MCP-1 Protein Research Articles

MiR-21, MiR-29a, GATA4, and MEF2c Expression Changes in Endothelin-1 and Angiotensin II Cardiac Hypertrophy Stimulated Isl-1+Sca-1+c-kit+ Porcine Cardiac Progenitor Cells In Vitro.

Cost- and time-intensive porcine translational disease models offer great opportunities to test drugs and therapies for pathological cardiac hypertrophy and can be supported by porcine cell culture models that provide further insights into basic disease mechanisms. Cardiac progenitor cells (CPCs) residing in the adult heart have been shown to differentiate in vitro into cardiomyocytes and could contribute to cardiac regeneration. Therefore, it is important to evaluate their changes on the cellular level caused by disease. We successfully isolated Isl1+Sca1+cKit+ porcine CPCs (pCPCs) from pig hearts and stimulated them with endothelin-1 (ET-1) and angiotensin II (Ang II) in vitro. We also performed a cardiac reprogramming transfection and tested the same conditions. Our results show that undifferentiated Isl1+Sca1+cKit+ pCPCs were significantly upregulated in GATA4, MEF2c, and miR-29a gene expressions and in BNP and MCP-1 protein expressions with Ang II stimulation, but they showed no significant changes in miR-29a and MCP-1 when stimulated with ET-1. Differentiated Isl1+Sca1+cKit+ pCPCs exhibited significantly higher levels of MEF2c, GATA4, miR-29a, and miR-21 as well as Cx43 and BNP with Ang II stimulation. pMx-MGT-transfected Isl1+Sca1+cKit+ pCPCs showed significant elevations in MEF2c, GATA4, and BNP expressions when stimulated with ET-1. Our model demonstrates that in vitro stimulation leads to successful Isl1+Sca1+cKit+ pCPC hypertrophy with upregulation of cardiac remodeling associated genes and profibrotic miRNAs and offers great possibilities for further investigations of disease mechanisms and treatment.

411. Does an Early Cytokine Response During Ebola Virus Disease Improve the Duration of Survival in Rhesus Macaques?

BackgroundEbola virus disease results in a severe cytokine release resulting in organ failure and disseminated intravascular coagulation, often leading to death. An early post-exposure immune response may improve outcomes but that remains poorly characterized. Therefore, we evaluated select serum cytokine markers of immune activation in nonhuman primates (NHPs) for their association with duration of survival.MethodsThis was a post-hoc analysis of an interventional supportive care NHP study in which 13 rhesus macaques were inoculated intramuscularly with a target dose of 1000 PFU Zaire ebolavirus (Kikwit). We measured cytokines with a Luminex MAGPIX panel at baseline and daily starting day 3 post-exposure until euthanasia. Based on human clinical data, 10 cytokines and proteins were included in our analysis: IL-1ra, IL-6, IL-10, GM-CSF, MCP-1, MIP-1α, MIP-1β, IFN-γ, TNF-α, and C-reactive protein levels. After NHPs were divided into two groups by k-means clustering, we developed Kaplan–Meier curves for time to death (Figure 1). We visually explored Pearson’s correlation and kinetics of serum cytokines and log10viral load (Figure 1; Figure 2). We fitted cox regression models with each cytokine to evaluate the risk of early disease for each cytokine log10 level or log2-fold change. We performed a sensitivity analysis for MIP-1β centering the data at dpe 0.ResultsAmong NHPs with temperature data, 83% (N = 10) developed fevers (>3 SD baseline) from dpe 3 to 4.The macrophage markerMIP-1β was associated with an increased risk of early death (per log10pg/mL increase, HR= 52.83 at dpe 3, adjusted P = 0.045). Surprisingly, this association was also observed at dpe 0 (HR= 36.88 at dpe 0, adjusted P = 0.044). Other cytokine levels or changes were not associated with an increased hazard of death.ConclusionOur findings did not support a role for early systemic cytokine release in improving survival. However, elevated baseline levels of the MIP-1β may predispose NHPs to early death from EVD. This finding could represent a target for therapeutic strategies and should be further researched. Disclosures All authors: No reported disclosures.

