Monocyte Chemoattractant Protein-1 mRNA Research Articles

Short-Chain Fatty Acids Suppress MCP-1 and Cellular Aging Induced by Lipopolysaccharides under High-Concentration Glucose

Background: High glucose levels, as observed in diabetic patients, enhances the inflammatory response of Porphyromonas gingivalis-Lipopolysaccharide (Pg-LPS) in various cell types and may thus worsen atherosclerosis and other inflammatory diseases. Short-Chain Fatty Acids (SCFAs) have been suggested to defend against inflammation and atherosclerosis, but, it is unknown whether SCFAs also affect aging, which accelerates atherosclerosis. We investigated the inhibitory effects of SCFAs on an aging marker, i.e., telomere length, and an atherogenic factor, Monocyte Chemoattractant Protein-1 (MCP-1), in Human Umbilical Vein Endothelial Cells (HUVECs) exposed to Pg-LPS under high glucose. Methods: We treated HUVECs with the three SCFAs acetic acid, propionic acid and butyric acid and then stimulated the cells with 0.1 µg/mL Pg-LPS + high (20 mM) glucose. We then measured the MCP-1 mRNA expression and telomere length by real-time RT-PCR. Results: Acetic acid or propionic acid at ≥0.5 mM and butyric acid at ≥0.25 mM suppressed the enhanced expression of MCP-1 mRNA induced by Pg-LPS + high glucose to the level in unstimulated HUVECs. The NF-κB activation, which promotes the transcription of MCP-1 mRNA, and MCP-1 protein production enhanced by Pg-LPS + high glucose were suppressed by acetic acid. The telomere length of HUVECs exposed to Pg-LPS + high glucose was significantly decreased by 30% compared to that in unstimulated HUVECs. Acetic acid or propionic acid at 1 mM and butyric acid 0.5 mM restored the telomere length to levels comparable to those in unstimulated HUVECs. Conclusion: SCFAs may reduce the atherogenic risk and aging risk of periodontal disease in individuals with diabetes mellitus.

Group IVA Phospholipase A2 in Collagen-Producing Cells Promotes High-Fat Diet-Induced Infiltration of Inflammatory Cells into the Liver by Upregulating the Expression of MCP-1.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic inflammation and fibrosis due to excessive fat accumulation. Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that infiltrates inflammatory cells into the liver during the development of NASH. Our previous studies demonstrated that a systemic deficiency of group IVA phospholipase A2 (IVA-PLA2), an enzyme that contributes to the production of lipid inflammatory mediators, protects mice against high-fat diet-induced hepatic fibrosis and markedly suppresses the CCl4-induced expression of MCP-1 in the liver. However, it remains unclear which cell types harboring IVA-PLA2 are involved in the elevated production of MCP-1. Hence, the present study assessed the types of cells responsible for IVA-PLA2-mediated production of MCP-1 using cultured hepatic stellate cells, endothelial cells, macrophages, and hepatocytes, as well as cell-type specific IVA-PLA2 deficient mice fed a high-fat diet. A relatively specific inhibitor of IVA-PLA2 markedly suppressed the expression of MCP-1 mRNA in cultured hepatic stellate cells, but the suppression of MCP-1 expression was partial in endothelial cells and not observed in monocytes/macrophages or hepatocytes. In contrast, a deficiency of IVA-PLA2 in collagen-producing cells (hepatic stellate cells), but not in other types of cells, reduced the high-fat diet-induced expression of MCP-1 and inflammatory cell infiltration in the liver. Our results suggest that IVA-PLA2 in hepatic stellate cells is critical for hepatic inflammation in the high-fat diet-induced development of NASH. This supports a potential therapeutic approach for NASH using a IVA-PLA2 inhibitor targeting hepatic stellate cells.

Inhibitory Effects of Eicosapentaenoic Acid on Vascular Endothelial Growth Factor-Induced Monocyte Chemoattractant Protein-1, Interleukin-6, and Interleukin-8 in Human Vascular Endothelial Cells.

Vascular endothelial growth factor (VEGF) induces monocyte chemoattractant protein-1 (MCP-1) and plays an important role in vascular inflammation and atherosclerosis. We investigated the mechanisms of VEGF-induced MCP-1 expression and the effects of eicosapentaenoic acid (EPA) in human umbilical vein endothelial cells (HUVECs). Real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that VEGF enhanced MCP-1 gene expression and protein secretion in HUVECs. Western immunoblot analysis revealed that VEGF induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor (NF)-κB (IκB). Treatment with pharmacological inhibitors of p38 MAPK (SB203580) or NF-κB (BAY11-7085) significantly suppressed VEGF-induced MCP-1 in HUVECs. EPA inhibited VEGF-induced MCP-1 mRNA, protein secretion, phosphorylation of p38 MAPK, and the translocation of phospho-p65 to the nucleus. Additionally, VEGF also stimulated gene expressions of interleukin (IL)-6 and IL-8, which were suppressed by SB203580, BAY11-7085, and EPA. The present study has demonstrated that VEGF-induced activation of MCP-1, IL-6, and IL-8 involves the p38 MAPK and NF-κB signaling pathways and that EPA inhibits VEGF-induced MCP-1, IL-6, and IL-8 via suppressing these signaling pathways. This study supports EPA as a beneficial anti-inflammatory and anti-atherogenic drug to reduce the VEGF-induced activation of proinflammatory cytokine and chemokines.

