Wenjuan Yao

TSG (2,3,4’ ,5-tetrahydroxystilbene 2-O-β-D-glucoside) suppresses induction of pro-inflammatory factors by attenuating the binding activity of nuclear factor-κB in microglia

BackgroundInduction of pro-inflammatory factors is one of the characteristics of microglia activation and can be regulated by numerous active components of Chinese traditional herbs. Suppression of pro-inflammatory factors is beneficial to alleviate microglia-mediated cell injury. The present study aims to investigate the effect and possible mechanism of 2,3,4’,5-tetrahydroxystilbene 2-O-β-D-glucoside (TSG) on LPS-mediated induction of pro-inflammatory factors in microglia.MethodsWestern blot, ELISA, and Hoechst 33258 were used to measure the protein expression, TNF-α/IL-6 content, and apoptotic nuclei, respectively. The mRNA level was measured by real time-PCR. Nitric oxide (NO) content, lactate dehydrogenase (LDH) content, and NF-κB binding activity were assayed by commercial kits.ResultsTSG reduced iNOS protein expression as well as TNF-α, IL-6, and NO content in LPS-stimulated BV-2 cells. TSG attenuated the increase in apoptotic nuclei, caspase-3 cleavage, and LDH content induced by BV-2 cell-derived conditioned medium in primary hippocampal neurons. Mechanistic studies showed that TSG reduced the mRNA level of iNOS, TNF-α, and IL-6. TSG failed to suppress IκB-α degradation, NF-κB phosphorylation and nuclear translocation, and ERK1/2, JNK, and p38 phosphorylation. TSG, however, markedly reduced the binding of NF-κB to its DNA element. Chromatin immunoprecipitation (ChIP) assays confirmed that TSG reduced NF-κB binding to the iNOS promoter. These findings were ascertained in primary microglia where the LPS-induced increase in iNOS expression, NO content, apoptotic nuclei, and NF-κB binding to its DNA element were diminished by TSG.ConclusionsThese studies demonstrate that TSG attenuates LPS-mediated induction of pro-inflammatory factors in microglia through reducing the binding activity of NF-κB. This might help us to further understand the pharmacological role of TSG in inflammatory response in the central nervous system.

Methylene Blue Attenuates iNOS Induction Through Suppression of Transcriptional Factor Binding Amid iNOS mRNA Transcription

Inducible nitric oxide synthase (iNOS) critically contributes to the development of endotoxin‐mediated inflammation. It can be induced by cytokines or endotoxins via distinct signaling pathways. Lipopolysaccharide (LPS) triggers iNOS expression through activation of the inhibitor of κB‐α (IκB‐α)‐nuclear factor κB (NF‐κB) cascade, whereas interferon‐γ (IFN‐γ) acts primarily through Janus kinase (JAK)‐signal transducer and activator of transcription 1 (STAT1). Methylene blue (MB), an agent used clinically to treat numerous ailments, has been shown to reduce NO accumulation through suppression of iNOS activity. But it remains unclear whether MB affects iNOS induction. This knowledge gap is addressed in the present study using cultured cells and endotoxemic mice. With mouse macrophages, MB treatment prevented the LPS‐ and/or IFN‐γ‐stimulated iNOS protein expression. Real‐time PCR experiments showed that iNOS mRNA transcription was robustly blocked by MB treatment. The inhibitory effect of MB on iNOS expression was confirmed in vivo in endotoxemic mice. Further analysis showed that MB had no significant effect on IκB‐α degradation and NF‐κB or STAT1 phosphorylation in LPS/IFN‐γ‐stimulated cells. The nuclear transport of active NF‐κB or STAT1 was also not affected by MB treatment. But MB treatment markedly reduced the binding of NF‐κB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that MB reduced NF‐κB and STAT1 bindings to iNOS promoter inside the cell. These studies show that MB attenuates transcriptional factor binding amid iNOS mRNA transcription, providing further insight into the molecular mechanism of MB in disease therapy. J. Cell. Biochem. 116: 1730–1740, 2015.

