Intercellular Adhesion Molecule-1 Protein Expression Research Articles

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

The Alterations and Significance of Intercellular Adhesion Molecule-1 in Mouse Brainstem During Herpes Simplex Virus Type 1-Induced Facial Palsy.

Herpes simplex virus type 1 (HSV-1) results in the development of Bell's pals but still, the pathophysiology of the facial nerve paralysis is still not fully studied. The main objective is to establish an animal model of type 1 herpes simplex virus (HSV-1)-induced face paralysis in the mouse and to investigate the pattern of changes in intercellular adhesion molecule -1(ICAM-1) expression in the facial nucleus of the brain stem in mice with facial paralysis as well as the effects of glucocorticoids on intercellular adhesion molecule -1(ICAM-1) expression. A total of 170 4-week-old Balb/c male mice were randomly divided into the virus inoculation group (n = 135), saline control group (n = 26), and blank control group (n = 9). Mice in the virus inoculation group that showed facial paralysis were divided into A, B, and C subgroups. The A group did not receive any treatments, the B group received methylprednisolone sodium succinate (MPSS) intervention, and the C group received MPSS + RU486 treatment. The mouse model of facial paralysis was established by inoculating HSV-1 to the skin at the back of the ears. The facial nerve function of mice was assessed, and real-time PCR and western blot were used to assess ICAM-1 expression in the facial nucleus of the brain stem in mice with facial paralysis after drug intervention. In the virus inoculation group, 95 mice (55.88%) showed varying degrees of facial paralysis symptoms within 2-5days after inoculation. The ICAM-1 gene and protein expression levels remained at low levels in the facial nucleus of the brain stem of mice in the saline group, which showed no significant difference compared to the normal control group (P > 0.05). However, for mice of the virus inoculation group, ICAM-1 expression increased at 6h after the occurrence of facial paralysis and peaked after 2days, differing significantly from the blank control group (P < 0.01). ICAM-1 expression subsequently decreased gradually. In the HSV-1 + MPSS group, ICAM-1 protein expression decreased significantly on the 2nd day after facial paralysis. In the HSV-1 + MPSS + RU486 group, MPSS inhibition of ICAM-1 protein expression was reduced. The results suggested that ICAM-1 is involved in the pathological processes by which HSV-1 induces facial paralysis in mice, and the treatment effects of MPSS for Bell's palsy can be achieved by the inhibition of MCP-1.

Thymosin beta-4 improves endothelial function and reparative potency of diabetic endothelial cells differentiated from patient induced pluripotent stem cells

BackgroundPrior studies show that signature phenotypes of diabetic human induced pluripotent stem cells derived endothelial cells (dia-hiPSC-ECs) are disrupted glycine homeostasis, increased senescence, impaired mitochondrial function and angiogenic potential as compared with healthy hiPSC-ECs. In the current study, we aimed to assess the role of thymosin β-4 (Tb-4) on endothelial function using dia-hiPSC-ECs as disease model of endothelial dysfunction.Methods and resultsUsing dia-hiPSC-ECs as models of endothelial dysfunction, we determined the effect of Tb-4 on cell proliferation, senescence, cyto-protection, protein expression of intercellular adhesion molecule-1 (ICAM-1), secretion of endothelin-1 and MMP-1, mitochondrial membrane potential, and cyto-protection in vitro and angiogenic potential for treatment of ischemic limb disease in a mouse model of type 2 diabetes mellitus (T2DM) in vivo. We found that 600 ng/mL Tb4 significantly up-regulated AKT activity and Bcl-XL protein expression, enhanced dia-hiPSC-EC viability and proliferation, limited senescence, reduced endothelin-1 and MMP-1 secretion, and improved reparative potency of dia-hiPSC-ECs for treatment of ischemic limb disease in mice with T2DM. However, Tb4 had no effect on improving mitochondrial membrane potential and glycine homeostasis and reducing intercellular adhesion molecule-1 protein expression in dia-hiPSC-ECs.ConclusionsTb-4 improves endothelial dysfunction through enhancing hiPSC-EC viability, reducing senescence and endothelin-1 production, and improves angiogenic potency in diabetes.

MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY

Abstract Background and Aims Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4). Conclusion DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.

Expression of ICAM-1 in placental tissues and the association between its gene polymorphisms and pathogenesis of preeclampsia.

