MCP-1 Gene Expression Research Articles

S100A9 induces nociceptive pain but is not involved in allodynia in acute experimental synovitis

Purpose: Synovitis-associated pain is an important aspect of arthritis pathology. Several inflammatory mediators released by the synovium have been implicated in the regulation of pain, including S100A8 and S100A9 which may regulate pain either via direct stimulation of TLR4 on the nerve endings in the synovium or via stimulation at the site of the dorsal root ganglia (DRG), thereby enabling an increased phagocyte infiltration, which may cause sensitization. We aimed to elucidate the role of S100A9 in the pain response after induction of an acute synovitis using streptococcal cell walls (SCW) as a trigger, comparing S100A9-/- mice and their WT controls. Methods: Synovial biopsies of 25 patients with early symptomatic OA were studied by micro-array analysis and subsequent functional annotation clustering (FAC). Patients with pain at t=5 years after biopsy isolation according to WOMAC score, were compared to those without pain. Acute synovitis was induced by a single i.a. injection of SCW in the knee joint of C57Bl6 (WT) mice and S100A9-/- mice, control mice received an i.a. saline injection. Serum S100A8/A9 levels were investigated by ELISA and expression of S100A8 and S100A9 in synovium and DRG by immunohistochemistry. Joint swelling and cell influx was assessed by 99mTc accumulaiton and histology, respectively. Pain response were investigated Incapacitance Tester (weight bearing), Catwalk (gait analysis) and von Frey’s filaments (mechanical allodynia). Gene expression of inflammatory mediators and neuron activation markers in DRG were determined by q-PCR. Monocyte influx and protein expression of NAV1.7 and GAP43 was monitored by immunohistochemistry. Results: In synovial biopsies of early symptomatic OA patients, approximately 750 genes were upregulated in patients that had pain 5 years after inclusion compared to patients without pain. Functional annotation clustering showed that inflammatory genes were highly significantly enriched (p=6,4*10-12). Among the upregulated genes were the alarmins S100A8 and S100A9. In mice, a single i.a. injection of SCW resulted in increased synovial expression of S100A8 and S100A9 and subsequent increased serum S100A8/A9 levels (2.6-fold, P < 0.001) 1 day p.i., which returned to basal levels at 7 days p.i. The increased expression of S100A9 did not contribute to the development of inflammation since joint swelling and cell influx were similar in WT and S100A9-/- mice 1 day p.i. Using the Incapacitance Tester, WT mice showed a marked and significant decrease in percentage of weight bearing on the SCW injected hindpaw (28%) compared to saline injection (47%, P < 0.001) 1 day p.i., whereas S100A9-/- mice did not. In addition, gait analysis showed that the stand-phase of the unaffected paws were significantly increased in WT mice 1 day p.i., which will reduce the load on the inflamed paw, while in S100A9-/- mice these parameters were not altered. No difference in mechanical allodynia was observed, both mouse strains showed a similar reduction of paw withdrawal threshold (4.2 and 4.5 fold decrease respectively). Analysis of DRG showed no increased phagocyte infiltration after SCW injection as determined by S100A8 and S100A9 immunohistochemistry and no change in gene expression of MCP-1, KC, IL-1β or TNF was measured. In addition, nu change in F4/80 staining was seen in both WT and S100A9-/- mice. However, expression of neuron activation markers NAV1.7, ATF3 and GAP43 were significantly increased at 1 day after SCW injection in WT mice as compared to saline injected mice (P = 0.022, 0.004 and 0.030, respectively) while SCW injection in S100A9-/- mice did not show increased expression, which is in line in with the reduced pain response observed earlier in S100A9-/- mice. Immunohistochemically the difference in NAV1.7 expression in the DRG was further confirmed at protein level. Conclusions: These findings show that S100A9, which is released from the synovium upon inflammation, is an important mediator of inflammatory nociceptive pain response in the knee, rather than by being involved in peripheral sensitization. During the acute phase of inflammation S100A8/A9 is likely regulated via direct activation of TLR4 on nerve endings in the synovium and not via increased infiltration of phagocytes in the DRG.

