Expression Of Inflammatory Proteins Research Articles

Effects of endurance exercise on serum inflammatory cytokine level and kidney structure in a rat diabetes model

Diabete mellitus (DM) is becoming a global health problem. Whilst many studies have previously focused on the therapeutic effects of exercise on diabetes, insufficient data exist on its effectiveness on disease prevention. The present study was designed to evaluate the effects of endurance exercises on kidney injury and on the expression of metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and transforming growth factor (TGF)-β1. Type 2 diabetic rat model was created followng 8 weeks of feeding on high fat diet, followed by injection with streptozotocin (STZ; 30 mg/kg). A total of three different intensity endurance exercises, including low-intensity exercise (8 m/min and 0˚ slope), moderate-intensity exercise (15 m/min and 5˚ slope) and high-intensity exercise (20 m/min and 10˚ slope), were arranged during this process. Oral glucose tolerance (OGTT) and oral sucrose tolerance tests (OSTT) were performed in all rats 1 week after STZ injection. Serum interleukin-6 and tumor necrosis factor-α levels were measured using ELISA. After OGTT, all rats were sacrificed and kidney samples were removed for hematoxylin and eosin staining and western blot analyzes. Urea and ureatinine levels, representative of renal function, were estimated by using automatic biochemical analyzer. Rats in the DM group showed severe impaired glucose tolerance, which was alleviated in the moderate-intensity exercise (ME) and the high-intensity exercise (HE) groups. Inflammatory cytokines were also significantly reduced rats in the ME group compared with those in the DM group. No difference in renal function, MMP-9/TIMP-1 and TGF-β1 expression was observed. In addition, rats in the DM group exhibited glomerular enlargement with structural renal abnormalities, whilst those in the ME and HE groups showed improved symptoms. To conclude, no increased expression of inflammatory cytokines and renal fibrotic proteins could be observed in the present rat model of type-2 DM, but evident structural abnormalities can be observed in the kidneys. Medium-intensity endurance exercise can reduce serum inflammatory cytokine levels and prevent aberrant changes in renal structures.

Eicosapentaenoic acid modulates endothelial function and inflammatory protein expression from pulmonary and vascular tissues following cytokine challenge

Background and Aims: Endothelial cell (EC) dysfunction is characterized by reduced nitric oxide (NO) bioavailability and contributes to inflammation and atherosclerosis. Eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), reduced cardiovascular (CV) events in high-risk patients (REDUCE-IT) but the mechanisms are not fully understood. We therefore evaluated the effects of EPA on expression of proteins and NO bioavailability in both umbilical vein and pulmonary arterial ECs under conditions of inflammation.

Modic changes are associated with activation of intense inflammatory and host defense response pathways – molecular insights from proteomic analysis of human intervertebral discs

Patients with modic changes (MC) form a distinct clinical subset with reports of higher intensity of pain, poor clinical and surgical outcomes and higher incidence of recurrence. MC also is an independent risk factor for increased post-operative surgical site infection. This study aimed to investigate the biological changes at molecular level, in discs with MCs. We also aim to identify biological biomarkers and potential targets for molecular therapy. Experimental analysis MATERIALS AND METHODS: Nucleus pulposus (NP) from 24 patients undergoing microdiscectomy for disc herniation [14 discs with MC and 10 without modic changes (NMC)] were procured. The overall expression of proteins, biological processes, protein-protein and metabolite interactions were analysed and compared. Host defense response proteins (HDRPs) and immunological pathways activated in patients with MC were documented and analysed. Label-free proteomic approach with stringent filters revealed a total of 208 proteins in MC and 193 in NMC groups. 45 proteins were specific to MC; 30 to NMC and 163 common to both. Downregulated proteins in MC belonged to components of extracellular matrix such as collagens (COL- 6A1, 6A2, 6A3, 11A1, 12A1, and 20A1), and proteoglycans (versican (VCAN), and biglycan (BGN)). Inflammatory molecules [plasminogen (PLG), angiogenin (ANG), fibroblast growth factor-binding protein 2 (FGFBP2), tetranectin (CLEC3B), cartilage acidic protein 1(CRTAC1), kininogen (KNG-1), chitinase-3-like protein 2 (CHI3L2), and ferritin (FTL) were expressed only in the MC group. The significantly altered pathways in MC included Fc Fragment of IgG Receptor IIIa (FCGR3A)-mediated phagocytosis, regulation of Toll-like receptors (TLR) by endogenous ligand, neutrophil and platelet degranulation. 50 HDRPs were identified in the study, 14 of which were specific to MC and included acute phase reactants, antimicrobial peptides, complement cascade proteins, inflammatory molecule and stress response proteins. Metabolite-protein interaction analysis revealed a significant interaction between 19 proteins, specifically involving ubiquitin mediating proteasome degradative pathway and an association with the metabolite-glutamic acid in the MC group. Accumulation of glutamic acid in MC discs was confirmed by quantitative amino acid analysis using High-performance liquid chromatography. Our study confirms that MC represents an intense inflammatory status and activation of host defense response and immunological pathways. Downstream effects leading to ubiquitin mediated proteasomal degradation of ECM proteins and the resulting metabolites such as glutamic acid could cause excessive pain and needs further investigation. We have documented the expression of inflammatory molecules, immune mechanisms and host defense response proteins which throw molecular insights into the pathological mechanisms of MC. Further, ubiquitin mediated proteasomal degradation and accumulation of glutamate in discs with MC might serve as targets for molecular therapy.

