Expression Of Inflammatory Proteins Research Articles

Sodium aescinate inhibits microglia activation through NF-κB pathway and exerts neuroprotective effect.

Background: Microglia are resident immune cells of the central nervous system that sense environmental changes and maintain central nervous system homeostasis. Dysfunctional microglia produce toxic mediators that lead to neuronal death. Recent studies suggest that Sodium Aescinate has a neuroprotective effect. However, it is unclear whether Sodium Aescinate exerts neuroprotective effects by inhibiting activation of microglia. Method: Traumatic brain injury and lipopolysaccharide neuroinflammation model were used to evaluate the microglia activation in vivo. BV2 and primary microglia cells were used to assess the microglia activation in vitro. Molecular docking technique was used to predict the binding energy of Sodium Aescinate to NF-κB signaling pathway proteins. Result: Sodium Aescinate inhibited microglial activation in-vivo and in-vitro. Sodium Aescinate inhibited the activation of microglia in Traumatic brain injury and lipopolysaccharide mouse models. Sodium Aescinate also inhibited the expression of inflammatory proteins in BV2 and primary microglia cells. Western blot experiment showed that SA inhibited the activation of NF-κB pathway in BV2 and primary microglia cells. Molecular docking results also showed that Sodium Aescinate had a better affinity with the core protein of the NF-κB pathway. Western blot identified that SA inhibited activation of NF-κB pathway. In Traumatic brain injury model and conditioned medium experiment, Sodium Aescinate pretreatment inhibited inflammation and protected neuron. Conclusion: Our study confirmed that the protection effects of Sodium Aescinate on neurons by inhibiting microglia activation through NF-κB pathway.

A placenta-on-a-chip model to determine the regulation of FKBPL and galectin-3 in preeclampsia

Preeclampsia is a pregnancy-specific cardiovascular disorder, involving significant maternal endothelial dysfunction. Although inappropriate placentation due to aberrant angiogenesis, inflammation and shallow trophoblast invasion are the root causes of preeclampsia, pathogenic mechanisms are poorly understood, particularly in early pregnancy. Here, we first confirm the abnormal expression of important vascular and inflammatory proteins, FK506-binding protein-like (FKBPL) and galectin-3 (Gal-3), in human plasma and placental tissues from women with preeclampsia and normotensive controls. We then employ a three-dimensional microfluidic placental model incorporating human umbilical vein endothelial cells (HUVECs) and a first trimester trophoblast cell line (ACH-3P) to investigate FKBPL and Gal-3 signaling in inflammatory conditions. In human samples, both circulating (n = 17 controls; n = 30 preeclampsia) and placental (n ≥ 6) FKBPL and Gal-3 levels were increased in preeclampsia compared to controls (plasma: FKBPL, p < 0.0001; Gal-3, p < 0.01; placenta: FKBPL, p < 0.05; Gal-3, p < 0.01), indicative of vascular dysfunction in preeclampsia. In our placenta-on-a-chip model, we show that endothelial cells are critical for trophoblast-mediated migration and that trophoblasts effectively remodel endothelial vascular networks. Inflammatory cytokine tumour necrosis factor-α (10 ng/mL) modulates both FKBPL and Gal-3 signaling in conjunction with trophoblast migration and impairs vascular network formation (p < 0.005). Our placenta-on-a-chip recapitulates aspects of inappropriate placental development and vascular dysfunction in preeclampsia.

Sensory Receptor, Inflammatory, and Apoptotic Protein Expression in the Bladder Urothelium of Patients with Different Subtypes of Interstitial Cystitis/Bladder Pain Syndrome.

The aim of this study was to investigate the expression levels of sensory receptors, inflammatory proteins, and pro-apoptotic proteins in the urothelium of non-Hunner's interstitial cystitis (NHIC) bladders of patients with different clinical and cystoscopic phenotypes. The urothelia from the bladders of 52 NHIC patients were harvested. The expression of sensory receptors, including TRPV1, TRPV4, TRPA1, H1-receptors, and sigma-1 receptors; the inflammatory proteins p38 and tryptase; and the pro-apoptotic proteins, such as caspase-3, BAD, and BAX in the urothelium, were investigated using immunohistochemistry and Western blotting. We compared the expression levels of these proteins in NHIC subtypes according to IC symptom scores, visual analog scores of bladder pain, maximal bladder capacity, glomerulation grades, and combined maximal bladder capacity and glomerulations after cystoscopic hydrodistention. The expression levels of TRPV1, TRPV4, sigma-1, P38, tryptase, caspase-3, and BAD were significantly increased in the urothelium of IC/BPS patients compared with the expression levels in the controls. TRPV1 was significantly associated with IC symptom severity. However, no significant differences in sensory receptor expression in the IC/BPS bladders with different bladder conditions were detected. Inflammatory and pro-apoptotic protein expression levels in the urothelium were similar among the IC/BPS subgroups. This study concluded that IC/BPS patients with frequency and bladder pain complaints have higher levels of urothelial sensory receptors, and inflammatory and pro-apoptotic proteins. The expression levels of these sensory receptors, inflammatory proteins, and pro-apoptotic proteins are not significantly different among IC/BPS bladders with different conditions.

