Wanchun Sun

Lipopolysaccharide induces endothelial cell apoptosis via activation of Na+/H+ exchanger 1 and calpain-dependent degradation of Bcl-2

The calcium-dependent protease calpain is involved in lipopolysaccharide (LPS)-induced endothelial injury. The activation of Na(+)/H(+) exchanger (NHE) is responsible to increase intracellular Ca(2+) (Ca(i)(2+)) in cardiovascular diseases. Here we hypothesized that activation of NHE mediates LPS-induced endothelial cell apoptosis via calcium-dependent calpain pathway. Our results revealed that LPS-induced increases in NHE activity are dependent on NHE1 in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with LPS increased the NHE1 activity in a time-dependent manner associated with the increased Ca(i)(2+), which resulted in enhanced calpain activity as well as HUVECs apoptosis via NHE1-dependent degradation of Bcl-2.

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis.

A20 promotes Brucella intracellular growth via inhibition of macrophage cell death and activation.

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of macrophage activation and apoptosis in tumor necrosis factor receptor1 (TNFR1) signaling pathway. Brucella infection can induce A20 expression in macrophages. Here, we hypothesize that A20 promotes Brucella intracellular growth via inhibition of activation and apoptosis of macrophages. To test this hypothesis, we stably incorporated mouse A20-shRNA into the RAW264.7 cells by lentiviral gene transfer to successfully knockdown A20. A20-deficient RAW264.7 cells were subsequently challenged with Brucella abortus and colony formation units (CFUs) of bacteria, TNFα production, NF-kB activation, macrophages apoptosis and cell death were evaluated. The A20 knockdown was shown to effectively promote B. abortus-stimulated TNFα release, NF-kB activation and macrophage cell death, which suppressed B. abortus intracellular replication. Unexpectedly, deficiency of A20 failed to lead to B. abortus-induced macrophage apoptosis. A20 deficiency coupled NF-kB inhibition promoted caspase-8 dependent B. abortus-induced macrophage apoptosis. These findings provide a novel mechanism by which Brucella intracellular growth within macrophages occurs through up-regulation of A20 thereby limiting activation and macrophages cell death.

17-Hydroxy-jolkinolide A inhibits osteoclast differentiation through suppressing the activation of NF-κB and MAPKs.

Osteoclasts (OC) are bone-specific multinucleated giant cells (MNCs) derived from the monocyte/macrophage hematopoietic lineage cells. Inhibiting osteoclast formation is considered as an effective therapeutic approach for the treatment of the pathological bone loss. In this study, we investigated effects of 17-hydroxy-jolkinolide A (HJA), an ent-abietane diterpenoid isolated from the dried root of Euphorbia fischeriana, on osteoclastogenesis induced by RANKL. The results showed that HJA significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). HJA also prevented bone resorption by mature osteoclasts in a dose-dependent manner. In addition, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (Cts K) and MMP-9, was significantly inhibited by HJA. Furthermore, HJA also significantly inhibited RANKL-induced activation of NF-κB and phosphorylation of MAPK. Our results indicate that HJA has an inhibitory role in the bone loss by preventing osteoclast formation as well as its bone resorptive activity. Therefore, HJA may be useful as a therapeutic reagent for bone loss-associated diseases.

The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.

MicroRNA-125b-5p suppresses Brucella abortus intracellular survival via control of A20 expression

BackgroundBrucella may establish chronic infection by regulating the expression of miRNAs. However, the role of miRNAs in modulating the intracellular growth of Brucella remains unclear.ResultsIn this study, we show that Brucella. abortus infection leads to downregulation of miR-125b-5p in macrophages. We establish that miR-125b-5p targets A20, an inhibitor of the NF-kB activation. Additionally, expression of miR-125b-5p decreases A20 expression in B. abortus-infected macrophages and leads to NF-kB activation and increased production of TNFα. Furthermore, B. abortus survival is attenuated in the presence of miR-125b-5p.ConclusionsThese results uncover a role for miR-125b-5p in the regulation of B. abortus intracellular survival via the control of A20 expression.

Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

Aim:To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.Methods:Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na+/H+ exchanger 1 (NHE1) and calpain, intracellular free Ca2+level ([Ca2+]i), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE−/−) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.Results:LPS treatment increased NHE1 activity and [Ca2+]i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca2+-dependent protease calpain. Amiloride (1−10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca2+]i. and calpain activity. In the presence of the Ca2+ chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE−/− mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression.Conclusion:LPS stimulates NHE1 activity, increases [Ca2+]i, and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.

Tatarinan O, a lignin-like compound from the roots of Acorus tatarinowii Schott inhibits osteoclast differentiation through suppressing the expression of c-Fos and NFATc1

Osteoclasts (OC) are large multinucleated cells derived from monocyte/macrophage precursors. Suppressing osteoclastogenesis is considered as an effective therapeutic approach to erosive bone disease. The root of Acorus tatarinowii Schott, a well-known traditional Chinese medicine was used to treat rheumatosis and other inflammatory disease. However, the effects of tatarinan O (TO), one of the lignin-like compounds isolated from the roots of Acorus tatarinowii Schott during bone development are still unclear. In the present study, we explored the effect of TO on RANKL-induced osteoclastogenesis in vitro. TO was found to suppress osteoclast differentiation from RANKL-stimulated mouse bone marrow macrophages (BMMs) without significant cytotoxicity. TO also dose-dependently suppressed bone resorption activity of mature osteoclasts. Additionally, TO apparently inhibited the expression of osteoclastic marker genes, such as MMP-9, Cts K and TRAP. Furthermore, our results showed that TO decreased RANKL-induced expression of c-Fos and NFATc1 without influencing NF-κB activation and MAPK phosphorylation. Hence, for the first time we revealed that TO dose-dependently inhibited osteoclastogenesis from RANKL-stimulated mouse BMMs via decreasing the expression of NFATc1 and c-Fos.

HJB-1, a 17-hydroxy-jolkinolide B derivative, inhibits LPS-induced inflammation in mouse peritoneal macrophages.

Jolkinolide B (JB) and 17-hydroxy-JB (HJB) are diterpenoids from plants and it has been reported that the presence of a C-17 hydroxy group in JB significantly enhances the anti-inflammatory potency of JB. In this study, two HJB derivatives HJB-1 and HJB-2 were generated by the chemical modification of a 17-hydroxy group of HJB. HJB-1 more effectively inhibited TNF-α, IL-1β and IL-6 release in LPS-stimulated mouse peritoneal macrophages. In addition, HJB-1 reduced LPS-induced mRNA expression of TNF-α, IL-1β, IL-6, COX-2 and iNOS in a concentration-dependent manner, but did not alter IL-10 mRNA expression. LPS-induced NF-κB activation and MAPK phosphorylation were also effectively inhibited by HJB-1. These results demonstrate that HJB-1 exerts anti-inflammatory effects on LPS-activated mouse peritoneal macrophages by inhibiting NF-κB activation and MAPK phosphorylation and modification of a 17-hydroxy group of HJB may enhance the anti-inflammatory potency of HJB derivatives.

The Protective Effects of HJB-1, a Derivative of 17-Hydroxy-Jolkinolide B, on LPS-Induced Acute Distress Respiratory Syndrome Mice

Acute respiratory distress syndrome (ARDS),which is inflammatory disorder of the lung, which is caused by pneumonia, aspiration of gastric contents, trauma and sepsis, results in widespread lung inflammation and increased pulmonary vascular permeability. Its pathogenesis is complicated and the mortality is high. Thus, there is a tremendous need for new therapies. We have reported that HJB-1, a 17-hydroxy-jolkinolide B derivative, exhibited strong anti-inflammatory effects in vitro. In this study, we investigated its impacts on LPS-induced ARDS mice. We found that HJB-1 significantly alleviated LPS-induced pulmonary histological alterations, inflammatory cells infiltration, lung edema, as well as the generation of inflammatory cytokines TNF-α, IL-1β and IL-6 in BALF. In addition, HJB-1 markedly suppressed LPS-induced IκB-α degradation, nuclear accumulation of NF-κB p65 subunit and MAPK phosphorylation. These results suggested that HJB-1 improved LPS-induced ARDS by suppressing LPS-induced NF-κB and MAPK activation.