Gene Expression Levels Of IL-6 Research Articles

Azilsartan Suppressed LPS-Induced Inflammation in U937 Macrophages through Suppressing Oxidative Stress and Inhibiting the TLR2/MyD88 Signal Pathway.

Background and purpose: Lipopolysaccharide (LPS) is an important factor that induce severe inflammation, resulting in multiple types of diseases. It is reported that LPS-induced inflammation is related to the activation of the NF-κB signal pathway and reactive oxygen species (ROS)-induced oxidative stress. Azilsartan, an angiotensin II type 1 (AT1) receptor blocker, has been licensed as a new generation of Sartan antihypertensive drugs. However, the effects of azilsartan in LPS-induced inflammation have not been reported before. The present study aims to investigate the anti-inflammatory effects of azilsartan on LPS-stimulated macrophages and explore the underlying mechanism. Methods: The release of lactic dehydrogenase (LDH), secretion of HMGB-1, and concentrations of IL-6, IL-1β, MCP-1, MMP-2, MMP-9, and PGE2 were evaluated using the enzyme-linked immunosorbent assay (ELISA). The gene expression levels of IL-6, IL-1β, MCP-1, MMP-2, MMP-9, and COX-2 were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Western blot analysis was used to detect the protein expression level of COX-2, Nrf2, TLR2, MyD-88, and NF-κB. The level of ROS was determined using the dihydroethidium (DHE) staining assay. The activity of NF-κB was evaluated using the luciferase activity assay. Results: The release of LDH, HMGB-1, IL-6, IL-1β, MCP-1, MMP-2, MMP-9, and PGE2 was significantly promoted by LPS stimulation, whereas it was greatly suppressed by azilsartan. The upregulated COX-2, TLR2, MyD-88, and NF-κB in the LPS-treated macrophages were significantly downregulated by azilsartan. Interestingly, the expression level of Nrf2 was elevated by azilsartan. On the contrary, ROS levels were greatly increased by LPS but suppressed by azilsartan. Mechanistically, it was found that azilsartan suppressed LPS-induced activation of the TLR2/Myd-88/NF-κB signaling pathway. Conclusion: Azilsartan might suppress LPS-induced inflammation in U937 macrophages through suppressing oxidative stress and inhibiting the TLR/MyD88 signal pathway.

Ferrostatin-1 alleviates angiotensin II (Ang II)- induced inflammation and ferroptosis in astrocytes

Background and purposeInflammation and ferroptosis in astrocytes can be induced by external injuries, which results in excessive production of inflammatory factors and further injury on neurons. Alleviating ferroptosis might be an effective way to protect the brain from external injuries. The present study aims to explore the protective effects of Ferrostatin-1 against ferroptosis induced by Angiotensin II and the underlying mechanism. MethodsThe mouse primary astrocytes were isolated from the cortices of mice. The astrocytes were stimulated using 10 µM angiotensin II in the presence or absence of 1 or 2 μM Ferrostatin-1. The gene expression levels of AT1R, IL-6, IL-1β, COX-2, GFAP, and GPx4 were evaluated using qRT-PCR. Western Blot was used to determine the protein levels of AT1R, COX-2, GFAP, GPx4, Nrf2, and HO-1 and ELISA was used to detect the concentrations of IL-6, IL-1β, and PGE2. The ROS levels were evaluated using DHE staining and the reduced GSH level was determined using GSH detection kits. ResultsThe expression levels of AT1R, IL-6, IL-1β, COX-2, and GFAP in the astrocytes were significantly elevated by stimulation with Ang II and greatly suppressed by the introduction of Ferrostatin-1 in a dose-dependent manner. The promoted ROS level and inhibited GSH level in the astrocytes by the stimulation with Ang II were significantly reversed by Ferrostatin-1. Down-regulated GPx4, Nrf2, and HO-1 in the astrocytes induced by Ang II were extremely up-regulated by the treatment of Ferrostatin-1 in a dose-dependent manner. ConclusionFerrostatin-1 alleviates angiotensin II (Ang II)- induced inflammation and ferroptosis by suppressing the ROS levels and activating the Nrf2/HO-1 signaling pathway.