Extracellular vesicles as a potential therapeutic for age-related macular degeneration.

Age-related macular degeneration is a major global cause of central visual impairment and severe vision loss. With an aging population, the already immense economic burden of costly anti-vascular endothelial growth factor treatment is likely to increase. In addition, current conventional treatment is only available for the late neovascular stage of age-related macular degeneration, and injections can come with potentially devastating complications, introducing the need for more economical and risk-free treatment. In recent years, exosomes, which are nano-sized extracellular vesicles of an endocytic origin, have shown immense potential as diagnostic biomarkers and in the therapeutic application, as they are bestowed with characteristics including an expansive cargo that closely resembles their parent cell and exceptional ability of intercellular communication and targeting neighboring cells. Exosomes are currently undergoing clinical trials for various conditions such as type 1 diabetes and autoimmune diseases; however, exosomes as a potential therapy for several retinal diseases have just begun to undergo scrutinizing investigation with little literature on age-related macular degeneration specifically. This article will focus on the limited literature available on exosome transplantation treatment in age-related macular degeneration animal models and in vitro cell cultures, as well as briefly identify future research directions. Current literature on exosome therapy using age-related macular degeneration rodent models includes laser retinal injury, N-methyl-N-nitrosourea, and royal college of surgeon models, which mimic inflammatory and degenerative aspects of age-related macular degeneration. These have shown promising results in preserving retinal function and morphology, as well as protecting photoreceptors from apoptosis. Exosomes from their respective cellular origins may also act by regulating the expression of various inflammatory cytokines, mRNAs, and proteins involved in photoreceptor degeneration pathways to exert a therapeutic effect. Various findings have also opened exciting prospects for the involvement of cargo components in remedial effects on the damaged macula or retina.

Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect.

The noble gas argon has the potential to protect neuronal cells from cell death. So far, this effect has been studied in treatment after acute damage. Preconditioning using argon has not yet been investigated. In this study, human neuroblastoma SH-SY5Y cells were treated with different concentrations of argon (25%, 50%, and 74%; 21% O2, 5% CO2, balance nitrogen) at different time intervals before inflicting damage with rotenone (20 µM, 4 hours). Apoptosis was determined by flow cytometry after annexin V and propidium iodide staining. Surface expressions of Toll-like receptors 2 and 4 were also examined. Cells were also processed for analysis by western blot and qPCR to determine the expression of apoptotic and inflammatory proteins, such as extracellular-signal regulated kinase (ERK1/2), nuclear transcription factor-κB (NF-κB), protein kinase B (Akt), caspase-3, Bax, Bcl-2, interleukin-8, and heat shock proteins. Immunohistochemical staining was performed for TLR2 and 4 and interleukin-8. Cells were also pretreated with OxPAPC, an antagonist of TLR2 and 4 to elucidate the molecular mechanism. Results showed that argon preconditioning before rotenone application caused a dose-dependent but not a time-dependent reduction in the number of apoptotic cells. Preconditioning with 74% argon for 2 hours was used for further experiments showing the most promising results. Argon decreased the surface expression of TLR2 and 4, whereas OxPAPC treatment partially abolished the protective effect of argon. Argon increased phosphorylation of ERK1/2 but decreased NF-κB and Akt. Preconditioning inhibited mitochondrial apoptosis and the heat shock response. Argon also suppressed the expression of the pro-inflammatory cytokine interleukin-8. Immunohistochemistry confirmed the alteration of TLRs and interleukin-8. OxPAPC reversed the argon effect on ERK1/2, Bax, Bcl-2, caspase-3, and interleukin-8 expression, but not on NF-κB and the heat shock proteins. Taken together, argon preconditioning protects against apoptosis of neuronal cells and mediates its action via Toll-like receptors. Argon may represent a promising therapeutic alternative in various clinical settings, such as the treatment of stroke.

Anti-inflammatory, procollagen, and wound repair properties of topical insulin gel.

Diabetes mellitus is associated with impaired wound healing. The topical use of insulin is a promising therapy because it may favor all phases of the wound healing process. This study aimed to investigate the therapeutic outcomes of insulin gel in wounds of hyperglycemic mice. After diabetes induction, a 1-cm2 full-thickness wound was created on each animal's dorsum. The lesions were treated daily for 14 days with insulin gel (insulin group) or vehicle gel without insulin (vehicle group). Tissue samples were extracted on days 4, 7, 10, and 14 after the creation of the lesion. The samples were analyzed with hematoxylin/eosin and Sirius red staining, immunohistochemistry, Bio-Plex immunoassays, and western blotting. Insulin gel favored re-epithelialization at day 10 and increased the organization and deposition of collagen. Additionally, it modulated the expression of cytokines (interleukin (IL)-4 and IL-10) and increased the expression of arginase I, VEGF receptor 1, and VEGF on day 10. Activation of the insulin signaling pathway occurred via IRβ, IRS1, and IKK on day 10 and activation of Akt and IRS1 on day 14. These results suggested that insulin gel improved wound healing in hyperglycemic mice by modulating the expression of inflammatory factors, growth factors, and proteins of the insulin signaling pathway.