Leptin Replacement Therapy Improves Endothelial Function and Vascular Inflammation in a Mouse Model of Acquired Lipodystrophy Associated with Antiretroviral Treatment by Regulating Nox1‐Derived ROS and C‐C Chemokine Receptor Type 5

Highly active antiretroviral therapy (HAART) has been used at suppressing HIV replication, as a side effect HAART promotes lipodystrophy, which is a metabolic disorder characterized by an abnormal adipose tissue distribution, reduced leptin plasma levels and vascular complications. Leptin replacement therapy (LRT) is currently used to treat metabolic dysfunctions in patients diagnosed with congenital lipodystrophy. Here, we sought to investigate whether LRT restores vascular function and reduces inflammation in mice treated with the antiretroviral agent, ritonavir. Four weeks of ritonavir reduced body weight [control (C): 28.4±0.5 vs. Rit: 24.4 ± 0.2g*, *P&lt;0.05], fat mass (C: 10 ± 0.4 vs. Rit: 4.5 ± 0.9 %*, *P&lt;0.05) and leptin plasma levels (C: 4.7 ± 0.05 vs. Rit: 2.0 ± 0.36 ng/mL*, *P&lt;0.05) confirming that it induces acquired lipodystrophy. Acquired lipodystrophy impaired endothelial function, increased plasma lipid peroxidation and vascular H2O2, and ROS producing enzymes (Nox1 and NoxA1). Furthermore, acquired lipodystrophy was associated with vascular inflammation characterized by elevation of NLRP3, Caspase‐1, IL‐1β, MCP‐1, F4/80, CCR5 and CCL5 gene expression. ROS scavenging via tempol (SOD mimetic, 100 μmol/l) or GKT771 (Nox1 inhibitor, 10 μmol/l) restored endothelial function. LRT (10μg/day/7 days, osmotic mini‐pump), at the end of the 3‐week ritonavir, restored endothelial function, reduced vascular oxidative stress and Nox1 and NOXA1 gene expression and vascular inflammation. Nox1 deficiency (Nox1−/−) protected mice from acquired lipodystrophy‐induced endothelial dysfunction and reduced inflammatory markers including CCR5 and CCL5. In order to analyze whether vascular inflammation is occurring via CCR5/CCL5, we induced lipodystrophy in CCR5 deficient mice (CCR5−/−). These mice were protected from acquired lipodystrophy‐induced endothelial dysfunction and vascular inflammation. Moreover, deleting endothelial leptin signaling via specific deletion of leptin receptor (LepR) in endothelial cells (LepR−/−EC mice) promoted endothelial dysfunction, which was reverted by pre‐incubating aortic rings with Nox1 inhibitor (GKT771) in vitro and increased inflammatory markers. Furthermore, LepR−/−EC mice were resistant to the vascular protective effects of LRT. Taken all together, LRT restores endothelial function and reduces vascular inflammation in acquired lipodystrophy associated with HAART via endothelium dependent reduction in Nox1‐derived ROS and CCR5/CCL5. Taken together these data support a key role for endothelial leptin signaling in the control of vascular tone and inflammation and suggest that LRT could represent a potential new avenue for the treatment of vascular diseases associated with acquired lipodystrophy.Support or Funding InformationWork supported by 17POST33410363 and 1K99HL140139‐01A1 to TBN and 1R01HL13030101 to EJ.BdC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Active Cushing Disease Is Characterized by Increased Adipose Tissue Macrophage Presence.

Although glucocorticoids (GCs) have potent anti-inflammatory actions, patients with hypercortisolism due to Cushing disease (CD) have increased circulating proinflammatory cytokines that may contribute to their insulin resistance and cardiovascular disease. The mechanisms and tissues that account for the increased systemic inflammation in patients with CD are unknown. To determine whether chronic endogenous GC exposure due to CD is associated with adipose tissue (AT) inflammation in humans. Abdominal subcutaneous AT samples from 10 patients with active CD and 10 age-, sex-, and body mass index‒matched healthy subjects were assessed for macrophage infiltration and mRNA expression of proinflammatory cytokines. Using immunohistochemistry, AT samples were analyzed for the expression of vimentin, caspase, CD3, CD4, CD8, CD11c, CD20, CD31, CD56, CD68, and CD163. Quantitative PCR was used to assess the mRNA gene expression of arginase, CD11b, CD68, EMR-1, IL-6, IL-10, MCP-1, and TNF-α. Immunohistochemistry revealed higher mean percentage infiltration of CD68+ macrophages and CD4+ T lymphocytes, increased mean area of CD11c+ M1 macrophages, higher number of CD11c+ crownlike structures, and decreased vimentin in the AT of patients with active CD compared with controls. PCR revealed no differences in mRNA expression of any analyzed markers in patients with CD. Chronic exposure to GCs due to CD increases the presence of AT macrophages, a hallmark of AT inflammation. Hence, AT inflammation may be the source of the systemic inflammation seen in CD, which in turn may contribute to obesity, insulin resistance, and cardiovascular disease in these patients.