Abstract 010: Neutrophil Gelatinase Associated Lipocalin From Immune Cells is Involved in Renal Damages Induced by Mineralocorticoid Excess

Introduction: Neutrophil Gelatinase-Associated Lipocalin (NGAL) (or lipocalin 2) is a novel mineralocorticoid biotarget in the cardiovascular system. NGAL is also described as an acute renal lesion biomarker and NGAL serum concentration is associated with the severity of renal damages patients with a chronic kidney disease (CKD). Lipocalin2 (Lcn2) gene invalidation in a CKD mouse model protects from proteinuria and renal lesions. Objective: Since the immune system is involved in renal inflammation and fibrosis process, we hypothesized that the production of NGAL/Lcn2 by immune cells plays an important role in the deleterious mineralocorticoid effects in the kidney. Methods: The role of Lcn2 produced by immune cells was analyzed using full Lcn2 knock out mice (Full KO) and mice depleted for Lcn2 in their immune cells by bone marrow transplantation (BMT lcn2 KO). These mice were challenged with uni-Nephrectomy, Aldosterone 200μg/kg/day, Salt 1% (NAS model) during 6 weeks. Results: NAS induced a significant increase in the expression (relative values, mean±SEM, compared to 1 in the control samples, p<0.05, n=6-9) of proinflammatory proteins such as MCP-1 (5.00±0.26), TNF-α (8.58±0.31), OPN (1.60±0.07), RANTES (3.98±0.20) and extracellular matrix proteins such as collagen IV (1.94±0.10), α-SMA (2.04±0.13) and fibronectin (3.38±0.20) in the kidney of WT mice. This effect was blunted in both KO mouse models (Full KO and BMT KO). Kidney fibrosis was induced by NAS (relative values, mean±SEM, compared to 1 in the control samples, p<0.05, n= 6-9) (5.98±0.19). this increase was fully prevented in Full KO NAS mice and partly corrected in BMT KO NAS mice. Macrophages isolated from Full Lcn2 KO or WT mice were co-treated with aldosterone (10 -8 M) and NaCl (40mM). In WT macrophages, mRNA expression (relative values, mean±SEM, compared to 1 in the control samples, p<0.05, n=6) of Lcn2 (22.06±1.36) as well as a the RANTES/CCl5 chemokine (10.88±0.88) is increased. The increase of RANTES/CCl5 was fully blunted in Lcn2 KO macrophages. Conclusion: NGAL produced by immune cells plays a critical role in renal interstitial fibrosis and inflammation induced by mineralocorticoid excess. The role of RANTES/CCl5 in the renal NGAL-mineralocorticoid pathways is currently analyzed.

Electrical stimulation promotes the angiogenic potential of adipose-derived stem cells

Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.

Ligustrazine nanoparticles nano spray's activation on Nrf2/ARE pathway in oxidative stress injury in rats with postoperative abdominal adhesion.

Postoperative abdominal adhesions formation is considered a significant clinical entity implicating the healing process following major pelvic and abdominal surgery, with serious clinical complications and need for substantial health care expenditures. However, setting a physical barrier between the damage site and the neighboring tissues is a convenient and highly valid way to minimize or prevent peritoneal adhesions. The present experimental study evaluated the preventive effect of ligustrazine nanoparticles nano spray (LNNS) on postoperative abdominal adhesion in rats and explored its mechanism. Sixty male Sprague Dawley (SD) rats were randomly divided into sham operation group, control group, sodium hyaluronate group and low, medium, and high dose LNNS groups. All groups were prepared with abdominal adhesion models except for the sham operation group. The models were made by opening the abdominal cavity to and filing the serosa in ileocecal junction. The abdominal cavity of rats in the sham operation group were only opened and sutured. The wound surface of rats in the sodium hyaluronate group, low, medium, and high dose LNNS groups were sprayed with sodium hyaluronate gel (0.5 mL/kg) and LNNS (2.5, 5, and 10 mL/kg). Rats in each group were sacrificed 7 days later. Degree of adhesion was evaluated by naked eyes and the pathological sections were scored afterwards. The collagen synthesis in adhesion tissues was detected by Masson's trichrome stain, and the activities of reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD) and malondialdehyde (MDA) in peritoneal fluid were detected with the method of chromogenic substrate. Levels of TNF-α and IL-1β in serum, and the protein levels of MCP-1 and MMP-9 in adhesion tissues were detected by ELISA and. immunohistochemistry respectively. RT-PCR and Western blot were utilized to identify the expression levels of Nrf2, heme-oxygenase-1, NQO1 mRNA and protein in adherent intestinal tissues. Compared with the control group, the incidence of postoperative abdominal adhesions decreased in the low, medium and high dose LNNS groups, while the expression of SOD in the peritoneal fluid significantly increased. The expression levels of ROS, MDA and NO were reduced remarkably (P<0.05), so were the expression levels of serum TNF-α and IL-1β (P<0.01) and the expression of MCP-1 protein in adhesion tissues. The MMP-9 protein expression, and Nrf2, heme-oxygenase-1, NQO1 mRNA and protein expressions increased. LNNS with medium or high dose can significantly reduce the incidence of postoperative abdominal adhesions, the mechanism of which may be the activation of Nrf2/ARE pathway, resulting in the up-regulation of Nrf2, heme-oxygenase-1, NQO1 and mRNA expression, as well as the levels of TNF-α and IL-1β in peripheral blood and the expression of MCP-1 protein in adhesion tissues. Meanwhile, the content of MMP-9 protein in adhesion tissues were raised, and oxidative stress and inflammatory response are released.

