Weijun Wu

Hydrogen Sulfide Attenuates Inflammatory Hepcidin by Reducing IL-6 Secretion and Promoting SIRT1-Mediated STAT3 Deacetylation.

AIMS Anemia of inflammation is quite prevalent in hospitalized patients with poor prognosis. Concerns about the effectiveness and safety of iron supplementation have arisen, driving the demand for alternative therapies. Induction of hepatic hepcidin, the master hormone of iron homeostasis, causes anemia under inflammatory conditions. Previous studies indicated that hydrogen sulfide (H2S), the third gasotransmitter and a well-known regulator of inflammation, may inhibit the secretion of inflammatory cytokines. We thus investigated the effect of H2S on inflammatory hepcidin induction. RESULTS H2S suppressed lipopolysaccharide (LPS)-induced hepcidin production and regulated iron homeostasis in mice by decreasing serum interleukin-6 (IL-6) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) activation; similar results were obtained in Huh7 cells exposed to conditioned medium from LPS-challenged THP-1 macrophages. Intriguingly, we found H2S also attenuated hepcidin levels in Huh7 cells and mouse primary hepatocytes in a sirtuin 1 (SIRT1)-dependent manner. By promoting SIRT1 expression and stabilizing SIRT1-STAT3 interactions, H2S ameliorated IL-6-induced STAT3 acetylation, resulting in reduced hepcidin production. Inhibition and silencing of SIRT1 diminished H2S-mediated suppression of hepcidin, as opposed to SIRT1 activation and overexpression. Consistent results were observed in vivo. Furthermore, knockout of cystathionine γ-lyase (CSE), an endogenous H2S synthase, exaggerated inflammatory hepcidin expression in mice. INNOVATION For the first time, we elucidated the effects and possible mechanisms of H2S on inflammatory hepcidin and established a novel regulatory link between SIRT1 and hepcidin. CONCLUSION Our work demonstrates that H2S attenuates inflammation-induced hepatic hepcidin via multipathways and suggests new treatment strategies for anemia of inflammation.

Novel Therapeutic Effects of Leonurine On Ischemic Stroke: New Mechanisms of BBB Integrity

Stroke is a leading cause of morbidity and mortality globally. Leonurine (also named SCM-198), a compound extracted from Herba leonuri, was effective on the prevention of various cardiovascular and brain diseases. The purpose of this study was to explore the possible therapeutic potential of SCM-198 against ischemia reperfusion injury and underlying mechanisms. In the in vivo transient middle cerebral artery occlusion (tMCAO) rat model, we found that treatment with SCM-198 could decrease infarct volume and improve neurological deficit by protecting against blood-brain barrier (BBB) breakdown. In the in vitro model of cell oxygen-glucose deprivation and reoxygenation (OGD/R), consistent results were obtained with decreased reactive oxygen species (ROS) production and maintained the BBB integrity. Further study demonstrated that SCM-198 increased the expression of histone deacetylase- (HDAC-) 4 which could inhibit NADPH oxidase- (NOX-) 4 and matrix metalloproteinase- (MMP-) 9 expression, resulting in the elevation of tight junction proteins, including claudin-5, occludin, and zonula occluden- (ZO-) 1. These results indicated SCM-198 protected BBB integrity by regulating the HDAC4/NOX4/MMP-9 tight junction pathway. Our findings provided novel insights into the protective effects and mechanisms of SCM-198 on ischemic stroke, indicating SCM-198 as a new class of potential drug against acute onset of ischemic stroke.

