Heat shock protein-27 attenuates foam cell formation and atherogenesis by down-regulating scavenger receptor-A expression via NF-κB signaling

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE^?/? mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (? 34%; p < 0.005) and uptake (? 38%, p < 0.05). rHSP27 reduced SR-A mRNA (? 39%, p = 0.02), total protein (? 56%, p = 0.01) and cell surface (? 53%, p < 0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p < 0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE^?/? and ApoE^?/? SR-A^?/? mice fed with a high fat diet were treated for 3 weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE^?/? mice by 39% and 36% (p < 0.05), respectively, but not in ApoE^?/?SR-A^?/? mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.     

Adiponectin knockout accentuates high fat diet-induced obesity and cardiac dysfunction: Role of autophagy

Adiponectin (APN), an adipose-derived adipokine, offers cardioprotective effects although the precise mechanism of action remains unclear. This study was designed to examine the role of APN in high fat diet-induced obesity and cardiac pathology. Adult C57BL/6 wild-type and APN knockout mice were fed a low or high fat diet for 22 weeks. After 40 day feeding, mice were treated with 2 mg/kg rapamycin or vehicle every other day for 42 days on respective fat diet. Cardiomyocyte contractile and Ca^2 + transient properties were evaluated. Myocardial function was evaluated using echocardiography. Dual energy X-ray absorptiometry was used to evaluate adiposity. Energy expenditure, metabolic rate and physical activity were monitored using a metabolic cage. Lipid deposition, serum triglyceride, glucose tolerance, markers of autophagy and fatty acid metabolism including LC3, p62, Beclin-1, AMPK, mTOR, fatty acid synthase (FAS) were evaluated. High fat diet intake induced obesity, systemic glucose intolerance, cardiac hypertrophy, dampened metabolic ability, cardiac and intracellular Ca^2 + derangements, the effects of which were accentuated by APN knockout. Furthermore, APN deficiency augmented high fat diet-induced upregulation in the autophagy adaptor p62 and the decline in AMPK without affecting high fat diet-induced decrease in LC3II and LC3II-to-LC3I ratio. Neither high fat diet nor APN deficiency altered Beclin-1. Interestingly, rapamycin negated high fat diet-induced/APN-deficiency-accentuated obesity, cardiac hypertrophy and contractile dysfunction as well as AMPK dephosphorylation, mTOR phosphorylation and p62 buildup. Our results collectively revealed that APN deficiency may aggravate high fat diet-induced obesity, metabolic derangement, cardiac hypertrophy and contractile dysfunction possibly through decreased myocardial autophagy.     

A semi-synthetic derivative of artemisinin,artesunate inhibits prostaglandin E2 production in LPS/IFNγ-activated BV2 microglia

Artesunate is a semi-synthetic derivative of artemisinin used to treat malaria, and has been shown to possess anti-inflammatory activity. In this study, we have investigated the effect of artesunate on PGE₂ production/COX-2 protein expression in LPS + IFNγ-activated BV2 microglia. To further understand the mechanism of action of this compound, we investigated its interference with NF-κB and p38 MAPK signalling pathways. PGE₂ production was determined using EIA, while protein expressions of inflammatory targets like COX-2, mPGES-1, IκB, p38 and MAPKAPK2 were evaluated using western blot. An NF-κB-bearing luciferase reporter gene assay was used to test the effect of artesunate on NF-κB-mediated pro-inflammatory gene expression in HEK293 cells stimulated with TNFα (1 ng/ml). Artesunate (2 and 4 μM), significantly (p <0.01) suppressed PGE₂ production in LPS + IFNγ-activated BV2 microglia. This effect was found to be mediated via reduction in COX-2 and mPGES-1 proteins. Artesunate also produced significant inhibition of TNFα and IL-6 production in activated BV2 microglia. Further investigations showed that artesunate (0.5–4 μM) significantly (p <0.001) reduced NF-κB-driven luciferase expression, and inhibited IκB phosphorylation and degradation, through inhibition of IKK. Artesunate inhibited phosphorylation of p38 MAPK and its substrate MAPKAPK2 following stimulation of microglia with LPS + IFNγ. Taken together, we have shown that artesunate prevents neuroinflammation in BV2 microglia by interfering with NF-κB and p38 MAPK signalling.     