Raf-1/CK2 and RhoA/ROCK signaling promote TNF-α-mediated endothelial apoptosis via regulating vimentin cytoskeleton

Both RhoA/ROCK and Raf-1/CK2 pathway play essential roles in cell proliferation, apoptosis, differentiation, and multiple other common cellular functions. We previously reported that vimentin is responsible for TNF-α-induced cell apoptosis. Herein, we investigated the regulation of RhoA/ROCK and Raf-1/CK2 signaling on vimentin filaments and endothelial apoptosis mediated by TNF-α. Treatment with TNF-α significantly induced the activation of RhoA and ROCK, and the expression of ROCK1. RhoA deficiency could obviously inhibit ROCK activation and ROCK1 expression induced by TNF-α. Both RhoA deficiency and ROCK activity inhibition (Y-27632) greatly inhibited endothelial apoptosis and preserved cell viability in TNF-α-induced human umbilical vein endothelial cells (HUVECs). Also vimentin phosphorylation and the remodeling of vimentin or phospho-vimentin induced by TNF-α were obviously attenuated by RhoA suppression and ROCK inhibition. TNF-α-mediated vimentin cleavage was significantly inhibited by RhoA suppression and ROCK inhibition through decreasing the activation of caspase3 and 8. Furthermore, TNF-α treatment greatly enhanced the activation of Raf-1. Suppression of Raf-1 or CK2 by its inhibitor (GW5074 or TBB) blocked vimentin phosphorylation, remodeling and endothelial apoptosis, and preserved cell viability in TNF-α-induced HUVECs. However, Raf-1 inhibition showed no significant effect on TNF-α-induced ROCK expression and activation, suggesting that the regulation of Raf-1/CK2 signaling on vimentin was independent of ROCK. Taken together, these results indicate that both RhoA/ROCK and Raf-1/CK2 pathway are responsible for TNF-α-mediated endothelial cytotoxicity via regulating vimentin cytoskeleton.

RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton

Graphical abstract Figure. No Caption available. &NA; The RhoA/ROCK signaling pathway regulates cell morphology, adhesion, proliferation, and migration. In this study, we investigated the regulatory role of RhoA/ROCK signaling on PDGF‐BB‐mediated smooth muscle phenotypic modulation and vascular remodeling and clarified the molecular mechanisms behind these effects. PDGF‐BB treatment induced the activation of RhoA, ROCK, PDGF‐R&bgr;, and the expression of PDGF‐R&bgr; in HA‐VSMCs (human aortic vascular smooth muscle cells). PDGF‐R&bgr; inhibition and RhoA suppression blocked PDGF‐BB‐induced RhoA activation and ROCK induction. In addition, PDGF‐BB‐mediated cell proliferation and migration were suppressed by PDGF‐R&bgr; inhibition, RhoA suppression, and ROCK inhibition, suggesting that PDGF‐BB promotes phenotypic modulation of HA‐VSMCs by activating the RhoA/ROCK pathway via the PDGF receptor. Moreover, suppressing both ROCK1 and ROCK2 blocked cell cycle progression from G0/G1 to S phase by decreasing the transcription and protein expression of cyclin D1, CDK2, and CDK4 via JNK/c‐Jun pathway, thus reducing cell proliferation in PDGF‐BB‐treated HA‐VSMCs. ROCK1 deletion, rather than ROCK2 suppression, significantly inhibited PDGF‐BB‐induced migration by reducing the expression of vimentin and preventing the remodeling of vimentin and phospho‐vimentin. Furthermore, ROCK1 deletion suppressed vimentin by inhibiting the phosphorylation of Smad2/3 and the nuclear translocation of Smad4. These findings suggested that ROCK1 and ROCK2 might play different roles in PDGF‐BB‐mediated cell proliferation and migration in HA‐VSMCs. In addition, PDGF‐BB and its receptor participated in neointima formation and vascular remodeling by promoting cell cycle protein expression via the JNK pathway and enhancing vimentin expression in a rat balloon injury model; effects that were inhibited by treatment with fasudil. Together, the results of this study reveal a novel mechanism through which RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.