Establishment of inflammatory model of bovine mammary epithelial cells induced by lipoteichoic acid

The mammary gland of the cow is particularly susceptible to infections of a wide range of pathogenic bacteria, including both Gram-positive and Gram-negative bacteria. The endotoxins of these pathogenic bacteria include peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS), and they are the pathogen-associated molecular patterns (PAMPs) to induce mastitis. Cow mastitis is a detrimental factor in dairy farming industry. Lipoteichoic acid (LTA) is the main component of Staphylococcus aureus cell wall and the key cytotoxic factor causing inflammation. The aims of our work was to establish inflammatory model of study procedures were approved by the Animal Care and Use Committee of the Sumy National Agricultural University, Sumy, Ukraine, and the Henan Institute of Science and Technology, Xinxiang, China, and performed in accordance with the animal welfare and ethics guidelines.
 The BMECs harvested from mid-lactation dairy cow milk were isolated by our laboratory. Briefly, the base medium for this cell is DMEM/F-12 (Gibco, USA, cat.12400-024). The complete growth medium included 10% fetal bovine serum (Biological Industries, Israel, cat.04-011-1A/B), DMEM/F-12, and 10 ng/mL epidermal growth factor (Sigma, USA, cat. E4127). Cells were maintained at 37℃in an incubator containing 5% CO2. When cells grew to 80% confluency, the cells were rinsed twice with PBS, and then the primary mammary epithelial cells were trypsinized with 0.25% trypsin plus 0.02% EDTA and passaged. In this study, one inflammatory bovine mammary epithelial cell (BMEC) model was established by infecting the cells with LTA. The BMEC viability induced by LTA were evaluated. The expressions of pro-inflammatory cytokines (TNF-α and IL-6) were measured by ELISA and RT- qPCR. The results showed that the treatment of BMECs with LTA at 20 ng/μL for 24 h obviously improved TNF-α and IL-6 protein and gene expression levels. The establishment of the model will play an important role in the screening of anti-inflammatory drugs and the study of the mechanism of action in the future.

Immunomodulatory effects of Nd:YAG (1064 nm) and diode laser (810 nm) wavelengths to LPS-challenged human gingival fibroblasts

ObjectiveHuman gingival fibroblasts (hGFs) are involved in inflammatory responses to bacteria by recognizing pathogen-associated molecular patterns. In search of host modulation strategies to increase LPS tolerance, Low level laser therapy (LLLT) has been suggested as an alternative treatment that reduces periodontal tissue inflammation. In this study, we investigate whether 810 nm (diode) and 1064 nm (Nd:YAG) laser wavelengths, modulate pro-inflammatory responses to LPS challenges in hGFs. DesignPrimary hGFs were challenged with Porphyromonas gingivalis LPS and irradiated with either Diode (810 nm) or with Nd:YAG (1064 nm) lasers. Cell cultures were examined for cell proliferation by MTT assay and IL-6 and IL-8 expression by qPCR at 24, 48 and 72 h. IL-6 and IL-8 protein levels were detected via ELISA. ResultsNaïve hGF populations irradiated with both Diode 810 nm and Nd:YAG 1064 nm lasers demonstrated cellular proliferation (p < 0.05), but LLLT did not affect cellular viability in LPS-challenged cells. IL-6 and IL-8 gene expression levels revealed significant anti-inflammatory effects of irradiation with both examined wavelengths on hGFs challenged with P. gingivalis LPS. Protein levels of these cytokines were increased by LPS challenge. Treatment with LLLT inhibited this increase for both wavelengths evaluated in the study at a statistically significant level particularly for the first 48 h. ConclusionsThe present study demonstrates a modulatory effect of LLLT using both 810 nm diode and Nd:YAG 1064 nm lasers in gingival fibroblasts by decreasing the production of IL-6, IL-8 in response to LPS.

Ginsenoside Rg3 alleviates inflammation in a rat model of myocardial infarction via the SIRT1/NF-κB pathway.