Astragaloside IV ameliorates renal injury in streptozotocin-induced diabetic rats through inhibiting NF-κB-mediated inflammatory genes expression

Accumulating evidence suggests that inflammatory processes are involved in the development of diabetic nephropathy (DN). However, there are no effective interventions for inflammation in the diabetic kidneys. Here, we tested the hypothesis that Astragaloside IV(AS-IV), a novel saponin purified from Astragalus membranaceus (Fisch) Bge, ameliorates DN in streptozotocin (STZ)-induced diabetic rats through anti-inflammatory mechanisms. Diabetes was induced with STZ (65 mg/kg) by intraperitoneal injection in rats. Two weeks after STZ injection, rats were divided into three groups (n = 8/each group), namely, diabetic rats, diabetic rats treated with AS-IV at 5 and 10 mg kg^?1 d^?1, p.o., for 8 weeks. The normal rats were chosen as nondiabetic control group (n = 8). The rats were sacrificed 10 weeks after induction of diabetes. AS-IV ameliorated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. Renal NF-κB activity, as wells as protein and mRNA expression were increased in diabetic kidneys, accompanied by an increase in mRNA expression and protein content of TNF-α, MCP-1 and ICAM-1 in kidney tissues. The α₁-chain type IV collagen mRNA was elevated in the kidneys of diabetic rats. All of these abnormalities were partially restored by AS-IV. AS-IV also decreased the serum levels of TNF-α, MCP-1 and ICAM-1 in diabetic rats. These findings suggest that AS-IV, a novel anti-inflammatory agent, attenuated DN in rats through inhibiting NF-κB mediated inflammatory genes expression.     

β1-Adrenergic blockers exert antioxidant effects,reduce matrix metalloproteinase activity,and improve renovascular hypertension-induced cardiac hypertrophy

Hypertension induces left-ventricular hypertrophy (LVH) by mechanisms involving oxidative stress and unbalanced cardiac matrix metalloproteinase (MMP) activity. We hypothesized that β₁-adrenergic receptor blockers with antioxidant properties (nebivolol) could reverse hypertension-induced LVH more effectively than conventional β₁-blockers (metoprolol) when used at doses that exert similar antihypertensive effects. Two-kidney one-clip (2K1C) hypertension was induced in male Wistar rats. Six weeks after surgery, hypertensive and sham rats were treated with nebivolol (10 mg kg⁻¹ day⁻¹) or metoprolol (20 mg kg⁻¹ day⁻¹) for 4 weeks. Systolic blood pressure was monitored weekly by tail-cuff plethysmography. LV structural changes and fibrosis were studied in hematoxylin/eosin- and picrosirius-stained sections, respectively. Cardiac MMP levels and activity were determined by in situ zymography, gel zymography, and immunofluorescence. Dihydroethidium and lucigenin-derived chemiluminescence assays were used to assess cardiac reactive oxygen species (ROS) production. Nitrotyrosine levels were determined in LV samples by immunohistochemistry and green fluorescence and were evaluated using the ImageJ software. Cardiac protein kinase B/Akt (AKT) phosphorylation state was assessed by Western blot. Both β-blockers exerted similar antihypertensive effects and attenuated hypertension-induced cardiac remodeling. Both drugs reduced myocyte hypertrophy and collagen deposition in 2K1C rats. These effects were associated with lower cardiac ROS and nitrotyrosine levels and attenuation of hypertension-induced increases in cardiac MMP-2 levels and in situ gelatinolytic activity after treatment with both β-blockers. Whereas hypertension increased AKT phosphorylation, no effects were found with β-blockers. In conclusion, we found evidence that two β₁-blockers with different properties attenuate hypertension-induced LV hypertrophy and cardiac collagen deposition in association with significant cardiac antioxidant effects and MMP-2 downregulation, thus suggesting a critical role for β₁-adrenergic receptors in mediating those effects. Nebivolol is not superior to metoprolol, at least with respect to their capacity to reverse hypertension-induced LVH.     