A new terpenoid from Olea europaea L. leaves and potential aptitude of different leaf extracts as anti-inflammatory agents

Background The olive tree (Olea europaea L.) has played a significant role in medical history, being used to relieve a wide range of illnesses in traditional medicine. The value of olive leaves came from their medicinal and nutritional properties. In recent times, it has been proven that there is a potential role of leaves in cancer prevention and inflammatory illnesses.Objective The purpose of this study was to look at the chemical makeup of methylene chloride fraction and also to evaluate the chemopreventive properties and the anti-inflammatory activity of different olive leave fractions using hepa1c1c7 cells and RAW264.7 macrophages activated by lipopolysaccharides, respectively.Materials and methods Air-dried powdered olive leaves were defatted with hexane, and the marc was then soaked in 80% methanol and successively extracted with CH2Cl2, EtOAc, and n-BuOH. Phytochemical investigation of CH2Cl2 fraction was done. The chemopreventive effect was determined in vitro using a hepa1c1c7 human carcinoma cell line, and the anti-inflammatory was carried out using RAW264.7 macrophages.Results and conclusion One novel compound, identified and given the name oleuropeinone (1), was obtained together with two known compounds, blumenol A (2) and megaritolactonol (3). Spectroscopic data such as 1H and 13C NMR were used to identify their chemical structures. The CH2Cl2 fraction is the most effective fraction to inhibit inflammatory markers inducible nitric oxide synthase and COX-2 protein expression in RAW264.7 macrophages. Moreover, it has a potent effect in inducing the chemopreventive marker NQO1 protein expression.

Protective mechanisms of Leontopodium leontopodioides extracts on lipopolysaccharide-induced acute kidney injury viathe NF-κB/NLRP3 pathway.

Sepsis-induced uncontrolled systemic inflammatory response syndrome (SIRS) is a critical cause of multiple organ failure. Acute kidney injury (AKI) is one of the most serious complications associated with an extremely high mortality rate in SIRS, and it lacked simple, safe, and effective treatment strategies. Leontopodium leontopodioides (Willd.) Beauv (LLB) is commonly used in traditional Chinese medicine for the treatment of acute and chronic nephritis. However, it remains unclear whether lipopolysaccharide (LPS) affects LPS-induced AKI. To identify the molecular mechanisms of LLB in LPS-induced HK-2 cells and mice, LLB was prepared by extraction with 70% methanol, while a lipopolysaccharide (LPS)-induced HK-2 cell model and an AKI model were established in this study. Renal histopathology staining was performed to observe the morphology changes. The cell supernatant and kidney tissues were collected for determining the levels of inflammatory factors and protein expression by ELISA, immunofluorescence, and Western blot. The results indicated that LLB significantly reduced the expression of IL-6 and TNF-α in LPS-induced HK-2 cells, as well as the secretion of IL-6, TNF-α, and IL-1β in the supernatant. The same results were observed in LPS-induced AKI serum. Further studies revealed that LLB remarkably improved oxidative stress and apoptosis based on the content of MDA, SOD, and CAT in serum and TUNEL staining results. Notably, LLB significantly reduced the mortality due to LPS infection. Renal histopathology staining results supported these results. Furthermore, immunofluorescence and Western blot results confirmed that LLB significantly reduced the expression of the protein related to the NF-κB signaling pathway and NLRP3, ASC, and Caspase-1 which were significantly increased through LPS stimulation. These findings clearly demonstrated the potential use of LLB in the treatment of AKI and the crucial role of the NF-κB/NLRP3 pathway in the process through which LLB attenuates AKI induced by LPS.

PS-C19-3: HYDROCHLOROTHIAZIDE WAS NOT ASSOCIATED WITH PHOTOSENSITIVE OR PHOTOTOXIC REACTIONS AND DNA-DAMAGE IN VIVO AND IN VITRO (THE HCTOX-STUDY)