Inhibition of Experimental Corneal Neovascularization by the Tight Junction Protein ZO-1.

Purpose: To explore the effects of the tight junction protein zonula occludens 1 (ZO-1) on experimental corneal neovascularization (CNV). Methods: CNV models were established in the left eyes of BALB/c mice using NaOH. Anti-ZO-1 neutralizing antibody was topically applied to the burnt corneas after modeling thrice a day for 1 week. CD31 expression was analyzed to calculate the ratio of CNV number to area using a corneal whole-mount fluorescent immunohistochemical assay. Messenger ribonucleic acid (mRNA) and protein expression levels of ZO-1, vascular endothelial growth factor (VEGF), interleukin (IL)-1β, IL-6, IL-8, IL-18, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), phosphorylated protein kinase C (pPKC), and clusterin in burned corneas were detected by reverse transcriptase polymerase chain reaction (PCR) and western blot analyses. Infiltration of neutrophils, macrophages, and progenitor cells was examined by flow cytometry. Results: CNV was obviously greater in 45 s than in 15 s alkali injury group. In another experiment, CNV was obviously greater in the ZO-1 antibody group than in the vehicle-treated group. Corneal mRNA and protein expression levels of VEGF, IL-1β, IL-6, IL-8, IL-18, and MCP-1 were significantly higher in the ZO-1 antibody group than in the control group. Infiltration of neutrophils, macrophages, and progenitor cells was significantly greater in the ZO-1 antibody group than in the control group. TNF-α expression was much higher in 45 s than in 15 s alkali injury group. However, protein expression of pPKC and clusterin was much lower in 45 s than in 15 s alkali injury group. Conclusions: Anti-ZO-1 neutralizing antibody-treated mice exhibited enhanced alkali-induced CNV through enhanced intracorneal infiltration of progenitor and inflammatory cells.

Epstein-Barr Virus Promotes Inflammatory Cytokine Production in Human Gingival Fibroblasts

BackgroundPeriodontitis is one of the most common chronic oral inflammatory diseases. Over the past decade, herpes viruses, particularly Epstein-Barr virus (EBV), have been considered promising pathogenic candidates for periodontitis. However, the specific mechanism by which EBV contributes to the development of periodontitis is still unknown. This study aimed to explore the mechanism of EBV underlying the inflammatory response in human gingival fibroblasts (HGFs). Materials and methodsHGFs were stimulated with different concentrations of EBV (104, 105, 106, 107, and 108 DNA copies/mL) for 0, 8, 24, or 48 hours. The mRNA levels of interleukin (IL)-1β, tumour necrosis factor-α (TNF-α), IL-8, monocyte chemoattractant protein-1 (MCP-1), and Toll-like receptor 9 (TLR9) were measured using quantitative real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assays (ELISAs) were performed for determining the mRNA and protein levels of IL-1β, TNF-α, IL-8, and MCP-1. Real-time PCR and ELISA were performed to determine the protein levels of IL-1β, TNF-α, IL-8, and MCP-1. Activation of the TLR9/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway was evaluated using western blotting. ResultsThe expressions of IL-1β, TNF-α, IL-8, and MCP-1 were significantly upregulated in HGFs under EBV stimulation in a concentration- and time-dependent manner. EBV promoted TLR9 and MyD88 expression and induced NF-κB transcription. On the contrary, the upregulation of these factors and the activation of NF-κB pathway were drastically inhibited by TLR9 antagonists. ConclusionsOur findings demonstrate that EBV promotes the production of inflammatory cytokines IL-1β and TNF-α and chemokines IL-8 and MCP-1 in HGFs through the TLR9/MyD88/NF-κB pathway.

Effect of Flavonoids on MCP-1 Expression in Human Coronary Artery Endothelial Cells and Impact on MCP-1-Dependent Migration of Human Monocytes.

The monocyte chemoattractant protein-1 (MCP-1), also known as chemokine (CC motif) ligand 2 (CCL2), is involved in the formation, progression, and destabilization of atheromatous plaques. Flavonoids, found in fruits and vegetables, have been associated with various health-promoting properties, including antioxidant, anti-inflammatory, and cardioprotective effects. In the present study, the flavonoids quercetin, kaempferol, and luteolin, but not cannflavin A, were shown to substantially inhibit interleukin (IL)-1β-induced MCP-1 mRNA and protein expression in human coronary artery endothelial cells (HCAEC). At the functional level, conditioned medium (CM) from IL-1β-stimulated HCAEC caused an increase in the migration of THP-1 monocytes compared with CM from unstimulated HCAEC. However, this induction was suppressed when IL-1β-treated HCAEC were coincubated with quercetin, kaempferol, or luteolin. The functional importance of MCP-1 in IL-1β-induced monocyte migration was supported by experiments showing that neutralization of MCP-1 in the CM of IL-1β-treated HCAEC led to a significant inhibition of migration. In addition, a concentration-dependent induction of monocyte migration in the presence of recombinant MCP-1 was demonstrated. Collectively, the flavonoids quercetin, kaempferol, and luteolin were found to exert potential antiatherogenic effects in HCAEC, challenging further studies with these compounds.

