Tatarinan N inhibits osteoclast differentiation through attenuating NF-κB, MAPKs and Ca2+-dependent signaling.

Osteoclasts are multinucleated cells that originate from hemopoietic stem cells. Targeting over activated osteoclasts is thought to be an effective therapeutic approach to osteoporosis. In a previous study, we reported that Tatarinan O, a lignin-like compound, suppressed RANKL-induced osteoclastogenesis. In this study, we further examined the effects on osteoclast formation of three lignin-like compounds including Tatarinan N (TN), Tatarinan U (TU) and Tatarinan V (TV), all containing a common structure of asarone. We found that only TN suppressed RANKL-induced osteoclast differentiation, bone resorption pit formation and F-acting ring formation. TU and TV did not influence RANKL-induced osteoclastogenesis. We also found that TN dose-dependently inhibited the expression of osteoclastogenesis-associated genes, including TRAP, cathepsin K and MMP-9. Furthermore, we found that TN down-regulated the key transcription factor NFATc1 and c-Fos by preventing the activation of NF-κB and phosphorylation of MAPKs including ERK1/2 and p38 but not JNK. TN attenuated calcineurin expression via suppression of the Btk-PLCγ2 cascade and reduction of intracellular Ca2+, modulating NFATc1 activation. Taking together, our results indicated that TN might have therapeutic potential for osteoporosis.

Tert-butylhydroquinone promotes angiogenesis and improves heart functions in rats after myocardial infarction.

BACKGROUND: Hypertension is an increased risk of heart failure and acute myocardial infarction (MI). Tert-butylhydroquinone (tBHQ), as an antioxidant, shows multiple cardioprotective actions including the reduction in blood pressure. The aim of this study was to investigate whether and how tBHQ improves heart functions in rats. METHODS: The MI model was established in WKY and spontaneously hypertensive rats (SHRs) by ligation of left anterior descending coronary artery. Akt phosphorylation was examined by western blot in human umbilical vein endothelial cells (HUVECs) or in rats. Angiogenesis was assessed by immunohistochemistry and immunofluorescence. Heart function was determined by echocardiography. RESULTS: tBHQ increased Akt phosphorylation, promoted cell proliferations and migrations in HUVECs, which were abolished by Akt inhibitor wortmannin. In SHRs following MI, administration of tBHQ significantly increased Akt phosphorylation, promoted angiogenesis, reduced infarct size, and improved heart functions after 14 postoperative days. Importantly, these in vivo effects of tBHQ were ablated by wortmannin in SHRs. CONCLUSION: tBHQ via Akt activation promotes ischemia-induced angiogenesis and improves heart functions in hypertensive rats. In perspectives, the application of tBHQ should be considered in patients with ischemic diseases such as MI and stroke.

Lovastatin upregulates microRNA-29b to reduce oxidative stress in rats with multiple cardiovascular risk factors.

AIMS: Proteasome-linked oxidative stress is believed to cause endothelial dysfunction, an early event in cardiovascular diseases (CVD). Statin, as HMG-CoA reductase inhibitor, prevents endothelial dysfunction in CVD. However, the molecular mechanism of statin-mediated normalization of endothelial function is not completely elucidated. METHODS AND RESULTS: Lovastatin time/dose-dependently increased miR-29b expression and decreased proteasome activity in cultured human umbilical vein endothelial cells (HUVECs). Anti-miR-29b or overexpression of PA200 abolished lovastatin-induced inhibition of proteasome activity in HUVECs. In contrast, pre-miR-29b or PA200 siRNA mimics these effects of lovastatin on proteasome activity. Lovastatin inhibited oxidative stress induced by multiple oxidants including ox-LDL, H2O2, TNFα, homocysteine thiolactone (HTL), and high glucose (HG), which were reversed by inhibition of miR-29b in HUVECs. Ex vivo analysis indicated that lovastatin normalized the acetylcholine-induced endothelium-dependent relaxation and the redox status in isolated rat aortic arteries exposure to multiple cardiovascular risk factors. In vivo analysis revealed that administration of lovastatin remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats with hyperglycemia, dyslipidemia, and hyperhomocysteinemia, as well as increased miR-29b expressions, reduced PA200 protein levels, and suppression of proteasome activity in aortic tissues. CONCLUSION: Upregulation of miR-29b expression is a common mechanism contributing to endothelial dysfunction induced by multiple cardiovascular risk factors through PA200-dependent proteasome-mediated oxidative stress, which is prevented by lovastatin.

Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo.

BACKGROUND: GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation. METHODS: Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization. RESULTS: We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin. CONCLUSIONS: Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

Activation of activator protein 2 alpha by aspirin alleviates atherosclerotic plaque growth and instability in vivo.

AIMS: Aspirin has been used for the secondary prevention and treatment of cardiovascular disease for several decades. We investigated the roles of transcriptional factor activator protein 2α (AP-2α) in the beneficial effects of aspirin in the growth and vulnerability of atherosclerotic plaque. METHODS AND RESULTS: In mice deficient of apolipoprotein E (Apoe-/-), aspirin (20, 50 mg/kg/day) suppressed the progression of atherosclerosis in aortic roots and increased the plaque stability in carotid atherosclerotic plaques induced by collar-placement. In vivo lentivirus-mediated RNA interference of AP-2α reversed the inhibitory effects of aspirin on atherosclerosis in Apoe-/- mice. Mechanically, aspirin increased AP-2α phosphorylation and its activity, upregulated IkBα mRNA and protein levels, and reduced oxidative stress in cultured vascular smooth muscle cells. Furthermore, deficiency of AP-2α completely abolished aspirin-induced upregulation of IkBα levels and inhibition of oxidative stress in Apoe-/- mice. Clinically, conventional doses of aspirin increased AP-2α phosphorylation and IkBα protein expression in humans subjects. CONCLUSION: Aspirin activates AP-2α to upregulate IkBα gene expression, resulting in attenuations of plaque development and instability in atherosclerosis.

Berberine via suppression of transient receptor potential vanilloid 4 channel improves vascular stiffness in mice.

Berberine, as an alkaloid found in many Chinese herbs, improves vascular functions in patients with cardiovascular diseases. We determined the effects of berberine in hypertension and vascular ageing, and elucidated the underlying mechanisms. In isolated aortas, berberine dose-dependently elicited aortic relaxation. In cultured cells, berberine induced the relaxation of vascular smooth muscle cells (VSMCs). Overexpression of transient receptor potential vanilloid 4 (TRPV4) channel by genetic approaches abolished the berberine-induced reduction in intracellular Ca(2+) concentration in VSMCs and attenuated berberine-elicited vessel dilation in mice aortas. In deoxycorticosterone acetate (DOCA)-induced hypertensive model, treatment of mice with berberine or RN-1734, a pharmacological inhibitor of TRPV4, significantly decreased systemic blood pressure (BP) in control mice or mice infected with an adenovirus vector. However, berberine-induced effects of lowering BP were reversed by overexpressing TRPV4 in mice by infecting with adenovirus. Furthermore, long-term administration of berberine decreased mean BP and pulse BP, increased artery response to vasodilator and reduced vascular collagen content in aged mice deficient in apolipoprotein E (Apoe-KO), but not in Apoe-KO old mice with lentivirus-mediated overexpression of TRPV4 channel. In conclusion, berberine induces direct vasorelaxation to lower BP and reduces vascular stiffness in aged mice through suppression of TRPV4.

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-1ß and IL-6 release in LPS-stimulated mouse peritoneal macrophages. In addition, HJB-1 reduced LPS-induced mRNA expression of TNF-α, IL-1ß, 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.

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 Ca(2+)level ([Ca(2+)](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 [Ca(2+)](i) in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca(2+)-dependent protease calpain. Amiloride (1-10 µmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca(2+)](i). and calpain activity. In the presence of the Ca(2+) 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 [Ca(2+)](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.

[High level expression, purification and cytotoxicity of IL-10(18-57)-PE40].

The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.