 was used to activated quiescent hepatic stellate cell (qHSC) into activated HSC (aHSC). Hematoxylin-eosin evaluated hepatic fibrosis in vivo, and the distribution of α-smooth muscle actin (α-SMA) or HMGB1 was detected by immunohistochemistry or immunofluorescence. The expressions of SIRT7, autophagy related proteins, and HSC activation-related proteins were detected by Western blot. Immunoprecipitation detected the acetylation level of HMGB1. Lysine mutants of HMGB1 were constructed in vitro to explore the acetylation sites of HMGB1.ResultsHepatocyte autophagy and activation levels were enhanced in CCl₄ group or aHSC group, and the acetylation level of HMGB1 was increased. Nuclear transfer of HMGB1 occurred in aHSC, and HMGB1was mainly distributed in cytoplasm. The expression of SIRT7 in CCl₄ group or aHSC group was most significantly decreased, and knockdown of SIRT7 leads to increased levels of HSCs autophagy and activation. Overexpression of SIRT7 or interference of HMGB1 alone in aHSC can reduce the level of autophagy and activation of aHSC. However, continued overexpression of SIRT7 in shHMGB1-aHSC could not reduce the autophagy and activation levels of aHSC. Among the 11 Flag-HMGB1 mutants, the acetylation level of K86R-Flag-HMGB1 was the lowest. The acetylation level of K86R-Flag-HMGB1 did not change due to SIRT7 downregulation.ConclusionThis study proved that SIRT7 can directly target the K86R site of HMGB1 and participate in regulating the expression and distribution of HMGB1, thus affecting the autophagy and activation level of HSCs.     

Adenovirus-mediated delivery of interferon-γ gene inhibits the growth of nasopharyngeal carcinoma

Background

Epidermal growth factor receptor (EGFR) is suggested to predict the radiosensitivity and/or prognosis of human esophageal squamous cell carcinoma (ESCC). The objective of this study was to investigate the efficacy of Nimotuzumab (an anti-EGFR monoclonal antibody) on ESCC radiotherapy (RT) and underlying mechanisms.

Methods

Nimotuzumab was administrated to 2 ESCC cell lines KYSE30 and TE-1 treated with RT. Cell growth, colony formation and apoptosis were used to measure anti-proliferation effects. The method of RNA interference was used to investigate the role of insulin-like growth factor binding protein-3 (IGFBP-3) in ESCC cells radiosensitivity treated with Nimotuzumab. In vivo effect of Nimotuzumab on ESCC radiotherapy was done using a mouse xenograft model.

Results

Nimotuzumab enhanced radiation response of KYSE30 cells (with high EGFR expression) in vitro, as evidenced by increased radiation-inhibited cell growth and colony formation and radiation-mediated apoptosis. Mechanism study revealed that Nimotuzumab inhibited phosphorylated EGFR (p-EGFR) induced by EGF in KYSE30 cells. In addition, knockdown of IGFBP-3 by short hairpin RNA significantly reduced KYSE30 cells radiosensitivity (P<0.05), and even after the administration of Nimotuzumab, the RT response of IGFBP-3 silenced KYSE30 cells was not enhanced (P>0.05). In KYSE30 cell xenografts, Nimotuzumab combined with radiation led to significant tumor growth delay, compared with that of radiation alone (P=0.029), and also with IGFBP-3 up-regulation in tumor tissue.

Conclusions

Nimotuzumab could enhance the RT effect of ESCC cells with a functional active EGFR pathway. In particular, the increased ESCC radiosensitivity by Nimotuzumab might be dependent on the up-regulation of IGFBP-3 through EGFR-dependent pathway.