The effect of FTY720 at different doses and time-points on LPS-induced acute lung injury in rats

We sought to assess the protective effect of different doses of Fingolimod (FTY720) in a rat model of acute lung injury (ALI) induced by intratracheal instillation of lipopolysaccharide (LPS) and explored the underlying mechanisms. The ALI model was established in rats and different doses of FTY720 (0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, or 2 mg/kg) were injected intraperitoneally. Lung computed tomography and blood gas analyses were performed at 6 h, 24 h, and 48 h after intraperitoneal injection, and the lung tissues were extracted to prepare paraffin sections for histopathological examination. The levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) were detected by ELISA, and the expressions of inflammatory pathway proteins in each group were measured by Western blot analysis. A single intraperitoneal injection of FTY720 inhibited LPS-induced NF-κB activation, reduced the level of inflammatory cytokines, and decreased the infiltration of inflammatory cells. Moreover, it alleviated lung tissue injury, as shown by marked attenuation of pulmonary oedema and improved arterial partial pressure of oxygen (PaO2) and the general condition of ALI rats. In conclusion, our results demonstrate the protective effect of FTY720 against LPS-induced ALI. The underlying mechanism of the protective effect may involve inhibition of LPS-induced activation of NF-κB and regulation of the inflammatory pathway to alleviate barrier dysfunction of alveolar capillaries.

Gingko biloba abrogate lead-induced neurodegeneration in mice hippocampus: involvement of NF-κB expression, myeloperoxidase activity and pro-inflammatory mediators.

Neuroimmune alterations have important implication in the neuropsychiatric symptoms and biochemical changes associated with lead-induced neurotoxicity. It has been suggested that inhibition of neuroinflammatory-mediated lead-induced neurotoxicity by phytochemicals enriched with antioxidant activities would attenuate the deleterious effects caused by lead. Hence, this study investigated the neuroinflammatory mechanism behind the effect of Ginkgo biloba supplement (GB-S) in lead-induced neurotoxicity in mice brains. Mice were intraperitoneally pretreated with lead acetate (100mg/kg) for 30min prior the administration of GB-S (10 and 20mg/kg, i.p.) and ethylenediaminetetraacetic acid (EDTA) (50mg/kg, i.p.) for 14 consecutive days. Symptoms of neurobehavioral impairment were evaluated using open field test (OFT), elevated plus maze (EPM), and tail suspension test (TST) respectively. Thereafter, mice brain hippocampi were sectioned for myeloperoxidase activity (MPO), pro-inflammatory cytokine (TNF-α and IL-6) estimation and inflammatory protein (NF-κB) expression. Furthermore, histomorphormetric studies (Golgi impregnation and Cresyl violet stainings) were carried out. GB-S (10 and 20mg/kg) significantly restores neurobehavioral impairments based on improved locomotion, reduced anxiety- and depressive-like behavior. Moreover, GB-S reduced the MPO activity, inhibits TNF-α, IL-6 release, and downregulates NF-κB immunopositive cell expression in mice hippocampus. Histomorphometrically, GB-S also prevents the loss of pyramidal neuron in the hippocampus. The endpoint of this findings suggest that GB-S decreases neuropsychiatric symptoms induced by lead acetate through mechanisms related to inhibition of release of pro-inflammatory mediators and suppression of hippocampal pyramidal neuron degeneration in mice.

