PTGS2 Expression Research Articles

SYVN1 attenuates ferroptosis and alleviates spinal cord ischemia–reperfusion injury in rats by regulating the HMGB1/NRF2/HO-1 axis

BackgroundThe ferroptosis of neurons is an important pathological mechanism of spinal cord ischemia reperfusion injury (SCIRI). Previous studies showed that synoviolin 1 (SYVN1) is a good prognostic marker of neurodegenerative diseases, but its mechanism is still unclear. This study aims to explore the role of SYVN1 in the ferroptosis of neurons and to clarify its internal mechanism. MethodsRat primary spinal cord neurons were treated with oxygen-glucose deprivation (OGD) for 1, 4 or 8 h, and then cell viability, ROS and MDA levels, glutathione peroxidase (GSH-Px) activity, and the expression of ferroptosis-related proteins GPX4, FTH1 and PTGS2 were detected. OGD/R-induced neurons were transfected with pcDNA-SYVN1 or si-HMGB1, and then cell functions were detected. Transmission electron microscope (TEM) was used to detect cell ferroptosis. The interplay between SYVN1 and high mobility group box 1 (HMGB1) was confirmed with Co-immunoprecipitation (Co-IP) assay. The stability of HMGB1 was measured by ubiquitination assay. Also, cells were treated with pcDNA-SYVN1 or together with ubiquitination inhibitor MG132, as well as treated with pcDNA-SYVN1 and pcDNA-HMGB1 or together with NRF2 activator dimethyl fumarate (DMF), and then Western blotting was used to detect the expression of HMGB1, nuclear NRF2 and HO-1 proteins. In addition, SD rats were occluded left common carotid artery and aortic arch to establish a SCIRI rat model. And rats were injected intrathecal with adenovirus-mediated SYVN1 overexpression vector (Ad-SYVN1, 2 μL, virus titer 5 × 1013 transduction unit [TU]/mL) to overexpress SYVN1. The motion function of rats was quantified using the Basso Rat Scale (BMS) for Locomotion. The ferroptosis and the number of neurons in the spinal cord tissue of rats were detected. ResultsSYVN1 overexpression inhibited ferroptosis of SCIRI rats and OGD/R-treated primary spinal cord neurons, and down-regulated the expression of HMGB1. In terms of mechanism, the binding of SYVN1 and HMGB1 promoted the ubiquitination and degradation of HMGB1, and negatively regulated the expression of HMGB1. Moreover, under OGD/R conditions, MG132 treatment or HMGB1 overexpression eliminated the inhibitory effect of SYVN1 overexpression on the ferroptosis of neurons and the activation of the NRF2/HO-1 pathway, and DMF treatment abolished the inhibition of HMGB1 overexpression on the NRF2/HO-1 pathway. Finally, in vivo experiments showed that SYVN1 overexpression could alleviate the spinal cord ischemia–reperfusion injury in rats by down-regulating HMGB1 and promoting the activation of the NRF2/HO-1 pathway. ConclusionSYVN1 regulates ferroptosis through the HMGB1/NRF2/HO-1 axis to prevent spinal cord ischemia–reperfusion injury.

Effect of acute PM2.5 exposure on PTGS2 and RNA m6A modification

Particulate matter 2.5 (PM2.5) is a prevalent risk factor in many diseases, but its molecular mechanism remains ambiguous and may be diverse. RNA m6A is an important epigenetic modification that regulates gene expression at the post-transcriptional level. Some previous animal exposure studies found that PM2.5 exposure up-regulated m6A RNA methylation in the lung, but there is no research on m6A RNA methylation in humans from PM2.5 exposure now. Here, in the present experiment, we performed a panel study of 65 students at the Chinese research academy of environmental sciences (CRAES) with 3 rounds of follow-up visits from August 2021 to January 2022. We examined m6A RNA modification profiles of peripheral blood mononuclear cells (PBMCs) from subjects after low and high concentrations of ambient PM2.5 exposure. We applied a linear mixed-effect (LME) model to investigate the association between PM2.5 exposure and global m6A RNA methylation and PTGS2 level in peripheral blood. We found that increased levels of global m6A RNA methylation and PTGS2 level were associated with higher PM2.5 exposure. Among the methylated mRNAs, PTGS2 was hyper-methylated after high concentrations of PM2.5 exposure, which coincided with the increased expression of PTGS2 mRNA. In the present study, we determined that PM2.5 exposure promoted RNA m6A modification, and PTGS2 in peripheral blood could serve as a novel regulatory factor of inflammation induced by PM2.5 exposure. Furthermore, RNA m6A modification may contribute to the altered expression of PTGS2 induced by PM2.5 exposure. Our finding provided a new perspective for the prevention and treatment of PM2.5 exposure-induced adverse health effects.