Lipoic acid effects on established atherosclerosis

AimsAlpha-lipoic acid (LA) is a commonly used dietary supplement that exerts anti-oxidant and anti-inflammatory effects in vivo and in vitro. We investigated the mechanisms by which LA may confer protection in models of established atherosclerosis.Main methodsWatanabe heritable hyperlipidemic (WHHL) rabbits were fed with high cholesterol chow for 6 weeks and then randomized to receive either high cholesterol diet alone or combined with LA (20 mg/kg/day) for 12 weeks. Vascular function was analyzed by myography. The effects of LA on T cell migration to chemokine gradients was assessed by Boyden chamber. NF-κB activation was determined by measuring translocation and electrophoresis migration shift assay (EMSA).Key findingsLA decreased body weight by 15 ± 5% without alterations in lipid parameters. Magnetic Resonance Imaging (MRI) analysis demonstrated that LA reduced atherosclerotic plaques in the abdominal aorta, with morphological analysis revealing reduced lipid and inflammatory cell content. Consistent with its effect on atherosclerosis, LA improved vascular reactivity (decreased constriction to angiotensin II and increased relaxation to acetylcholine and insulin), inhibited NF-κB activation, and decreased oxidative stress and expression of key adhesion molecules in the vasculature. LA reduced T cell content in atherosclerotic plaque in conjunction with decreasing ICAM and CD62L (l-selectin) expression. These effects were confirmed by demonstration of a direct effect of LA in reducing T cell migration in response to CCL5 and SDF-1 and decreasing T cell adhesion to the endothelium by intra-vital microscopy.SignificanceThe present findings offer a mechanistic insight into the therapeutic effects of LA on atherosclerosis.     

Rho-kinase inhibition reverses impaired Ca2+ handling and associated left ventricular dysfunction in pressure overload-induced cardiac hypertrophy

Recent studies have implicated a relationship between RhoA/ROCK activity and defective Ca²⁺ homeostasis in hypertrophic hearts. This study investigated molecular mechanism underlying ROCK inhibition-mediated cardioprotection against pressure overload-induced cardiac hypertrophy, with a focus on Ca²⁺ homeostasis.Cardiac hypertrophy model was established by performing transverse aortic constriction (TAC) in 8-week-old male rats. Groups were assigned as SHAM, TAC and TAC + Fas (rats undergoing TAC and treated with fasudil). Rats in the TAC + Fas group were administered fasudil (5 mg/kg/day), and rats in the SHAM and TAC groups were treated with vehicle for 10 weeks. Electrophysiological recordings were obtained from isolated left ventricular myocytes and expression levels of proteins were determined using western blotting. Rats in the TAC group showed remarkable cardiac hypertrophy, and fasudil treatment significantly reversed this alteration. TAC + Fas myocytes showed significant improvement in reduced contractility and Ca²⁺ transients. Moreover, these myocytes showed restoration of slow relaxation rate and Ca²⁺ reuptake. Although L-type Ca²⁺ currents did not change in TAC group, there was a significant reduction in the triggered Ca²⁺ transients which was reversed either by long-term fasudil treatment or incubation of TAC myocytes with fasudil. The hearts of rats in the TAC group showed a significant decrease in ROCK1, ROCK2, RyR2 protein levels and p-PLB^S16/T17/SERCA2 ratio and increase in RhoA expression and MLC phosphorylation. However, fasudil treatment largely reversed TAC-induced alterations in protein expression.Thus, our findings indicate that upregulation of the RhoA/ROCK pathway is significantly associated with cardiac hypertrophy-related Ca²⁺ dysregulation and suggest that ROCK inhibition prevents hypertrophic heart failure.     

A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a pharmaceutical preparation of Salvia miltiorrhiza, on cardiac myocytes and fibroblasts with respect to the viability, proliferation, and collagen synthesis in these cells under various conditions. A cardiac myocyte line, H9c2, and primarily cultured fibroblasts from rat hearts were incubated with CP over a broad concentration range (50–800 μg/ml) under normal cultures, conditions of ischemia (serum-free culture), and stimulation by angiotensin II (AII, 100 nM), hydrogen peroxide (H₂O₂, 50–200 μM), or tumor necrosis factor α (TNFα, 40 ng/ml) for 24–48 h. Cell growth, apoptosis, DNA and collagen synthesis, and expression of relevant genes were assessed via cell number study, morphological examination, Annexin-V staining, flow-cytometry, [³H]-thymidine or [³H]-proline incorporation assay, and Western blotting analysis. It was found that (1) at therapeutic (50 μg/ml) and double therapeutic (100 μg/ml) concentrations, CP did not significantly affect normal DNA synthesis and cell growth in these cardiac cells, while at higher (over 4-fold therapeutic) concentrations (200–800 μg/ml), CP decreased DNA synthesis and cell growth and increased cell death; (2) CP treatment (50 μg/ml) significantly inhibited TNFα-induced apoptosis in myocytes, with 12.3±1.46% cells being apoptosis in CP treatment group and 37.0±7.34% in the control (p<0.01), and simultaneously, expression of activated (phosphorylated) Akt protein was increased by about 2 folds in the CP-treated cells; and (3) in cultured fibroblasts, CP significantly reduced AII-induced collagen synthesis in a concentration-dependent manner (by ～50% and ～90% reduction of AII-induced collagen synthesis at 50 and 100 μg/ml, respectively). Thus, Salvia miltiorrhiza preparation CP is physiologically active on cardiac cells. The actions by CP to reduce apoptotic damage in myocytes and collagen synthesis in fibroblasts may help to preserve the heart function and reduce heart failure risk. The actions by CP to inhibit DNA synthesis and cell growth, which occurred at over therapeutic doses, may weaken the ability of heart repair. Further studies are needed to identify the chemical compounds in this herbal product that are responsible for these observed physiological effects.     