The aim of this study was to explore the expression of intercellular adhesion molecule-1 (ICAM-1) in placental tissues of patients with preeclampsia, and to elucidate the association between its polymorphisms and pathogenesis of preeclampsia. A total of 100 preeclampsia patients (Preeclampsia group) and 100 normal puerperae (Control group) were selected as research objects. The protein expression of ICAM-1 in placental tissues was detected via Western blotting and immunohistochemical staining. The single nucleotide polymorphisms (SNPs) rs134568, rs128343, and rs201931 in the promoter region of ICAM-1 were typed via conformation difference gel electrophoresis. Chi-square test was used to detect whether the distribution frequency of ICAM-1 genotype was in agreement with Hardy-Weinberg equilibrium. The associations of ICAM-1 alleles and polymorphic sites with pathogenesis of preeclampsia were analyzed as well. Finally, the correlation between GG genotype of ICAM-1 rs134568 and clinicopathological features of preeclampsia was analyzed. The protein expression of ICAM-1 in placental tissues was significantly higher in Preeclampsia group than that in Control group (p<0.05). ICAM-1 SNPs rs134568, rs128343 and rs201931 all met Hardy-Weinberg equilibrium (p>0.05). According to gene correlation analysis, ICAM-1 rs134568 polymorphism and alleles were associated with the pathogenesis of preeclampsia (p<0.05). However, ICAM-1 rs128343 and rs201931 polymorphisms and alleles had no associations with the pathogenesis of preeclampsia (p>0.05). Besides, systolic blood pressure, serum creatinine level and plasma albumin level showed no statistically significant differences between people with GG genotype of ICAM-1 rs134568 in Preeclampsia group and those in Control group (p>0.05). ICAM-1 expression increased significantly in placental tissues of patients with preeclampsia. In addition, rs134568 in the promoter region of ICAM-1 was associated with the pathogenesis of preeclampsia.

α-Conidendrin inhibits the expression of intercellular adhesion molecule-1 induced by tumor necrosis factor-α in human lung adenocarcinoma A549 cells

α-Conidendrin is a lignan isolated from Taxus wallichiana and other species. In the present study, we demonstrated that α-conidendrin inhibited the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) at an IC50 value of 40–60 μM in human lung adenocarcinoma A549 cells. α-Conidendrin decreased ICAM-1 protein and mRNA expression levels at concentrations of 40–100 μM in TNF-α-stimulated A549 cells. The TNF-α-induced mRNA expression of vascular cell adhesion molecule-1, E-selectin, and cyclooxygenase-2 was also reduced by α-conidendrin. In the TNF-α-induced nuclear factor κB (NF-κB) signaling pathway, α-conidendrin did not influence the translocation of the NF-κB subunit RelA from the cytoplasm to the nucleus at concentrations up to 100 μM. A chromatin immunoprecipitation assay revealed that α-conidendrin at 100 μM reduced the binding of RelA to the ICAM-1 promoter in response to a stimulation with TNF-α. Collectively, these results indicated that α-conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter, thereby reducing ICAM-1 transcription.

Protective effect of magnesium sulfate on cranial nerves in preeclampsia rats through NF-κB/ICAM-1 pathway.

The aim of this study was to investigate the protective effect of magnesium sulfate (MgSO4) on the cranial nerves of preeclampsia (PE) rats through the nuclear factor-κB (NF-κB)/intercellular adhesion molecule-1 (ICAM-1) pathway. A total of 30 pregnant rats were randomly divided into three groups, including control group, model group, and treatment group, with 10 rats in each group. Systolic blood pressure was measured at 13 d, 15 d, and 19 d. The apoptosis level in brain tissues was detected via Western blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Protein expression of genes was detected using immunohistochemical staining. Moreover, the messenger ribonucleic acid (mRNA) expressions of NF-κB and ICAM-1 in brain tissues were determined through Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Systolic blood pressure exhibited significant differences among the three groups at 15 d and 19 d of gestational age (p<0.05). At 15 d of gestational age, systolic blood pressure was significantly higher in model group than that of control group (p<0.05). However, it was slightly lower in treatment group than model group (p<0.05). At 19 d of gestational age, systolic blood pressure was significantly higher in model group than control group (p<0.05). However, it decreased remarkably in treatment group when compared with model group (p<0.05). In treatment group, systolic blood pressure at 19 d was significantly lower than that at 15 d (p<0.05). Subsequent Western blotting revealed that the protein expression of B-cell lymphoma-2 (Bcl-2) in brain tissues decreased evidently, whereas the expression of Bcl-2 associated X protein (Bax) increased significantly in model group compared with control group, showing statistically significant differences (p<0.01). The protein expression of Bcl-2 in brain tissues increased significantly, while the expression of Bax declined remarkably in treatment group compared with model group (p<0.01). The number of apoptotic cells in model group and treatment group increased significantly compared with that in control group, with the largest in model group (p<0.05). However, it remarkably declined in treatment group compared with model group (p<0.05). These results suggested that MgSO4 treatment could significantly reduce neuronal apoptosis in PE rats. According to the results of immunohistochemistry, the protein expressions of NF-κB and ICAM-1 in brain tissues were significantly higher in model group and treatment group than those in control group (p<0.05). However, they were significantly lower in treatment group than model group (p<0.05). RT-PCR results manifested that the mRNA expressions of NF-κB and ICAM-1 in brain tissues exhibited evident differences among the three groups (p<0.05). Model group and treatment group showed significantly up-regulated mRNA expressions of NF-κB and ICAM-1 in brain tissues compared with control group (p<0.05). The highest mRNA expression was observed in model group. However, treatment group exhibited remarkably decreased mRNA expressions of NF-κB and ICAM-1 in brain tissues compared with model group (p<0.05). MgSO4 exerts a protective effect on cranial nerves of PE rats by inhibiting the NF-κB/ICAM-1 signaling pathway.