Abstract 3169: The therapeutically inflamed tumor microenvironment drives melanoma progression

Abstract Even the most therapy responsive melanomas eventually evade immune checkpoint inhibition. Interestingly, initial responses seem to benefit from localized inflammation, but this can become prohibitive when treating advanced disease. Subsequently, there is an urgent need to elucidate how inflammation impacts the tumor microenvironment (TME). Here, inhibition of EGFR and Ras/Raf/MEK is shown to drive the release of a pro-inflammatory secretome that activates the TME to promote progression. This secretome was enriched with immune recruiting cytokines and members of the transforming growth factor beta (TGFβ) superfamily. Both healthy and cancer associated fibroblasts (CAFs) were hyperactivated in response to the inflamed secretome with induction of the inflammatory marker, alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ receptor type I (TGFβRI) using SB431542 or clinically relevant TEW-7197, diminished CAF αSMA phenotype, disrupted TGFβ and phospo-SMAD2/3 signaling, and blunted CAF expression of tumor promoting extracellular matrix and adhesion molecules. Invasion of melanoma spheroids was enhanced ~2.5-fold in CAF coculture transwells. However, treatment with MEK inhibitor AZD6244 and TEW-7197 only limited invasion of mixed melanoma and CAF multi-cell spheroids in vitro. Prompted by this, the extracellular matrix of patient-derived and melanoma xenograft tumors was analyzed for inflammatory protein expression. Micrometastatic foci were flanked by peri-tumor αSMA+ CAFs and enriched for inflammatory Tenascin-C (TNC) at the invasive front. The analysis of human melanoma tissue microarrays corroborated the experimentally observed expression of αSMA CAFs and TNC which statistically increase with malignant tumor grade. Conversely, the anti-invasive Decorin (DCN), decreased in malignant melanoma while collagen-1 increased during late stage tumor fibrosis. These findings prompted the use of ex vivo human skin organ cultures (SOCs) to define how cell matrix impacted melanoma. Addition of DCN to the SOC eliminated melanoma vertical invasion whereas pathological TNC promoted progression. Thus, a BRAFV600E resistant patient-derived metastatic cell line was used to establish intrasplenic xenografts that were treated after one week using AZD6244 and TEW-7197. Both monotherapy and combination therapy were well tolerated and resulted in reduced splenic tumor burden, αSMA staining, and halted metastasis to the liver. Citation Format: Andrew M. Bradshaw, Erica Kuo, Jelena Grahovac, Kyle Sylakowski, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3169.

Photoreceptor Cell Calcium Dysregulation and Calpain Activation Promote Pathogenic Photoreceptor Oxidative Stress and Inflammation in Prodromal Diabetic Retinopathy

This study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1-/-) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2months. Cytosolic Ca2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA invitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy.

Biomarkers after Controlled Inhalation Exposure to Exhaust from Hydrogenated Vegetable Oil (HVO).

Hydrogenated vegetable oil (HVO) is a renewable diesel fuel used to replace petroleum diesel. The organic compounds in HVO are poorly characterized; therefore, toxicological properties could be different from petroleum diesel exhaust. The aim of this study was to evaluate the exposure and effective biomarkers in 18 individuals after short-term (3 h) exposure to HVO exhaust and petroleum diesel exhaust fumes. Liquid chromatography tandem mass spectrometry was used to analyze urinary biomarkers. A proximity extension assay was used for the measurement of inflammatory proteins in plasma samples. Short-term (3 h) exposure to HVO exhaust (PM1 ~1 µg/m3 and ~90 µg/m3 for vehicles with and without exhaust aftertreatment systems, respectively) did not increase any exposure biomarker, whereas petroleum diesel exhaust (PM1 ~300 µg/m3) increased urinary 4-MHA, a biomarker for p-xylene. HVO exhaust from the vehicle without exhaust aftertreatment system increased urinary 4-HNE-MA, a biomarker for lipid peroxidation, from 64 ng/mL urine (before exposure) to 141 ng/mL (24 h after exposure, p < 0.001). There was no differential expression of plasma inflammatory proteins between the HVO exhaust and control exposure group. In conclusion, short-term exposure to low concentrations of HVO exhaust did not increase urinary exposure biomarkers, but caused a slight increase in lipid peroxidation associated with the particle fraction.