SIRPα/CD47 axis controls the maintenance of transplant tolerance sustained by myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature hematopoietic precursors known to suppress immune responses. Interaction of SIRP alpha (SIRPα), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response regulating macrophages and dendritic cells functions. We previously described that MDSC expressing SIRPα accumulated after transplantation and maintained kidney allograft tolerance. However, the role of the SIRPα/CD47 axis on MDSC function remained unknown. Here, we found that blocking SIRPα or CD47 with monoclonal antibodies (mAbs) induced differentiation of MDSC into myeloid cells overexpressing MHC class II, CD86 costimulatory molecule and increased secretion of macrophage-recruiting chemokines (eg, MCP-1). Using a model of long-term kidney allograft tolerance sustained by MDSC, we observed that administration of blocking anti-SIRPα or CD47 mAbs induced graft dysfunction and rejection. Loss of tolerance came along with significant decrease of MDSC and increase in MCP-1 concentration in the periphery. Graft histological and transcriptomic analyses revealed an inflammatory (M1) macrophagic signature at rejection associated with overexpression of MCP-1 mRNA and protein in the graft. These findings indicate that the SIRPα-CD47 axis regulates the immature phenotype and chemokine secretion of MDSC and contributes to the induction and the active maintenance of peripheral acquired immune tolerance.

Hyperglycemia induces NF-κB activation and MCP-1 expression via downregulating GLP-1R expression in rat mesangial cells: inhibition by metformin.

Hyperglycemia impairs glucagon-like peptide-1 receptor (GLP-1R) signaling in multiple cell types and thereby potentially attenuates the therapeutic effects of GLP-1R agonists. We hypothesized that the downregulation of GLP-1R by hyperglycemia might reduce the renal-protective effects of GLP-1R agonists in diabetic nephropathy (DN). In this study, we examined the effects of high glucose on the expression of GLP-1R and its signaling pathways in the HBZY-1 rat mesangial cell line. We found that high glucose reduced GLP-1R messenger RNA (mRNA) levels in HBZY-1 cells and in the renal cortex in db/db mice comparing with control groups. In consistence, GLP-1R agonist exendin-4 induced CREB phosphorylation was attenuated by high glucose but not low glucose treatment, which is paralleled with abrogated anti-inflammatory functions in HBZY-1 cells linked with nuclear factor-κB (NF-κB) activation. In consistence, GLP-1R inhibition aggravated the high glucose-induced activation of NF-κB and MCP-1 protein levels in cultured HBZY-1 cells while overexpression of GLP-1R opposite effects. We further proved that metformin restored high glucose-inhibited GLP-1R mRNA expression and decreased high glucose evoked inflammation in HBZY-1 cells. On the basis of these findings, we conclude that high glucose lowers GLP-1R expression and leads to inflammatory responses in mesangial cells, which can be reversed by metformin. These data support the rationale of combinative therapy of metformin with GLP-1R agonists in DN.

Synthesis and biological evaluation of (1,2,4)triazole[4,3-a]pyridine derivatives as potential therapeutic agents for concanavalin A-induced hepatitis

A series of (1,2,4)triazole[4,3-a]pyridine (TZP) derivatives have been designed and synthesized. Compound 8d was identified as having the most potent inhibitory activity on NO release in response to lipopolysaccharide (LPS) stimulation and inhibition of the migration induced by MCP-1 protein on RAW264.7 macrophages. Based on the screening data, an immunofluorescence assay and a real-time qPCR assay were conducted, indicating that compound 8d suppressed NF-κB p65 translocation and expression of inflammatory genes by concanavalin A (Con A)-induced RAW264.7 macrophages. More importantly, 8d also exhibited potent efficacy, alleviating Con A-induced hepatitis by downregulating the levels of plasma alanine transaminase (ALT), aspartate transaminase (AST) and inflammatory infiltration in a mouse autoimmune hepatitis (AIH) model. In addition, the flow cytometry (FCM) data showed that compound 8d inhibited the accumulation of MDSCs in the liver of Con A-induced mice. These findings raise the possibility that compound 8d might serve as a potential agent for the treatment of AIH.