Administration of methyl palmitate prevents non-alcoholic steatohepatitis (NASH) by induction of PPAR-α

Background and aimsThe lack of valid therapeutic approach that can ameliorate the manifestations of NASH is a barrier to therapeutic development. Therefore, we investigate the novel role of Methyl Palmitate (MP) in preventing NASH and the possible mechanism involved. Methods50 Male C57BL/6 J mice were randomly divided into 5 groups (n = 10). The control group was fed control diet; model group was fed MCD diet; MP 1 group was fed MCD diet supplemented with MP (75 mg/kg/day); MP 2 group was fed MCD plus MP diet (150 mg/kg/day); and MP 3 group was fed MCD plus MP diet (300 mg/kg/day). Histological staining’s, and commercially available kits for serum ALT and AST and hepatic contents of TG, TC, MDA, SOD, and GSH were used to assess NASH. Furthermore, relative liver protein and gene expression levels were determined by Western Blot and qPCR, respectively. ResultsMice fed MCD diet developed NASH, which was markedly improved by MP in a dose-dependent manner. MP treatment improved hepatic content of TG, TC, MDA, SOD and GSH and serum levels of ALT and AST. In vivo studies showed that MP treatment activated PPARα expression, that in turns, promoted β-oxidation protein and gene expressions, suppressed TNFα, MCP1, TGFβ1 and Colla1 protein and gene expression levels, contributing to the prevention of NASH. ConclusionsOur results indicated that MP could successfully prevent NASH. This effect of MP was mediated through induction of PPARα pathway. This study provides a novel therapeutic target that plays pivotal role in the prevention of NASH.

Leptin decreases circulating inflammatory IL-6 and MCP-1 in mice.

Leptin influences inflammation and immune response. Dose-dependent effects of leptin on biomarkers of inflammation have not been studied in vivo, so far. Leptin-deficient low-density lipoprotein receptor (LDLR) knockout (LDLR-/- ;ob/ob) female mice were treated with three different leptin doses or saline for 12 weeks. The effect of leptin on plasma interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 concentrations and Il-6 and Mcp-1 mRNA expression in vivo were assessed. Macrophage infiltration in epididymal adipose tissue (epiAT) after leptin treatment was determined by quantitative immunohistochemical analysis. Aortic root atherosclerotic lesions were analyzed by oil red O staining. Mean plasma IL-6 and MCP-1 decreased significantly in the 3.0 mg/kg BW/day group as compared to control mice (both P < 0.01). Messenger RNA expression of Il-6 and Mcp-1 was significantly down-regulated by leptin treatment in different adipose tissues in vivo. Characteristic crown-like structures formed by adipose tissue macrophages were significantly reduced by leptin treatment in epiAT. Recombinant leptin dose-dependently diminished plaque area in the aortic root. Leptin administration within the subphysiological to physiological range diminishes circulating pro-inflammatory IL-6 and MCP-1. Reduction of Il-6 and Mcp-1 gene expression in adipose tissue, as well as decreased adipose tissue macrophage infiltration might contribute. © 2018 BioFactors, 45(1):43-48, 2019.

Proteoglycan 4 deficiency protects against glucose intolerance and fatty liver disease in diet-induced obese mice

ObjectiveProteoglycan 4 (Prg4) has emerged from human association studies as a possible factor contributing to weight gain, dyslipidemia and insulin resistance. In the current study, we investigated the causal role of Prg4 in controlling lipid and glucose metabolism in mice. MethodsPrg4 knockout (KO) mice and wild-type (WT) littermates were challenged with an obesogenic high-fat diet (45% of total calories as fat) for 16 weeks. To further stimulate the development of metabolic alterations, 10% fructose water was provided starting from week 13. ResultsPrg4 deficiency only tended to reduce diet-induced body weight gain, but significantly improved glucose handling (AUC: −29%; p < 0.05), which was also reflected by a tendency towards a reduced HOMA-IR score (−49%; p = 0.06 as compared to WT mice). This coincided with lower hepatic expression of glycolysis (Gck: −30%; p < 0.05) and lipogenesis (Acc: −21%; p < 0.05 and Scd1: −38%; p < 0.001) genes, which translated in significantly lower hepatic triglyceride levels (−56%; p < 0.001) in Prg4 KO mice as compared to WT mice. Prg4 KO mice likely had lower glucose utilization by skeletal muscle as compared to WT mice, judged by a significant reduction in the genes Glut4 (−29%; p < 0.01), Pfkm (−21%; p < 0.05) and Hk2 (−39%; p < 0.001). Moreover, Prg4 KO mice showed a favorable white adipose tissue phenotype with lower uptake of triglyceride-derived fatty acids (−46%; p < 0.05) and lower gene expression of inflammatory markers Cd68, Mcp1 and Tnfα (−65%, −81% and −63%, respectively; p < 0.01) than WT mice. ConclusionPrg4 KO mice are protected from high-fat diet-induced glucose intolerance and fatty liver disease.

