Tannic acid alleviates lipopolysaccharide­induced H9C2 cell apoptosis by suppressing reactive oxygen species­mediated endoplasmic reticulum stress.

Sepsis­induced myocardial dysfunction is one of the features of multiple organ dysfunction in sepsis, which is associated with extremely high mortality and is characterized by impaired myocardial compliance. To date, there are few effective treatment options available to cure sepsis. Tannic acid (TA) is reportedly protective during sepsis; however, the underlying mechanisms by which TA protects against septic heart injury remain elusive. The present study investigated the potential effects and underlying mechanisms of TA in alleviating lipopolysaccharide (LPS)­induced H9C2 cardiomyocyte cell apoptosis. H9C2 cells were treated with LPS (15 µg/ml), TA (10 µM) and TA + LPS; control cells were treated with medium only. Apoptosis was measured using flow cytometry, reverse transcription­quantitative PCR (RT­qPCR) and western blot analysis. Additionally, the levels of cellular reactive oxygen species (ROS), malondialdehyde and nicotinamide adenine dinucleotide phosphate were evaluated. Western blotting and RT­qPCR were also employed to detect the expression levels of endoplasmic reticulum (ER) stress­associated functional proteins. The present findings demonstrated that TA reduced the degree of LPS­induced H9C2 cell injury, including inhibition of ROS production and ER stress (ERS)­associated apoptosis. ERS­associated functional proteins, including activating transcription factor 6, protein kinase­like ER kinase, inositol­requiring enzyme 1, spliced X box­binding protein 1 and C/EBP­homologous protein were suppressed in response to TA treatment. Furthermore, the expression levels of ERS­associated apoptotic proteins, including c­Jun N­terminal kinase, Bax, cytochrome c, caspase­3, caspase­12 and caspase­9 were reduced following treatment with TA. Additionally, the protective effects of TA on LPS­induced H9C2 cells were partially inhibited following treatment with the ROS inhibitor N­acetylcysteine, which demonstrated that ROS mediated ERS­associated apoptosis and TA was able to decrease ROS­mediated ERS­associated apoptosis. Collectively, the present findings demonstrated that the protective effects of TA against LPS­induced H9C2 cell apoptosis may be associated with the amelioration of ROS­mediated ERS. These findings may assist the development of potential novel therapeutic methods to inhibit the progression of myocardial cell injury.

Oxidized low-density lipoprotein induces inflammatory responses in cultured human mast cells via Toll-like receptor 4.

BACKGROUND/AIMS: Oxidized low-density lipoprotein (ox-LDL) is a powerful atherogen. Toll-like receptor 4 (TLR4) has a pathophysiological role in regulating inflammatory responses and atherosclerosis. Mast cells can infiltrate into the atheromatous plaque and secrete various pro-inflammatory cytokines, which significantly amplify the atherogenic processes and promote plaque vulnerability. Small interfering RNA (siRNA) is an effective method to silence the target genes. We evaluated whether ox-LDL-induced inflammation depended in part on the activation of TLR4-dependent signaling pathways in a cultured human mast cell line (HMC-1). METHOD: HMC-1 cells were cultured, and treated with ox-LDL, TLR4-specific siRNA, or inhibitors of phosphorylation of mitogen-activated protein kinase (MAPKs), and nuclear factor-κB (NF-κB), a critical mediator of inflammation. The expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) was measured subsequently. RESULTS: Ox-LDL increased the expression of TLR4 and secretion of MCP-1, TNF-α and IL-6. Moreover, ox-LDL stimulated the translocation of NF-κB, from the cytoplasm to nucleus. Additionally, phosphorylation of MAPK was greatly increased. These ox-LDL-induced alterations were significantly attenuated by pretreatment with TLR4-specific siRNA. CONCLUSION: Ox-LDL induced inflammatory responses in cultured HMC-1 cells including NF-κB nuclear translocation and phosphorylation of MAPKs, a process mediated in part by TLR4.

Selenium attenuates adriamycin-induced cardiac dysfunction via restoring expression of ATP-sensitive potassium channels in rats.

The possible mechanism of adriamycin (ADR) and/or selenium (Se) deficiency-induced cardiac dysfunction, and cardioprotective effects of Se against ADR-induced cardiac toxicity were investigated in this study. Cardiac function was evaluated by plasma brain natriuretic peptide level and echocardiographic and hemodynamic parameters. Cardiac glutathione peroxidase (GPx) activity was assessed spectrophotometrically. Expression of ATP-sensitive potassium channels (KATP) subunits-SUR2A and Kir6.2-were examined by real-time PCR and Western blotting. The results showed that cardiac function and cardiac GPx activity decreased remarkably after administration of ADR or Se deficiency; more dramatic impairment of cardiac function and cardiac GPx activity were observed after co-administration of ADR and Se deficiency. Mechanically, it is novel for us to find down-regulation of KATP subunits gene expression in cardiac tissue after administration of ADR or Se deficiency, and more significant inhibition of cardiac KATP gene expression was identified after co-administration of ADR and Se deficiency. Furthermore, cardiac toxicity of ADR was found alleviated by Se supplementation, accompanied by restoring of cardiac GPx activity and cardiac KATP gene expression. These results indicate that decreased expression of cardiac KATP is involved in adriamycin and/or Se deficiency-induced cardiac dysfunction; Se deficiency exacerbates adriamycin-induced cardiac dysfunction by future inhibition of KATP expression; Se supplementation seems to protect against adriamycin-induced cardiac dysfunction via restoring KATP expression, showing potential clinical application in cancer chemotherapy.