Distinct Roles For ROCK1 and ROCK2 in the Regulation of Oxldl-Mediated Endothelial Dysfunction

Background/Aims: This study used Rho-associated protein kinase (ROCK) isoform-selective suppression or a ROCK inhibitor to analyze the roles of ROCK1 and ROCK2 in regulating endothelial dysfunction triggered by oxidized low-density lipoprotein (oxLDL). Methods: ROCK1 or ROCK2 expression in human umbilical vein endothelial cells (HUVECs) was suppressed by small interfering RNA (siRNA). HUVECs were pretreated with 30 μM Y27632 (pan ROCK inhibitor) for 30 min before exposure to 200 μg/mL oxLDL for an additional 24 h. Cell viability was determined by the MTT assay, and cell apoptosis was evaluated by the TUNEL assay. Protein expression and phosphorylation were assessed by Western blot analysis. The morphology of total and phosphorylated vimentin (p-vimentin) and the co-localization of vimentin with vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were detected by the immunofluorescence assay. The adhesion of promonocytic U937 cells to HUVECs was observed by light microscopy. Results: ROCK2 suppression or Y27632 treatment, rather than ROCK1 deletion, effectively reduced endothelial cell apoptosis and preserved cell survival. ROCK2 suppression exhibited improved vimentin and p-vimentin cytoskeleton stability and decreased vimentin cleavage by attenuating caspase-3 activity. In addition, increased p-vimentin expression induced by oxLDL was significantly inhibited by ROCK2 deletion or Y27632 treatment. In contrast, ROCK1 suppression showed no obvious effects on the vimentin cytoskeleton, but significantly regulated the expression of adhesion molecules. Endothelial ICAM-1 or VCAM-1 expression induced by oxLDL was obviously inhibited by ROCK1 suppression or Y27632 treatment. Moreover, the expression of ICAM-1 induced by oxLDL could also be reduced by ROCK2 suppression. Furthermore, ROCK2 deficiency or Y27632 treatment inhibited the redistribution of adhesion molecules and their co-localization with vimentin caused by oxLDL. These effects resulted in the significant inhibition of monocyte-endothelial adhesion induced by oxLDL. Conclusion: The results of this study support the novel concept that ROCK1 is involved in oxLDL-induced cell adhesion by regulating adhesion molecule expression, whereas ROCK2 is required for both endothelial apoptosis and adhesion by regulating both the vimentin cytoskeleton and adhesion molecules. Consequently, ROCK1 and ROCK2 have distinct roles in the regulation of oxLDL-mediated endothelial dysfunction.

Macrophage differentiation induced by PMA is mediated by activation of RhoA/ROCK signaling

In order to investigate the effects of RhoA/ROCK signaling in macrophage differentiation, we used 100 ng/mL PMA to induce macrophage differentiation from U937 cells in vitro. The observation of cell morphology and the expression of CD68 and SR-A were performed to confirm the differentiation induced by PMA. Western blot analysis showed that the expression of ROCK1 and ROCK2 and the phosphorylation of MYPT1 were significantly increased after PMA treatment. Pulldown assay showed that the activation of RhoA was obviously enhanced when U937 cells were treated with PMA. In order to further demonstrate whether RhoA/ROCK signaling could mediate the macrophage differentiation induced by PMA, we successfully suppressed the expression of RhoA, ROCK1 and ROCK2 by performing siRNA technology in U937 cells, respectively. The macrophage differentiation and the expression of CD68 and SR-A were significantly inhibited by the suppression of RhoA, ROCK1 or ROCK2 in PMA-induced U937 cells, indicating that the macrophage differentiation induced by PMA is associated with RhoA/ROCK signaling pathway. In addition, we pretreated U937 cells with Y27632 (ROCK inhibitor, 20 μM) for 30 min and then observed the macrophage differentiation induced by PMA. The result illustrated that Y27632 pretreatment obviously inhibited PMA-induced differentiation and the expression of CD68 and SR-A. In conclusion, the activation of RhoA/ROCK signaling is responsible for the macrophage differentiation induced by PMA.