Dual-specificity phosphatases regulate mitogen-activated protein kinase signaling in adipocytes in response to inflammatory stress

Obesity is a strong predictor of heart disease, insulin resistance, and type II diabetes. Chronic, low-grade inflammation links obesity and insulin resistance through mitogen-activated protein kinase (MAPK) signaling pathways. Upstream kinases activate MAPK signaling, while MAPK-specific dual-specificity phosphatases (DUSPs) act as key modulators and controllers of MAPK deactivation (i.e. dephosphorylation). Using tumor necrosis factor α (TNFα) in 3 T3-L1 adipocytes as a model of inflammation, we report that TNFα-mediated induction of Dusp1, Dusp8 and Dusp16 modulated the transient regulation of MAPK (i.e., ERK, JNK, and p38) phosphorylation and subsequent inflammatory gene expression. All three MAPKs examined were phosphorylated in preadipocytes and adipocytes in response to TNFα, where signaling magnitude and duration were phenotype-specific. Moreover, TNFα increased mRNA abundance of DUSPs in preadipocytes and adipocytes in a phenotype-specific manner, concomitant with dephosphorylation of MAPKs. RNA interference (RNAi)-mediated knockdown of Dusp1, Dusp8 and Dusp16 increased signaling magnitude and duration of ERK, JNK, and p38 that subsequently resulted in significant increases in MAPK-dependent inflammatory gene expression of MCP-1, IL-6, and Cox-2 in response to TNFα. This study highlights important roles for DUSPs as integral components of MAPK signaling and adipocyte inflammatory gene expression.

Alveolar Macrophage Chemokine Secretion Mediates Neutrophilic Lung Injury in Nox2-Deficient Mice.

Acute lung injury (ALI), developing as a component of the systemic inflammatory response syndrome (SIRS), leads to significant morbidity and mortality. Reactive oxygen species (ROS), produced in part by the neutrophil NADPH oxidase 2 (Nox2), have been implicated in the pathogenesis of ALI. Previous studies in our laboratory demonstrated the development of pulmonary inflammation in Nox2-deficient (gp91phox-/y) mice that was absent in WT mice in a murine model of SIRS. Given this finding, we hypothesized that Nox2 in a resident cell in the lung, specifically the alveolar macrophage, has an essential anti-inflammatory role. Using a murine model of SIRS, we examined whole-lung digests and bronchoalveolar lavage fluid (BALf) from WT and gp91phox-/y mice. Both genotypes demonstrated neutrophil sequestration in the lung during SIRS, but neutrophil migration into the alveolar space was only present in the gp91phox-/y mice. Macrophage inflammatory protein (MIP)-1α gene expression and protein secretion were higher in whole-lung digest from uninjected gp91phox-/y mice compared to the WT mice. Gene expression of MIP-1α, MCP-1, and MIP-2 was upregulated in alveolar macrophages obtained from gp91phox-/y mice at baseline compared with WT mice. Further, ex vivo analysis of alveolar macrophages, but not bone marrow-derived macrophages or peritoneal macrophages, demonstrated higher gene expression of MIP-1α and MIP-2. Moreover, isolated lung polymorphonuclear neutrophils migrate to BALf obtained from gp91phox-/y mice, further providing evidence of a cell-specific anti-inflammatory role for Nox2 in alveolar macrophages. We speculate that Nox2 represses the development of inflammatory lung injury by modulating chemokine expression by the alveolar macrophage.

NADPH Oxidase Nox5 Aggravates Renal Injury in Akita Mouse Model of Diabetic Nephropathy

Background: Renal reactive oxygen species (ROS) play an important role in mediating kidney injury in diabetes. Increasing evidence suggests that the pro-oxidant enzyme, Nox5 plays a significant role in human diabetic nephropathy (DN). Nox5 is present in humans and rabbits but not in mice or rats. Thus, there is a paucity of information about Nox5 in conventional animal models of DN. We examined the role of Nox5 in the insulin deficient diabetic Akita mice model using human inducible transgenic mice that express Nox5 selectively in endothelial cells (VEcad+Nox5+) or in mesangial cells (SM22+Nox5+). We also examined the endogenous expression of Nox5 in a high fat fed rabbit model of kidney disease. Methods: At week 10 mice were culled and kidneys were removed for the assessment of structural damage as well as gene and protein expression of markers of inflammation, fibrosis and oxidative stress. Protein expression of Nox5 and its localization in glomerular cells (endothelial and mesangial cells) were examined in transgenic mice by immunostaining. We also examined expression of pro-fibrotic gene in high fat fed rabbits by next generation sequencing (NGS) and RT-PCR and renal injury by histochemistry. Results: Expression of Nox5 was confirmed in glomerular endothelial and mesangial cells of transgenic mice. Diabetes induced increase in glomerulosclerosis, gene and protein expression of fibronectin and MCP-1 as well as nitrotyrosine were further increase in both diabetic Nox5 transgenic mice. Moreover, increased expression of Nox5 in high fat fed rabbits vs. normal diet fed rabbits was associated with increased expression of fibronectin, CTGF, collagen IV and VCAM-1 as well as increased mesangial expansion in the kidney. Conclusions: These findings in both transgenic mice model (endothelial and mesangial cells) suggest that Nox5 plays a significant role in mediating renal injury in diabetes. Disclosure J.C. Jha: None. A. Dai: None. M.E. Cooper: Advisory Panel; Self; AbbVie Inc.. Research Support; Self; Boehringer Ingelheim GmbH. Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Speaker's Bureau; Self; Eli Lilly and Company. Advisory Panel; Self; Merck Sharp &amp; Dohme Corp., AstraZeneca. Research Support; Self; Novo Nordisk A/S. Advisory Panel; Self; Novartis AG. R.M. Touyz: None. C. Kennedy: Research Support; Self; Prometic Life Sciences Inc.. K. Jandeleit-Dahm: None.