Objective: Pharmacoepidemiologic studies associated the use of HCT with an increased risk of skin cancer, resulting in a decrease of HCT prescriptions, in turn leading to worsening of blood pressure therapy in a significant proportion of patients. However, whether HCT causes skin cancer remains elusive. Hence, we aimed to examine the photosensitive and phototoxic potential of HCT in vivo in a randomized, placebo-controlled trial. To further enlighten the pathophysiologic mechanisms of carcinogenesis and phototoxicity caused by HCT in vitro, we conducted a series of laboratory experiments. Design and method: A randomized, double-blind, placebo-controlled clinical trial to assess the phototoxic properties of HCT was conducted, assigning 30 healthy normotensive adult volunteers in a 2:1 ratio to either HCT 25 mg daily or placebo once daily for 15 days. The skin photosensitivity by phototesting, office blood pressure, serum 25-hydroxyvitamin D (25(OH)D) status and urinary excretion of thymidine-dimers, i.e. cyclobutan-dimers by ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) following whole-body irradiation were assessed. To further assess the pathophysiologic mechanisms of possibly HCT induced photosensitivity, human keratinocytes (HaCaT) were incubated with HCT and then irradiated with UV-B radiation (311 nm one burst of 100 J/cm2). rt-PCR-testing and western blots were performed to analyze reactive oxygen species, inflammation, and carcinogenesis. Results: All 30 participants were adherent to the protocol, which was confirmed by UHPLC-HRMS analysis of serum and plasma. Skin photosensitivity to exposure of UV-A and UV-B radiation did not change in both groups (HCT vs. placebo: MED-UVB change 0.0 J/cm2 vs. 0.2 J/cm2; p = 0.06). No thymidine-dimers were detectable in either group. Systolic blood pressure decreased in both groups (-5.2 mmHg vs. 5.4 mmHg; p = 0.94), as did diastolic blood pressure (-4.3 mmHg vs. -1.9 mmHg; p = 0.34). Serum 25(OH)D increased in both groups (3.1 ng/ml vs. 1.2 ng/ml; p = 0.52). HCT in combination with a single high-intensity bursts of UV-B radiation was not associated with increased expression of inflammatory proteins, chronic inflammation and reactive oxygen species. Conclusions: HCT was not associated with increased photosensitivity for UV-A or UV-B radiation in healthy volunteers. Moreover, no relevant DNA-damages as measured by UHPLC-HRMS were detectable in either group. The combination of HCT treatment and a single burst of UV-B did not increase inflammatory markers. HCT appeared to be safe in healthy volunteers and was not associated with photosensitivity or DNA-damages in vivo.

Does IFITM3 link inflammation to tumorigenesis?

Uncontrolled chronic inflammation, in most cases due to excessive cytokine signaling through their receptors, is known to contribute to the development of tumorigenesis. Recently, it has been reported that the antiviral membrane protein interferon-induced transmembrane protein 3 (IFITM3), induced by interferon signaling as part of the inflammatory response after viral infection, contributes to the development of B-cell malignancy. The unexpected oncogenic signaling of IFITM3 upon malignant B cell activation elucidated the mechanism by which the uncontrolled expression of inflammatory proteins contributes to leukemogenesis. In this review, the potential effects of inflammatory cytokines on upregulation of IFITM3 and its contribution to tumorigenesis are discussed. [BMB Reports 2022; 55(12): 602-608].

1192: COVID SEPSIS GENE EXPRESSION SUBPHENOTYPES MANIFEST DIFFERENTIAL PLASMA PROTEIN SIGNATURES

Introduction: Transcriptome-derived sepsis subphenotypes, termed ‘adaptive’, ‘inflammopathic’ and ‘coagulopathic’, have been reliably identified in sepsis cohorts, however plasma proteomics in these groups have not been well characterized. We hypothesized that inflammatory and vascular injury markers would be elevated in the inflammopathic and coagulopathic groups compared to the adaptive group. Methods: We prospectively enrolled and obtained blood from 130 inpatients with COVID19-related sepsis. Severity was classified by NIH ordinal scale. Gene expression analysis was performed by Nanostring nCounter (Inflammatix). Inflammatory proteins interleukin (IL)-6, IL8, IL10, IL1RA, IL1RL1, and IFNg and vascular markers ANGPT2, sICAM, vWF, ADAMTS13, and protein C were measured with OLINK proximity extension assay. Clinical variables were compared by chi-square and protein levels were compared using ANOVA with Bonferroni adjustment. Results: The transcriptomic classifier identified 32% (41) inflammopathic, 50% (65) adaptive and 18% (24) coagulopathic subjects. The inflammopathic group had more patients requiring mechanical ventilation (39% vs 9% vs 21%; p < 0.001) and higher 90-day mortality (32% vs 8% vs 13%, p = 0.016). Inflammatory cytokines IL8 and IL10 were significantly higher in inflammopathic compared to adaptive (p=0.038 and p=0.017 respectively), but not compared to coagulopathic (p>0.99 and p=0.24, respectively). Both the inflammopathic and coagulopathic groups expressed higher IL1RL1 and interferon-gamma compared to adaptive (IL1RL1; p< 0.001, p=0.002, IFNg; p=0.007, p=0.001). Plasma IL6 and IL1RA did not differ between groups, nor did many vascular proteins. The inflammopathic group expressed higher sICAM (p=0.049 vs adaptive) and lower ADAMTS13 compared to the adaptive group, and the coagulopathic group did not differ in its vascular protein expression. Conclusions: Transcriptomic subphenotypes are present in COVID-19 sepsis at similar proportions to non-COVID-19 sepsis. Inflammopathic subjects manifested higher severity of illness at admission, higher expression of inflammatory proteins and higher mortality. Markers of vascular injury did not distinguish the coagulopathic group. Integrating RNA and protein expression may offer new insights to host immune dysregulation during COVID sepsis.