426-P: ROCK2 Regulates Monocytic Migration and Cell-to-Cell Adhesion in Vascular Endothelial Cells

A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the RhoA/Rho-kinase signaling pathway and has been shown to induce the expression of chemokines and cell adhesion molecules (CAMs). LPA levels are elevated in the circulation of patients with acute coronary syndrome and have also been detected inside human atherosclerotic specimens. However, the involvement of Rho-kinase isoforms (i.e., ROCK1, ROCK2) in LPA-mediated expression of chemokines and CAMs in endothelial cells is not fully understood. In this study, we investigated isoform-specific roles of Rho-kinase in LPA-mediated MCP-1 and E-selectin expression using human aortic endothelial cells (HAEC). Exposure of HAEC to LPA resulted in an increase of MCP-1 and E-selectin expression at both mRNA and protein levels. Rho-kinase inhibitor abolished MCP-1 and E-selectin protein expression, indicating that LPA induces MCP-1 and E-selectin expression via Rho-kinase activation. From a mechanistic standpoint, phosphorylation of IκBα, a well-characterized initial step in NF-κB activation, was decreased by Rho-kinase inhibition in the LPA-stimulated cells. Furthermore, using isoform specific siRNAs, we found that Rho-kinase isoform ROCK2 is the key mediator of LPA-induced MCP-1 and E-selectin expression. Consistently, ROCK2 but not ROCK1 controlled LPA-induced monocytic migration and adhesion in functional assays using HAECs and human monocytic cells (THP-1). In the present study, we demonstrate that endothelial ROCK2 mediates monocyte migration and cell adhesion induced by lipid mediator. In order to further explore endothelial-specific role of ROCK2 in vivo, we generated endothelial ROCK2 knockout mouse (EC-ROCK2KO) by mating of ROCK2 flox mice with VE-cadherin-cre mice. Further investigation using HFD (high-fat diet) mouse model is ongoing. ROCK2 could be an important therapeutic target against atherosclerosis. Disclosure Y. Takeda: None. K. Matoba: Research Support; Self; Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Sanofi, Shionogi &amp; Co., Ltd., Takeda Pharmaceutical Company Limited. D. Kawanami: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Y. Nagai: None. K. Utsunomiya: None. Funding Japan Society for the Promotion of Science; Uehara Memorial Foundation; MSD Life Science Foundation

Baked corn (Zea mays L.) and cooked common bean (Phaseolus vulgaris L.) chips improved enzymatic biomarkers and alleviated inflammation during chronic colitis in vivo (P06-063-19)

ObjectivesThe aim of this research was to evaluate the anti-inflammatory properties of an oven-baked nixtamalized corn (Zea mays L.)/cooked common bean (Phaseolus vulgaris L.) chip in a model of chronic colitis in vivo. The hypothesis was that the chip consumption would prevent colon barrier disruption and improve enzymatic biomarkers by the modulation of infiltration and adhesion of inflammatory cells. MethodsA 70% corn and 30% bean chip (7030C) was evaluated, using dextran sodium sulfate (DSS, 2% v/v) as chemical inductor of colitis. After 1-week of acclimatization, 36 CD-1 male mice (6–8 weeks age) were randomly divided into 5 groups: G1 (negative control, fed with basal diet, BD, and water); G2 (positive control, BD + DSS), G3 (100 g 7030C/kg body weight, BW/day + BD), G4 (200 g 7030C/kg BW + BD) and G5 (300 g 7030C/kg BW + BS). The G2-G5 groups were administered DSS every other week, during 5-weeks.After the mice were euthanized, BW and disease activity index (DAI) were recorded. Liver, colon, and spleen were collected, weighed and analyzed for histology. Colonic myeloperoxidase (MPO)/fecal b-glucuronidase (GLUC) activities were also quantified, as well as fecal/cecal metabolites. The colonic mRNA expression of inflammation-associated genes was conducted using a gene inflammation profiler array. ResultsDSS increased DAI up to 2 units, BW loss was 10–17%, and induced colon shortening 10–15%. Compared to G2, G4 exhibited significantly (p < 0.05) lower DAI (0.75 ± 0.01), spleen relative weight (0.003 ± 0.0001) and colon weight/length ratio (0.045 ± 0.008). The histological analysis showed that the chip consumption prevented colonic barrier damage. G4 displayed the lowest MPO and GLUC among all DSS-induced groups (0.004 ± 0.0004 mU/mg colon; 0.44 ± 0.01 mmol/min/g feces, respectively), and the lowest seric content of MCP-1 protein. Amid all the quantified metabolites, the chip consumption significantly reduced the fecal/cecal content of acetic acid, while butyric and propionic increased at the end of the study. Inflammation gene expression was modulated by the chip consumption. ConclusionsOur results suggest that the consumption of this chip might alleviate chronic colitis symptoms because of a protective effect in the gut barrier function and the modulation of infiltration of inflammatory cells. Funding SourcesThe funding received by CONACyT, CONCyTEQ-Mexico and NIFA-USDA-HATCH are appreciated.