Tetramethylpyrazine inhibits keratitis and neovascularization induced by corneal alkali burn by suppressing the TLR4/NF-κB pathway activation and NLRP1/NLRP3 inflammasomes in rats

The role and related mechanisms of tetramethylpyrazine (TMP) in corneal alkali burn in rats were expected to be explored in this article. After construction of corneal alkali burn rat models, TMP eye drops were given four times daily for consecutive 7 days. H&E staining was utilized for observing the histopathological changes of corneas on the 3rd and 7th days of treatment; immunohistochemistry for detecting the Nestin protein expression changes; qRT-PCR for determining the expression changes of genes correlated with neovascularization [C-X-C Motif Chemokine Ligand 1 (CXCL-1), vascular endothelial growth factor A (VEGFA) and CD31] and inflammation-related factors [monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α), and IL-6]; Western blot for testing NLR Family Pyrin Domain Containing 1 (NLRP1)/NLRP3 inflammasomes and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway-related protein expression changes. All above trials were completed based on rat corneal tissue. TMP ameliorated the pathological damage in alkali-burned rat corneal tissue. Specifically, TMP treatment decreased Nestin-positive cell expression and the CXCL-1, VEGFA and CD31 mRNA expression in alkali-burned rat corneal tissue dose-dependently. TMP also down-regulated the IL-1β, TNF-α, MCP-1 and IL-6 mRNA expression and inhibited the NLRP1, caspase-1, NLRP3, pro-IL-1β and mature IL-1β protein expression in the alkali-burned rat corneal tissue. In addition, TMP treatment down-regulated the myeloid differentiation factor 88 (MyD88) and TLR4 protein expression and decreased the p–NF–κB p65/NF-κB p65 ratio in the alkali-burned rat corneal tissue. The mechanism of TMP relieving the inflammatory response and inhibiting neovascularization caused by corneal alkali burn in rats might have a correlation with the suppression of acitivation of TLR4/NF-κB pathway and NLRP1/NLRP3 inflammasomes in rats.

Ectodomain Shedding by ADAM17 Increases the Release of Soluble CD40 from Human Endothelial Cells under Pro-Inflammatory Conditions.

Homozygosity for the C allele of the -1T>C single nucleotide polymorphism (SNP) of the CD40 gene (rs1883832) is associated with susceptibility to coronary heart disease (CHD), enhanced CD40 expression, and shedding. The disintegrin metalloprotease ADAM17 can cleave various cell surface proteins. This study investigates an association between ADAM17-mediated CD40 shedding and inflammation in CC genotype human endothelial cells. Human umbilical vein endothelial cells (HUVEC) carrying the CC genotype were stimulated with soluble CD40 ligand (sCD40L) or tumor necrosis factor-α (TNFα). Messenger RNA and protein expression were determined with standard methods. Levels of high sensitive c-reactive protein (hs-CRP), interleukin-6 (IL-6), and sCD40 in plasma samples from patients with CHD were assessed using ELISA. ADAM17 surface abundance was elevated following stimulation with CD40L and TNFα just as its regulator iRhom2. Inhibition of ADAM17 prevented TNFα-induced sCD40 and soluble vascular cell adhesion molecule-1 release into the conditioned medium and reinforced CD40 surface abundance. Secondary to inhibition of ADAM17, stimulation with CD40L or TNFα upregulated monocyte chemoattractant protein-1 mRNA and protein. Levels of sCD40 and the inflammatory biomarkers hs-CRP and IL-6 were positively correlated in the plasma of patients with CHD. We provide a mechanism by which membrane-bound CD40 is shed from the endothelial cell surface by ADAM17, boosting sCD40 formation and limiting downstream CD40 signaling. Soluble CD40 may represent a robust biomarker for CHD, especially in conjunction with homozygosity for the C allele of the -1T>C SNP of the CD40 gene.

The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting monocyte chemoattractant protein 1 (MCP1). However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, the N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regulated MCP1 expression in MSCs through the m6A modification. Specifically, the expression of METTL16 in MSCs decreased gradually and was negatively correlated with the expression of MCP1 after coculture with monocytes. Knocking down METTL16 markedly enhanced MCP1 expression and the ability to recruit monocytes. Mechanistically, knocking down METTL16 decreased MCP1 mRNA degradation, which was mediated by the m6A reader YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2). We further revealed that YTHDF2 specifically recognized m6A sites on MCP1 mRNA in the CDS region and thus negatively regulated MCP1 expression. Moreover, an in vivo assay showed that MSCs transfected with METTL16 siRNA showed greater ability to recruit monocytes. These findings reveal a potential mechanism by which the m6A methylase METTL16 regulates MCP1 expression through YTHDF2-mediated mRNA degradation and suggest a potential strategy to manipulate MCP1 expression in MSCs.