Pharyngeal Inflammation on Positron Emission Tomography/Magnetic Resonance Imaging Before and After Obstructive Sleep Apnea Treatment.

Rationale: There is upper airway inflammation in patients with obstructive sleep apnea (OSA), which reduces with continuous positive airway pressure (CPAP) therapy. Objectives: Validate the use of positron emission tomography (PET)/magnetic resonance imaging (MRI) to quantify metabolic activity within the pharyngeal mucosa of patients with OSA against nasal lavage proteomics and assess the impact of CPAP therapy. Methods: Adults with OSA underwent [18F]-Fluoro-2-deoxy-D-glucose PET/MRI of the neck before and 3 months after initiating CPAP. Nasal lavage samples were collected. Inflammatory protein expression from samples was analyzed using the Olink platform. Upper airway imaging segmentation was performed. Target-to-background ratio (TBRmax) was calculated from target pharyngeal maximum standard uptake values (SUV) and personalized background mean SUV. Most-diseased segment TBRmax was identified per participant at locations with the highest PET avidity. Correlation analysis was performed between baseline TBRmax and nasal lavage proteomics. TBRmax was compared before and after CPAP using linear mixed-effect models. Results: Among 38 participants, the baseline mean age was 46.3 years (standard deviation [SD], 12.5), 21% were female, the mean body mass index was 30.9 kg/m2 (SD, 4.6), and the mean respiratory disturbance index measured by peripheral arterial tonometry was 31 events/h (SD, 16.4). There was a significant positive correlation between pharyngeal mucosa most-diseased segment TBRmax and nasal lavage proteomic inflammation (r = 0.41 [P < 0.001, false discovery rate = 0.002]). Primary analysis revealed a reduction in the most-diseased segment TBRmax after a median of 2.91 months of CPAP therapy (-0.86 [standard error (SE) ± 0.30; P = 0.007]). Stratified analysis by smoking status revealed a significantly decreased most-diseased segment TBRmax after CPAP therapy among never-smokers but not among ever-smokers (-1.01 [SE ± 0.39; P = 0.015] vs. -0.64 [SE ± 0.49; P = 0.201]). Conclusions: CPAP therapy reduces metabolic activity measured by PET/MRI within the upper airway of adults with OSA. Furthermore, PET/MRI measures of upper airway metabolic activity correlate with a noninvasive marker of inflammation (i.e., nasal lavage inflammatory protein expression).

IL-1β enhances cell viability and decreases 5-FU sensitivity in novel colon cancer cell lines derived from African American patients.

In the U.S., African Americans (AAs) present with the highest incidence and mortality rates for Colorectal Cancer (CRC). When compared to Caucasian American (CA) patients, AAs also have reduced response to the first line standard of care chemotherapeutic agent 5-Fluorouracil (5-FU). Previously, we observed differential gene expression between the two populations, suggesting that colon tumors from AA patients display a decreased antitumor immune response and an increased expression of genes encoding proteins involved in inflammatory processes, such as Interleukin-1β (IL-1β). Here, we investigate the role of IL-1β in modifying chemotherapeutic response and altering expression of proteins in novel AA and well-established CA colon cancer cell lines. RNA sequencing analysis was performed to detect expression of genes involved in inflammation in AA and CA colon cancer cells. The effects of IL-1β on 5-FU response was evaluated by assessing cell viability (MTS assay) and apoptosis (flow cytometry analysis) following treatment with 5-FU alone or in combination with the cytokine. Further, we used an IL-1 receptor antagonist (IL-1Ra) to inhibit IL-1β-induced effects on 5-FU sensitivity and NF-kB pathway activation. AA colon cancer cell lines present significant increase in expression of genes IL1R2 (373-fold change (FC), IRAK1 (3.24 FC), IKBKB, (5.33 FC) NF-KB IA (5.95 FC), MYD88, (3.72 FC), IRAK3 (161 FC), TRAF5 (4.1 FC). A significant decrease in the response to 5-FU treatment, as well as a significant increase in phosphorylation of IκBα and secretion of IL-8, was seen following IL-1β treatment, in both AA and CA cell lines. Finally, treatment with IL-1Ra was able to reverse the effects induced by IL-1β, by increasing the cells sensitivity to 5-FU. IL-1Ra also inhibited phosphorylation of IκBα and IL-8 secretion. Our results suggest a differential expression of inflammatory genes and proteins that might regulate the different response to IL-1β between AA and CA colon cancer cell lines. Our data also demonstrates that IL-1β is involved in modulating 5-FU response in both AA and CA colon cancer cell lines. Further investigation of these mechanisms might help elucidate the differences seen in incidence, mortality and response to therapy in AA colon cancer patients.