Inflammation serves an important role in myocardial infarction (MI). Ginsenoside Rg3 (Rg3), an activator of sirtuin 1 (SIRT1), has been identified to elicit anti-inflammatory effects via the NF-κB pathway. However, the function of Rg3 in MI remains unknown. In the present study, a MI rat model was established by coronary artery ligation and treated with Rg3 to explore whether Rg3 could inhibit inflammation in MI rats by inhibiting the SIRT1/NF-κB pathway. At 28 days post-MI, it was identified that Rg3 not only decreased the ST-segment ECG values in MI rats, but also significantly decreased serum LDH, CK-MB and cTnI levels in MI rats. In addition, Rg3 also significantly decreased serum tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 levels and increased serum IL-10 levels in MI rats. In the heart tissues of the MI rats, Rg3 attenuated myocardial pathological changes and cell apoptosis caused by MI, decreased the gene expression levels of TNF-α, IL-1β and IL-6, but increased the gene expression level of IL-10. In addition, the expression levels of the SIRT1 and transcription factor RelB proteins were significantly increased following Rg3 treatment, and the expression level of p-p65/p65 protein was significantly decreased in the heart tissues of MI rats with Rg3 treatment compared with that in heart tissues of MI rats without Rg3 treatment. In conclusion, Rg3 alleviates inflammation in a rat model of MI via the SIRT1/NF-κB pathway.

Kartogenin mediates cartilage regeneration by stimulating the IL-6/Stat3-dependent proliferation of cartilage stem/progenitor cells

A decrease in the number of endogenous stem cells in cartilage is regarded as the cause of cartilage degeneration. Kartogenin (KGN) is known to induce chondrogenesis of cartilage stem/progenitor cells (CSPCs). Using CSPCs isolated from rat cartilage, we analysed changes in the transcriptome after treatment with KGN in vitro. An animal model of destabilization of the medial meniscus (DMM) was then used to identify the effect of intra-articular (IA) KGN injection on CSPC proliferation in vivo. Here, we demonstrated that KGN promoted the proliferation of CSPCs isolated from cartilage. The percentage of G2-M phase cells in the KGN-treated group reached over 10%, nearly twice that in the control group. Transcriptomic profiling of rat CSPCs revealed significant changes in KGN-treated samples compared to control samples. The gene expression levels of IL-6 and its coreceptor Gp130 were much higher in the KGN-treated group than in the control group. Phosphorylation of the IL-6 downstream molecule Stat3 was enhanced via KGN stimulation. The DMM animal model showed increased articular cartilage thickness after IA KGN injection. IHC staining also demonstrated upregulation of Stat3 phosphorylation and enhanced distribution of CD44+/CD105+ cells in cartilage following IA KGN injection. Thus, our data suggested that KGN promoted cartilage regeneration at least partially by stimulating IL-6/Stat3-dependent proliferation.

Upregulation of TNF-α and IL-6 induces preterm premature rupture of membranes by activation of ADAMTS-9 in embryonic membrane cells

AimTo investigate the role of thrombospondin motifs 9 (ADAMTS9) in preterm premature rupture of membranes (pPROM). Materials and methodsADAMTS9 levels were measured in amnion cells from 24 patients of different groups (preterm vs. full-term birth, with vs. without PROM). ADAMTS9 was suppressed in human amnioblasts to investigate its effects on embryonic membrane cells and inflammation-induced cell damage. Pregnant mouse models were used to assess whether inflammation regulates ADAMTS9 by upregulating TNF-α and IL-6, contributing to the preterm birth occurrence. Key findingsWe found that ADAMTS9 protein and gene expression levels significantly differed among various groups (pPROM > full-term PROM > preterm non-PROM > full-term non-PROM). After ADAMTS9 suppression in human amnioblast WISH cells, TNF-α- and IL-6-induced apoptosis was decreased. In addition, TNF-α, IL-6, and ADAMTS9 protein and gene expression levels were increased in the embryos of mice treated with LPS compared with controls. In agreement, the rate of preterm birth was higher in the LPS group compared with controls. SignificanceTaken together, these in vitro and in vivo findings suggest that TNF-α and IL-6 secreted by macrophages during inflammation regulate ADAMTS9 and induce pPROM.