Calcineurin inhibitors reduce NFAT-dependent expression of antifungal pentraxin-3 by human monocytes.

Calcineurin (CN) inhibitors are effective clinical immunosuppressants but leave patients vulnerable to potentially fatal fungal infections. This study tested the hypothesis that CN inhibition interferes with antifungal immune defenses mediated by monocytes. We showed that NFAT is expressed by human monocytes, and is activated by exposure to fungal ligands. We confirmed that NFAT translocation potently activated target gene transcription using a human monocytic reporter cell line. Inhibition of CN‐NFAT by cyclosporine A significantly reduced monocyte production of TNF‐α, IL‐10, and MCP‐1 proteins in response to pattern recognition receptor ligands as well as to Aspergillus fumigatus conidia. Moreover, we revealed that human monocytes express the antifungal protein pentraxin‐3 under control of NFAT. In conclusion, clinical CN inhibitors have the potential to interfere with the novel NFAT‐dependent pentraxin‐3 pathway as well as antifungal cytokine production in human monocytes, thereby impeding monocyte‐mediated defenses against fungal infection in immune‐suppressed patients.

Ginsenoside Rh2 Ameliorates Doxorubicin-Induced Senescence Bystander Effect in Breast Carcinoma Cell MDA-MB-231 and Normal Epithelial Cell MCF-10A.

The anthracycline antibiotic doxorubicin is commonly used antineoplastic drug in breast cancer treatment. Like most chemotherapy, doxorubicin does not selectively target tumorigenic cells with high proliferation rate and often causes serve side effects. In the present study, we demonstrated the cellular senescence and senescence associated secretory phenotype (SASP) of both breast tumor cell MDA-MB-231 and normal epithelial cell MCF-10A induced by clinical dose of doxorubicin (100 nM). Senescence was confirmed by flattened morphology, increased level of beta galactose, accumulating contents of lysosome and mitochondrial, and elevated expression of p16 and p21 proteins. Similarly, SASP was identified by highly secreted proteins IL-6, IL-8, GRO, GM-CSF, MCP-1, and MMP1 by antibody array assay. Reciprocal experiments, determined by cell proliferation and apoptosis assays and cell migration and cell invasion, indicated that SASP of MDA-MB-231 cell induces growth arrest of MCF-10A, whereas SASP of MCF-10A significantly stimulates the proliferation of MDA-MB-231. Interestingly, SASP from both cells powerfully promotes the cell migration and cell invasion of MDA-MB-231 cells. Treatment with the natural product ginsenoside Rh2 does not prevent cellular senescence or exert senolytic. However, SASP from senescent cells treated with Rh2 greatly attenuated the above-mentioned bystander effect. Altogether, Rh2 is a potential candidate to ameliorate this unwanted chemotherapy-induced senescence bystander effect.

Cytokines and Chemokines in Pediatric Appendicitis: A Multiplex Analysis of Inflammatory Protein Mediators.