     

Glycyrrhizic acid promotes sciatic nerves recovery in type 1 diabetic rats and protects Schwann cells from high glucose-induced cytotoxicity

The present study aims to investigate the therapeutic effect and mechanism of glycyrrhizic acid (GA) in diabetic peripheral neuropathy (DPN). GA significantly mitigated nerve conduction velocity (NCV) deficit and morphological abnormality and reduced high-mobility group box-1 (HMGB1) expression in the sciatic nerves of diabetic rats independent of blood glucose and body weight. Notably, GA alleviated the increase of HMGB1 and the decrease of cell viability in high glucose-stimulated RSC96 cells. Furthermore, GA obviously reduced the concentration of inflammatory cytokines in the sciatic nerves of diabetic rats and supernatants of high glucose-exposed RSC96 cells, then restored the decreased expression levels of nerve growth factor (NGF) and neuritin-1, and the increased expression levels of cleaved caspase-3 and neuron-specific enolase. Additionally, GA markedly inhibited receptor for advanced glycation end products (RAGE) expression, p38MAPK phosphorylation, and the nuclear translocation of NF-κBp65 in diabetic rats and high glucose-exposed RSC96 cells. The promotional effect of high glucose in RSC96 cells was diminished following Hmgb1 siRNA treatment. Our findings indicate that GA may exert neuroprotection on DPN by suppressing HMGB1, which lead to extenuation of inflammation response, balance of NGF, neuritin-1 and caspase-3, as well as inactivation of RAGE/p38MAPK/NF-κBp65 signaling pathway.     

A dual regulatory role of apurinic/apyrimidinic endonuclease 1/redox factor-1 in HMGB1-induced inflammatory responses

Apurinic/apyrimidinic endonuclease 1/Redox factor-1 (APE1) is a multifunctional protein involved in reduction-oxidation regulation. High-mobility group box 1 (HMGB1) is released by necrotic cells and various inflammatory stimuli, acting as an inflammatory marker in sepsis and autoimmune diseases. Here, we report the dual regulatory role of APE1 in inflammatory signaling to extracellular HMGB1 or in the release of endogenous HMGB1 in human monocytes/macrophages. Forced cytoplasmic overexpression of APE1 profoundly attenuated the upregulation of HMGB1-mediated reactive oxygen species generation, cytokine secretion, and cyclooxygenase-2 expression by primary monocytes and macrophage-like THP-1 cell lines. In addition, HMGB1-induced activation of p38 and c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2, was strongly abrogated by the overexpression of APE1. The activation of apoptosis signal-regulating kinase 1 was required for both the p38 and JNK activation challenge with HMGB1. The extracellular release of HMGB1 by activated macrophages was inhibited by APE1 transfection. Small interfering RNA (siRNA) knockdown of endogenous APE1 impaired HMGB1-mediated cytokine expression and MAPK activation in THP-1 cells. HMGB1 stimulation induced the translocation of APE1 to the nucleus of the cell. In addition, APE1 silencing via siRNA transfection inhibited both the nuclear and cytoplasmic expression of APE1. These data identify APE1 as a novel dual regulator of inflammatory signaling to HMGB1 by human monocytes/macrophages. The modulation of cytosolic APE1 expression might be useful as a potential therapeutic modality for the treatment of inflammatory or autoimmune diseases.     

High-mobility group box-1 inhibition stabilizes intestinal permeability through tight junctions in experimental acute necrotizing pancreatitis

Background

In acute necrotizing pancreatitis (ANP), bacterial translocation (BT) from the gastrointestinal tract is the essential pathogenesis in the development of septic complications. Although high-mobility group box-1 (HMGB1) is associated with BT and organ dysfunction in ANP, the mechanism of HMGB1 in the intestinal barrier dysfunction and BT has not been well addressed. In this study, we intend to address the role of HMGB1 in ANP involving BT and intestinal barrier dysfunction.

Methods

Experimental ANP was achieved in male Sprague–Dawley rats through a retrograde injection of taurocholate into the common biliopancreatic duct following a laparotomy operation. HMGB1 blockade intervention was conducted with a subcutaneous injection of anti-HMGB1 antibody immediately before the laparotomy procedure. Twenty-four hours after ANP induction, pancreatic and intestinal tissues and blood samples were collected for a histopathological assessment and lipid peroxidation or glutathione (GSH) evaluation. AP-induced barrier dysfunction was determined by an intestinal permeability assessment. Tight junction proteins and autophagy regulators were investigated by western blotting, immunohistological analysis and confocal immunofluorescence imaging.