Effect of TNF\u03b1 stimulation on expression of kidney risk inflammatory proteins in human umbilical vein endothelial cells cultured in hyperglycemia

We recently identified a kidney risk inflammatory signature (KRIS), comprising 6 TNF receptors (including TNFR1 and TNFR2) and 11 inflammatory proteins. Elevated levels of these proteins in circulation were strongly associated with risk of the development of end-stage kidney disease (ESKD) during 10-year follow-up. It has been hypothesized that elevated levels of these proteins in circulation might reflect (be markers of) systemic exposure to TNFα. In this in vitro study, we examined intracellular and extracellular levels of these proteins in human umbilical vein endothelial cells (HUVECs) exposed to TNFα in the presence of hyperglycemia. KRIS proteins as well as 1300 other proteins were measured using the SOMAscan proteomics platform. Four KRIS proteins (including TNFR1) were down-regulated and only 1 protein (IL18R1) was up-regulated in the extracellular fraction of TNFα-stimulated HUVECs. In the intracellular fraction, one KRIS protein was down-regulated (CCL14) and 1 protein was up-regulated (IL18R1). The levels of other KRIS proteins were not affected by exposure to TNFα. HUVECs exposed to a hyperglycemic and inflammatory environment also showed significant up-regulation of a distinct set of 53 proteins (mainly in extracellular fraction). In our previous study, circulating levels of these proteins were not associated with progression to ESKD in diabetes.

Study on the Immune Escape Mechanism of Acute Myeloid Leukemia With DNMT3A Mutation.

DNA (cytosine-5)-methyltransferase 3A (DNMT3A)-mutated acute myeloid leukemia (AML) has a poor prognosis, but the exact mechanism is still unclear. Here, we aimed to explore the mechanism of immune escape in AML with DNMT3A mutation. We constructed a DNMT3A knockout clone and DNMT3A-R882H-mutated clones. RNA-seq results showed that transcription factors and macrophage inflammatory proteins were significantly downregulated in the DNMT3A mutant clones. KEGG enrichment and gene set enrichment analysis (GSEA) showed that a large number of genes were enriched in inflammatory immune-related pathways, such as the toll-like receptor signaling pathway. Therefore, we co-cultured AML cells with macrophages. The DNMT3A-mutated AML cells attenuated M1 macrophage polarization and resisted its killing effect in vitro and in vivo. In xenografts, the tumor volumes in the experimental group were significantly larger than those in the control group, and the proportion of M2 macrophages was significantly higher. After the co-culture, the increase in pro-inflammatory cytokine expression in the mutant cells was significantly lower than that in the control group, while that in immunosuppressive factors was not significantly different. In co-cultivated supernatants, the concentration of inflammatory factors in the experimental group was significantly lower than that in the control group, while that of immunosuppressive factors was significantly higher. Resistin significantly promoted the expression of inflammatory proteins in AML cells. It relieved the inhibitory effect of DNMT3A mutation, promoted the phenotypic recovery of the co-cultured macrophages, eliminated resistance, and regulated the immune microenvironment. Thus, resistin may serve as an ancillary drug for patients with DNMT3A-mutated AML.