Effect of Silencing FMOD on Proliferation and Migration of H322 Cells and Its Mechanism

To investigate the regulation of fibromodulin (FMOD) on proliferation, adhesion and migration of non-small cell lung cancer cell line H322, and discuss its action mechanism. H322 cells were randomly divided into control group, small interfering RNA (siRNA) silencing FMOD ( FMOD siRNA) group and control siRNA (Con siRNA) group. FMOD siRNA and Con siRNA were transfected into H322 cells. The cell viability of each group was detected by CCK-8 method. The adhesion ability of cells was detected by fluorescein diacetate (FDA) fluorescent staining. The cell migration ability was detected by Transwell method. Real time-PCR was used to detect the mRNA expressions of Cyclin D1, intercellular adhesion molecule -1 （ICAM-1）, E-cadherin, FMOD, transforming growth factor-β (TGF-β), Smad2, Smad3, Smad4 and Smad7 in cells. The protein expressions of Cyclin D1, ICAM-1, E-cadherin, FMOD, TGF-β1, Smad2, Smad3, Smad4 and Smad7 were detected by Western blot. Compared with the Con siRNA group, the cell viability, cell adhesion and migration ability of the FMOD siRNA group were decreased, and the difference was statistically significant ( P<0.01). There was no significant difference between the control group and the Con siRNA group. Real time-PCR and Western blot results showed that the mRNA and protein expression levels of Cyclin D1, ICAM-1, TGF-β1, Smad2, Smad3 and Smad4 were decreased in FMOD siRNA group, compared with Con siRNA group, while the mRNA and protein expression levels of E-cadherin and Smad7 are elevated. Silencing of the FMOD gene significantly reduces the proliferation, adhesion and migration of H322 cells, which may be conducted by inhibiting the TGF-β/Smad signaling pathway.

548-P: Baicalin Attenuates Fibrosis Process in Human Renal Proximal Tubular Cells in Hyperglycemic Condition

Background: Baicalin, isolated from Scutellaria baicalensis, has been reported to have anti-inflammatory effect by inhibiting expression of cell adhesion molecules and migration of leukocytes in vascular cells. However, there is a limited evidence on its anti-inflammatory effect in hyperglycemia-associated milieu in other cells. Hyperglycemia and its associated factors are known to induce diabetic nephropathy by exacerbating inflammation, especially, in proximal convoluted tubule. Herein, we investigated whether baicalin exerts anti-inflammatory and anti-fibrosis effects on human renal proximal tubular cells in hyperglycemia induced conditions. Method: HK-2 cell was maintained in normal glycemic (5.78mM glucose) or in hyperglycemic (30mM glucose) condition with or without advanced glycation end-products (AGE) and tumor necrosis factor alpha (TNFα). In each condition, baicalin was treated at 10uM. Cell viability was determined by MTT assay. Gene and protein expression of multiple inflammatory and fibrosis markers including toll-like receptor 2 (TLR2), TLR4, fibronectin, collagen IV α1 (ColIVα1), collagen IV α2 (ColIVα2), transforming growth factor β (TGFβ), intercellular adhesion molecule 1 (ICAM1), and vascular adhesion molecule 1 (VCAM1) were evaluated by PCR and WB, respectively. Results: Baicalin had no effect on cell viability. Baicalin significantly decreased gene expression of ColIVα2 in response to TNFα stimulation in normal glycemic condition (p=0.038). In hyperglycemic condition, baicalin decreased gene expressions of ICAM1 (p=0.049) and VCAM1 (p=0.049) significantly in AGE treated cells; and it significantly decreased gene expressions of TGFβ (p=0.038) and ICAM1 (p=0.017) in TNFα treated cells. Baicalin also decreased protein expression of ICAM1 in response to AGE (p=0.048) and TNFα (p=0.040) stimulation in hyperglycemic condition. Conclusion: Baicalin attenuated fibrosis process by hyperglycemia associated factors in HK-2 cell. Disclosure J. Nam: None. J. Kim: None. K. Park: None. S. Lee: None. J. Nam: None. J. Park: None. S. Park: None. Y. Kim: None. C. Ahn: None.