ETS1 Ameliorates Hyperoxia-Induced Bronchopulmonary Dysplasia in Mice by Activating Nrf2/HO-1 Mediated Ferroptosis.

Bronchopulmonary dysplasia (BPD) is associated with hyperoxia-induced oxidative stress-associated ferroptosis. This study examined the effect of E26 oncogene homolog 1 (ETS1) on oxidative stress-associated ferroptosis in BPD. Hyperoxia-induced A549 cells and neonatal mice were used to establish BPD models. The effects of ETS1 on hyperoxia-induced ferroptosis-like changes in A549 cells were investigated by overexpression of ETS1 plasmid transfection and erastin treatment. Glucose consumption, lactate production, and NADPH levels were assessed by the glucose, lactate, and NADP+/NADPH assay kits, respectively. The potential regulatory relationship between ETS1 and Nrf2/HO-1 was examined by treating hyperoxia-induced A549 cells with the Nrf2 inhibitor ML385. ETS1 effect on the Nrf2 promoter was explored by dual-luciferase reporter and chromatin immunoprecipitation assay. The effect of ETS1 on the symptoms of BPD mice was examined by injecting an adenovirus overexpressing ETS1. ETS1 overexpression increased hyperoxia-induced cell viability, glucose consumption, lactate production, and NADPH levels and reduced inflammation and apoptosis in A549 cells. In animal experiments, ETS1 overexpression prevented weight loss, airway enlargement, and reductions in radial alveolar counts in BPD mice, while reducing the mean linear intercept, mean alveolar diameter and inflammation. ETS1 overexpression suppressed PTGS2 and CHAC1 expression, reduced ROS, MDA and ferrous iron (Fe2+) production and increased GSH levels in hyperoxia-induced A549 cells and BPD mice. In addition, ETS1 can bind to the Nrf2 promoter region and thus promote Nrf2 transcription. ETS1 overexpression increased the mRNA and protein levels of Nrf2, HO-1, xCT, and GPX4 in hyperoxia-induced A549 cells and BPD mice. In hyperoxia-induced A549 cells, erastin and ML385 treatment abolished the effect of ETS1 overexpression. ETS1 is important in oxidative stress-related ferroptosis in a hyperoxia-induced BPD model, and the effect is partially mediated by the Nrf2/HO-1 axis.

Unbiased differential proteomic profiling between cancer-associated fibroblasts and cancer cell lines

Cancer-associated fibroblasts (CAFs) are a key component of tumors. We aimed to profile the proteome of cancer cell lines representing three common cancer types (lung, colorectal and pancreatic) and a representative CAF cell line from each tumor type to gain insight into CAF function and novel CAF biomarkers. We used isobaric labeling, liquid chromatography and mass spectrometry to evaluate the proteome of 9 cancer and 3 CAF cell lines. Of the 9460 proteins evaluated, functional enrichment analysis revealed an upregulation of N-glycan biosynthesis and extracellular matrix proteins in CAFs. 85 proteins had 16-fold higher expression in CAFs compared to cancer cells, including previously known CAF markers like fibroblast activation protein (FAP). Novel overexpressed CAF biomarkers included heat shock protein β-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1). SiRNA knockdown of the genes encoding these proteins did not reduce contractility in lung CAFs, suggesting they were not crucial to this function. Immunohistochemical analysis of 30 tumor samples (10 lung, 10 colorectal and 10 pancreatic) showed restricted HSPB6 and PTGS1 expression in the stroma. Therefore, we describe an unbiased differential proteome analysis of CAFs compared to cancer cells, which revealed higher expression of HSPB6 and PTGS1 in CAFs. Data are available via ProteomeXchange (PXD040360). SignificanceCancer-associated fibroblasts (CAFs) are highly abundant stromal cells present in tumors. CAFs are known to influence tumor progression and drug resistance. Characterizing the proteome of CAFs could give potential insights into new stromal drug targets and biomarkers. Mass spectrometry-based analysis comparing proteomic profiles of CAFs and cancers characterized 9460 proteins of which 85 proteins had 16-fold higher expression in CAFs compared to cancer cells. Further interrogation of this rich resource could provide insight into the function of CAFs and could reveal putative stromal targets. We describe for the first time that heat shock protein β-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1) are overexpressed in CAFs compared to cancer cells.