Results

ANP developed as indicated by microscopic parenchymal necrosis and fat necrosis, which were associated with intestinal mucosal barrier dysfunction. HMGB1 inhibition played a protective role in intestinal mucosal barrier dysfunction, protected against microbiome changes in ANP, and relieved intestinal oxidative stress. Additionally, HMGB1 inhibition attenuated intestinal permeability; preserved the expression of TJs, such as claudin-2 and occludin; and decreased autophagy. Furthermore, the autophagy regulator LC3 and TJ protein claudin-2 were both upregulated in ANP according to dual immunofluorescence analysis.

Conclusion

HMGB1 inhibition ameliorated the severity of experimental ANP though beneficial effects on BT, mainly involving in TJ function.

     

Dopamine D1-like receptor activation depolarizes medium spiny neurons of the mouse nucleus accumbens by inhibiting inwardly rectifying K currents through a cAMP-dependent protein kinase A-independent mechanism

Dopamine/cAMP signaling has been reported to mediate behavioral responses related to drug addiction. It also modulates the plasticity and firing properties of medium spiny neurons (MSNs) in the nucleus accumbens (NAc), although the effects of cAMP signaling on the resting membrane potential (RMP) of MSNs has not been specifically defined. In this study, activation of dopamine D1-like receptors (D1Rs) by SKF-38393 elicited membrane depolarization and inward currents in MSNs from the NAc core of 14–17 day-old mice. Similar results were obtained following stimulation of adenylyl cyclase (AC) activity with forskolin or application of exogenous cAMP. Forskolin occluded SKF-38393's effects, thus indicating that D1R action is mediated by AC/cAMP signaling. Accordingly, AC blockade by SQ22536 significantly inhibited the responses to SKF-38393. Effects elicited by D1R stimulation or increased cAMP levels were unaffected by protein kinase A (PKA) or protein kinase C (PKC) blockade and were not mimicked by the Epac agonist, 8CPT-2Me-cAMP. Responses to forskolin were also not significantly modified by cyclic nucleotide-gated (CNG) channel blockade. Forskolin-induced membrane depolarization was associated with increased membrane input resistance. Voltage-clamp experiments revealed that forskolin and SKF-38393 effects were due to inhibition of resting K⁺ currents exhibiting inward rectification at hyperpolarized potentials and a reversal potential (around −90 mV) that shifted with the extracellular K⁺ concentration. Forskolin and D1R agonist effects were abolished by the inward rectifier K⁺ (Kir)-channel blocker, BaCl₂. Collectively, these data suggest that stimulation of postsynaptic D1Rs in MSNs of the NAc core causes membrane depolarization by inhibiting Kir currents. This effect is mediated by AC/cAMP signaling but it is independent on PKA, PKC, Epac and CNG channel activation, suggesting that it may stem from cAMP's direct interaction with Kir channels. D1R/cAMP-mediated excitatory effects may influence the generation of output signals from MSNs by facilitating their transition from the quiescent down-state to the functionally active up-state.     

Circulating Insulin-Like Growth Factor Binding Proteins (IGFBPs) 1 and 2 Induced in Vitamin C-Deficient or Fasted Guinea Pigs Inhibit IGF-I Action in Cultured Cells

Abstract

Collagen gene expression and proteoglycan synthesis are decreased in vitamin C-deficient guinea pigs losing weight and in fasted guinea pigs receiving ascorbate. Sera from such guinea pigs contain an insulin-like growth factor (IGF)-I-reversible inhibitor of collagen, proteoglycan and DNA synthesis and elevated levels of 29 and 35-kDa IGF binding proteins (IGFBPs). We now have identified the induced proteins as IGFBPs 1 and 2 and investigated their role as inhibitors. Guinea pig sera were treated with antibodies to IGFBPs 1 and 2 and antibody-IGFBP complexes were removed by passage through a Protein A-Sepharose column. Inhibitor content of fasted and scorbutic sera, and Protein A pass-through fractions derived from them, was assessed by their level of stimulation of DNA and collagen synthesis in 3T3 cells, compared to analogously treated normal guinea pig serum. Removal of IGFBP-1 from scorbutic serum reversed inhibition of collagen and DNA synthesis by more than half but removal of IGFBP-2 was less effective. Removal of both IGFBPs reversed inhibition almost completely. Similar results were obtained with fasted guinea pig serum. Conversely, purified rat IGFBPs 1 and 2 inhibited DNA and collagen synthesis in cells cultured in normal guinea pig serum or IGF-I-stimulated DNA synthesis, with IGFBP-1 being more potent. Thus, IGFBP-1 and, to a lesser extent IGFBP-2, cause inhibition of IGF-I action by sera from fasted and scorbutic guinea pigs and may inhibit collagen gene expression in vivo.     