HDAC4 regulates vascular inflammation via activation of autophagy

Aims Angiotensin II (Ang II) causes vascular inflammation, leading to vascular endothelial cell dysfunction, and is associated with the development of cardiovascular diseases. Therefore, interventions in inflammation may contribute to the reduction of cardiovascular diseases. Here, we aim to demonstrate that HDAC4, one of class IIa family histone de-acetylases (HDACs) members, promotes autophagy-dependent vascular inflammation. Methods and results By loss-of-function approaches, our study provides the first evidence that HDAC4 mediates Ang II-induced vascular inflammation in vitro and in vivo. In response to the Ang II, HDAC4 expression is up-regulated rapidly, with increased autophagic flux and inflammatory mediators in vascular endothelial cells (VECs). In turn, HDAC4 deficiency suppresses activation of autophagy, leading to reduced inflammation in Ang II-induced VECs. Consistently, using autophagy inhibitor or silencing LC3-II also alleviates vascular inflammation. Furthermore, HDAC4 regulates autophagy via facilitating transcription factor forkhead box O3a (FoxO3a) de-acetylation, thereby to increase its transcriptional activity. Loss of HDAC4 in VECs results in inhibition of FoxO3a de-acetylation to block its transcriptional activity, leading to downregulation of the downstream FoxO3a target, and hence reduces autophagy and vascular inflammation. FoxO3a silencing using siRNA approach significantly inhibits activation of autophagy. Finally, knockdown of HDAC4 in Ang II-infused mouse models ameliorates vascular inflammation, suggesting that inhibitor of HDAC4 may be potential therapeutics for vascular diseases associated with inflammation. Conclusion These results suggest that HDAC4-mediated FoxO3a acetylation regulates Ang II-induced autophagy activation, which in turn plays an essential role in causing vascular inflammation.

Endogenous hydrogen sulfide regulates histone demethylase JMJD3-mediated inflammatory response in LPS-stimulated macrophages and in a mouse model of LPS-induced septic shock

Graphical abstract Figure. No Caption available. Abstract Overproduction of inflammatory mediators contributes to uncontrolled inflammation during endotoxin shock. Cystathionine‐&ggr;‐lyase (CSE), an enzyme involved in hydrogen sulfide (H2S) biosynthesis, has potential anti‐inflammatory activity in a variety of inflammatory diseases. Jumonji domain‐containing protein 3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase, has been implicated in macrophage activation, but its function in CSE‐mediated anti‐inflammatory activities remains unknown. In the present study CSE was found to be upregulated in macrophages and mouse lipopolysaccharide (LPS) challenge models. LPS stimulation also enhanced the activation of JMJD3 and decreased H3K27me3 levels. JMJD3 knockdown upregulated H3K27me3 levels and attenuated the LPS‐mediated inflammatory response. CSE knockout amplified the inflammatory cascade by increasing JMJD3 expression in septic mice. Similarly, enhanced production of inflammatory mediators by macrophages was mitigated by CSE overexpression via inhibition of JMJD3 expression. This is the first report indicating that inflammation enhanced CSE/H2S system biosynthesis, that in turn attenuated the LPS‐triggered inflammatory response by regulating JMJD3 expression. Thus, the CSE/H2S system represents an epigenetic‐based modification mechanism to prevent uncontrolled inflammation.

ZYZ-168 alleviates cardiac fibrosis after myocardial infarction through inhibition of ERK1/2-dependent ROCK1 activation

Selective treatments for myocardial infarction (MI) induced cardiac fibrosis are lacking. In this study, we focus on the therapeutic potential of a synthetic cardio-protective agent named ZYZ-168 towards MI-induced cardiac fibrosis and try to reveal the underlying mechanism. ZYZ-168 was administered to rats with coronary artery ligation over a period of six weeks. Ecocardiography and Masson staining showed that ZYZ-168 substantially improved cardiac function and reduced interstitial fibrosis. The expression of α–smooth muscle actin (α-SMA) and Collagen I were reduced as was the activity of matrix metalloproteinase 9 (MMP-9). These were related with decreased phosphorylation of ERK1/2 and expression of Rho-associated coiled-coil containing protein kinase 1 (ROCK1). In cardiac fibroblasts stimulated with TGF-β1, phenotypic switches of cardiac fibroblasts to myofibroblasts were observed. Inhibition of ERK1/2 phosphorylation or knockdown of ROCK1 expectedly reduced TGF-β1 induced fibrotic responses. ZYZ-168 appeared to inhibit the fibrotic responses in a concentration dependent manner, in part via a decrease in ROCK 1 expression through inhibition of the phosphorylation status of ERK1/2. For inhibition of ERK1/2 phosphorylation with a specific inhibitor reduced the activation of ROCK1. Considering its anti-apoptosis activity in MI, ZYZ-168 may be a potential drug candidate for treatment of MI-induced cardiac fibrosis.