Objectives We aimed to demonstrate the potential of precision medicine to describe the inflammatory landscape present in children with suspected appendicitis. Our primary objective was to determine levels of seven inflammatory protein mediators previously associated with intra-abdominal inflammation (C-reactive protein—CRP, procalcitonin—PCT, interleukin-6 (IL), IL-8, IL-10, monocyte chemoattractant protein-1—MCP-1, and serum amyloid A—SAA) in a cohort of children with suspected appendicitis. Subsequently, using a multiplex proteomics approach, we examined an expansive array of novel candidate cytokine and chemokines within this population. Methods We performed a secondary analysis of targeted proteomics data from Alberta Sepsis Network studies. Plasma mediator levels, analyzed by Luminex multiplex assays, were evaluated in children aged 5-17 years with nonappendicitis abdominal pain (NAAP), acute appendicitis (AA), and nonappendicitis sepsis (NAS). We used multivariate regression analysis to evaluate the seven target proteins, followed by decision tree and heat mapping analyses for all proteins evaluated. Results 185 children were included: 83 with NAAP, 79 AA, and 23 NAS. Plasma levels of IL-6, CRP, MCP-1, PCT, and SAA were significantly different in children with AA compared to those with NAAP (p < 0.001). Expansive proteomic analysis demonstrated 6 patterns in inflammatory mediator profiles based on severity of illness. A decision tree incorporating the proteins CRP, ferritin, SAA, regulated on activation normal T-cell expressed and secreted (RANTES), monokine induced by gamma interferon (MIG), and PCT demonstrated excellent specificity (0.920) and negative predictive value (0.882) for children with appendicitis. Conclusions Multiplex proteomic analyses described the inflammatory landscape of children presenting to the ED with suspected appendicitis. We have demonstrated the feasibility of this approach to identify potential novel candidate cytokines/chemokine patterns associated with a specific illness (appendicitis) amongst those with a broad ED presentation (abdominal pain). This approach can be modelled for future research initiatives in pediatric emergency medicine.

Glycosaminoglycan from Apostichopus japonicus induces immunomodulatory activity in cyclophosphamide-treated mice and in macrophages.

This study was designed to systematically elucidate the immunomodulation effect of glycosaminoglycan from Apostichopus japonicus (AHG) in cyclophosphamide (CY)-induced immunosuppression model and potential mechanism responsible for the activation of macrophages. The results showed that the treatment with AHG could increase natural killer (NK) cell cytotoxicity, carbon clearance and marker enzymes activities in CY-induced immunosuppression mice, indicating that the innate immunity experienced recovery to some extent. Moreover, CY-induced reductions in thymus and spleen indices, serum levels of cytokines, immunoglobulins and hemolysin, as well as the ratio of spleen lymphocyte subsets were recovered by AHG, suggesting that AHG could improve the adaptive immunity through cellular immunity and humoral immunity. Delightedly, it was found that AHG at 10 mg/kg body weight could restore the CY-induced immunosuppression in mice to normal level on both innate and adaptive immunity. Furthermore, AHG also promoted both the expression of NO, TNF-α, IL-6, IL-1β, IL-18 and MCP-1 protein and related mRNA in macrophages. It was revealed that AHG activated macrophages through the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor-B (NF-κB). In conclusion, AHG exerts remarkable immunomodulatory activities in both innate and adaptive immune system. These findings should have great value for further study on the immunopotentiating mechanisms of this biomacromolecule.

Effect of nanoformulated copper/zinc superoxide dismutase on chronic ethanol-induced alterations in liver and adipose tissue.

We previously reported that nanoformulated copper/zinc superoxide dismutase (Nano) attenuates non-alcoholic fatty liver disease and adipose tissue (AT) inflammation in obese animals. Here, we sought to determine whether Nano treatment attenuates alcohol-associated liver disease (AALD) and AT inflammation in alcohol-fed mice. We pre-treated E-47cells (HepG2 cells that over-express CYP2E1) with native- or nano-superoxide dismutase (SOD) for 6h, followed by treatment with ethanol and/or linoleic acid (LA), a free fatty acid. For invivo studies, male C57BL/6 mice were fed the Lieber-DeCarli control or ethanol liquid diet for 4 weeks. The mice received Nano once every 2 days during the last 2 weeks of ethanol feeding. Our invitro studies revealed that Nano pretreatment reduced LA+ethanol-induced oxidative stress in E-47cells. Our invivo experiments showed that ethanol-fed Nano-treated mice had 22% lower hepatic triglyceride levels than mice fed ethanol alone. Nano-treated ethanol-fed mice also had 2-fold lower levels of Cd68 and similarly reduced levels of Ccl2 and Mmp12 mRNAs, than in untreated ethanol-fed mice. We also noted that ethanol feeding caused a remarkable increase in hepatic and/or plasma MCP-1 and CCR2 protein, which was blunted in ethanol+Nano-treated animals. The hepatic content of SREBP-1c, a transcription factor that promotes lipogenesis, was higher in ethanol-fed mice than controls but was attenuated in ethanol+Nano-treated animals. Further, livers of ethanol+Nano-treated mice had significantly higher levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) than both control and ethanol-fed mice. In AT, the levels of Il6 mRNA, a hepatoprotective cytokine, and that of Arg1, a marker of anti-inflammatory macrophages, were significantly increased in ethanol+Nano-treated mice compared with control mice. Our data indicate that Nano treatment attenuates ethanol-induced steatohepatitis and that this effect is associated with an apparent activation of AMPK signaling. Our data also suggest that Nano induces Arg1 and Il6 expression in AT, suggesting anti-inflammatory effects in this tissue.