Chemopreventive Effects of Cranberry Extracts on Colitis-Associated Colon Cancer

Abstract Objectives Colon cancer is the third most common cancer in the U.S., and inflammatory bowel disease (IBD) patients are at a greater risk of developing colon carcinoma. Cranberries (Vaccinium macrocarpon) are a commonly grown crop in North America which contain constituents relevant to human health, including flavonoids, phenolics and terpenoids. Recently, we have demonstrated the chemopreventive effects of whole cranberry fruits against colitis and colon cancer in murine models. However, the secondary metabolites in cranberry fruit could play different contributions against colon carcinogenesis. Thus, comparing nonpolar (ECE) and polar extracts (DPE) of cranberries to determine their efficacy on colon carcinogenesis is desired. Methods Selected polyphenols and other constituents were determined using established methods: quantitative 1H NMR methods by Bruker Assure-RMS for triterpenoids, HPLC-DAD for anthocyanins and flavonol glycosides and proanthocyanidins by DMAC assay. Over half of ECE was made up of triterpenes and sterols and trace amount of proanthocyanidins. Over 70% of DPE was polyphenol and trace amount of triterpenes. Higher free-radical scavenging activity was found in DPE than ECE. Results Next, we determined the chemopreventive effects of ECE and DPE extracts against colitis-associated colon cancer in azoxymethane (AOM) and dextran sulfate sodium (DSS)-treated mice. It was observed that oral administration of ECE and DPE extracts at physiologically achievable doses significantly suppressed the development of colitis-associated colon cancer, evidenced by decreased tumor incidence, multiplicity and burden, compared to the control group, while minimum side-effect was observed. ECE treatment also reversed the shortening of colon caused by colitis. qRT-PCR results showed that gene expression levels of proinflammatory cytokines IL-1, IL-6 and TNF-α were significantly attenuated by ECE and DPE interventions, in comparison to the control group. Conclusions In conclusion, our findings demonstrate that the novel cranberry extracts may offer an efficacious, safe and inexpensive means to prevent colonic tumorigenesis in humans, particularly for individuals with chronic inflammation. Funding Sources USDA-NIFA, Leo and Anne Albert Charitable Trust, UMass Cranberry Health Research Center/UMass President's S&amp;T Initiative, and NIH.

Extra-Virgin Olive Oil from Apulian Cultivars and Intestinal Inflammation.

Inflammatory bowel disease (IBD) is a multifactorial intestinal disorder characterized by chronic intestinal inflammation. The etiology of IBD is still unclear, although genetic, environmental and host factors have been associated to the disease. Extra-virgin olive oil (EVO) is a central component of the Mediterranean diet and it decreases chronic inflammation by interfering with arachidonic acid and NF-κB signaling pathways. Specifically, the different components of EVO are able to confer advantages in terms of health in their site of action. For instance, oleic acid displays a protective effect in liver dysfunction and gut inflammation, whereas phenolic compounds protect colon cells against oxidative damage and improve the symptoms of chronic inflammation in IBD. Given the biological properties of EVO, we investigated whether its administration is able to confer protection in a mouse model of dextrane sodium sulfate (DSS)-induced colitis. Four EVO cultivars from the Apulian Region of Italy, namely Ogliarola (Cima di Bitonto), Coratina, Peranzana and Cima di Mola, respectively, were used. Administration of EVO resulted in reduced body weight loss in our colitis model. Furthermore, mice treated with Ogliarola, Coratina and Cima di Mola EVO displayed a reduction of rectal bleeding and IL-1β, TGFβ, IL-6 gene expression levels. Furthermore, Ogliarola, Coratina and Peranzana EVO administration ameliorated intestinal permeability and histopathological features of inflammation. Our data further validate the well-known positive effects of EVO supplementation in promoting human health and suggest the bona fide contribution of EVO in preventing onset and reducing progression of intestinal inflammation.

Investigation of IL-10 Gene Expression and rs1554286 SNP in Behçet's Disease

Objective: Behcet's disease (BD) is a chronic, recurrent and inflammatory disorder characterized by oral and genital aphthous ulcerations, uveitis, skin lesions and skin pathergy reaction. There are many genetic studies focused on determining BD etiology. In this study, we aimed to investigate IL-10 gene expression and rs1554286 single nucleotide polymorphism (SNP) which is located in IL-10 gene. Material and Method: In this study, the blood specimens of BD patients (n=45) and healthy controls (HC) (n=28) were collected for RNA and DNA extraction. We genotyped rs1554286 from the DNA samples (45 BD, 28 HC) using real time PCR. We identified the IL-10 gene expression levels from cDNA samples of patients with sufficient sample amounts (29 patients with BD and 23 HCs). Results: We compared the expression levels of IL-10 gene and also genotyping analysis, and no significant difference was observed between BD patients and HC. Conclusion: In our study, which compared IL-10 gene expression and SNP genotyping between Behcet patients and healthy controls, no difference was found between the two groups. Since this may have occurred due to the low number of samples in our study, studies with higher sample numbers are needed.