The Protective Effects of a Synthetic Geranyl Acetophenone in a Cellular Model of TNF-α-Induced Pulmonary Epithelial Barrier Dysfunction.

Alveolar epithelial barrier dysfunction contributes to lung edema and can lead to acute lung injury (ALI). The features include increased epithelial permeability, upregulation of inflammatory mediators and downregulation of junctional complex molecules; these changes are often induced by inflammation. tHGA is an acetophenone analogue with therapeutic potential in asthma. Its therapeutic potential in ALI is presently unknown. Herein, the effects of tHGA on epithelial barrier dysfunction were determined in TNF-α-induced human alveolar epithelial cells. The anti-inflammatory properties of tHGA were assessed by monocyte adhesion assay and analysis of MCP-1 and ICAM-1 expression. The epithelial barrier function was assessed by paracellular permeability and transepithelial electrical resistance (TEER) assays, and analysis of junctional complex molecules expression. To elucidate the mechanism of action, the effects of tHGA on the NF-κB and MAPK pathways were determined. Gene and protein expression were analyzed by RT-PCR and Western blotting or ELISA, respectively. tHGA suppressed leukocyte adhesion to TNF-α-induced epithelium and reduced MCP-1 and ICAM-1 gene expression and secretion. tHGA also increased TEER readings, reduced epithelial permeability and enhanced expression of junctional complex molecules (zona occludens-1, occludin and E-cadherin) in TNF-α-induced cells. Correspondingly, the NF-κB, ERK and p38 MAPK pathways were also inhibited by tHGA. These findings suggest that tHGA is able to preserve alveolar epithelial barrier function in response to acute inflammation, via its anti-inflammatory activity and stabilization of epithelial barrier integrity, mediated by NF-κB, ERK and p38 MAPK signaling.

Exercise Training Favorably Modulates Gene and Protein Expression That Regulate Arterial Cholesterol Content in CETP Transgenic Mice.

Aerobic exercise training (AET) improves the reverse cholesterol transport (RCT) in cholesteryl ester transfer protein-transgenic (CETP-tg) mice. We aimed at investigating the role of AET in the expression of genes and proteins involved in lipid flux in the aorta and macrophages of CETP-tg mice. Three-month-old male mice were randomly divided into trained (T; treadmill 15 m/min; 30 min/day) and sedentary (S) groups. After 6 weeks, peritoneal macrophages and the aortic arch were obtained immediately (0 h) or 48 h after the last exercise session. mRNA was determined by RT-qPCR, protein levels by immunoblot and 14C-cholesterol efflux determined in macrophages. AET did not change body weight, plasma cholesterol, triglycerides, glucose and CETP activity. In macrophages, at time 0 h, a higher expression of genes that encode PPAR gamma, ABCA-1 and a lower expression of MCP-1 and IL-10, was observed in T as compared to S. After 48 h, lower expressions of MCP-1 and PPAR gamma genes were observed in T mice. Increase in ABCA-1, SR-BI and IL-6 and decrease of LOX-1, MCP-1, TNF and IL-10 gene expression was observed in the aorta of T compared to S mice (0 h) and LOX-1 and MCP-1 remained diminished after 48 h. The protein level of MCP-1 and SR-BI in the aortic arch was unchanged in T animals after 48 h as compared to S, but LOX-1 was reduced confirming data of gene expression. The apo A-I and the HDL2 mediated-cholesterol efflux (8 and 24 h) were not different between T and S animals. In the presence of CETP, AET positively influences gene expression in the arterial wall and macrophages of CETP-tg mice contributing to the RCT and prevention of atherosclerosis. These changes were perceptible immediately after the exercise session and were influenced by the presence of CETP although independent of changes in its activity. Reductions in gene and protein expression of LOX-1 were parallel and reflect the ability of exercise training in reducing the uptake of modified LDL by the arterial wall macrophages.

Down-regulation of magnesium transporting molecule, claudin-16, as a possible cause of hypermagnesiuria with the development of tubulo-interstitial nephropathy.