Oral contraceptive pill (OCP) treatment alters the gene expression of intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) in polycystic ovary syndrome (PCOS) women compared to drug-naive PCOS women

BackgroundPolycystic ovary syndrome (PCOS) presents clinical symptoms of menstrual abnormalities, excessive hair growth (hirsutism), scalp hair loss, acne and infertility. Metabolic abnormalities such as obesity, insulin resistance, glucose intolerance and cardiovascular problems constitute an essential part of PCOS, all of which can have significant long-term health consequences. Low-grade chronic inflammation demonstrated by persistent moderately elevated serum levels of inflammatory and coagulatory markers plays a critical role in the pathogenesis of PCOS. Oral contraceptive pills (OCPs) constitute the mainstay of pharmacologic therapy for women with PCOS to regularize cyclicity and ameliorate androgen excess. On the other hand, OCP use is associated with various venous thromboembolic and proinflammatory events in the general population. PCOS women always carriers the increased lifetime risk of these events. The studies on the effect of OCPs on inflammatory, coagulation and metabolic parameters in PCOS are less robust. Therefore in this study, we investigated and compared the messenger RNA (mRNA) expression profiles of genes implicated in inflammatory and coagulation pathways between drug-naive and OCP-treated PCOS women. The selected genes include intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1). Furthermore, the correlation between the selected markers and various metabolic indices in the OCP group has also been explored.MethodThe relative amounts of ICAM-1, TNF-α, MCP-1 and PAI-1 mRNA in peripheral blood mononuclear cells from 25 drug-naive PCOS subjects (controls) and 25 PCOS subjects who received OCPs containing 0.03 mg-ethinyl-estradiol and 0.15 mg-levonorgestrel for at least six months (cases) were estimated using real-time qPCR. The statistical interpretation was conducted using SPSS version 20.0 (SPSS, Inc, Chicago, IL), Epi Info version 2002 (Disease Control and Prevention Centres, Atlanta, GA) and GraphPad Prism 5 (GraphPad Software, La Jolla, CA) software.ResultSix months of OCP therapy enhanced the expression of inflammatory genes viz ICAM-1, TNF-α and MCP-1 mRNA in PCOS women by 2.54, 2.05 and 1.74 folds, respectively, in this study. However, PAI-1 mRNA in the OCP group showed no significant increase. Furthermore, in cases, ICAM-1 mRNA expression positively correlated with body mass index (BMI) (p = 0.01), fasting insulin (p = 0.01), insulin 2 h p = 0.02), glucose 2 h (p = 0.01) and triglycerides (p = 0.01). TNF-α mRNA expression positively correlated with fasting insulin (p = 0.0007). MCP-1 mRNA expression positively correlated with (BMI) (p = 0.002).ConclusionOCPs helped reduce clinical hyperandrogenism and regularise menstrual cycles in women with PCOS. However, OCP use was associated with increased fold expression of inflammatory markers which positively correlated with metabolic abnormalities.

The Effect of TGFβ1 in Adipocyte on Inflammatory and Fibrotic Markers at Different Stages of Adipocyte Differentiation.

Transforming growth factor beta (TGFβ) is a versatile cytokine. Although a profibrotic role of TGFβ is well established, its effect on tissue inhibitor of metalloproteinase (TIMPs) and inflammatory mediators are incompletely described. This study investigates the profibrotic and pro-inflammatory role of TGFβ1 during adipocyte differentiation. NIH3T3L1 cells were used for the in vitro study and were differentiated by adding a standard differentiation mix either with rosiglitazone (R-Diff) or without (S-Diff). Recombinant TGFβ1 (2 ng/mL) was added to the undifferentiated preadipocyte during the commitment stage and at the terminal differentiation stage. TGFβ1 treatment significantly decreased adiponectin mRNA at both early commitment (>300 fold) and terminal differentiated cells [S-Diff (~33%) or R-Diff (~20%)]. TGFβ1 upregulated collagen VI mRNA and its regulators connective tissue growth factor (CCN2/CTGF), TIMP1 and TIMP3 mRNA levels in undifferentiated preadipocytes and adipocytes at commitment stage. But in the terminal differentiated adipocytes, changes in mRNA and protein of collagen VI and TIMP3 mRNA were not observed despite an increase in CCN2/CTGF, TIMP1 mRNA. Although TGFβ1 upregulated interleukin-6 (IL6) and monocyte chemoattractant protein-1 (MCP1) mRNA at all stages of differentiation, decreased tumor necrosis factor-α (TNFα) mRNA was observed early in adipocyte differentiation. This study highlights the complex role of TGFβ1 on extracellular matrix (ECM) remodeling and inflammatory markers in stimulating both synthetic and inhibitory markers of fibrosis at different stages of adipocyte differentiation.

Role of adipose tissue-derived cytokines in the progression of inflammatory breast cancer in patients with obesity