Histone demethylase JMJD3 regulates fibroblast-like synoviocyte-mediated proliferation and joint destruction in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an immune‐mediated disease with the characteristics of progressive joint destruction, deformity, and disability. Epigenetic changes have been implicated in the development of some autoimmune disorders, resulting in an alteration of gene transcription. Here, we investigated how Jumonji C family of histone demethylases (JMJD3) regulated the proliferation and activation of fibroblast‐like synoviocytes (FLSs), which are involved in RA joint destruction and pathologic process. The JMJD3 expression and proliferation markers in RA‐FLS were higher than those in healthy‐FLS and were upregulated in platelet‐derived growth factor (PDGF)‐induced FLS. Elevated JMJD3 promoted the proliferation and migration of FLS. Treatment with JMJD3 small interfering RNA or inhibitor glycogen synthase kinase (GSK) J4 led to decreased proliferation and migration of FLS. Interestingly, induction of proliferating cell nuclear antigen (PCNA), a major player of the cell‐cycle regulation, was correlated with trimethylated lysine 27 in histone H3 loss around the gene promoters. The knockdown of JMJD3 abolished PCNA expression in PDGF‐induced FLS and further inhibited cell proliferation and migration, sug gesting that JMJD3/PCNA played a crucial role in aspects of FLS proliferation and migration. In vivo, the ability of GSK J4 to hinder collagen‐induced arthritis (CIA) in DBA/1 mice was evaluated. We found that GSK J4 markedly attenuated the severity of arthritis in CIA mice. The therapeutic effects were associated with ameliorated joint swelling and reduced bone erosion and destruction. This study revealed how JMJD3 integrated with epigenetic processes to regulate RA‐FLS proliferation and invasion. These data suggested that JMJD3 might contribute to rheumatoid synovial hyperplasia and have the potential as a novel therapeutic target for RA.—Jia, W., Wu, W., Yang, D., Xiao, C., Su, Z., Huang, Z., Li, Z., Qin, M., Huang, M., Liu, S., Long, F., Mao, J., Liu, X., Zhu, Y. Z. Histone demethylase JMJD3 regulates fibroblast‐like synoviocyte‐mediated proliferation and joint destruction in rheumatoid arthritis. FASEB J. 32, 4031–4042 (2018). www.fasebj.org

Endogenous Hydrogen Sulfide Ameliorates NOX4 Induced Oxidative Stress in LPS-Stimulated Macrophages and Mice

Background/Aims: Sepsis is a severe and complicated syndrome that is characterized by dysregulation of host inflammatory responses and organ failure. Cystathionine-γ-lyase (CSE)/ hydrogen sulfide (H2S) has potential anti-inflammatory activities in a variety of inflammatory diseases. NADPH oxidase 4 (Nox4), a member of the NADPH oxidases, is the major source of reactive oxygen species (ROS) and its expression is increased in sepsis, but its function in CSE-mediated anti-inflammatory activities remains unknown. Methods: Macrophages were either transfected with CSE, Nox4 siRNA or transduced with lentiviral vector encoding CSE or Nox4, and then stimulated with lipopolysaccharide (LPS). The expression of inflammatory mediators and signaling pathway activation were measured by quantitative PCR (qPCR), ELISA, and immunoblotting. LPS-induced shock severity in WT, Nox4 knockdown and CSE knockout (CSE-/-) mice was assessed. Results: Here we showed that CSE and Nox4 were upregulated in macrophage and mouse in response to LPS. After LPS stimulation, the inflammatory responses were significantly ameliorated by lentiviral Nox4 shRNA knockdown, but were exacerbated by lentiviral overexpressing Nox4. Furthermore, Nox4 mediated inflammation through PI3K/Akt and p-p38 mitogen-activated protein kinase signal pathway. Notably, CSE knockout served to amplify the inflammatory cascade by increasing Nox4-ROS signaling activation in septic mice and macrophage. Similarly, the enhanced production of inflammatory mediators by macrophages was reduced by CSE overexpression. Conclusion: Thus, we demonstrated that CSE/H2S attenuated LPS-induced sepsis against oxidative stress and inflammation damage probably largely through mediated Nox4 pathway.