Association Between EphA1 and Tumor Microenvironment in Gastric Carcinoma and its Clinical Significance.

BackgroundWith the growing global burden of gastric carcinoma (GC) and the urgent need for biomolecular targeted therapies, this study aimed to elucidate the relationship between EphA1 and the tumor microenvironment (focusing primarily on the key inflammatory cytokines IL-6 and tumor angiogenic cytokine VEGF) to identify a new potential therapeutic target.Material/MethodsIHC and qRT-PCR were performed to quantify the protein and gene expression levels of EphA1, IL-6, and VEGF in normal mucosal tissues, carcinoma tissues, and paracarcinomatous tissues from 57 GC patients. Spearman’s rank correlation test was performed to determine the relationship between EphA1, IL-6, and VEGF expression levels. The relationships of EphA1 with clinicopathologic parameter and survival in GC patients were also evaluated.ResultsThe protein and gene expression levels of EphA1 were all attenuated gradually from carcinoma tissues to paracarcinomatous tissues and then to normal mucosal tissues in GC patients. Additionally, significant correlations between the overexpression of EphA1 with aggressive clinicopathological features and shorter survival time of GC patients were verified. In particular, we found a significant positive correlation between the expression of EphA1 and tumor microenvironment hallmark proteins IL-6 and VEGF in carcinoma tissues and paracarcinomatous tissues.ConclusionsEphA1 can promote the occurrence and development of GC by its selective high expression in cancer tissues and its relationship with malignant clinical features and prognosis of GC patients. The underlying potential mechanism appears to involve enhancement of the tumor microenvironment, which via drives the expression of tumor microenvironment hallmark proteins IL-6 and VEGF.

Androgen Suppresses Hyperosmolarity-Induced Inflammatory Mediators in Human Corneal Epithelial Cells.

To investigative the effects of sex steroids on hyperosmolar stress-induced proinflammatory cytokine expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8, and IL-6, and on the mitogen-activated protein kinase pathway in immortalized human corneal epithelial cells (hCECs). Immortalized hCECs were cultured with keratinocyte-free medium until reaching 80% confluency with either 10 M dihidrotestosteron (DHT) or 10 M 17-β-estradiol, and then, the medium was changed to hyperosmolar for various time points. After hyperosmolar treatment, a real-time polymerase chain reaction was performed to show the TNF-α, IL-8, and IL-6 gene expression levels in hCECs. In addition, the treated cells were lysed, and Western blot analysis was applied for phosphorylated and nonphosphorylated forms of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2). hCECs viability was measured with Annexin V/propidium iodide. Pretreatment with 10 M DHT or 17-β-estradiol inhibited the high osmolarity-induced expression of TNF-α, IL-8, and IL-6. The upregulation of p-ERK, p-JNK, and p-p38 with high osmolarity was inhibited partially by DHT, but 17-β-estradiol pretreatment only affected p-p38 for a short time interval. In addition, DHT increased cell viability of hCECs under hyperosmolar conditions. Our results demonstrated that DHT and 17-β-estradiol decreased the proinflammatory cytokine gene expression levels which were stimulated by high osmolarity in immortalized hCECs. The mitogen-activated protein kinase signaling pathway is partially involved in the regulatory effects of DHT on hCECs. These findings may contribute to the etiologic role and therapeutic implications of sex steroids in certain ocular surface diseases.

Anti-Inflammatory Effects of Potassium Iodide on SDS-Induced Murine Skin Inflammation

Potassium iodide (KI), initially derived from seaweed in the early 19th century, is used for treating sporotrichosis in dermatological practice. KI has also been used to treat several noninfectious inflammatory skin diseases. However, the mechanisms underlying the improvement in such skin diseases remain unknown, and KI is not used widely. Thus, although KI is an old drug, physicians may not prescribe it frequently because they lack knowledge about it. Although KI is very inexpensive and causes few side effects, it has been superseded by new powerful and expensive drugs, such as biological agents. We applied 3% KI topically to areas of inflammation induced by SDS in mice. The levels of IL-1 and TNF-α gene expression were reduced, whereas that of IL-10 gene expression was increased. Small interfering RNA that was designed to reduce IL-10 gene expression levels was injected into the same mice, and the anti-inflammatory effects of KI were not observed. Thus, the pharmacologic action of KI is based on its anti-inflammatory effects caused by the increase in IL-10 levels. This information would increase dermatologists' awareness of KI as an efficacious and cost-effective treatment.