BackgroundInflammatory breast cancer (IBC) represents a deadly aggressive phenotype of breast cancer (BC) with a unique clinicopathological presentation and low survival rate. In fact, obesity represents an important risk factor for BC. Although several studies have identified different cellular-derived and molecular factors involved in IBC progression, the role of adipocytes remains unclear. Cancer-associated adipose tissue (CAAT) expresses a variety of adipokines, which contribute to tumorigenesis and the regulation of cancer stem cell (CSC). This research investigated the potential effect of the secretome of CAAT explants from patients with BC on the progression and metastasis of the disease.MethodsThis study established an ex-vivo culture of CAAT excised from IBC (n = 13) vs. non-IBC (n = 31) patients with obesity and profiled their secretome using a cytokine antibody array. Furthermore, the quantitative PCR (qPCR) methodology was used to validate the levels of predominant cytokines at the transcript level after culture in a medium conditioned by CAAT. Moreover, the impact of the CAAT secretome on the expression of epithelial-mesenchymal transition (EMT) and cells with stem cell (CSC) markers was studied in the non-IBC MDA-MB-231 and the IBC SUM-149 cell lines. The statistical differences between variables were evaluated using the chi-squared test and unpaired a Student’s t-test.ResultsThe results of cytokine array profiling revealed an overall significantly higher level of a panel of 28 cytokines secreted by the CAAT ex-vivo culture from IBC patients with obesity compared to those with non-IBC. Of note, interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemo-attractant protein 1 (MCP-1) were the major adipokines secreted by the CAAT IBC patients with obesity. Moreover, the qPCR results indicated a significant upregulation of the IL-6, IL-8, and MCP-1 mRNAs in CAAT ex-vivo culture of patients with IBC vs. those with non-IBC. Intriguingly, a qPCR data analysis showed that the CAAT secretome secretions from patients with non-IBC downregulated the mRNA levels of the CD24 CSC marker and of the epithelial marker E-cadherin in the non-IBC cell line. By contrast, E-cadherin was upregulated in the SUM-149 cell.ConclusionsThis study identified the overexpression of IL-6, IL-8, and MCP-1 as prognostic markers of CAAT from patients with IBC but not from those with non-IBC ; moreover, their upregulation might be associated with IBC aggressiveness via the regulation of CSC and EMT markers. This study proposed that targeting IL-6, IL-8, and MCP-1 may represent a therapeutic option that should be considered in the treatment of patients with IBC.

Scutellarin alleviates lipopolysaccharide-induced renal injury via mediating cysteine-rich protein 61-connective tissue growth factor-nephroblastoma overexpressed gene 1 expression to inhibit nuclear factor-κB signaling pathway

To explore the protective effect and mechanism of scutellarin (Scu) on sepsis associated-acute kidney injury (SA-AKI). (1) In vivo experiment: 36 male C57BL/6 mice were divided into normal saline (NS) control group, lipopolysaccharide (LPS) induced SA-AKI model group (LPS group), 20 mg/kg Scu control group (Scu 20 control group), and 5, 10, 20 mg/kg Scu pretreatment groups by random number table with 6 mice in each group. The SA-AKI model was reproduced by intraperitoneal injection of 10 mg/kg LPS. The NS control group was injected with NS intraperitoneally. The Scu pretreatment groups were intraperitoneally injected with different doses of Scu every day before LPS injection for 1 week. Scu 20 control group was injected with 20 mg/kg Scu for 1 week. After 24 hours of LPS treatment, mice in each group were sacrificed, kidney tissues were collected, and kidney injury was detected by hematoxylin-eosin (HE) staining. Western blotting was used to detect the protein expression levels of nuclear factor-κB (NF-κB) signaling pathway related molecules, apoptosis-related proteins and cysteine-rich protein 61-connective tissue growth factor-nephroblastoma overexpressed gene 1 (CCN1). (2) In vitro experiment: human renal tubular epithelial cell line HK-2 was cultured in vitro and used for experiment when the cells fused to 80%. In the cells without LPS treatment and after 100 g/L LPS treatment, pcDNA3.1-CCN1 and small interfering RNA (siRNA) CCN1 sequence were transfected to overexpress and inhibit CCN1 expression, respectively, to observe whether CCN1 was involved in NF-κB signaling pathway activation and apoptosis. In addition, 100g/L LPS and 20 μmol/L Scu were added into HK-2 cells transfected with and without CCN1 siRNA to investigate the mechanism of protective effect of Scu on LPS-induced HK-2 cells injury. (1) The results of in vivo experiment: the renal function of SA-AKI mice induced by LPS was significantly decreased, and had kidney histological damage and severely damaged renal tubules. Scu could alleviate renal function and histological damage in a dose-dependent manner. Western blotting results showed Scu could reduce the protein expression of NF-κB signaling pathway related molecules and CCN1 in the renal tissue, and had a significant alleviating effect on apoptosis, indicating that CCN1 was involved in NF-κB signaling pathway activation and apoptosis. (2) The results of in vitro experiment: in HK-2 cells not treated with LPS, CCN1 overexpression had no effect on apoptosis related protein and pro-inflammatory factors of NF-κB signaling pathway. In HK-2 cells treated with LPS, overexpression of CCN1 significantly inhibited the mRNA expressions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1), with significant differences as compared with cells stimulated only by LPS [IL-1β mRNA (2-ΔΔCT): 3.20±0.57 vs. 4.88±0.69, TNF-α mRNA (2-ΔΔCT): 2.99±0.44 vs. 5.00±0.81, MCP-1 mRNA (2-ΔΔCT): 2.81±0.50 vs. 5.41±0.75, all P < 0.05], and the apoptosis-related protein was significantly down-regulated. However, when siRNA was used to inhibit the expression of CCN1, the mRNA expressions of pro-inflammatory factors were significantly increased as compared with cells stimulated only by LPS [IL-1β mRNA (2-ΔΔCT): 6.01±1.13 vs. 4.88±0.69, TNF-α mRNA (2-ΔΔCT): 5.15±0.86 vs. 5.00±0.81, all P < 0.05], and apoptosis-related protein was significantly up-regulated. In the LPS-induced HK-2 cells, the mRNA expressions of pro-inflammatory factors were significantly down-regulated after Scu treatment as compared with cells stimulated only by LPS [IL-1β mRNA(2-ΔΔCT): 2.55±0.50 vs. 6.15±1.04, TNF-α mRNA (2-ΔΔCT): 2.58±0.40 vs. 3.95±0.52, MCP-1 mRNA (2-ΔΔCT): 2.64±0.44 vs. 6.21±0.96, all P < 0.05], and apoptosis-related protein was also significantly reduced. When the expression of CCN1 was inhibited by siRNA, the protective effect of Scu on cells was weakened, which showed that the mRNA expressions of pro-inflammatory factors in cells was significantly up-regulated compared with the cells without inhibition of CCN1 expression [IL-1β mRNA (2-ΔΔCT): 5.34±0.76 vs. 2.55±0.50, TNF-α mRNA (2-ΔΔCT): 3.66±0.54 vs. 2.58±0.40, MCP-1 mRNA (2-ΔΔCT): 5.15±0.79 vs. 2.64±0.44, all P < 0.05], and the expression of apoptosis related protein was also significantly up-regulated. Scu could protect the renal function in SA-AKI mice, and the protective effect is associated with NF-κB signaling pathway and CCN1. Thus, Scu could alleviate LPS-induced kidney injury by regulating the NF-κB signaling pathway.