Lycium barbarum polysaccharide attenuates cardiac hypertrophy, inhibits calpain-1 expression and inhibits NF-κB activation in streptozotocin-induced diabetic rats.

Cardiac hypertrophy is one of the key structural changes that occurs in diabetic cardiomyopathy. Previous studies have indicated that the activation of NF-κB by calpain-1, a Ca2+-dependent cysteine protease, serves an important role in cardiac hypertrophy. The aim of the present study was to assess the effect of 30 and 60 mg/kg Lycium barbarum polysaccharide (LBP) treatment, the major active ingredient extracted from Lycium barbarum, on cardiac hypertrophy in streptozotocin (STZ) induced diabetic rats. In addition, the present study examined the possible underlying mechanisms of this effect by assessing calpain-1 expression and the NF-κB pathway. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was determined by reverse transcription-quantitative PCR. Western blotting was used to detect the protein expressions of calpain-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and toll-like receptor-4 (TLR-4) in the heart tissue. The results revealed that compared with non-diabetic rats, diabetic rats exhibited cardiac hypertrophy. Cardiac hypertrophy was defined by the following: Dysfunction of the cardiac hemodynamics, an increase in the ratios of left ventricular weight/body weight and heart weight/body weight and the increased expressions of ANP and BNP, which serve as hypertrophic markers in cardiac tissue. However, all of these changes were attenuated in diabetic rats treated with LBP. In addition, the protein expression of calpain-1 was increased in the heart tissue of diabetic rats compared with that of non-diabetic rats, where it was inhibited by LBP. LBP also decreased the protein expression of certain inflammatory mediators, IL-6, TNF-α, ICAM-1, VCAM-1 and TLR-4 in diabetic heart tissue. Furthermore, LBP treatment reduced the production of reactive oxygen species, upregulated the protein expression of endothelial nitric oxide synthase and downregulated the protein expression of inducible nitric-oxide synthase. Additionally, LBP increased the protein expression of p65, the subunit of NF-κB and inhibitory protein кB-α in the cytoplasm and reduced p65 expression in the nucleus. In conclusion, LBP improves cardiac hypertrophy, inhibits the expression of calpain-1 and inhibits the activation of NF-κB in diabetic rats.

Radix Kansui Stir-Fried with Vinegar Reduces Radix Kansui-Related Hepatotoxicity in Mice via Mitochondrial Pathway.

To investigate the mechanism of Radix Kansui (RK) stir-fried with vinegar (VRK) decreased hepatotoxicity in mice. According to a random number table, 40 mice were randomly divided into negative control group (0.5% carboxymethylcellulose sodium, 20 mL/kg), positive control group (0.1% mixture of carbon tetrachloride in soybean oil, 20 mL/kg), RK group (the ethyl acetate extracts of RK, 250 g crude drug/kg) and VRK group (the ethyl acetate extracts of VRK, 250 g crude drug/kg) with 10 mice per group. All mice were administered orally by gavage daily for 7 continuous days. The morphology of liver tissues was examined to assess the liver injury by a transmission electron microscope. Hepatocyte apoptosis in vivo was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling (TUNEL) assay. Immunohistochemical technique was adopted to detect the expression of particular antiapoptotic and proapoptotic proteins in the mitochondrial pathways, including B-cell lymphoma (Bcl-2) and caspase-3, as well as the expression of inflammatory mediators, including nuclear factor kappa B (NF- κ B) and intercellular adhesion molecule-1 (ICAM-1). Liver injury and hepatocyte apoptosis were observed in RK mice, and the liver injury were significantly reduced in VRK-treated mice. In immunohistochemistry study, compared with the negative control group, RK inhibited dramatically the Bcl-2 protein expression and significantly increased the expression of caspase-3, NF- κ B and ICAM-1 (all P&lt;0.01). Compared with the RK group, VRK group induced significant increase on Bcl-2 protein expression, and decreased the caspase-3, NF- κ B and ICAM-1 protein expression (P&lt;0.05 or P&lt;0.01). The mechanism of reduced hepatotoxicity of VRK may be associated with the reduced inflammation, regulation of antiapoptotic and proapoptotic mediators in the mitochondrial pathway.

1, 25-Dihydroxyvitamin D3 activates Apelin/APJ system and inhibits the production of adhesion molecules and inflammatory mediators in LPS-activated RAW264.7 cells.