Two cAMP-dependent pathways differentially regulate exocytosis of large dense-core and small vesicles in mouse β-cells

It has been reported that cAMP regulates Ca^{2 +} -dependent exocytosis via protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac) in neurons and secretory cells. It has, however, never been clarified how regulation of Ca^{2 +} -dependent exocytosis by cAMP differs depending on the involvement of PKA and Epac, and depending on two types of secretory vesicles, large dense-core vesicles (LVs) and small vesicles (SVs). In this study, we have directly visualized Ca^{2 +} -dependent exocytosis of both LVs and SVs with two-photon imaging in mouse pancreatic β-cells. We found that marked exocytosis of SVs occurred with a time constant of 0.3 s, more than three times as fast as LV exocytosis, on stimulation by photolysis of a caged-Ca^{2 +} compound. The diameter of SVs was identified as ∼80 nm with two-photon imaging, which was confirmed by electron-microscopic investigation with photoconversion of diaminobenzidine. Calcium-dependent exocytosis of SVs was potentiated by the cAMP-elevating agent forskolin, and the potentiating effect was unaffected by antagonists of PKA and was mimicked by the Epac-selective agonist 8-(4-chlorophenylthio)-2'- O -methyl cAMP, unlike that on LVs. Moreover, high-glucose stimulation induced massive exocytosis of SVs in addition to LVs, and photolysis of caged cAMP during glucose stimulation caused potentiation of exocytosis with little delay for SVs but with a latency of 5 s for LVs. Thus, Epac and PKA selectively regulate exocytosis of SVs and LVs, respectively, in β-cells, and Epac can regulate exocytosis more rapidly than PKA.     

下调人高迁移率族蛋白1对人前列腺癌细胞PC-3的影响

目的研究小干扰RNA(siRNA)抑制高迁移率族蛋白1(HMGB1)基因对PC-3侵袭和转移的影响。方法构建真核表达载体Pgenesil-1/HMGB1siRNA,转染PC-3细胞系,通过RT-PCR和Western blot检测HMGB1的mRNA及蛋白;通过细胞划痕、transwell和明胶酶谱分析检测PC-3体外侵袭能力。结果成功构建siRNA表达载体Pgene-sil-1/HMGB1siRNA,可使PC-3细胞HMGB1mRNA和蛋白表达水平显著降低(P<0.05),并能有效抑制PC-3体外侵袭和转移活性(P<0.05)。结论应用siRNA技术能有效的抑制基因的表达,同时也能有效抑制PC-3细胞的体外侵袭和转移,为肿瘤的生物学治疗提供了新思路。     

Effect of high mobility group box 1 on Toll-like receptor 9 in B cells in myeloperoxidase-ANCA-associated vasculitis

Abstract

High mobility group box 1 (HMGB1) played pathogenic role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Recent findings demonstrated that Toll-like receptor 9 (TLR9) was involved in B cell tolerance breaking of autoimmune disease, including AAV. Here, we investigated the effect of HMGB1 on TLR9 in B cells of AAV. In the present work, patients with myeloperoxidase (MPO)-AAV in active stage were recruited. Intracellular TLR9 expression in various B cell subpopulations of the whole blood was detected by flow cytometry and the correlation with clinical data was analysed. Our results showed that intracellular TLR9 expression in B cells, memory B cells and plasmablasts correlated with erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP). In particular, TLR9 expression in plasma cells correlated with ESR, CRP, serum creatinine, eGFR, and Birmingham Vasculitis Activity Score. To further explore the effect of HMGB1 on B cell, peripheral blood mononuclear cells (PBMCs) from AAV patients were isolated. After stimulated with HMGB1, TLR9 expression in various B cell subpopulations and proliferation ratio of live B cells were analysed by flow cytometry. We found that TLR9 expression in plasma cells and the proliferation ratio of live B cells by HMGB1 stimulation were significantly upregulated compared with the control group. Therefore, TLR9 expression in plasma cells was associated with disease activity of MPO-AAV. HMGB1 could enhance TLR9 expression in plasma cells and B cell proliferation. These indicated a role of HMGB1 on TLR9 in B cells in MPO-AAV, which would provide potential clues for intervention strategies.     