Antiatherosclerotic effects of corilagin via suppression of the LOX-1/MyD88/NF-κB signaling pathway in vivo and in vitro.

Corilagin, a natural polyphenol compound isolated from Phyllanthus urinaria L., exerts various pharmacological effects, such as antihyperglycemic, antitumor, and antioxidative stress properties, but the mechanisms underlying the antiatherosclerotic effects of corilagin have not been entirely elucidated. In the present study, we investigated the antiatherosclerotic effects of corilagin using a high-fat diet (HFD)-induced atherosclerotic rabbit model and ox-LDL-induced vascular smooth muscle cells (VSMCs) and explored the underlying molecular mechanisms. The serum lipid levels were measured through an enzymatic colorimetric assay. A histological analysis of rabbit aortas was performed after hematoxylin-eosin and oil red O staining. The proliferation of ox-LDL-induced VSMCs was detected using MTT assays, and the migration of cells was determined by wound scratch assays. In addition, the mRNA and protein expression levels of lectin-like ox-LDL receptor-1 (LOX-1), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays. Our results indicate that corilagin significantly reduced the serum levels of TC, TG and LDL-C, increased the HDL-C levels, decreased the intimal thickening in the thoracic aorta, and reduced the formation of foam cells in an HFD-induced rabbit atherosclerosis model. Moreover, corilagin suppressed the proliferation and migration of ox-LDL-induced VSMCs and reduced LOX-1, MyD88, NF-κB, MCP-1, and TNF-α mRNA and protein expression in vivo and in vitro. These data demonstrate that corilagin exerts antiatherosclerotic effects in vivo and in vitro and that the mechanisms may be closely associated with downregulation of the LOX-1/MyD88/NF-κB pathway.

Total flavonoids from Lagerstroemia speciosa (L.) Pers inhibits TNF-α-induced insulin resistance and inflammatory response in 3T3-L1 adipocytes via MAPK and NF-кB signaling pathways

Adipocyte tissue inflammation orchestrates the occurrence and development of diseases induced by obesity. Here, the effects of Lagerstroemia speciosa (L.) Pers total flavonoids (LTF) on TNF-α-induced insulin resistance (IR) and inflammatory response in 3T3-L1 adipocytes and underlying mechanisms were studied. Cell glucose uptake test confirmed that TNF-α could induce IR in 3T3-L1 adipocytes. The sensitivity of 3T3-L1 adipocytes to insulin was increased by LTF treatment. The phosphorylation of IRS1 and Akt in TNF-α-treated 3T3-L1 adipocytes was markedly restored by LTF treatment. The results revealed that interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) mRNA expression was elevated in TNF-α-administered 3T3-L1 adipocytes, whereas the mRNA expression of adiponectin (ADPN) was considerably decreased. When treated with 50 μg/mL LTF, the IL-6 and MCP-1 mRNA expression levels were drastically reduced, and the ADPN mRNA expression level was significantly raised. Moreover, the concentration of IL-6 in the supernatant of the medium was decreased after LTF treatment compared with the model group. Western blotting results showed that the phosphorylation of p38, JNK, and ERK of mitogen-activated protein kinase (MAPK) signaling pathways and IкB of NF-кB signaling pathway were inhibited. It suggested that LTF improves TNF-α-induced IR and inflammatory response in 3T3-L1 adipocytes via inhibiting MAPK and NF-кB signaling pathways.

Plasma SiOx:H Nanocoatings to Enhance the Antibacterial and Anti-Inflammatory Properties of Biomaterials.