1, 25-Dihydroxyvitamin D3 (1, 25(OH)2D3), an active form of vitamin D3, plays a crucial role in the mitigation of inflammation damage. Recent studies have revealed that apelin and its receptor (apelin/APJ system) could significantly ameliorate LPS-induced inflammation-response. This investigation aimed to appraise the effects of 1, 25(OH)2D3 on the apelin/APJ system and production of adhesion molecules and inflammatory mediators in LPS-activated RAW264.7 macrophage cells. Murine RAW264.7 cells were pretreated with 1, 25(OH)2D3, followed stimulation with LPS (1 μg/mL) for 24 h. The effect of 1, 25(OH)2D3 on LPS-induced cell injury was determined by MTT assay, whereas, enzyme-linked immunosorbent assay (ELISA), qPCR and western blotting were used to evaluate cytokine production and apelin/APJ system expression. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein expression were measured by flow cytometry. The levels of IL-1β, IL-6, and TNF-α cytokines were significantly increased by incubation with LPS. LPS also increased the protein expression of adhesion molecules, including VCAM-1 and ICAM-1. However, pretreatment with 1, 25(OH)2D3 markedly inhibited LPS-induced production of inflammatory cytokines and adhesion molecules. Moreover, we found that 1, 25(OH)2D3 could induced the apelin/APJ system expression. Further experiments demonstrated the significant increase of apelin/APJ system expression at both the protein and mRNA levels in LPS-activated cells when pretreated with 1, 25(OH)2D3. Taken together, our results indicated that 1, 25(OH)2D3 confers an anti-inflammatory effect through a likely mechanism involving a reduction in pro-inflammatory mediators and adhesion molecules via up-regulation of the apelin/APJ system in RAW264.7 cells.

Simvastatin Improves Cardiac Hypertrophy in Diabetic Rats by Attenuation of Oxidative Stress and Inflammation Induced by Calpain-1-Mediated Activation of Nuclear Factor-κB (NF-κB).

BackgroundSimvastatin, an HMG-CoA reductase inhibitor, has been reported to exert multiple protective effects on the cardiovascular system. However, the molecular mechanism remains to be examined. The present study was designed to study the effects of simvastatin on cardiac hypertrophy in diabetic rats and to explore its potential mechanism.Material/MethodsSprague-Dawley rats were assigned into a control (Con) group, a streptozotocin (STZ) group, and a STZ+simvastatin (STZ+SIM) group. The level of reactive oxygen species (ROS) was measured by using dihydroethidium (DHE) staining. The protein expressions of p65, IκBα, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), calpain-1, and endothelial nitric oxide synthase (eNOS) were examined by Western blot analysis. qPCR was used to detect the levels of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP).ResultsSimvastatin improved the cardiac hypertrophy of diabetic rats, as demonstrated by decreases in the ratios of left ventricular weight/body weight (LVW/BW) and heart weight/body weight (HW/BW) and by the downregulation of mRNA expression of BNP and ANP in the heart tissue. Simvastatin decreased the protein expressions of VCAM-1, ICAM-1, IL-6, and TNF-α, increased eNOS protein expression, and limited an increase in ROS levels in the heart tissue. Simvastatin increased IκBα protein expression in cytoplasm and inhibited the translocation of p65, the subunit of nuclear factor-κB (NF-κB) to the nucleus from the cytoplasm of the heart tissue. Furthermore, simvastatin attenuated the activity of calpain and calpain-1 protein expression in heart tissue.ConclusionsSimvastatin attenuates cardiac hypertrophy in diabetic rats, which might be due to the attenuation of oxidative stress and inflammation induced by calpain-1-mediated activation of NF-κB.

Adipose-Derived Stem Cell Transplantation Inhibits Vascular Inflammatory Responses and Endothelial Dysfunction in Rats with Atherosclerosis.

PurposeThis study aimed to investigate the effect of adipose-derived stem cell (ADSC) transplantation on atherosclerosis (AS) and its underlying mechanisms.Materials and MethodsIn our study, rat AS model was established, and ADSCs were isolated and cultured. Atherosclerotic plaque and pathological symptoms of thoracic aorta were measured by Oil Red O staining and Hematoxylin-Eosin staining, respectively. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by an automatic biochemical analyzer. Expressions of vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), aortic endothelin-1 (ET-1), interleukin-6 (IL-6), c-reactive protein (CRP), and tumor necrosis factor α (TNF-α) were measured by enzyme linked immunosorbent assay, VEGF, VCAM-1, ICAM-1, ET-1, respectively, and NF-κB p65 mRNA expressions were detected by quantitative real-time polymerase chain reaction. Protein expressions of VEGF, VCAM-1, ICAM-1, ET-1, NF-κB p65, p-NF-κB p65, and IκBα were measured by western blot. Moreover, NF-κB p65 expression was measured by immunofluorescence staining.ResultsADSC transplantation alleviated the pathological symptoms of aortic AS. ADSC transplantation decreased the levels of TC, TG, and LDL-C and increased serum HDL-C level. Meanwhile, ADSC transplantation decreased the levels of IL-6, CRP, and TNF-α in AS rats. Moreover, the expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were decreased by ADSC transplantation. ADSC transplantation inhibited phosphorylation of NF-κB p65 and promoted IκBα expression in AS rats.ConclusionOur study demonstrated that ADSC transplantation could inhibit vascular inflammatory responses and endothelial dysfunction by suppressing NF-κB pathway in AS rats.