Dexmedetomidine exerts cerebral protective effects against cerebral ischemic injury by promoting the polarization of M2 microglia via the Nrf2/HO-1/NLRP3 pathway

Introduction

Cerebral ischemic injury is associated with long-term disability. Dexmedetomidine (Dex) can exert neuroprotective effects on cerebral ischemic/reperfusion injury. The present study explored the mechanism of Dex in cerebral ischemic injury.

Materials and methods

To this end, the permanent middle cerebral artery occlusion (p-MCAO) mouse model was established and treated with Dex or/and Nrf2 inhibitor ML385. Subsequently, microglia were subjected to oxygen–glucose deprivation (OGD) in sugar-free environment and thereafter treated with Dex, Nrf2 inhibitor, and NLRP3 lentiviral overexpression vector, respectively.

Results

Dex alleviated the neurobehavioral deficit of p-MCAO mice, reduced brain water content, relieved pathological changes, and reduced cerebral infarction size. Dex promoted the polarization of microglia from M1 to M2, thus ameliorating oxidative stress and inflammatory responses. Our results showed that Dex promoted M2-polarization of microglia in vivo and in vitro by promoting HO-1 expression via Nrf2 nuclear import. Moreover, the Nrf2/HO-1 axis inhibited the activation of NLRP2 inflammasome and NLRP3 overexpression reversed the effect of Dex.

Conclusion

In conclusion, Dex promoted M2-polarization of microglia and attenuated oxidative stress and inflammation, and thus protected against cerebral ischemic injury by activating the Nrf2/HO-1 pathway and inhibiting NLRP3 inflammasome.

     

Inhibition of adenylate cyclase attenuates muscarinic Ca signaling by a PKA-independent mechanism in rat carotid body Type I cells

Carotid body (CB) Type I cells respond to hypoxia by releasing excitatory and inhibitory neurotransmitters. This mechanism leads to increased firing of the carotid sinus nerve (CSN) which alters breathing to maintain blood gases within the physiological range. Acetylcholine targets both muscarinic and nicotinic receptors in the rat CB, acting postsynaptically on CSN and presynaptically on Type I cells. Muscarinic Ca²⁺ signaling is inhibited by the activation of G_i-coupled receptors including histamine H3 receptors. Here inhibition of adenylate cyclase with SQ22536 mimicked H3 receptor activation. Using Ca²⁺ imaging techniques it was observed that inhibition of muscarinic Ca²⁺ signaling was independent of protein kinase A (PKA) as PKA inhibitors H89 and KT5720 were without effect on the muscarinic Ca²⁺ response. By contrast the Epac (exchange protein activated by cAMP) inhibitor brefeldin A inhibited muscarinic Ca²⁺ signaling whereas the Epac activator 8-pCPT-2′-O-Me-cAMP-AM potentiated Ca²⁺ signaling.     

MicroRNA-142-3p Inhibits Chondrocyte Apoptosis and Inflammation in Osteoarthritis by Targeting HMGB1