Effect of peroxisome proliferator activated receptor γ agonist on angiotensin converting enzyme 2 mRNA expression in monocyte-derived macrophages of essential hypertensive patients.

OBJECTIVE: To study the effect of peroxisome proliferator activated receptor γ (PPAR-γ) agonist on the angiotensin converting enzyme 2 (ACE2) mRNA expression in monocyte-derived macrophages of essential hypertensive patients. METHODS: Totally 57 essential hypertensive patients were randomly divided into three groups: conventional treatment group (n=18), telmisartan group (n=19), and benazepril group (n=20); 20 patients with normal blood pressure were also selected as the control group. Monocyte-derived macrophages were isolated from blood samples of patients in all four groups. The expression of ACE2 mRNA in monocyte-derived macrophages was detected by RT-PCR before treatment and 4 and 12 weeks after treatment. RESULTS: Four and 12 weeks after treatment, the systolic pressure and diastolic pressure of telmisartan group and benazepril group were significantly lower than that of the conventional treatment group (all P<0.01), and the systolic pressure and diastolic pressure of telmisartan group were significantly lower than that of the benazepril group(both P<0.01) .The expression of ACE2 mRNA in monocyte-derived macrophages were significantly lower in essential hypertensive patients than that in control group (P<0.01). After having been treated for 4 weeks and 12 weeks, the expression of ACE2 mRNA in monocyte-derived macrophages of hypertensive patients in telmisartan and benazepril groups were significantly higher than that in conventional treatment group (all P<0.01), and the expression of ACE2 mRNA in telmisartan group was significantly higher than that in benazepril group (both P<0.01). CONCLUSION: PPAR-γ agonist could increase the ACE2 mRNA expression in monocyte-derived macrophages of essential hypertensive patients.

[Effects of alpha-linolenic acid on inflammation and oxidative stress in the diabetic rats].

OBJECTIVE: To investigate the effects of alpha-linolenic acid (ALA) on inflammation and oxidative stress in the diabetic rats. METHODS: An experimental type 2 diabetes mellitus model was induced by feeding male SD rats with diet of high fat for 4 weeks and then injected them intraperitoneally with streptozocin (STZ) at 30 mg/kg. Then the animals were randomly divided into three groups (n = 10): control group, diabetic group and ALA group. Four weeks later, tumor necrosis factor (TNF)-a, soluble P-selectin (sP-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), nitric oxide (NO) production, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the serum were determined. RESULTS: Inflammatory agents including TNF-alpha, sP-selectin and sICAM-1 increased in diabetic rats to compare with control group. Treatment with ALA significantly decreased TNF-alpha, sP-selectin and slCAM-1 to compare with diabetic group. Furthermore, compared with control group, serum MDA production increased whereas NO production, SOD and CAT activities decreased in diabetic rats. Treatment with ALA reduced MDA production, increased NO production, promoted SOD and CAT activities compared with diabetic group. CONCLUSION: These results indicate that diet rich in ALA exerted the anti-inflammatory and anti-oxidative effects in diabetic rats, which may be beneficial to the prevention and treatment of diabetes.

[Expression and purification of truncated fragment of extracellular segment of sodium pump alpha3 subunit in Escherichia coli by in-fusion technology].

OBJECTIVE: To construct prokaryotic expression system for expressing, purifying and identifying truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology. METHODS: According to the conservative sequence of M1-M2 and M3-M4 extra-cellular gene fragments of sodium pump a3 subunit, which published in GenBank, a serial of primers and gene fragments was designed, and directly synthesized to fuse the above two gene fragments. The fusion gene was fused with gene-specific primers by PCR, and then fusion gene fragment was fused into the single stranded homology regions of vector pGEX-6P-1 by in-fusion cloning to construct recombinant vector pGEX-Trf-alpha3 (Truncated fragment of extracellular segment of sodium pump alpha3 subunit, Trf-alpha3). After DH10bac was transferred with it, the pGEX-Trf-alpha3 plasmid was purified and identified by PCR and sequenced. Then the recombinant plasmid pGEX-Trf-alpha3 was expressed in E. coli BL21 cells, inducted by IPTG. GST-Trf-alpha3 fusion protein was purified with Glutathione Sepharose 4B purifying system and analyzed by SDS-PAGE. RESULTS: The results of PCR and sequencing demonstrated that the M1-M2 and M3-M4 extra-cellular gene was inserted in plasmid pGEX-6P-1 vector successfully. And the sequence was correct. Protein sequence analysis showed that the GST-Trf-alpha3 fusion protein was consisted of 262 amino-acid residues. Relative molecular mass in theory was 33.22 X 10(3). The amount of recombinant protein was 10% of the total bacteria protein. The soluble fusion protein was about 80.8%. After affinity purification, the purity of GST-Trf-alpha3 fusion protein was over 95%. There was some extent binding activity between GST-Trf-alpha3 fusion protein and ouabain, but the activity was very low. CONCLUSION: Prokaryotic expression system for expressing truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology had been constructed. The purified method had also established. High purified GST-Trf-alpha3 fusion protein was obtained. These have found the foundation of further study on its biological function and potential pharmacology function.