IL-1β-induced ICAM-1 and IL-8 expression/secretion of dental pulp cells is differentially regulated by IRAK and p38

Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine involved in the acute and chronic inflammatory processes of dental pulp. Intercellular adhesion molecule-1 (ICAM-1) and IL-8 are two major inflammatory mediators. However, the role of interleukin-1 receptor-associated kinases (IRAKs) signaling pathways in responsible for the inflammatory effects of IL-1β on dental pulp cells is not clear. Cultured human dental pulp cells were exposed to IL-1β with/without pretreatment and co-incubation with IRAK1/4 inhibitor or SB203580 (p38 inhibitor). IRAK-1 phosphorylation was evaluated by immunno fluorescent staining. The protein expression of ICAM-1 and IL-8 were tested by western blotting. The secretion of soluble ICAM-1 (sICAM-1) and IL-8 was measured by enzyme-linked immunosorbant assay (ELISA). IL-1β stimulated IRAK-1 phosphorylation of pulp cells within 120min of exposure. IRAK1/4 inhibitor attenuated the IL-1β-induced ICAM-1, but not IL-8 protein expression. IRAK1/4 inhibitor also prevented the IL-1β-induced sICAM-1, but not IL-8 secretion. SB203580 showed little effect on IL-1β-induced sICAM-1 secretion, but effectively inhibited its induction of IL-8 secretion in pulp cells. The Results reveal the important role of IL-1β in pulpal inflammatory responses via stimulation of IL-8 and ICAM-1 expression and secretion. Moreover, IL-1β-induced effects on IL-8 and ICAM-1 are differentially regulated by IRAK1/4 and p38 signaling in dental pulp cells. Blocking of IRAKs and p38 signaling may have potential to control inflammation of dental pulp in the future.

Alterations in intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in human endothelial cells.

Alterations of Endothelial cells (ECs) play a critical role in different pathogenesis of many serious human diseases, and dysfunction of the vascular endothelium is an indicator for human disorders. Endothelial dysfunction is considered to be an early indicator for atherosclerosis, which is characterised by overexpression of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Hydrogen peroxide (H2O2) released via neutrophils is an important mediator of endothelial cell function. Ambient production of superoxide anion (O2-) and subsequently H2O2 at low levels is critical for regulating endothelial cell functions and proliferation. In this study, we investigated the effects of H2O2 on the expression of adhesion molecules VCAM-1 and ICAM-1 in cultured human umbilical vein endothelial cells (HUVECs). Intracellular superoxide anion production was detected by using p-Nitro Blue Tetrazolium (NBT) assay. Our results showed that administration of 100μM of H2O2 on HUVECs for 2, 6, 12 and 24 h induced a time-dependent increase in ICAM-1 and VCAM-1 mRNA and protein expression levels with a significant increase observed from 6 h. HUVECs exposed to H2O2 exhibit increased O2-, suggesting that H2O2 induced oxidative stress may be a reasonable for atherosclerosis. This increase can be reduced by the flavonoid, N-acetyl cysteine (NAC). The modulation of endothelial cell function through this mechanism may underlie the contribution of H2O2 to the development of vascular disease.

Effect of electroacupuncture preconditioning on hippocampal IκB-α/NF-κB/ICAM-1 signaling pathway during cerebral ischemia-reperfusion in mice