Cellular sources of interleukin-6 and associations with clinical phenotypes and outcomes in pulmonary arterial hypertension

The pro-inflammatory cytokine interleukin (IL)-6 has been associated with outcomes in small pulmonary arterial hypertension (PAH) cohorts composed largely of patients with severe idiopathic PAH (IPAH). It is unclear whether IL-6 is a marker of critical illness or a mechanistic biomarker of pulmonary vascular remodelling. We hypothesised that IL-6 is produced by pulmonary vascular cells and sought to explore IL-6 associations with phenotypes and outcomes across diverse subtypes in a large PAH cohort.IL-6 protein and gene expression levels were measured in cultured pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) from PAH patients and healthy controls. Serum IL-6 was measured in 2017 well-characterised PAH subjects representing each PAH subgroup. Relationships between IL-6 levels, clinical variables, and mortality were analysed using regression models.Significantly higher IL-6 protein and gene expression levels were produced by PASMCs than by PAECs in PAH (p<0.001), while there was no difference in IL-6 between cell types in controls. Serum IL-6 was highest in PAH related to portal hypertension and connective tissue diseases (CTD-PAH). In multivariable modelling, serum IL-6 was associated with survival in the overall cohort (hazard ratio 1.22, 95% CI 1.08-1.38; p<0.01) and in IPAH, but not in CTD-PAH. IL-6 remained associated with survival in low-risk subgroups of subjects with mild disease.IL-6 is released from PASMCs, and circulating IL-6 is associated with specific clinical phenotypes and outcomes in various PAH subgroups, including subjects with less severe disease. IL-6 is a mechanistic biomarker, and thus a potential therapeutic target, in certain PAH subgroups.

Extracellular Vesicles Shedding Promotes Melanoma Growth in Response to Chemotherapy

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.

Dexmedetomidine inhibits the lipopolysaccharide-stimulated inflammatory response in microglia through the pathway involving TLR4 and NF-κB.

To investigate the effects of dexmedetomidine (DEX) on lipopolysaccharide (LPS)-induced neuroinflammation in BV2 microglia. BV2 microglial cells were treated with various concentrations of DEX (0, 1, 10, and 100 ng/mL) for 1 hour, and then incubated in the presence or absence of 0.1 μg/mL LPS for 24 hours. Cell viability was assessed by Cell Counting Kit-8 assays. The expression levels of IL-1β, IL-6, and TNF-α were determined using enzyme-linked immunosorbent assay (ELISA). The expressions of TLR4 and NF-кB were detected by western blotting. Moreover, BV2 microglial cells were transfected with small interfering RNA (siRNA) specific for TLR4 (si-TLR4 group) or negative control siRNA (si-NC group) for 24 hours, followed by exposing to 0.1 μg/mL LPS for 24 hours. TLR4, IL-1β, IL-6, and TNF-α expressions were detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR). There were no significant differences in cell viability with the different treatments. Compared with the control group, LPS markedly increased the release of IL-6, TNF-α, IL-1β, TLR4, and NF-κB, but these increases were significantly attenuated by pretreatment with 10 or 100 ng/mL DEX in a dose-dependent relationship, but not with 1 ng/mL DEX. Gene expression levels of IL-1β, IL-6, and TNF-α were obviously upregulated in si-NC group and si-TLR4 group when cells were exposed to 0.1 μg/mL LPS for 24 hours. Meanwhile, si-TLR4 group had significantly lower IL-1β, IL-6, and TNF-α expressions than si-NC group. The anti-inflammatory effects of DEX on LPS-induced BV2 microglia may be mediated through pathway involving TLR4 and NF-κB.