Expression of IL-32 modulates NF-κB and p38 MAP kinase pathways in human esophageal cancer

BackgroundEsophageal cancer is the seventh leading cause of cancer death in males in USA, and there is a strong link has been demonstrated between inflammation and esophageal cancer, interleukin (IL)-32 is a recently described pro-inflammatory cytokine characterized by the induction of nuclear factor NF-κB activation, the p38MAPK also plays an important role in key cellular processes related to inflammation and cancer. We investigated whether the IL-32 expression may be involved in esophageal carcinogenesis through modulates the activity of NF-κB and p-p38 MAPK.MethodMalignant esophageal tissue and blood samples were obtained from 65 operated untreated patients, normal samples was obtained from 35 patients operated for other reasons as control. IL-32 expression visualized by immunohistochemistry, Real time RT–PCR for IL-32 mRNA expression, NF-κB phosphorylation and phosphorylated p38mapk were analyzed by immunoblotting, ELISA for further detection IL-32 and cytokines (TNF-α, IL-1β, IL-6 and IL-8) concentration in the patient’s sera.ResultsIL-32 expression was increased in immunohistochemical staining for malignant esophageal tissue and it’s correlated with the relative expression level of IL-32 mRNA P = 0.007, the P-NF-κB level elevated in tumor tissue compared with control and no difference in the total NF-κB level P = 0.003 while the IL-32 up-regulated the P-pNF-κB in the esophageal tumor P = 0.005. There is increase in p-p38MAPK activation underlying IL-32 expression in tumor P = 0.004, but no change in total p38 MAPK in malignant esophagus. The plasma level of IL-32 expression was increased in malignant esophageal patients P = 0.01, with increased in the levels of the cytokines TNF-α, IL-6, and IL-1β P<0.05.ConclusionsUnderstanding the pathway of IL-32 expression to stimulate the secretion cytokines via the activation of NF-κB and up-regulation of p-p38MAPK may or may not prove to be a therapeutic target, or a biomarker, and future studies will finally answer this hypothesis generated.     

Effects of praeruptorin C on blood pressure and expression of phospholamban in spontaneously hypertensive rats

BackgroundThe traditional Chinese medicine Praeruptorin c (Pra-c) has many physiological and pharmacological effects, including antagonistic effects on blood pressure and calcium levels, maintenance of cellular calcium homeostasis, and improved cardiac systolic and diastolic function. It is potentially a novel and versatile drug for the treatment and prevention of cardiovascular diseases.ObjectiveTo explore the possible impact of Pra-c on blood pressure in SHR and its mechanism of action.Materials and methodsTwenty SHR were randomly divided into a Pra-c group [Pra-c was administered intragastrically, 20 mg kg⁻¹ d⁻¹, n = 10] or an untreated control group (n = 10), containing 10 age-matched SD rats. Each group of rats was followed for 8 weeks. Before and during the treatment, tail artery systolic blood pressure was measured using a tail-cuff every 2 weeks. After 8 weeks, the rats were sacrificed and RNA was extracted from homogenates of cardiac tissue. Tissue from the left ventricle was fixed, sectioned and H&E stained to assess possible changes in myocardial cell structure and morphology. Semi-quantitative RT-PCR was used to assess changes in phospholamban gene expression in treated and untreated rats.ResultsSHR treated with Pra-c for 8 weeks had a lower systolic pressure than untreated SHR (p < 0.05), two measures of cardiac damage, the heart mass index and left ventricle mass index (HMI and LVMI, respectively) were improved, and the level of PLB mRNA expression was lower in the untreated SHR group (p < 0.05).Discussion and conclusionWith continuous hypertension, SHR gradually formed or developed cardiac hypertrophy and fibrosis. Pra-c had a clear effect on blood pressure in SHR, and reversed SHR ventricular remodeling by upregulating the gene expression of sarcoplasmic reticulum PLB.     

Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

HDAC inhibitor-induced activation of NF-κB prevents apoptotic response of E1A + Ras-transformed cells to proapoptotic stimuli

HDAC inhibitors (HDACIs) are capable of suppressing the cell growth of tumour cells due to the induction of apoptosis and/or cell cycle arrest. This allows of considering HDACIs as promising agents for tumour therapy. The final outcome – apoptotic cell death or cell cycle arrest – depends on the type of tumour and cellular context. In this report, we addressed the issue by analysing effects produced in E1A + Ras-transformed MEF cells by HDAC inhibitors sodium butyrate (NaB), Trichostatin A (TSA) and some others. It has been shown that the HDACIs induced cell cycle arrest in E1A + Ras-transformed cells but not apoptosis. The antiapoptotic effect of HDACIs is likely to be a result of NF-κB-dependent signaling pathway activation. HDACI-induced activation of NF-κB takes place in spite of a deregulated PI3K/Akt pathway in E1A + Ras cells, suggesting an alternative mechanism for the activation of NF-κB based on acetylation. HDACI-dependent activation of NF-κB prevents the induction of apoptosis by cytostatic agent adriamycin and serum deprivation. Accordingly, suppression of NF-κB activity in HDACI-arrested cells by the chemical inhibitor CAPE or RelA-siRNA resulted in the induction of an apoptotic programme. Thus, our findings suggest that the activation of the NF-κB pathway in HDACI-treated E1A + Ras-transformed cells blocks apoptosis and may thereby play a role in triggering the programme of cell cycle arrest and cellular senescence.     

A monoterpene,unique component of thyme honeys,induces apoptosis in prostate cancer cells via inhibition of NF-κB activity and IL-6 secretion

We have previously demonstrated that Greek thyme honey inhibits significantly the cell viability of human prostate cancer cells. Herein, 15 thyme honey samples from several regions of Greece were submitted to phytochemical analysis for the isolation, identification and determination (through modern spectral means) of the unique thyme honey monoterpene, the compound trihydroxy ketone E-4-(1,2,4-trihydroxy-2,6,6-trimethylcyclohexyl)-but-3-en-2-one.We investigated the anti-growth and apoptotic effects of the trihydroxy ketone on PC-3 human androgen independent prostate cancer cells using MTT assay and Annexin V-FITC respectively. The molecular pathways involved to such effects were further examined by evaluating its ability to inhibit (a) the NF-κB phosphorylation (S536), (b) JNK and Akt phosphorylation (Thr183/Tyr185 and S473 respectively) and (c) IL-6 production, using ELISA method. The anti-microbial effects of the trihydroxy ketone against a panel of nine pathogenic bacteria and three fungi were also assessed.The trihydroxy ketone exerted significant apoptotic activity in PC-3 prostate cancer cells at 100 μM, while it inhibited NF-κB phosphorylation and IL-6 secretion at a concentration range 10⁻⁶–10⁻⁴ M. Akt and JNK signaling were not found to participate in this process. The trihydroxy ketone exerted significant anti-microbial profile against many human pathogenic bacteria and fungi (MIC values ranged from 0.04 to 0.57 mg/ml). Conclusively, the Greek thyme honey-derived monoterpene exerted significant apoptotic activity in PC-3 cells, mediated, at least in part, through reduction of NF-κB activity and IL-6 secretion and may play a key role in the anti-growth effect of thyme honey on prostate cancer cells.     