PurposeTo evaluate the antibacterial and anti-inflammatory properties of SiOx:H nanocoatings using a plasma-deposition technique.Materials and MethodsFour groups of SiOx:H nanocoatings were prepared by plasma nanocoating technique using different deposition gases and durations, specifically trimethylsilane (TMS) for groups A1 and A2 and a mixture of TMS and oxygen for groups B1 and B2. Changes in surface chemistry and physical properties were measured. Staphylococcus aureus and Streptococcus mutans were cultured on plasma SiOx:H nanocoatings to evaluate antibacterial and antibiofilm formation activities. Human gingival fibroblasts (HGFs) and HaCaT human keratinocytes were cultured and stimulated with tumor necrosis factor-α (TNF-α). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to evaluate anti-inflammatory properties, including the mRNA and protein levels of inflammatory mediators and proinflammatory cytokines.ResultsThe carbon content was dominant in group A nanocoatings and the oxygen and silicon elements were dominant in group B nanocoatings. Groups A2 and B2 were approximately threefold thicker than groups A1 and B1. The plasma SiOx:H nanocoatings decreased bacterial growth and biofilm formation by 30–70% (p < 0.05). Scanning electron microscopy (SEM) revealed damaged biofilm structures. Moreover, the antibacterial properties of group B were greater than group A, and the antibacterial properties of groups A2 and B2 were more effective than A1 and B1, respectively. CCK-8 assays revealed the plasma SiOx:H nanocoatings had good biocompatibility. Furthermore, under TNF-α-induced inflammation, the mRNA and protein levels of interleukin-6, interleukin-8, cyclooxygenase-2, and monocyte chemoattractant protein-1 were downregulated in the plasma SiOx:H nanocoating groups (p < 0.05).ConclusionPlasma SiOx:H nanocoatings exerted antibacterial and anti-inflammatory effects with excellent biocompatibility. Therefore, the plasma SiOx:H nanocoating technique has potential for implant materials and other medical devices.

Immunomodulatory effects of Inonotus obliquus polysaccharide on splenic lymphocytes infected with Toxoplasma gondii via NF-κB and MAPKs pathways

Context As a medicinal and edible fungus, Inonotus obliquus has been traditionally used to prevent and treat various ailments. Inonotus obliquus polysaccharide (IOP) isolated from I. obliquus processes many biological activities, our series of in vivo studies have shown that IOP protects against Toxoplasma gondii infection. Objective This study aimed to investigate the in vitro immunomodulatory effects and its mechanisms of IOP on mouse splenic lymphocytes infected with T. gondii. Materials and methods Mouse splenic lymphocytes were infected with T. gondii tachyzoites, and treated with different concentrations of IOP. The levels of cytokines and chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The expression of toll-like receptor 2 (TLR2) and TLR4, and the modulation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways were determined by Western blot. Results IOP significantly decreased the over-release of cytokine interleukin-1 beta (IL-1β), IL-4, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in supernatant from T. gondii-infected splenic lymphocytes. IOP also effectively inhibited the overexpression of cytokines and chemokine macrophage inflammatory protein-1 (MIP-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA. Furthermore, IOP down-regulated TLR2 and TLR4 expressions and inhibited the over-phosphorylation of NF-κB p65 and inhibitor κBα (IκBα) in NF-κB signaling pathway and p38, c-Jun N-terminal kinase (JNK) in MAPKs signaling pathway. By observing the effect of IOP on TNF-α secretion after pretreatment with specific inhibitors, it was further confirmed that IOP was involved in the regulation of NF-κB, p38, and JNK signaling pathways. Conclusions These data indicate that IOP can inhibit the excessive inflammatory response caused by T. gondii infection through modulating NF-κB, p38, and JNK signaling pathways, and thus plays the in vitro anti-T. gondii role.

Potentilla discolor ameliorates LPS-induced inflammatory responses through suppressing NF-κB and AP-1 pathways

Potentilla discolor Bunge (PD) is a traditional Chinese medicine which has been widely used for the treatment of various inflammatory diseases (e.g., diarrhea, fever and furuncle). However, few studies focused on its effect on classical inflammation. This study aimed to investigate the anti-inflammatory effect and potential mechanism of the ethanol extract of the whole herbs of PD (EPD) in lipopolysaccharide (LPS)-induced inflammatory models. The obtained results showed that EPD decreased supernatant NO, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in LPS-activated RAW264.7 cells and mouse peritoneal macrophages. Moreover, its effect on NO was attributed to the suppression of iNOS expression rather than its activity. At the transcriptional level, EPD suppressed iNOS, TNF-α and MCP-1 mRNA expressions in LPS-stimulated RAW264.7 cells. Further study showed that EPD didn’t affect the phosphorylation and degradation of IκBα, but yet impeded the nuclear translocation of p65 to inhibit NF-κB activation. Meanwhile, it also prevented JNK, ERK1/2 and p38 phosphorylation to dampen the activation of AP-1. In endotoxemia mouse model, EPD not only decreased interleukin-6, TNF-α and MCP-1 levels in serum, but also potently ameliorated diarrhea. These findings provide the theoretical basis for PD to treat inflammatory diseases, especially intestinal inflammation.