Zika Virus Induced More Severe Inflammatory Response Than Dengue Virus in Chicken Embryonic Livers

Dengue (DENV) and Zika virus (ZIKV) are important flaviviruses in tropical and subtropical regions, causing severe Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS) and microcephaly, respectively. The infection of both viruses during pregnancy were reported with adverse fetal outcomes. To investigate the effects of ZIKV and DENV infections on fetal development, we established an infection model in chicken embryos. Compared with DENV-2, the infection of ZIKV significantly retarded the development of chicken embryos. High viral loads of both DENV-2 and ZIKV was detected in brain, eye and heart 7 and 11 days post-infection, respectively. Interestingly, only ZIKV but not DENV-2 was detected in the liver. Even both of them induced apparent liver inflammation, ZIKV infection showed a more severe inflammatory response than DENV-2 infection based on the inflammation scores and the gene expression levels of IL-1β, TNF, IL-6, and TGFβ-2 in liver. Our results demonstrated that ZIKV induced more severe inflammatory response in chicken embryo liver compared to DENV-2, which might partially attribute to viral replication in liver cells. Clinicians should be aware of the potential liver injury associated with ZIKV infection in patients, especially in perinatal fetuses.

Protective Effects of Tyrosol Against DSS-Induced Ulcerative Colitis in Rats.

In this study, the effects of tyrosol were investigated in DSS-induced experimental ulcerative colitis model. For this purpose, rats were divided into five groups of seven rats in each: control group, colitis group (DSS-4%), tyrosol group (tyrosol 20mg/kg), sulfasalazine (sulfasalazine+DSS 100mg/kg), and treatment group (tyrosol+DSS 20mg/kg). In the study, the active substances were administered to all animals for a period of 21days. At the end of the study, malondialdehyde (MDA) levels increased (p < 0.001); GSH level (p < 0.05) along with GSH.Px (p < 0.01) and CAT (p < 0.001) activities decreased in the DSS-induced colitis group. However, with the administration of tyrosol, MDA and GSH levels along with GSH.Px and CAT activities came to the same levels as the control group. In the colitis group, an increase occurred in IL-6, COX-2, and NF-κB parameters, which created a significant difference compared to the control group (p < 0.001). Similarly, TNF-α levels also significantly increased with the administration of DSS (p < 0.05) which created a significant difference compared to the control group, while there was no difference among the other groups. As for the Nrf-2 data, it decreased with the administration of DSS which created a significant difference compared to the control group (p < 0.05), while there was no difference in other groups. In the colitis-induced group, IL-6, COX-2, and NF-κB gene expression levels also similarly increased but returned to the normal levels with the administration of tyrosol. In the histopathological scoring, the negativity that increased with the administration of DSS returned to the normal levels with the administration of tyrosol+DSS. In conclusion, according to the data obtained, tyrosol fixed the destruction picture in the DSS-induced colitis model, giving rise to thought that it has a protective effect.

SUN-113 Adipocyte Morphology And Cytokine Gene Expression In Adipose Tissue From Patients Before And After Weight Loss From Bariatric Surgery

Multiple studies have implicated the accumulation of adipose tissue macrophages (ATMs) in the inflammatory response in obesity. In particular, increased secretion of pro-inflammatory cytokines is thought to cause local and systemic insulin resistance in a paracrine/endocrine fashion and is associated with obesity. It has been shown that ATMs accumulate in adipose tissue of obese individuals and increase in number with increasing mass. Yet, fasting also causes increased ATM accumulation. Numerous studies have shown that bariatric surgery reduces insulin resistance, cardiovascular disease and mortality independent of weight loss, effects thought to be related to the reduction in inflammation seen after bariatric surgery. However, the finding that pro-inflammatory gene expression decreases after human weight loss has been inconsistent across multiple studies. Here we have analyzed adipose tissue biopsies from five patients before and after undergoing sleeve gastrectomy to determine adipocyte morphology, pro-inflammatory cytokine gene expression, and macrophage infiltration of adipose tissue before and after weight loss. Levels of gene expression of pro-inflammatory markers IL-6, IL-1β and TNFα from subcutaneous adipose tissue were not significantly different in subjects before and three months after undergoing bariatric surgery despite significant weight loss in all subjects. Furthermore, gene expression levels of IL-10, an anti-inflammatory cytokine did not change after weight loss. Adipocyte morphology studies showed that adipocyte size appears to decrease after weight loss which is consistent with other studies. However, macrophage infiltration by immunofluorescence persisted in adipose tissue after bariatric surgery despite improved insulin sensitivity in these tissues and weight loss. These results indicate that macrophages are not only associated with the pro-inflammatory or obese state but they may play a distinct role in the setting of obesity and also in the setting of sustained weight loss. Thus, their classification as either pro-inflammatory “M1” or anti- inflammatory “M2” may not fully capture their heterogeneity or the scope of their metabolic function. Detailed studies looking at the activation state or metabolic phenotype of infiltrating macrophages before and after weight loss are needed. Our studies will provide insights into the role of inflammatory mediators after weight loss, in particular the metabolic role of the macrophage in during obesity and after extreme weight loss.