Objective To evaluate the effect of electroacupuncture (EA) preconditioning on hippocampal I-kappa B-α (IκB-α)/nuclear factor κB (NF-κB)/intercellular adhesion molecule-1 (ICAM-1) signaling pathway during cerebral ischemia-reperfusion (I/R)in mice. Methods A total of 120 healthy male C57BL/6 mice, aged 10-12 weeks, weighing 20-25 g, were divided into 4 groups (n=30 each)using a random number table method: control group (group C), cerebral I/R group (group I/R), preconditioning with EA at non-acupoint + cerebral I/R group (group S+ I/R) and preconditioning with EA at Baihui acupoint + cerebral I/R group (group E+ I/R). The cerebral I/R injury model was established by occlusion of bilateral common carotid arteries followed by reperfusion for 72 h in mice anesthetized with halothane or chloral hydrate in group I/R.Group S+ I/R received EA at the points 2 mm lateral to the acupoints of Baihui for 5 consecutive days, and then the cerebral I/R injury model was established.Group E+ I/R received EA at Baihui acupoints with a sparse-dense wave at an intensity of 1 mA and a frequency of 2 Hz/15 Hz for 30 min once a day for 5 consecutive days, and then the cerebral I/R injury model was established.Neurobehavioral score was assessed at 24 and 48 h of reperfusion.Then 5 mice in each group were sacrificed, and the hippocampal tissues were obtained and stained with haematoxylin and eosin for examination of the pathological changes in hippocampal CA1 region and for determination of the expression of IκB-α, NF-κB, ICAM-1, interleukin-6 (IL-6), IL-1β protein and mRNA by Western blot and real-time polymerase chain reaction, respectively. Results Compared with group C, neurobehavioral score was significantly increased, and the expression of hippocampal IκB-α, NF-κB, ICAM-1, IL-6 and IL-1β protein and mRNA was up-regulated in I/R, S+ I/R and E+ I/R groups (P 0.05). Compared with group S+ I/R, neurobehavioral score was significantly decreased, and the expression of hippocampal IκB-α, NF-κB, ICAM-1, IL-6 and IL-1β protein and mRNA was down-regulated in group E+ I/R (P<0.05). Conclusion The mechanism by which EA preconditioning attenuates cerebral I/R injury may be related to inhibiting activation of hippocampal IκB-α/NF-κB/ICAM-1 signaling pathway in mice. Key words: Electroacupuncture; Ischemia preconditioning; Brain ischemia; Reperfusion injury; I-kappa B proteins; NF-kappa B; Intercellular adhesion molecule-1

Neutrophil Extracellular Traps Induce Endothelial Cell Activation and Tissue Factor Production Through Interleukin-1α and Cathepsin G

Objective- Coronary artery thrombosis can occur in the absence of plaque rupture because of superficial erosion. Erosion-prone atheromata associate with more neutrophil extracellular traps (NETs) than lesions with stable or rupture-prone characteristics. The effects of NETs on endothelial cell (EC) inflammatory and thrombogenic properties remain unknown. We hypothesized that NETs alter EC functions related to erosion-associated thrombosis. Approach and Results- Exposure of human ECs to NETs increased VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA and protein expression in a time- and concentration-dependent manner. THP-1 monocytoid cells and primary human monocytes bound more avidly to NET-treated human umbilical vein ECs than to unstimulated cells under flow. Treatment of human ECs with NETs augmented the expression of TF (tissue factor) mRNA, increased EC TF activity, and hastened clotting of recalcified plasma. Anti-TF-neutralizing antibody blocked NET-induced acceleration of clotting by ECs. NETs alone did not exhibit TF activity or acceleration of clotting in cell-free assays. Pretreatment of NETs with anti-interleukin (IL)-1α-neutralizing antibody or IL-1Ra (IL-1 receptor antagonist)-but not with anti-IL-1β-neutralizing antibody or control IgG-blocked NET-induced VCAM-1, ICAM-1, and TF expression. Inhibition of cathepsin G, a serine protease abundant in NETs, also limited the effect of NETs on EC activation. Cathepsin G potentiated the effect of IL-1α on ECs by cleaving the pro-IL-1α precursor and releasing the more potent mature IL-1α form. Conclusions- NETs promote EC activation and increased thrombogenicity through concerted action of IL-1α and cathepsin G. Thus, NETs may amplify and propagate EC dysfunction related to thrombosis because of superficial erosion.

17β-Estradiol protects against lung injuries after brain death in male rats.

Brain death elicits microvascular dysfunction and inflammation, and thereby compromises lung viability for transplantation. As 17β-estradiol was shown to be anti-inflammatory and vascular protective, we investigated its effects on lung injury after brain death in male rats. Wistar rats were assigned to: sham-operation by trepanation only (SH, n = 7); brain death (BD, n = 7); administration of 17β-estradiol (280 μg/kg, iv) at 60 minutes after brain death (BD-E2, n = 7). Experiments were performed 180 minutes thereafter. Histopathological changes in the lung were evaluated by histomorphometry. Gene expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and endothelin-1 was measured by real-time polymerase chain reaction. Protein expression of NO synthases, endothelin-1, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), BCL-2, and caspase 3 was assessed by immunohistochemistry. Cytokines were quantified by enzyme-linked immunosorbent assay. Treatment with 17β-estradiol after brain death decreased lung edema and hemorrhage (p < 0.0001), and serum levels of cytokine-induced neutrophil chemoattractant-1 (CINC-1; p = 0.0020). iNOS (p < 0.0001) and VCAM-1 (p < 0.0001) also diminished at protein levels, while eNOS accumulated (p = 0.0002). However, gene expression of iNOS, eNOS, and endothelin-1 was comparable among groups, as was protein expression of endothelin-1, ICAM-1, BCL-2, and caspase 3. 17β-Estradiol effectively reduces lung injury in brain-dead rats mainly due to its ability to regulate NO synthases. Thus, the drug may improve lung viability for transplantation.