Sodium houttuyfonate effectively treats acute pulmonary infection of Pseudomonas aeruginosa by affecting immunity and intestinal flora in mice.

Pseudomonas aeruginosa is a major nosocomial pathogen that frequently causes ventilator-associated pneumonia in specific populations. Sodium houttuyfonate (SH) has shown mild antibacterial activity against P. aeruginosa in vitro, but the mechanism of potent antimicrobial activity of SH against P. aeruginosa infection in vivo remains unclear. Here, using the mouse pneumonia model induced by P. aeruginosa nasal drip to explore the therapeutic effects of SH. We found that SH exhibits dose-dependent therapeutic effects of reducing P. aeruginosa burden and systemic inflammation in pneumonia mice. SH ameliorates inflammatory gene expression and production of inflammatory proteins, such as interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB) and toll-like receptor 4 (TLR4), associated with the TLR4/NF-κB pathway in mice with P. aeruginosa pneumonia. Furthermore, we analyzed the intestinal flora of mice and found that compared with the model group, the abundance and diversity of beneficial bacterial flora of SH treatment groups increased significantly, suggesting that SH can improve the intestinal flora disorder caused by inflammation. In addition, SH improves alpha and beta diversity index and reduces species abundance differences of intestinal flora in pneumonia mice. Taken together, our presented results indicate that SH may effectively alleviate the acute pulmonary infection induced by P. aeruginosa by reducing the disturbance of regulating immunity and intestinal flora in mice.

Antioxidant, Anti-Inflammation and Antiaging Activities of Artocarpus altilis Methanolic Extract on Urban Particulate Matter-Induced HaCaT Keratinocytes Damage.

Particulate matter (PM) is one of the reasons that exacerbate skin diseases. Impaired barrier function is a common symptom in skin diseases, including atopic dermatitis, eczema and psoriasis. Herbal extracts rich in antioxidants are thought to provide excellent pharmacological activities; however, the anti-pollution activity of Artocarpus altilis extract (AAM) has not been investigated yet. The present study demonstrated that 5 μg/mL of AAM was considered to be a safe dose for further experiments without cytotoxicity. Next, we evaluated the anti-pollution activity of AAM through the PM-induced keratinocytes damage cell model. The results showed that AAM could reduce PM-induced overproduction of intracellular ROS and the final product of lipid peroxidation, 4-hydroxynonenal (4HNE). In addition, AAM not only reduced the inflammatory protein expressions, including tumor necrosis factor α (TNFα), TNF receptor 1 (TNFR1) and cyclooxygenase-2 (COX-2), but also balanced the aging protein ratio of matrix metalloproteinase (MMPs) and tissue inhibitors of metalloproteases (TIMPs) through downregulating the phosphorylation of mitogen-activated protein kinase (MAPK) signaling. For skin barrier protection, AAM could repair PM-induced barrier function proteins damage, including filaggrin, loricrin and aquaporin 3 for providing anti-aging bioactivity. In conclusion, AAM has the potential to be developed as an anti-pollution active ingredient for topical skin products to prevent skin oxidation, inflammation and aging, and restore the skin barrier function.

Single-cell profiling reveals mechanisms of uncontrolled inflammation and glycolysis in decidual stromal cell subtypes in recurrent miscarriage.