THE IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH THE TOLL-LIKE RECEPTORS

The present study aims to evaluate the effect of pre percutaneous coronary intervention (PCI) atorvastatin reload on peripheral monocyte of toll-like receptor 2 and toll like receptor 4 expression and their correlation with cardiac injury markers (troponin I and creatine kinase Muscle brain isoenzyme) in a patient with stable angina. A double-blind randomized prospective trial in which 60 stable angina patients scheduled for an elective PCI were randomly allocated into two groups after, control group: 30patients who received low dose atorvastatin 40 mg daily without reload. Atorvastatin reloads group: 30 patients who were already on the usual dose of atorvastatin with further 80 mg and 40 mg at 12 & 2 hrs. before PCI, respectively. TLR2 and TLR4 levels were assayed in peripheral monocyte by flow cytometry and cardiac troponin I, CK-MB, MCP-1 and HMG-box-1 protein were also measured before and at 4 hrs, 12 hrs after PCI. Stent implantation was associated with an elevation in TLR2 and TLR4 expression in peripheral monocyte in both study groups after stenting but significantly higher expression level was observed among the control group (05.0P) at 4 hr and 12 hr post PCI. Inflammatory cytokine (HMG Box-1 protein MCP-1) were remarkably elevated after stenting in both.study groups (005.0P) but significantly higher in control group as compared to the atorvastatin reload group (05.0P) also myocardial injury markers (CKMB, troponin I) were significantly higherin control group than atorvastatin reload group (05.0P).there is a positive correlation between TLR2 and TLR4 expression with serum level of cardiac troponin I and CK-MBAtorvastatin reload before coronary artery interventions attenuate toll-like receptor 2 and 4 expressions on peripheral monocyte and significantly reduce serum level of HMG-box-1protein, MCP-1 and cardiac injury markers (CK_MB and cardiac troponin I) and Increase peripheral monocyte expression of TLR2 and TLR4 associated with elevated serum level of cardiac injury markers (CK_MB and cardiac troponin I). How to Cite this Article Pubmed Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy. IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE RECEPTORS. SRP. 2019; 10(2): 53-59. doi:10.5530/srp.2019.2.10 Web Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy. IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE RECEPTORS. http://www.sysrevpharm.org/?mno=302644929 [Access: March 31, 2021]. doi:10.5530/srp.2019.2.10 AMA (American Medical Association) Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy. IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE RECEPTORS. SRP. 2019; 10(2): 53-59. doi:10.5530/srp.2019.2.10 Vancouver/ICMJE Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy. IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE RECEPTORS. SRP. (2019), [cited March 31, 2021]; 10(2): 53-59. doi:10.5530/srp.2019.2.10 Harvard Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy (2019) IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE RECEPTORS. SRP, 10 (2), 53-59. doi:10.5530/srp.2019.2.10 Turabian Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy. 2019. IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE Reviews in Pharmacy, 10 (2), 53-59. doi:10.5530/srp.2019.2.10 Chicago Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy. THE IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE RECEPTORS. Systematic Reviews in Pharmacy 10 (2019), 53-59. doi:10.5530/srp.2019.2.10 MLA (The Modern Language Association) Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy. THE IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE RECEPTORS. Systematic Reviews in Pharmacy 10.2 (2019), 53-59. Print. doi:10.5530/srp.2019.2.10 APA (American Psychological Association) Style Najah R. Hadi, Alaa Ghazi, Khalid I. Amber, Sahar A.Majeed, Hayder A. Al-Aubaidy (2019) IMPACT OF ATORVASTATIN RELOAD IN PATIENTS UNDERGOING PERCUTANEOUS CORONARY INTERVENTION AND ITS CORRELATION WITH TOLL-LIKE Reviews in Pharmacy, 10 (2), 53-59. doi:10.5530/srp.2019.2.10

Celastrol Ameliorates Inflammation in Human Retinal Pigment Epithelial Cells by Suppressing NF-κB Signaling.