Monocyte chemotactic protein-1 concentrations and expression of women with endometriosis undergoing IVF cycles

To research the local action of endometriosis on follicular inflammatory microenvironment of patients undergoing in vitro fertilization (IVF) cycles. A prospective case control study was organized at Kamal AL-Samarai IVF center (Baghdad/Iraq), January 2021 - May 2021. The study included (80) infertile women undergoing IVF cycles were split to two groups: Patients with endometriosis group (n = 40) and Patients without endometriosis group (n = 40). Follicular fluid (FF) and blood samples were gathered at the period of oocyte pick-up. Levels of MCP-1 mRNA were investigated with the reverse transcription polymerase chain reaction (RT-PCR) technique. Concentrations of cytokines (monocyte chemo attractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10)) were investigated in FF by sandwich enzyme-linked immunosorbent assay (ELISA). The mRNA expression of MCP-1 was significantly (P = 0.00) higher in patients with endometriosis group (1.95 ± 0.69) compared with patients without endometriosis group (0.40 ± 0.15). The studied cytokines concentrations in FF of women with endometriosis group were significantly (P = 0.00) higher than those without endometriosis group: MCP-1 (548.07 ± 199.43 pg/ml vs. 45.98 ± 18.72 pg/ml), TNF-α (912.15 ± 316.12 pg/ml vs. 261.89 ± 90.69 pg/ml), IL-10 (258.94 ± 68.82 pg/ml vs. 52.12 ± 14.46 pg/ml). The immune milieu of FF of patients with endometriosis is modified, which could explain the effect of endometriosis on infertility. Furthermore, elevated MCP-1 expression in endometriosis patients suggesting systemic inflammatory changes.

Impairment of Whole Kidney Blood Flow Autoregulation in the Transition from Acute to Chronic Kidney Disease in Ischemia‐Reperfusion Rats

Efficient autoregulation of renal blood flow (RBF) is an essential mechanism of renal microcirculation to protect against glomerular injury. Patients surviving an episode of acute kidney injury (AKI) develops chronic kidney disease (CKD). Renal ischemia-reperfusion (IR) is a major cause of AKI and triggers inflammation and reactive oxygen species (ROS) accumulation. We hypothesized that IR‑induced inflammation and ROS accumulation leads to a persistent reduction of RBF and impairment of RBF autoregulation contributing to CKD progression. Whole kidney RBF autoregulation was assessed in vivo in Sprague-Dawley male IR induced by bilateral arterial ischemia for 60 minutes followed by 24 hours, 1-week and 4‑weeks of reperfusion. Plasma creatinine increased in IR at 24 hours (3.9±0.2 vs. 1.0±0.1 mg/dl in sham, P<0.05, n=6-7/group) and week 1 (1.4±0.1 vs. 1.1±0.0 mg/dl in sham, P<0.05, n=7-8/group). Systolic blood pressure was similar between sham and IR over the 4 weeks post-IR (113-135 mmHg). IR reduced baseline RBF (5.8±0.5 vs. 9.1±0.5 ml/min.gkw in sham, P<0.05, n=6/group) at 24 hours and further decreased RBF at week 1 (3.7±0.7 vs. 9.1±1.1 ml/min.gkw in sham, n=5-6/group). At week 4, RBF was still significantly low in IR (6.9±0.4 vs. 10.4±0.3 ml/min.gkw in sham, P<0.05, n=6/group). RBF remained stable during step reduction of renal perfusion pressure (RPP) from 120 to 100 mmHg in all shams with autoregulatory index (AI) less than 0.2, indicating intact autoregulation. RBF autoregulation, however, was completely impaired in IR at 24 hours, week 1 and week 4 as RBF passively decreased during reduction of RPP, yielding AI of 0.8±0.2, 1.1±0.2 and 0.9±0.3, respectively. Preglomerular microvessels (PGMV) were collected for mRNA measurement (n=6-8/group). IR elevated mRNA expression of p67phox 7‑fold at 24 hours and 2‑fold at week 1 (P<0.05 vs. respective shams). IR also elevated gp91phox mRNA expression 5-fold at 24 hours and 2-fold at week 1 (P<0.05 vs. shams). IR tended to elevate p47phox mRNA expression at 24 hours and reached a significance at week 1 (2-fold, P<0.05 vs. sham). Inflammatory mediators, monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1), were also assessed in PGMV. MCP-1 mRNA expression was increased in IR with a peak of 10-fold at 24 hours and then decreased at week 1 but still elevated compared to shams (2-fold, P<0.05). VCAM-1 mRNA expression remained unchanged at 24 hours but increased 2-fold at week 1 (P<0.05). We also assessed profibrotic markers (TGF-β, collagen IV and fibronectin). TGF-β mRNA expression started to increase in IR at 24 hours (2-fold) and reached a significance at week 1 (3-fold, P<0.05 vs. sham) and further increased at week 4 (4-fold). IR also increased mRNA expression of collagen IV 5-fold at week 1 and 4-fold at week 4 (P<0.05). Fibronectin mRNA expression was also increased in IR 4-fold at week 1 and remained at the same level at week 4 (P<0.05). In conclusion, these results demonstrate that IR leads to a persistent reduction of RBF and impairment of RBF autoregulation in male rats and suggest that renal hemodynamic dysfunction might be associated with activation of NADPH oxidase, MCP-1 and TGF-β in PGMV of IR.