Zinc alloy-based bone internal fixation screw with antibacterial and anti-osteolytic properties

There is no targeted effective treatment for patients undergoing internal fixation surgery/two-stage total joint revision surgery with a high risk of postoperative infection and osteolysis, while the rate of reoperation due to infection and osteolysis remains high. In this study, we report a pioneering application of implants made of biodegradable Zn–Ag alloy with active antibacterial and anti-osteolytic properties in three classical animal models, illustrating antibacterial, anti-osteolysis, and internal fixation for fractures. The antibacterial activity of the Zn–2Ag alloy was verified in a rat femur osteomyelitis prevention model, while the anti-osteolytic properties were evaluated using a mouse cranial osteolysis model. Moreover, the Zn–2Ag based screws showed similar performance in bone fracture fixation compared to the Ti–6Al–4V counterpart. The fracture healed completely after 3 months in the rabbit femoral condyle fracture model. Furthermore, the underlying antibacterial mechanism may include inhibition of biofilm formation, autolysis-related pathways, and antibiotic resistance pathways. Osseointegration mechanisms may include inhibition of osteoclast-associated protein expression, no effect on osteogenic protein expression, and no activation of related inflammatory protein expression. The empirical findings here reveal the great potential of Zn–Ag-based alloys for degradable biomaterials in internal fixation surgery/two-stage total joint revision surgery for patients with a high risk of postoperative infection and osteolysis.

New use of old medicine: Nifedipine acts on the TRP family and inflammatory proteins in the treatment of chilblain

As a calcium antagonist, the mechanism of nifedipine for treating chilblain has not been reported. In the present study, we established the chilblain model by using −20 ℃ 95% ethanol to freeze the right back foot of SD rats, and investigated the effects of this drug. Hematoxylin–eosin (HE) examination indicated most of pannus in the skin tissue of chilblain rats had disappeared, and the local inflammatory cells were also greatly reduced when given nifedipine at 15.0 mg/kg/d. The enzyme-linked immunosorbent assay (ELISA) revealed that nifedipine inhibited release of inflammatory factors TNF-α, IL-6, IL-1β and VEGF in serum. The RT-PCR analysis showed that nifedipine down regulated mRNA levels of TRPC-6 and VEGF in skin tissue. Furthermore, immunohistochemical examination showed nifedipine inhibited expression of IL-1β, IL-6, and TNF-α inflammatory protein and further inhibited expression of TRP (transient receptor potential) family proteins TRPM-7, TRPC-1, TRPC-3 and TRPC-6 and reduced expression of VEGF in skin and relieved erythema and oedema. This study demonstrated that nifedipine as an old medicine can be new use for the treatment of chilblain by acting on TRPs family and inflammatory proteins.

Cirsilineol attenuates LPS-induced inflammation in both in vivo and in vitro models via inhibiting TLR-4/NFkB/IKK signaling pathway.

The anti-inflammatory activity of cirsilineol in in vivo condition was assessed by measuring the relative organ weight, lung dry/wet weight ratio, protein concentration, and infiltration of inflammatory cells in bronchoalveolar lavage fluid. We estimated the myeloperoxidase activity andlevels of cytokines, chemokines, and inflammatory markers to analyze the efficacy of cirsilineol against lipopolysaccharide (LPS)-induced lung inflammation. Furthermore, we quantified the gene expression of NFkB/IKK signaling molecules in cirsilineol-treated and untreated acute lung injury mice to confirm the anti-inflammatory property of cirsilineol. The lung histology was assessed with hematoxylin and eosin staining. Apart from in vivo experiments, in vitro tests with LPS-stimulated RAW 264.7 macrophages were also performed. Cell viability assay was performed in the presence and absence of LPS in RAW 264.7 macrophages to determine the cytotoxic effect of cirsilineol against macrophages. Reverse-transcription polymerase chain reaction (RT-PCR) analysis was done to analyze the gene expression of inflammatory markers in LPS-treated RAW 264.7 macrophages to prove that cirsilineol effectively inhibits inflammation in vitro. The results of our study prove that cirsilineol effectively inhibits inflammation in both in vivo and in vitro conditions. RT-PCR analysis results of NFkB/IKK signaling molecules clearly illustrate thatcirsilineol inhibited the expression of NFkB/IKK signaling protein andthereby prevented inflammation in in vivo condition, and it is further confirmed with the results of inflammatory protein expression in vitro model. The lung histopathological studies authentically confirm that cirsilineol potentially prevented the mice from LPS-induced lung inflammation.

Umbilical cord-derived MSC and hyperbaric oxygen therapy effectively protected the brain in rat after acute intracerebral haemorrhage.