Ribes orientale: A novel therapeutic approach targeting rheumatoid arthritis with reference to pro-inflammatory cytokines, inflammatory enzymes and anti-inflammatory cytokines

Ethnopharmacological relevanceThe roots of Ribes orientale (Family Grossulariaceae) have long been used as a folk remedy to treat rheumatism and joints pain in Northern Areas of Pakistan. Aim of the studyThe purpose of study was to observe the preventive efficacy of roots of Ribes orientale (RO) aqueous ethanolic extract (30:70) and its aqueous and n-butanol fractions in treating rheumatoid arthritis and to determine its possible mechanism of action. Material and methodsArthritis was evaluated in vitro using heat induced bovine serum albumin and egg albumin denaturation and membrane stabilizing assays at 50–6400 μg/ml concentration of extract/fractions whereas, in vivo arthritis was evaluated at 50, 100, 200 mg/kg doses of extract/fractions in formaldehyde model by measuring rat paw volume/diameter. Moreover, highest effective dose (200 mg/kg) of extract/fractions was evaluated in Freünd complete adjuvant (FCA) model. Arthritis was induced in Sprague Dawley rats by immunization with 0.1 ml FCA in left footpad. RO extract/fractions at 200 mg/kg were orally administered from day 0, 30 min prior to adjuvant injection and sustained for 28 days. Paw volume/diameter, arthritic score, body weight, and hematological (WBC, RBC, ESR, Hb and Platelet count) and biochemical (AST, ALT, ALP, urea, creatinine, CRP and RF) parameters were observed. The mRNA expression levels of COX-2, IL-1β, IL-6, NF-kB, TNF-α, IL-4 and IL-10 were measured by real time reverse transcription polymerase chain reaction (RT-PCR) whereas, PGE2 and TNF-α levels in serum samples were measured by Enzyme linked immunosorbent assay (ELISA). Furthermore, radiographs of hind paws and histological changes in ankle joint were analyzed in adjuvant injected rats. The anti-oxidant activity of plant extract and fractions was evaluated using DPPH and reducing power assays. In addition, phytochemistry, total phenolic and flavonoid contents, and HPLC analysis of most active fraction (aqueous fraction) were performed. ResultsResults showed that RO extract and fractions (notably aqueous fraction) significantly reduced protein denaturation and protected erythrocyte membrane in concentration dependent manner. Similarly, extract/fractions induced dose-dependent decrease in paw volume/diameter in the formaldehyde model. Plant extract and fractions significantly suppressed paw swelling and arthritic score, prevented cachexia and remarkably ameliorated hematological and biochemical changes. Furthermore, RO extract/fractions downregulated gene expression levels of PGE2, COX-2, IL-1β, IL-6, NF-kB and TNF-α whereas, upregulated those of IL-4 and IL-10, compared with FCA control rats. The radiographic and histopathologic improvement in joint architecture was also observed in RO treated rats. Piroxicam, used as reference drug, also significantly suppressed arthritis. Additionally, plant exhibited notable anti-oxidant activity and phytochemical analysis revealed the presence of flavonoids and polyphenols. ConclusionResults indicated that suppression of pro-inflammatory enzymes/cytokines, inhibition of protein denaturation, lysosomal membrane stabilizing abilities, and redox/free radical scavenging properties of RO extract and fractions support anti-arthritic and immunomodulatory property of Ribes orientale that might be due to its polyphenolic and flavonoid constituents. This suggests that Ribes orientale roots may be used as a therapeutic agent for treating human arthritis.