Do distinct subpopulations of decidual stromal cells (DSCs) exist and if so, are given subpopulations enriched in recurrent miscarriage (RM)? Three subpopulations of DSCs were identified from which inflammatory DSCs (iDSCs) and glycolytic DSCs (glyDSCs) are significantly enriched in RM, with implicated roles in driving decidual inflammation and immune dysregulation. DSCs play crucial roles in establishing and maintaining a successful pregnancy; dysfunction of DSCs has been considered as one of the key reasons for the development of RM. We collected 15 early decidual samples from five healthy donors (HDs) and ten RM patients to perform single-cell RNA sequencing (scRNA-seq). A total of 43 RM patients and 37 HDs were enrolled in the validation cohort. Non-immune cells and immune cells of decidual tissues were sorted by flow cytometry to perform scRNA-seq. We used tissue microarrays (TMA) to validate three distinct subpopulations of DSCs. The expression of inflammatory and glycolytic proteins by DSCs was validated by immunohistochemistry (IHC) and multiplex immunohistochemistry (mIHC). Different subsets of decidual NK (dNK) cells and macrophages were also validated by multicolor flow cytometry and mIHC. Cell ligand-receptor and spatial analyses between DSCs and immune cells were analyzed by mIHC. We classify the DSCs into three subtypes based on scRNA-seq data: myofibroblastic (myDSCs), inflammatory (iDSCs) and glycolytic (glyDSCs), with the latter two being significantly enriched in RM patients. The distribution patterns of DSC subtypes in the RM and HD groups were validated by mIHC. Single-cell analyses indicate that the differentiation of iDSCs and glyDSCs may be coupled with the degrees of hypoxia. Consequently, we propose a pathological model in which a vicious circle is formed and fueled by hypoxic stress, uncontrolled inflammation and aberrant glycolysis. Furthermore, our results show that the inflammatory SPP1+ macrophages and CD18+ dNK cells are preferentially increased in the decidua of RM patients. Cell ligand-receptor and mIHC spatial analyses uncovered close interactions between pathogenic DSCs and inflammatory SPP1+ macrophages and CD18+ NK cells in RM patients. The raw single-cell sequence data reported in this paper were deposited at the National Omics Data Encyclopedia (www.biosino.org), under the accession number OEP002901. The number of decidual samples for scRNA-seq was limited and in-depth functional studies on DSCs are warranted in future studies. Identification of three DSC subpopulations opens new avenues for further investigation of their roles in RM patients. This study was supported by the Strategic Priority Research Program (No. XDB29030302), Frontier Science Key Research Project (QYZDB-SSW-SMC036), Chinese Academy of Sciences; National Key Research and Development Program of China (2021YFE0200600), National Natural Science Foundation of China (No. 31770960), Shanghai Municipal Science and Technology Major Project (No. 2019SHZDZX02, HS2021SHZX001), and Shanghai Committee of Science and Technology (17411967800). All authors report no conflict of interest.

Abstract 9464: Adipocyte-Derived Fatty-Acid-Binding Protein 4 Impairs Human Dermal Microvascular Endothelial Cells Under Hypoxia Insults - A Mechanistic Clue to Impaired Wound Healing in Obesity

Introduction: Dermal white adipose tissue is found in the dermis of the skin, which may participate in the wound healing process. Obesity-mediated adipocyte hypertrophy has been related to adipose tissue hypoxia and chronic low-grade inflammation, which may impair wound healing by suppressing the vascularity. Fatty-acid-binding protein (FABP) 4 is a cytosolic fatty acid chaperone expressed in adipocytes, which may be involved in the pathogenesis of some vascular diseases and was found upregulated in skin tissue of obese animals. This study aimed to investigate the potential mechanistic impacts of adipocyte-derived FABP4 on vascular cells for wound healing under hypoxia insults. Methods and Results: Primary human dermal microvascular endothelial cells (HDMECs) and adipocytes were used. FABP4 and inflammatory protein expressions such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β were upregulated under the hypoxic conditions. Treatment with conditioned medium from adipocytes under the hypoxic conditions significantly impaired cell function including migration and tube formation abilities in HDMECs. Furthermore, FABP4 and inflammatory protein expressions were upregulated, and angiogenic signaling pathways such as phospho-AKT/hypoxia-inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF)/ stromal cell-derived factor (SDF)-1 were downregulated in these conditioned medium treated HDMECs. Interestingly, administration of FABP4 inhibitor could reverse the conditioned medium-induced cell damages in HDMECs. On the other hand, FABP4 could directly impair cell functions with lower angiogenic and higher inflammatory protein expressions in HDMECs under the hypoxic conditions. Conclusions: Our pilot findings suggested that adipocyte hypoxia might lead to the secretion of inflammatory proteins and FABP4. Adipocyte-derived FABP4 could impair the migration and tube formation abilities of HDMECs via upregulating TNF-α/IL-6/IL-1β and downregulating phospho-AKT/HIF-1α/VEGF/SDF-1 signal pathways. Our findings provide a theoretical basis to the potential impact of FABP4 as a novel therapeutic target on wound healing in obese subjects with adipose tissue hypoxia.

Abstract 13683: Eicosapentaenoic Acid (EPA) Decreases Endothelin Converting Enzyme-1 Expression and Improves Nitric Oxide Release in Pulmonary Endothelium Following Challenge With Air Pollutants