Inhibition of myeloid differentiation factor-2 attenuates obesity-induced cardiomyopathy and fibrosis

Obesity causes cardiovascular diseases, including cardiac hypertrophy and remodeling, via chronic tissue inflammation. Myeloid differentiation factor-2 (MD2), a binding protein of lipopolysaccharide, is functionally essential for the activation of proinflammatory pathways in endotoxin-induced acute inflammatory diseases. Here we tested the hypothesis that MD2 plays a central role in obesity-induced cardiomyopathy. Wildtype or MD2 knockout mice were fed with a high fat diet (HFD) or normal diet (Control) for total 16 weeks, and MD2 inhibitor L6H21 (20 mg/kg) or vehicle (1% CMC-Na) were administered from the beginning of the 9th week. HFD induced significant weight gain and cardiac hypertrophy, with increased cardiac fibrosis and inflammation. L6H21 administration or MD2 knockout attenuated HFD-induced obesity, inflammation and cardiac remodeling. In vitro exposure of H9C2 cells to high lipids induced cell hypertrophy with activated JNK/ERK and NF-κB pathways, which was abolished by pretreatment of MD2 inhibitor L6H21. Our results demonstrate that MD2 is essential to obesity-related cardiac hypertrophy through activating JNK/ERK and NF-κB-dependent cardiac inflammatory pathways. Targeting MD2 would be a therapeutic approach to prevent obesity-induced cardiac injury and remodeling.     

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca^2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca^2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca^2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca^2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.     

Quantification of theobromine and caffeine in saliva,plasma and urine via liquid chromatography–tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies

Targeted analyses of clinically relevant metabolites in human biofluids often require extensive sample preparation (e.g., desalting, protein removal and/or preconcentration) prior to quantitation. In this report, a single ultra-centrifugation based sample pretreatment combined with a designed liquid chromatography–tandem mass spectrometry (LC–MS/MS) protocol provides selective quantification of 3,7-dimethylxanthine (theobromine) and 1,3,7-trimethylxanthine (caffeine) in human saliva, plasma and urine samples. The optimized chromatography permitted elution of both analytes within 1.3 min of the applied gradient. Positive-mode electrospray ionization and a triple quadruple MS/MS instrument operated in multiple reaction mode were used for detection. ¹³C₃ isotopically labeled caffeine was included as an internal standard to improve accuracy and precision. Implementing a 20-fold dilution of the isolated low MW biofluid fraction prior to injection effectively minimized the deleterious contributions of all three matrices to quantitation. The assay was linear over a 160-fold concentration range from 2.5 to 400 μmol L^?1 for both theobromine (average R² 0.9968) and caffeine (average R² 0.9997) respectively. Analyte peak area variations for 2.5 μmol L^?1 caffeine and theobromine in saliva, plasma and urine ranged from 5 and 10% (intra-day, N = 10) to 9 and 13% (inter-day, N = 25) respectively. The intra- and inter-day precision of theobromine and caffeine elution times were 3 and <1% for all biofluids and concentrations tested. Recoveries for caffeine and theobromine ranged from 114 to 118% and 99 to 105% at concentration levels of 10 and 300 μmol L^?1. This validated protocol also permitted the relative saliva, plasma and urine distribution of both theobromine and caffeine to be quantified following a cocoa intervention.     

Synthesis and characterization of norbelladine,a precursor of Amaryllidaceae alkaloid,as an anti-inflammatory/anti-COX compound

Rising ROS and systemic inflammation is often a serious concern in many disease conditions including obesity. Therefore, compounds with both anti-oxidant and anti-inflammatory activities are considered beneficial in preventing/treating several human chronic diseases. Norbelladine is an amine compound, a precursor for Amaryllidaceae alkaloids (e.g., belladine, crinamine, lycorine, and galanthamine) found in plants traditionally used for treating a variety of human diseases. However, little information is available about its potential health effects. Therefore, the amine was first synthesized, and its anti-oxidant and anti-inflammatory activities were investigated in this study. Also, the potential effects of the amine on NF-κB activation were investigated due to the critical involvement of ROS in the activation. Norbelladine was synthesized with more than 60% yield, analyzed by a HPLC method, and verified using NMR spectroscopic method. Then, its radical scavenging activity was investigated using DPPH- and superoxide radical assays. At the concentration of 10 μM, norbelladine was a compound able to quench DPPH-radical by 31% (P < 0.05) and reduce superoxide radicals from xanthine oxidase by 33% (P < 0.05). At the concentration of 0.25 μM, the amine also inhibited both COX-1 and COX-2 enzymes by 51% and 25% (P < 0.05), respectively. Furthermore, at the concentration of 10 μM, norbelladine inhibited NF-κB activation by 23% (P < 0.05). In summary, the data suggests that norbelladine may be a compound to quench radicals, inhibit COX enzymes as well as suppress NF-κB activation at relatively lower concentrations.