Discovery of Leonuri and therapeutical applications: From bench to bedside

&NA; Despite several advances in percutaneous coronary intervention and the discovery of new drugs, the incidence of myocardial infarction and deaths due to cardiovascular diseases (CVD) has not decreased markedly in China. The quality of life is affected seriously, which further results in great social and family burden. Many drugs, from the century‐old aspirin to the newly FDA‐approved Byvalson, have been proven to be effective in the treatment and prevention of CVD. As clinically reported, those life‐saving drugs still have their side effects in regards to the narrow therapeutic indexes influenced by individual genetic variations. Herba Leonuri, also known as Chinese Motherwort, which are naturally present in plants and traditionally are used for the uterotonic action, postpartum blood stasis, breast pain as well as other gynecological disorders in China for thousands of years. Since the last two decades, our group has reported leonurine, a unique alkaloid found in Herba Leonuri, exhibits various bioactivities such as antioxidant, anti‐apoptotic effects, free radical scavenging and anti‐inflammatory effects, in addition to improving micro‐circulation. These bioactivities are related to the underlying mechanisms of ischemic heart diseases and cardiac fibrosis. Pharmacological studies have proven leonurine to be effective in treating CVD in various ways, particularly ischemic heart diseases. Besides the cardio protective effects, which are similar in the central nervous system, more specifically, inhibited mitochondrial reactive oxygen species production together with the restored mitochondrial function and redox state were observed in middle cerebral artery occlusion rats by leonurine treatment, which strongly reveals its neuroprotective effects and carries a therapeutic potential for recovery and prevention of stroke. Based on their mode of action, we propose that leonurine can be developed as drugs to treat ischemic heart diseases. Taking advantage of the most recent findings in pharmacological research including the effects of low toxicity and good pharmacokinetics characteristics, leonurine has a very attractive prospect of clinical application. Our recent promising pharmacological results may be able to eradicate the barrier hindering its sale on market. In sum, from bench to bedside is no longer a long way for leonurine.

Application of High-Performance Liquid Chromatography Coupled with Linear Ion Trap Quadrupole Orbitrap Mass Spectrometry for Qualitative and Quantitative Assessment of Shejin-Liyan Granule Supplements

A method for high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high-resolution mass spectrometry (HPLC-LTQ-Orbitrap MS) was developed and validated for the qualitative and quantitative assessment of Shejin-liyan Granule. According to the fragmentation mechanism and high-resolution MS data, 54 compounds, including fourteen isoflavones, eleven ligands, eight flavonoids, six physalins, six organic acids, four triterpenoid saponins, two xanthones, two alkaloids, and one licorice coumarin, were identified or tentatively characterized. In addition, ten of the representative compounds (matrine, galuteolin, tectoridin, iridin, arctiin, tectorigenin, glycyrrhizic acid, irigenin, arctigenin, and irisflorentin) were quantified using the validated HPLC-LTQ-Orbitrap MS method. The method validation showed a good linearity with coefficients of determination (r2) above 0.9914 for all analytes. The accuracy of the intra- and inter-day variation of the investigated compounds was 95.0–105.0%, and the precision values were less than 4.89%. The mean recoveries and reproducibilities of each analyte were 95.1–104.8%, with relative standard deviations below 4.91%. The method successfully quantified the ten compounds in Shejin-liyan Granule, and the results show that the method is accurate, sensitive, and reliable.

Cystathionine-γ-lyase ameliorates the histone demethylase JMJD3-mediated autoimmune response in rheumatoid arthritis

Cystathionine-γ-lyase (CSE), an enzyme associated with hydrogen sulfide (H2S) production, is an important endogenous regulator of inflammation. Jumonji domain-containing protein 3 (JMJD3) is implicated in the immune response and inflammation. Here, we investigated the potential contribution of JMJD3 to endogenous CSE-mediated inflammation in rheumatoid arthritis (RA). Upregulated CSE and JMJD3 were identified in synovial fibroblasts (SFs) from RA patients as well as in the joints of arthritic mice. Knocking down CSE augmented inflammation in IL-1β-induced SFs by increasing JMJD3 expression. In addition, CSE−/− mice with collagen-induced arthritis (CIA) developed severe joint inflammation and bone erosion. Conversely, overexpressing CSE inhibited JMJD3 expression by the transcription factor Sp-1 and was accompanied by reduced inflammation in IL-1β-treated SFs. Furthermore, JMJD3 silencing or the administration of the JMJD3 inhibitor GSK-J4 significantly decreased the inflammatory response in IL-1β-treated SFs, mainly by controlling the methylation status of H3K27me3 at the promoter of its target genes. GSK-J4 markedly attenuated the severity of arthritis in CIA mice. In conclusion, suppressing JMJD3 expression by the transcription factor Sp-1 is likely responsible for the ability of CSE to negatively modulate the inflammatory response and reduce the progression of RA.