This study tested the hypothesis that combined therapy with human umbilical cord‐derived mesenchymal stem cells (HUCDMSCs) and hyperbaric oxygen (HBO) was superior to either one on preserving neurological function and reducing brain haemorrhagic volume (BHV) in rat after acute intracerebral haemorrhage (ICH) induced by intracranial injection of collagenase. Adult male SD rats (n = 30) were equally divided into group 1 (sham‐operated control), group 2 (ICH), group 3 (ICH +HUCDMSCs/1.2 × 106 cells/intravenous injection at 3h and days 1 and 2 after ICH), group 4 (ICH +HBO/at 3 hours and days 1 and 2 after ICH) and group 5 (ICH +HUCDMSCs‐HBO), and killed by day 28 after ICH. By day 1, the neurological function was significantly impaired in groups 2‐5 than in group 1 (P < .001), but it did not differ among groups 2 to 5. By days 7, 14 and 28, the integrity of neurological function was highest in group 1, lowest in group 2 and significantly progressively improved from groups 3 to 5 (all P < .001). By day 28, the BHV was lowest in group 1, highest in group 2 and significantly lower in group 5 than in groups 3/4 (all P < .0001). The protein expressions of inflammation (HMGB1/TLR‐2/TLR‐4/MyD88/TRAF6/p‐NF‐κB/IFN‐γ/IL‐1ß/TNF‐α), oxidative stress/autophagy (NOX‐1/NOX‐2/oxidized protein/ratio of LC3B‐II/LC3B‐I) and apoptosis (cleaved‐capspase3/PARP), and cellular expressions of inflammation (CD14+, F4/80+) in brain tissues exhibited an identical pattern, whereas cellular levels of angiogenesis (CD31+/vWF+/small‐vessel number) and number of neurons (NeuN+) exhibited an opposite pattern of BHV among the groups (all P < .0001). These results indicate that combined HUCDMSC‐HBO therapy offered better outcomes after rat ICH.

Inflammatory, Thrombotic, and Diabetic Responses in Human Arterial Fibroblasts and Endothelial Cells are Regulated by SARS‐CoV‐2 Structural Proteins

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is responsible for many pathological processes, such as altered vascular disease development, dysfunctional thrombosis, and a heightened inflammatory state. Although many clinical reports support these observations, there has been limited experimental work to determine the underlying mechanisms and cellular processes induced by exposure to SARS‐CoV‐2 structural proteins. Importantly, these structural proteins are conserved amongst all current known SARS‐CoVs. Our objective was to investigate the effects of the Spike (S), Nucleocapsid (N), and Membrane‐Envelope (M) SARS‐CoV‐2 structural proteins on inflammation, thrombosis, and diabetic disease markers of human aortic adventitial fibroblasts and human umbilical vein endothelial cells. We hypothesized that short‐term exposure to SARS‐CoV‐2 structural proteins would result in increased expression of inflammatory, thrombotic, and diabetic proteins in both cell types, which would support a mechanism for altered vascular disease progression. To test this, the cells were incubated independently with the three SARS‐CoV‐2 proteins for one hour, after which we analyzed the expression of gC1qR, ICAM‐1, tissue factor, RAGE, GLUT‐4, thrombomodulin, PECAM‐1, and Connexin‐43 (in their respective cell types), using an ELISA approach. All cells were monitored for maintenance of typical culture parameters using a live/dead cell cytotoxicity assay and the MTT assay (for metabolic activity). We observed that each of these markers were significantly up‐regulated after exposure to SARS‐CoV‐2 structural proteins as compared to fibroblasts or endothelial cells that were not exposed to these proteins. Interestingly, the extent of the expression of these markers was sometimes significantly different for each of the SARS‐CoV‐2 structural proteins. This suggests that each of the SARS‐CoV‐2 proteins interacts with these cells through different mechanisms, however, more work will need to be undertaken to determine the mechanisms by which these proteins interact with cells. Thus, these results indicate that the cellular response of vascular cells towards SARS‐CoV‐2 structural proteins promotes inflammatory, thrombotic, and vascular dysfunction. However, these interactions are regulated by complex and possibly different cellular receptors/signal transduction pathways that should be explored further.