[Binding activity of polypeptide containing human Na+, K+-ATPase alpha1 subunit M1-M2 extracellular segment].

OBJECTIVE: To assess the binding activity of polypeptide containing human Na+, K+-ATPase alpha1 subunit M1-M2 extracellular segment (HES1 derivative). METHODS: HES1 derivative was synthesized by Fmoc method and purified by high-performance liquid chromatography-mass spectrometry, and its binding activity was identified by radioligand binding assay. RESULTS: 3H-ouabain and synthetic HES1 derivative showed some binding activity with the equilibrium dissociation constant (KD) of 24.58 nmol/L, with the the receptor density of 492.43 fmol x mg(-1) pro. and IC50 of 3.078 x 10(-7) mol/L. CONCLUSION: HES1 derivative can bind to ouabain and has the potential of becoming an effective therapeutic agent.

Involvement of ERK and AKT signaling in the growth effect of arginine vasopressin on adult rat cardiac fibroblast and the modulation by simvastatin.

Arginine vasopressin (AVP) has been shown to directly induce neonatal rat cardiac fibroblasts (CFs) proliferation, a major component involved in cardiac hypertrophy. Herein, we explored whether AVP is also a growth factor for adult rat CFs and, if so, whether the growth effect could be inhibited by simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. AVP significantly increased DNA synthesis in adult rat CFs by 73.5 +/- 5.1% (P < or = 0.05), an effect inhibited by V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin. AVP also activated extracellular signal-regulated kinase 1/2 (ERK1/2) as assessed by MBP phosphotransferase activity (5.1 +/- 0.6 fold over basal level, P < or = 0.05) and Western blot analysis, and effects were mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but abolished by inhibiting cellular PKC through chronic PMA incubation. In addition, AVP induced PKC activation (27.2 +/- 3.8% from a basal value of 9.3 +/- 0.7%, P < or = 0.05). AVP-induced increase in DNA synthesis could be attenuated by the specific inhibitors of ERK1/2 (PD98059), PI3K (LY294002), and AKT (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, HIMO). Simvastatin inhibited the effects of AVP on DNA synthesis, ERK1/2, and PKC activation in a dose-dependent manner. Phosphatidylinositol-3-kinase (PI3K)-dependent AKT activation induced by AVP was also inhibited by simvastatin. The effects of simvastatin on ERK1/2, PKC, and AKT activation and DNA synthesis could be reversed by mevalonate. These results support a growth-inducing effect of AVP on adult rat CFs through ERK and AKT signalings and the growth effect could be attenuated by simvastatin via inhibiting these two pathways.

[Inhibitory effects of rosiglitazone against endothelin-1-induced proliferation of rat cardiac myocytes: the role of PKC-c-fos pathway].

OBJECTIVE: To investigate the mechanism of rosiglitazone (RSG, the activator of peroxisome proliferators activated receptor lambda) for inhibiting endothelin-1 (ET-1)-induced neonatal rat cardiac myocyte hypertrophy and the role of protein kinase C (PKC) and c-fos. METHODS: In vitro cultured neonatal rat cardiac myocytes were treated with ET-1, phorbol ester (PMA, the PKC activator), ET-1+RSG, ET-1+chelerythrine (che, the PKC inhibitor), PMA+RSG, or without treatment (control), respectively. The effects of RSG on the protein content, (3)H-leucine incorporation, PKC activity and C-fos protein expression were observed in the cardiac myocytes stimulated with ET-1 or PMA. RESULTS: After two days of culture, the intracellular protein content in ET-1 group and PMA group were increased by 15% (339-/+15 microg/ml) and 13% (329-/+14 microg/ml) as compared with the control cells (290-/+13 microg/ml), respectively (P<0.01). Compared with the ET-1 group, cells treated with ET-1+10(-8) mol/L RSG, ET-1+10(-7) mol/L RSG, and ET-1+che showed decreased intracellular protein content by 10% (303-/+14 microg/ml, P<0.05), 12% (292-/+11 microg/ml, P<0.05), and 13% (291-/+12 microg/ml, P<0.01), respectively. The intracellular protein content in PMA+10(-7) mol/LRSG group was decreased by 10% (P<0.05) in comparison with the PMA group. RSG inhibited protein synthesis enhancement and increased (3)H-leucine incorporation induced by ET-1 and PMA, and antagonized the effects of ET-1 and PMA in promoting PKC activity and c-fos protein expression in the myocytes. CONCLUSION: The inhibitory effect of RSG on ET-1- or PMA-induced myocyte hypertrophy is associated with PKC-c-fos pathway.

[Activity identification of peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment in vitro].

In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.