Celastrol is a triterpenoid quinine methide that exerts important biological effects on a variety of disease models. In this study, we aim to assess the ability of celastrol to inhibit lipopolysaccharide (LPS)-induced inflammation in retinal pigment epithelial (RPE) cells. Primary cultures of human RPE (HRPE) cells and ARPE-19 cell lines were treated with celastrol alone or in combination with LPS. The cytotoxic effect of celastrol on RPE cells was determined by the CCK-8 assay. Protein and mRNA levels of inflammatory cytokines, including IL-6, IL-8, and MCP-1, were detected by flow cytometry or by real-time fluorescent quantitative PCR, respectively. The levels of phosphorylated intermediates in the NF-κB signaling pathway (such as IκBα/β and p65) and MAPK signaling pathway (p38MAPK, SAPK/JNK, and p42/p44MAPK) were detected by western blotting. Celastrol significantly inhibited LPS-induced expression of protein and mRNA expression levels encoding the proinflammatory cytokines, IL-6, IL-8, and MCP-1, in both HRPE and ARPE-19 cells. Cell viability and apoptosis assays revealed that celastrol had no apparent cytotoxic effect and it inhibited apoptosis of RPE cells at concentrations of less than 1 μM. Mechanistically, RPE cells that were pretreated with celastrol exhibited a substantial decrease in phosphorylation of the NF-κB pathway regulators, IKKα/β and IκBα, and subsequently inactivated P65, suggesting that celastrol ameliorates LPS-induced inflammation by suppressing the NF-κB signaling pathway. Our results provide evidence that celastrol is a potent anti-inflammatory agent in RPE cells and it may have potential applications in prevention and treatment of age-related macular degeneration.

Effects of ATP on expression of inflammatory factors in endothelial progenitor cells induced by LPS and the mechanisms

To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms. Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway. Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05). Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.

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.

Therapeutic Effect of the Substrate-Selective COX-2 Inhibitor IMMA in the Animal Model of Chronic Constriction Injury.

Enhancement of endocannabinoid signaling has emerged as an attractive strategy for the treatment of pain. In addition to the well-characterized hydrolytic pathways, cyclooxygenase-2 (COX-2) mediated oxygenation is thought to be an alternative route for endocannabinoid metabolism and therefore provides a new avenue for drug intervention. In this study, we examined the therapeutic effect of indomethacin morpholinamide (IMMA), a novel substrate-selective COX-2 inhibitor, in the chronic constriction injury (CCI) mouse model. Treatment with IMMA significantly alleviated hyperalgesia and mechanical allodynia demonstrated by increased thermal withdrawal latency in Hargreaves test and tactile thresholds in Von Frey test. Accumulation of astrocytes and microglia in spinal cord dorsal horn and infiltration of macrophages into the dorsal root ganglion and sciatic nerve were reduced by drug treatment. Co-administration of the CB2 receptor antagonist, but not the CB1 receptor antagonist partially reversed the inhibitory effect of IMMA on pain sensitivity and inflammatory infiltrates. IMMA downregulated the mRNA expression of TNF-α and IL-1β and the production of IL-6 and MCP-1 proteins in the ipsilateral sciatic nerve. The enhanced NF-κB DNA binding activity in the CCI mouse dorsal spinal cord was also significantly reduced, suggesting that inactivation of NF-κB contributes to the anti-inflammatory property of IMMA. However, different from the previous reports showing that IMMA can increase endocannabinoids without interfering with arachidonic acid metabolism, treatment with IMMA failed to elevate the endogenous levels of AEA and 2-AG, but significantly reduced the production of prostaglandin E2 (PGE2). Furthermore, the mRNA expression of enzymes involved in PGE2 production, COX-2 and prostaglandin E synthase 2 in the ipsilateral sciatic nerve was also suppressed by IMMA treatment. Taken together, these results suggested that IMMA might exert anti-nociceptive effects through multiple mechanisms which include, but are not limited to, CB2 receptor activation and reduced PGE2 production.