Depletion of Lysozyme M+ Cells Offers Cardiovascular, but not Cerebral, Protection from Noise‐Induced Stress

Background and Hypothesis Increased leukocyte extravasation in the aortic endothelium leads to a pro-oxidant and pro-inflammatory environment which facilitates the development of endothelial dysfunction. Moderate hypertension, endothelial dysfunction, and increases in oxidative stress are associated with exposure to the novel cardiovascular risk, noise pollution. Since we have previously established a role for myelomonocytes (LysM+ cells) in the deleterious effects of noise exposure, we hypothesized that partial ablation of these cells would protect from cardiovascular consequences. Methods LysM+ immune cells were depleted with diphtheria toxin (DTX) in LysMCre+/-iDTR+/- mice for 6 days prior to noise exposure (mean SPL; 72 dBA) and 4 days concurrently. Blood pressure was determined by tail cuff method. Endothelial dysfunction was measured with isometric tension recordings. Protein and gene analysis were carried out using Western Blot, dot blot, and rtPCR. Oxidative stress measurements included dihydroethidium HPLC and staining. Inflammatory cell infiltration was assessed via FACS. Astrocyte and microglia activation were measured by GFAP+ and Iba1+ staining. Results Ablation of LysM+ cells successfully normalized the blood pressure as well as aortic and retinal vessel endothelial function of mice exposed to 4 days of noise. Noise caused a significant increase in superoxide production in aortic and cardiac tissue. Mice with ablated LysM+ cells were protected from increased oxidative stress and its effects. The DTX +Noise group was protected from expression of inflammatory genes in aortic tissue. These effects were not seen in brain or cortical tissue, where microglia and astrocyte activation were increased and there was a general failure of protection from noise in inflammatory gene (NFκB2, CD40L, TNFα) and protein (CD68, LysM, CD40, IL6) expression. Noise, DTX, and DTX +Noise groups were all found to have heightened levels of stress hormones in plasma. Conclusion Ablation of myelomonocytic cells was successful in rescuing mice from the cardiovascular damage associated with noise exposure, suggesting that these cells play a critical role in the damage incurred upon noise exposure. However, this protection was only found in the vasculature and a distinctly different outcome was discovered in the brain, where a neuroinflammatory phenotype persisted. Heightened levels of hormones indicate that the stress response following noise exposure remains intact in the brain following immune ablation, but is not carried forth to the vasculature, both implicating LysM+ cells in the oxidative damage caused by noise exposure and also in playing an important role the hormone signaling resultant from stress reactions.

Influence of Bacterial Profiles in Cytokine and Clinical Features of Endodontic Disease

IntroductionWe verified the association between selected bacterial profiles and levels of cytokines, chemokines, and the expression of signs and symptoms of primary endodontic infection with apical periodontitis. MethodsSamples were collected from 21 root canals, and macrophages were stimulated for 24 hours. Tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-10, IL-12p70, interferon gamma, and chemokine (C-C motif) ligand 2 (CCL2) were measured using cytometric bead array. We investigated the overlapping networks between cytokines and chemokines with regression analysis. Checkerboard DNA-DNA hybridization was used to assess 40 target bacteria species. Using factor analysis, bacterial species aggregated in 2 factors. The association of bacteria species–based factors on cytokine and chemokine levels and clinical features was estimated with regression analysis. ResultsA negative relationship between IL-10 (anti-inflammatory cytokine) and CCL2, TNF-α, and IFN-γ (proinflammatory cytokines) (all P < .05) was observed. CCL2 was positively correlated with TNF-α (P < .01). Thirty-eight bacteria species were detected in primary endodontic infection with apical periodontitis. The first bacteria species–based factor was associated with the size of the radiolucent area (coefficient = 15.42) and tenderness to percussion/pain on palpation (coefficient = 20.79). The second factor was associated with CCL2 levels (coefficient = 1.28). ConclusionsDifferent bacterial profiles can be differentially related to the expression of inflammatory proteins and the experience of clinical features.