Dose and timing of normal saline resuscitation on endothelial glycocalyx in early septic shock

To observe the effect of different doses and timing of normal saline (NS) resuscitation combined with norepinephrine (NE) on endothelial glycocalyx in rabbits with early septic shock. Thirty New Zealand male rabbits were randomly divided into sham group, model group, 30 mL and 60 mL timely resuscitation groups (30 mL and 60 mL timely group), and 30 mL delayed resuscitation group (30 mL delayed group) with 6 rabbits in each group. The rabbit model of septic shock was reproduced by cecal ligation and puncture (CLP). The rabbits in sham group were only received abdominal cavity open without cecal and ligation. The rabbits in 30 mL and 60 mL timely groups and 30 mL delayed group were intravenous infused with 30 mL/kg or 60 mL/kg NS immediately or 1 hour after model reproduction for 1 hour, and the mean arterial pressure (MAP) was maintained over 75 mmHg (1 mmHg = 0.133 kPa) compared with intravenous pumping of 0.02-0.05 μg×kg-1×min-1 NE followed by 5 mL/h NS infusion till the end of the experiment. The rabbits in sham and model groups were only given 5 mL/h NS. The changes in arterial blood gas before and immediately after resuscitation were observed in three fluid resuscitation groups. The internal jugular vein blood was collected at 0, 3, 6 hours after model reproduction. The levels of syndecan-1 (polysaccharide envelope marker) in plasma were determined by enzyme linked immunosorbent assay (ELISA). The rabbits were sacrificed at 6 hours after model reproduction, and the lung tissue was harvested. Western Blot was used to determine the protein expressions of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase 2 (MMP-2) and syndecan-1. The positive expression of syndecan-1 in lung tissue was observed by immunohistochemical method. (1) Blood gas analysis: compared with the results before resuscitation, the levels of lactic acid (Lac) after resuscitation in three fluid resuscitation groups were significantly decreased, especially in 30 mL timely group; the central venous blood oxygen saturation (ScvO2) was significantly increased, especially in 30 mL delayed group. Oxygenation index (PaO2/FiO2) was improved in 30 mL timely and 30 mL delayed resuscitation groups, which was decreased in 60 mL delayed group. (2) Plasma marker: compared with sham group, plasma syndecan-1 level in model group was significantly increased with a time-dependent manner. Plasma syndecan-1 levels at 3 hours in 30 mL timely and 30 mL delayed groups were significantly decreased as compared with those of model group (ng/L: 138.0±2.4, 139.7±15.7 vs. 161.5±4.1, both P < 0.05), but it was significantly increased at 6 hours in 30 mL delayed group (ng/L: 213.1±19.4 vs. 206.4±15.5, P < 0.05). The plasma syndecan-1 levels at 3 hours and 6 hours in 60 mL timely group were significantly higher than those in model group (ng/L: 233.0±28.9 vs. 161.5±4.1, 252.3±27.2 vs. 206.4±15.5, both P < 0.05). (3) Protein expression in lung tissue: compared with sham group, the protein expressions of ICAM-1 and MMP-2 in lung tissue of model group were significantly increased, and syndecan-1 protein expression was significantly decreased. After 30 mL timely or 30 mL delayed resuscitation, the protein expressions of ICAM-1 and MMP-2 in lung tissue were significantly decreased, and syndecan-1 protein expression was significantly increased, especially in 30 mL timely group, which showed statistical differences as compared with those of model group (ICAM-1 protein: 0.56±0.09 vs. 1.04±0.05, MMP-2 protein: 0.83±0.15 vs. 1.06±0.06, syndecan-1 protein: 2.09±0.08 vs. 0.99±0.03, all P < 0.05). The change tendency of protein expressions in 60 mL timely group was opposite to the other two resuscitation groups. (4) Immunohistochemistry: the positive expression of syndecan-1 in lung tissues was significant in the sham group, and it was lowered in model group. The positive expression of syndecan-1 was increased after 30 mL timely or 30 mL delayed resuscitation, but further weakened in 60 mL timely group. The dose and timing of resuscitation with NS in septic shock can affect pulmonary vascular endothelial glycocalyx function. The timely resuscitation with 30 mL NS in combination with NE plays a protective effect on endothelial cell and glycocalyx. However, NS resuscitation which was not timely or excessive infusion can make the glycocalyx degradation more obvious, resulting in increased endothelial permeability, microcirculation damaged, thus aggravate lung injury.