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and joint inflammation in which microRNAs are significantly involved. Previous studies have reported that miR-142-3p is a novel mediator of inflammatory signaling pathways, but whether miR-142-3p regulates OA remains unknown. In this study, we aimed to investigate the potential role of miR-142-3p in OA and the underlying molecular mechanism. We showed that miR-142-3p was significantly reduced in the articular cartilage tissues from experimental OA mice. The expression of miR-142-3p was also decreased in chondrocytes treated with lipopolysaccharide (LPS) in vitro. Moreover, the overexpression of miR-142-3p significantly inhibited cell apoptosis, nuclear factor (NF)-kB, and the production of proinflammatory cytokines, including interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α induced by LPS. Interestingly, bioinformatics analysis demonstrated that high mobility group box 1 (HMGB1), an important inflammatory mediator of OA, was predicted as a target of miR-142-3p, which was validated by dual-luciferase reporter assay. The high expression of HMGB1 in chondrocytes induced by LPS was significantly inhibited by miR-142-3p overexpression. Furthermore, the restoration of HMGB1 markedly abrogated the effect of miR-142-3p. In OA mice, the overexpression of miR-142-3p by lentivirus-mediated gene transfer significantly inhibited HMGB1 expression, NF-kB signaling, and proinflammatory cytokines. Moreover, the overexpression of miR-142-3p significantly alleviated OA progression in OA mice in vivo. Taken together, our study suggests that miR-142-3p inhibits chondrocyte apoptosis and inflammation in OA by inhibiting the HMGB1-mediated NF-kB signaling pathway. The overexpression of miR-142-3p impedes the OA progression in mice in vivo indicating that miR-142-3p is a potential molecular target for OA treatment.     

Tetraspanin 1 inhibits TNFα-induced apoptosis via NF-κB signaling pathway in alveolar epithelial cells

Objective

Tetraspanin family plays an important role in the pathogenesis of cancer, but its role in lung fibrosis is unknown. To determine whether tetraspanin 1 (TSPAN1), a member of the family, may be involved in the pathogenesis of pulmonary fibrosis.

Methods

TNFα -stimulated human alveolar epithelial (A549) and alveolar epithelial type II cell (AT2) were treated in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). The expression of TSPAN1 was examined in vivo using the bleomycin-induced lung fibrosis model and tissue sample of IPF patients. Then we transfected the cells with TSPAN1 siRNA or plasmid and detected the expression changes of related proteins and cell apoptosis.

Results

In our study, we found that TSPAN1 was markedly down-regulated in lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and in bleomycin-induced pulmonary fibrosis in mice. We also found that TSPAN1 was significantly down-regulated in A549 and primary (AT2) cells following exposure to TNFα. Meanwhile, TSPAN1 inhibited p-IκBα, which attenuated nuclear NF-κB translocation and activation and inhibited apoptosis. We demonstrated that TSPAN1 reduced Bax translocation and caspase-3 activation, inhibited the apoptosis by regulating the NF-κB pathway in response to TNFα.

Conclusions

We conclude that TSPAN1 mediated apoptosis resistance of alveolar epithelial cells by regulating the NF-κB pathway. TSPAN1 may be a potential therapeutic target for pulmonary fibrosis or acute lung injury.

     

Effects of intermittent hypoxia training on leukocyte pyruvate dehydrogenase kinase 1 (PDK-1) mRNA expression and blood insulin level in prediabetes patients

Purpose

Intermittent hypoxia training/treatment (IHT) is an emerging therapeutic approach to alleviate chronic diseases, such as diabetes. The present study investigated the effects of IHT on blood leucocyte pyruvate dehydrogenase kinase 1 (PDK-1) mRNA expression and its relationship with the changes in blood insulin level.

Methods

Seven adult healthy volunteers and 11 prediabetic patients participated in this study. A 3-week course of IHT consisted of a 40-min session of 4 cycles of 5-min 12% O₂ and 5-min room air breathing per day, 3 sessions per week for 3 weeks (i.e., total 9 sessions of IHT). Plasma insulin levels and leukocyte PDK-1 mRNA expression were determined at various time points either under fasting condition or following oral glucose tolerance test (OGTT). Correlation between the IHT-induced changes in PDK-1 mRNA and insulin or glucose levels in the same serological samples was analyzed.

Results

At pre-IHT baseline, PDK-1 mRNA expression was two times higher in prediabetes than control subjects. IHT resulted in significant augmentation in PDK-1 mRNA expression (> twofold) in prediabetes at the end of 3-week IHT and remained elevated 1 month after IHT, which was correlated with a significantly reduced insulin release and lower blood glucose after glucose loading with OGTT.

Conclusion

IHT can trigger beneficial effects in normalizing blood insulin levels in prediabetic patients under oral glucose load, which were closely correlated with an enhanced mRNA expression of PDK-1 in leukocytes. Further clinical trials are warranted to validate the utility of IHT as a non-invasive complementary therapy against diabetes-associated pathologies.