Background: Air pollution particulate matter (PM) contributes to global mortality and chronic conditions, especially cardiovascular (CV) disease. Exposure to PM and cytokines like IL-6 cause endothelial cell (EC) dysfunction, leading to nitric oxide synthase (eNOS) uncoupling. The omega-3 fatty acid EPA improves eNOS coupling and reduces vascular inflammation. REDUCE-IT showed that treatment with icosapent ethyl (IPE), a purified EPA ethyl ester, reduced composite CV events. We compared the effects of EPA on pulmonary EC (PEC) function and protein expression following challenge with PMs versus IL-6. Methods: PECs were treated with EPA (40 μM) or equivolume vehicle for 2 h and then challenged with IL-6 (12 ng/mL) or PM (50 μg/mL) collected from urban environments (“urban PM”) for up to 8 h. Proteomic analysis was performed using LC/MS to measure relative protein expression. Significant ( p &lt;0.05) changes between treatment groups (&gt;1-fold) were analyzed by bioinformatic packages. Biological pathways were analyzed by gene set enrichment analysis (GSEA). In parallel, cells were stimulated with calcium ionophore to elicit nitric oxide (NO) and peroxynitrite (ONOO – ) release as assayed by tandem porphyrinic nanosensors; the NO/ONOO – ratio indicates eNOS coupling efficiency. Results: Challenge with IL-6 and urban PM caused eNOS uncoupling as evidenced by a decreased NO/ONOO – ratio of 35% (1.43 ± 0.04 vs. 2.19 ± 0.02, p &lt;0.001) and 50% (1.22 ± 0.07 vs. 2.42 ± 0.15 , p &lt;0.001), respectively, compared to control. EPA increased the NO/ONOO – ratio relative to both IL-6 (39%; 1.99 ± 0.11, p &lt;0.001) and urban PM (39%; 1.69 ± 0.14 , p &lt;0.05). Proteomics revealed that EPA decreased expression of endothelin converting enzyme-1 (ECE1) relative to both IL-6 (1.2-fold, p = 0.017) and urban PM (1.1-fold, p = 0.045). GSEA showed significant modulations to 18 and 35 proteins within the neutrophil degranulation pathway (GO:0043312) with EPA treatment relative to IL-6 and urban PM ( p &lt;0.0001 in both comparisons). Conclusion: EPA favorably reversed EC dysfunction and expression of inflammatory proteins and pathways following challenge with air pollution PMs similar to IL-6. These broad anti-inflammatory actions may contribute to reduced CV risk with EPA as observed in outcome trials.

PTP1B Inhibitor Claramine Rescues Diabetes-Induced Spatial Learning and Memory Impairment in Mice.

Accumulating clinical and epidemiological studies indicate that learning and memory impairment is more prevalent among people with diabetes mellitus (DM). PTP1B is a member of protein tyrosine phosphatase family and participates in a variety of pathophysiological effects including inflammatory, insulin signaling pathway, and learning and memory. This study was aimed to investigate the effects of CA, a specific inhibitor of PTP1B, on spatial learning and memory impairment in diabetic mice caused by high-fat diet and injection of streptozotocin. We found that the protein expressions of PTP1B increased in hippocampal CA1, CA3, and PFC regions of diabetic mice. Network pharmacology results showed that PTP1B might be one of the key targets between diabetes and cognitive dysfunction, and CA might alleviate DM-induced cognitive dysfunction. Animal experiments showed that CA ameliorated DM-induced spatial learning and memory impairment, and improved glucose and lipid metabolic disorders. Moreover, administration of CA alleviated hippocampal structure damage and enhanced the expressions of synaptic proteins, including PSD-95, SYN-1, and SYP in diabetic mice. Furthermore, CA treatment not only significantly down-regulated the expressions of PTP1B and NLRP3 inflammatory related proteins (NLRP3, ASC, Caspase-1, COX-2, IL-1β, and TNF-α), but also significantly up-regulated the expressions of insulin signaling pathway-related proteins (p-IRS1, p-PI3K, p-AKT, and p-GSK-3β) in diabetic mice. Taken together, these results suggested that PTP1B might be a targeted strategy to rescue learning and memory deficits in DM, possibly through inhibition of NLRP3 inflammasome and regulation of insulin signaling pathway.

Impact of Melatonin on RAW264.7 Macrophages during Mechanical Strain.

The concentration of melatonin is elevated during the night when patients mainly wear removable orthodontic appliances. Next to periodontal ligament fibroblasts and osteoblasts, macrophages react to mechanical strain with an increased expression of inflammatory mediators. Here, we investigated the impact of melatonin on RAW264.7 macrophages exposed to tensile or compressive strain occurring during orthodontic tooth movement in the periodontal ligament. Before exposure to mechanical strain for 4 h, macrophages were pre-incubated with different melatonin concentrations for 24 h, to determine the dependence of melatonin concentration. Afterwards, we performed experiments with and without mechanical strain, the most effective melatonin concentration (25 µM), and the melatonin receptor 2 (MT2) specific antagonist 4P-PDOT. The expression of inflammatory genes and proteins was investigated by RT-qPCR, ELISAs, and immunoblot. Both tensile and compressive strain increased the expression of the investigated inflammatory factors interleukin-1-beta, interleukin-6, tumor necrosis factor alpha, and prostaglandin endoperoxide synthase-2. This effect was inhibited by the addition of melatonin. Incubation with 4P-PDOT blocked this anti-inflammatory effect of melatonin. Melatonin had an anti-inflammatory effect on macrophages exposed to mechanical strain, independent of the type of mechanical strain. As inhibition was possible with 4P-PDOT, the MT2 receptor might be involved in the regulation of the observed effects.