Tubulo-interstitial nephropathy (TIN) is a critical pathological setting for the renal prognosis, and an increase in the urine magnesium excretion is a well-known characteristic feature as one of clinical parametets for the assessment of TIN. We examined the correlation between the development of TIN and the changes in the mRNA expression of renal magnesium-transporting molecules in rats with unilateral ureter obstruction (UUO). Ureter-ligated kidney was sampled at day-0 (control), day-1 (early phase) and day-7 (late phase). The development of TIN was assessed by immunohistochemistry and the real-time PCR of fibrosis-related genes (MCP-1: 105.1±14.8% on day-0, 132.9±25.7% on day-1, 302.7±32.7% on day-7, TGF-β: 101.1±7.6% on day-0, 93.6±4.1% on day-1, 338.9±20.7% on day-7) . The respective expressions of claudin-10, 14, 16, 19, and transient receptor potential (TRP) M6 as magnesium-transporting molecules were also studied. The expression of calcium sensing receptor (CaSR) as an inhibitory regulator of claudin-14 was additionally studied. The gene expression of claudin-16 was decreased in the late phase of UUO (100.2±2.9% at day-0, 90.3±6.3% at day-1, 36.4±1.6% at day-7) which was consistent with the increased urine magnesium excretion. Immunohistochemistry showed an apparent reduction of the immunoreactivity of claudin-16 in the late phase. The expression of TRPM6 was reduced even in the early phase. The immunohistochemistry and gene expression of MCP-1 and TGF-ß showed that TIN was not apparent in the early phase but was significant in the late phase of UUO. The density of peritubular capillaries was diminished in the late phase but not in the early phase. Expression of claudin-14 and CaSR was up- and down-regulated, respectively. Our findings may indicate that the characteristic hypermagnesiuria in TIN is principally caused by the dysfunction of magnesium reabsorption in the thick ascending limb of Henle resulting from a significant decrease in the claudin-16 expression. The down-regulation might be closely related to the development of TIN.

Human acellular amniotic membrane: A potential osteoinductive biomaterial for bone regeneration.

Human acellular amniotic membrane is an acellular, naturally extracellular matrix material with various bioactive factors, which applied in tissue engineering in clinic. Several studies have applied human acellular amniotic membrane in skin and ocular surface tissue engineering to enhance tissue regeneration. However, the application of human acellular amniotic membrane in bone tissue engineering was rarely investigated. The aim of the current study was to investigate the osteoinductivity, angiogenesis and the early molecular changes of human acellular amniotic membrane to bone regeneration. Our results showed that human acellular amniotic membrane with excellent biocompatibility was beneficial for bone marrow mesenchymal stem cells proliferation and osteogenic differentiation. In rat femoral defect model, the existence of human acellular amniotic membrane significantly improved bone regeneration in the defects. The gene expression of CXCR-4, MCP-1, OC and CatK which were connected with cells recruitment and bone remodeling, was enhanced in the defects implanted with human acellular amniotic membrane. The results of this study suggest that human acellular amniotic membrane is an osteoinductive biomaterial for bone regeneration.

Atp-Sensitive Potassium Channels Mediate the Cardioprotection of Panax Notoginseng Saponins against Myocardial Ischemia Reperfusion Injury and Inflammatory Reaction

Background Panax notoginseng saponins (PNS), the main active ingredient extracted from the Chinese herb SanQi, has antiapoptotic, antioxidant, and anti-inflammatory effects to protect the heart from myocardial ischemia/reperfusion injury (MIRI). ATP-sensitive potassium (KATP) channel plays an important role in pharmacological preconditioning against MIRI. However, its effect on inflammation induced by MIRI remains elusive. Thus, we hypothesise that PNS alleviates the inflammatory response induced by MIRI, and KATP channels play a crucial role in it. Methods The rat MIRI model was established by 40 min left anterior descending coronary artery occlusion followed by 60 min reperfusion. PNS was administered 30 min before ischemia, glibenclamide was administered 10 min before PNS. Cardiac function, myocardial histology, and MPO activity were evaluated. The levels of inflammatory mediators TNF-α and MCP-1 in myocardium were measured by ELISA. The protein and gene expression of KATP channel subunits were determined by Western blot analysis and real-time PCR. Results PNS pretreatment ameliorated cardiac function and myocardial pathological changes, decreased myocardial MPO activity and inhibited the gene and protein expression of TNF-α and MCP-1. Importantly, PNS increased the expression of the KATP channel. The non-selective KATP channel blocker glibenclamide could block the cardioprotection of PNS. Conclusion PNS preconditioning may reduce inflammation to due to MIRI via KATP channels. Acknowledgements Supported by a project grant from National Natural Science Foundation (NSFC) of China (Grant No. 81303256).

Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: Role of nitric oxide mediating pleiotropic effects.

Clopidogrel is commonly used in prevention and treatment of atherothrombosis. Some previous studies have suggested a pleiotropic effect of clopidogrel; however, when this drug causes platelet-independent effects on endothelial function remains unclear. To evaluate the influence of clopidogrel on inflammatory biomarkers and adhesion molecules in human endothelial cells and the role of nitric oxide (NO) in this process. TNF-α-induced human umbilical vein endothelial cells (HUVEC) were exposed to clopidogrel. Gene expression and protein expression of ICAM-1, P-selectin, IL-8, IL-6, and MCP-1 were evaluated by qPCR, flux cytometry, or milliplex technology. Expression of endothelial nitric oxide synthase (NOS3) and NO release were also evaluated. Influence of clopidogrel was further evaluated in NOS3 downregulated HUVEC by RNAi. Clopidogrel at 20μmol/L induced NO release in HUVEC after 24-hours treatment. Gene expressions of inflammatory markers IL-8 and MCP1 were reduced after clopidogrel treatment (P<.05); however, only MCP-1 remained reduced at protein level. IL-6 was not modified by clopidogrel treatment. Gene expression and protein expression of ICAM-1 were diminished by 24-hours clopidogrel exposure, whereas P-selectin was not modified. NOS3 downregulated HUVEC model revealed that ICAM-1 modification by clopidogrel is dependent of this via, whereas MCP-1 is modulated in an NO-independent form. Our results support new evidence for pleiotropic effects of clopidogrel on inflammation and endothelial function. Reduction in ICAM-1 and MCP-1 in human endothelium is an important extent of the use of this drug for treatment of cardiovascular diseases, and NO has an important role in this process.

The gut bacterium and pathobiont Bacteroides vulgatus activates NF-κB in a human gut epithelial cell line in a strain and growth phase dependent manner

The gut microbiota is increasingly implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) although the identity of the bacteria that underpin these diseases has remained elusive. The pathobiont Bacteroides vulgatus has been associated with both diseases although relatively little is known about how its growth and functional activity might drive the host inflammatory response. We identified an ATP Binding Cassette (ABC) export system and lipoprotein in B. vulgatus ATCC 8482 and B. vulgatus PC510 that displayed significant sequence similarity to an NF-κB immunomodulatory regulon previously identified on a CD-derived metagenomic fosmid clone. Interestingly, the ABC export system was specifically enriched in CD subjects suggesting that it may be important for colonization and persistence in the CD gut environment. Both B. vulgatus ATCC 8482 and PC510 activated NF-κB in a strain and growth phase specific manner in a HT-29/kb-seap-25 enterocyte like cell line. B. vulgatus ATCC 8482 also activated NF-κB in a Caco-2-NF-κBluc enterocyte like and an LS174T-NF-κBluc goblet cell like cell lines, and induced NF-κB-p65 subunit nuclear translocation and IL-6, IL-8, CXCL-10 and MCP-1 gene expression. Despite this, NF-κB activation was not coincident with maximal expression of the ABC exporter or lipoprotein in B. vulgatus PC510 suggesting that the regulon may be necessary but not sufficient for the immunomodulatory effects.

Frequency of MCP-1 (rs1024611) and CCR2 (rs1799864) gene polymorphisms and its effect on gene expression level in patients with AgP

ObjectiveTo identify the genetic risk markers of aggressive periodontitis (AgP), researchers focus on genetic components that regulate the immune response. Therefore the purpose of this study was to investigate genetic impact of monocyte chemoattractant protein (MCP)-1–2518A/G and CC chemokine receptor 2 (CCR2) −190G/A gene polymorphisms on AgP susceptibility and the effect of this polymorphism on MCP-1 gene expression in patients with AgP. Material and methodsA total of 215 subjects, 108 AgP and 107 periodontally healthy (H) were recruited in this cross-sectional study (NCT02817568). Gene polymorphisms of MCP-1–2518A/G and CCR2–190G/A were analyzed by a standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. MCP-1 messenger (m) RNA expression was measured using quantitative real-time (RT)-PCR in peripheral blood leukocytes from 26 AgP and 16H controls. Threshold cycles (Ct) values were obtained from the RT-PCR analysis based on SYBR Green detection and data was normalized via ΔCt. ResultsThere were no differences between AgP and H groups with regard to MCP-1 and CCR2 genotype distribution and allele frequencies (p>0.05). In contrast, the MCP-1 mRNA expression levels were higher in homozygous “AA” control subjects than having G+ genotype and AA homozygous AgP patients. ConclusionsIt can be concluded that MCP-1 and CCR2 polymorphisms are not associated with AgP in Turkish population. Although in AgP patients, there was AA genotype with MCP-1 mRNA expression it can be speculated that gene expression levels in peripheral blood may not reflect the cytokine/chemokine levels of local tissues.

The Chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of viral propagation and impacts immune function

BackgroundLianhuaqingwen Capsule (LH-C) is a traditional Chinese medicine (TCM) formula used to treat respiratory tract infectious diseases in Chinese. The aim of this study was to determine the antiviral activity of LH-C and its immunomodulatory effects on viral infection.MethodThe in vitro cytotoxicity and antiviral activity of LH-C was determined by MTT and Plaque reduction assays. Time course study under single-cycle virus growth conditions were used to determine which stage of viral replication was blocked. The effect of LH-C on the nuclear export of the viral nucleoprotein was examined using an indirect immunofluorescence assay. The regulation to different signaling transduction events and cytokine/chemokine expression of LH-C was evaluated using Western blotting and real-time RT-PCR. After virus inoculation, BALB/c mice were administered with LH-C of different concentrations for 5 days. Body-weight, viral titers and lung pathology of the mice were measured, the level of inflammatory cytokines were also examined using real-time RT-PCR.ResultsLH-C inhibited the proliferation of influenza viruses of various strain in vitro, with the 50% inhibitory concentration (IC50) ranging from 0.35 to 2 mg/mL. LH-C blocked the early stages (0–2 h) of virus infection, it also suppressed virus-induced NF-kB activation and alleviated virus-induced gene expression of IL-6, IL-8, TNF-a, IP-10, and MCP-1 in a dose-dependent manner. LH-C treatment efficiently impaired the nuclear export of the viral RNP. A decrease of the viral titers in the lungs of mice were observed in groups administered with LH-C. The level of inflammatory cytokines were also decreased in the early stages of infection.ConclusionsLH-C, as a TCM prescription, exerts broad-spectrum effects on a series of influenza viruses, including the newly emerged H7N9, and particularly regulates the immune response of virus infection. Thus, LH-C might be a promising option for treating influenza virus infection.

Pectin from transgenic flax shives regulates extracellular matrix remodelling in human skin fibroblasts

Abstract Pectin, a polysaccharide polymer from the plant cell wall, is an underestimated natural resource with many potential applications in the food and medical industries. Here we present, for the first time, the chemical composition of pectin obtained from flax shives, a by-product of flax fibre processing. The shives from transgenic flax overexpressing β-glucanase were analysed, revealing that genetic modification caused an increase in content of lignin, hemicellulose and pectin, without changes to cellulose, rearrangement of the structure of pectin and cellulose, a decrease in the content of phenolic compounds associated with the cell wall, and an increase in antioxidant capacity of the pectin CDTA fraction. The influence of pectin extract on the extracellular matrix remodelling process was verified. In fibroblast skin cells with induced oxidative stress, addition of pectin caused a reversal of the decrease of mRNA collagen genes, an increase of matrix metalloproteinase, interleukin 6 and MCP-1 gene expression, and a reduction in levels of TIMP-1 and SOCS-1 mRNA. The obtained results, in particular strong antioxidant properties of flax shives pectin from the CDTA-soluble fraction and its significant influence on genes participating in extracellular matrix remodelling, suggest the possible application of flax shives pectin in the wound healing process.

NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells.

Dysregulation of inflammatory responses is a hallmark of multiple diseases such as atherosclerosis and rheumatoid arthritis. As constitutively active transcription factors, NR4A nuclear receptors function to control the magnitude of inflammatory responses and in chronic inflammatory disease can be protective or pathogenic. Within this study, we demonstrate that TLR4 stimulation using the endotoxin lipopolysaccharide (LPS) rapidly enhances NR4A1–3 expression in human and murine, primary and immortalized myeloid cells with concomitant gene transcription and protein secretion of MIP-3α, a central chemokine implicated in numerous pathologies. Deficiency of NR4A2 and NR4A3 in human and murine myeloid cells reveals that both receptors function as positive regulators of enhanced MIP-3α expression. In contrast, within the same cell types and conditions, altered NR4A activity leads to suppression of LPS-induced MCP-1 gene and protein expression. An equivalent pattern of inflammatory gene regulation is replicated in TNFα-treated myeloid cells. We show that NF-κB is the critical regulator of NR4A1–3, MIP-3α, and MCP-1 during TLR4 stimulation in myeloid cells and highlight a parallel mechanism whereby NR4A activity can repress or enhance NF-κB target gene expression simultaneously. Mechanistic insight reveals that NR4A2 does not require DNA-binding capacity in order to enhance or repress NF-κB target gene expression simultaneously and establishes a role for NF-κB family member Relb as a novel NR4A target gene involved in the positive regulation of MIP-3α. Thus, our data reveal a dynamic role for NR4A receptors concurrently enhancing and repressing NF-κB activity in myeloid cells leading to altered transcription of key inflammatory mediators.