SARS-CoV-2 proteins regulate inflammatory, thrombotic and diabetic responses in human arterial fibroblasts

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for many pathological processes, including altered vascular disease development, dysfunctional thrombosis and a heightened inflammatory response. However, there is limited work to determine the underlying cellular responses induced by exposure to SARS-CoV-2 structural proteins. Thus, our objective was to investigate how human arterial adventitial fibroblasts inflammation, thrombosis and diabetic disease markers are altered in response to Spike, Nucleocapsid and Membrane-Envelope proteins. We hypothesized that after a short-term exposure to SARS-CoV-2 proteins, adventitial fibroblasts would have a higher expression of inflammatory, thrombotic and diabetic proteins, which would support a mechanism for altered vascular disease progression. After incubation, the expression of gC1qR, ICAM-1, tissue factor, RAGE and GLUT-4 was significantly up-regulated. In general, the extent of expression was different for each SARS-CoV-2 protein, suggesting that SARS-CoV-2 proteins interact with cells through different mechanisms. Thus, SARS-CoV-2 protein interaction with vascular cells may regulate vascular disease responses.

Overexpression of miR-365a-3p relieves sepsis-induced acute myocardial injury by targeting MyD88/NF-κB pathway.

Sepsis often leads to systemic multiple organ dysfunction, with the majority of deaths attributable to acute myocardial injury (AMI). In this study, we aimed to explore the functional role of miR-365a-3p in sepsis-induced AMI. The sepsis myocardial injury model was constructed using lipopolysaccharide (LPS) both in vitro and in vivo with selective regulation of miR-365a-3p expression. Real-time PCR or Western blot was employed to detect the expressions of miR-365a-3p, inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6), and inflammation-related proteins (nuclear factor-κB (NF-κB), I-κB, myeloid differentiation factor 88 (MyD88)) in myocardial tissues and cells. Also, cell counting kit-8 (CCK8) and flow cytometry assays were used to measure cardiomyocyte proliferation and apoptosis, respectively. Furthermore, the targeting relationship between miR-365a-3p and MyD88 was verified with the dual luciferase activity assay. miR-365a-3p was downregulated in LPS-induced myocardial injury model. miR-365a-3p overexpression attenuated cardiomyocyte apoptosis and suppressed the expressions of inflammatory cytokines and proteins. Inhibiting miR-365a-3p, however, produced the opposite effects. Mechanistically, miR-365a-3p targeted the 3'-untranslated region of MyD88, thereby inactivating MyD88-mediated NF-κB pathway. miR-365a-3p overexpression mitigated sepsis-mediated myocardial injury by inhibiting MyD88-mediated NF-κB activation.

Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in vitro by inhibiting NLRP3 Inflammasome activation

BackgroundMyocardial ischemia-reperfusion injury (MIRI) is the most common cause of death worldwide. The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the inflammatory response to MIRI. Dexmedetomidine (DEX), a specific agonist of α2-adrenergic receptor, is commonly used for sedation and analgesia in anesthesia and critically ill patients. Several studies have shown that dexmedetomidine has a strong anti-inflammatory effect in many diseases. Here, we investigated whether dexmedetomidine protects against MIRI by inhibiting the activation of the NLRP3 inflammasome in vitro.MethodsWe established an MIRI model in cardiomyocytes (CMs) alone and in coculture with cardiac fibroblasts (CFs) by hypoxia/reoxygenation (H/R) in vitro. The cells were treated with dexmedetomidine with or without MCC950 (a potent selective NLRP3 inhibitor). The beating rate and cell viability of cardiomyocytes, NLRP3 localization, the expression of inflammatory cytokines and NLRP3 inflammasome-related proteins, and the expression of apoptosis-related proteins, including Bcl2 and BAX, were determined.ResultsDexmedetomidine treatment increased the beating rates and viability of cardiomyocytes cocultured with cardiac fibroblasts. The expression of the NLRP3 protein was significantly upregulated in cardiac fibroblasts but not in cardiomyocytes after H/R and was significantly attenuated by dexmedetomidine treatment. Expression of the inflammatory cytokines IL-1β, IL-18 and TNF-α was significantly increased in cardiac fibroblasts after H/R and was attenuated by dexmedetomidine treatment. NLRP3 inflammasome activation induced the increased expression of cleaved caspase1, mature IL-1β and IL-18, while dexmedetomidine suppressed H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. In addition, dexmedetomidine reduced the expression of Bcl2 and BAX in cocultured cardiomyocytes by suppressing H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts.ConclusionDexmedetomidine treatment can suppress H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts, thereby alleviating MIRI by inhibiting the inflammatory response.