Exposure to Stress Alters Cardiac Gene Expression and Exacerbates Myocardial Ischemic Injury in the Female Murine Heart.

Mental stress is a risk factor for myocardial infarction in women. The central hypothesis of this study is that restraint stress induces sex-specific changes in gene expression in the heart, which leads to an intensified response to ischemia/reperfusion injury due to the development of a pro-oxidative environment in female hearts. We challenged male and female C57BL/6 mice in a restraint stress model to mimic the effects of mental stress. Exposure to restraint stress led to sex differences in the expression of genes involved in cardiac hypertrophy, inflammation, and iron-dependent cell death (ferroptosis). Among those genes, we identified tumor protein p53 and cyclin-dependent kinase inhibitor 1A (p21), which have established controversial roles in ferroptosis. The exacerbated response to I/R injury in restraint-stressed females correlated with downregulation of p53 and nuclear factor erythroid 2-related factor 2 (Nrf2, a master regulator of the antioxidant response system-ARE). S-female hearts also showed increased superoxide levels, lipid peroxidation, and prostaglandin-endoperoxide synthase 2 (Ptgs2) expression (a hallmark of ferroptosis) compared with those of their male counterparts. Our study is the first to test the sex-specific impact of restraint stress on the heart in the setting of I/R and its outcome.

Comparative interleukins and chemokines analysis of mice mesenchymal stromal cells infected with Mycobacterium tuberculosis H37Rv and H37Ra

Inflammatory pathways involving Mesenchymal stromal cells (MSCs) play an important role in Mycobacterium tuberculosis (Mtb) infection. H37Rv (Rv) is a standard virulent strain, however, H37Ra (Ra) is a strain with reduced virulence. Interleukins and chemokines production are known to promote inflammation resistance in mammalian cells and is recently reported to regulate mycobacterial immunopathogenesis via inflammatory responses. MSCs are very important cells during Mtb infection. However, the different expressions of interleukins and chemokines in the process of Mtb-infected MSCs between Ra and Rv remain unclear. We used the techniques of RNA-Seq, qRT-PCR, ELISA, and Western Blotting. We have shown that Rv infection significantly increased mRNA expressions of Mndal, Gdap10, Bmp2, and Lif, thereby increasing more differentiation of MSCs compared with Ra infection in MSCs. Further investigation into the possible mechanisms, we found that Rv infection enhanced more inflammatory response (Mmp10, Mmp3, and Ptgs2) through more activation of the TLR2-MAP3K1-JNK pathway than did Ra infection in MSCs. Further action showed that Rv infection enhanced more Il1α, Il6, Il33, Cxcl2, Ccl3, and Ackr3 production than did Ra infection. Rv infection showed more expressions of Mmp10, Mmp3, Ptgs2, Il1α, Il6, Il33, Cxcl2, Ccl3, and Ackr3 possibly through more active TLR2-MAP3K1-JNK pathway than did Ra infection in MSCs. MSCs may therefore be a new candidate for the prevention and treatment of tuberculosis.

Integration of metabolomics and network pharmacology to reveal the protective mechanism underlying Wogonoside in acute myocardial ischemia rats

Ethnopharmacological relevanceIn traditional medicine, both Scutellaria baicalensis Georgi (SBG) and the traditional formulas composed of it have been used to treat a wide range of diseases, including cancer and cardiovascular. Wogonoside (Wog) is the biologically active flavonoid compound extracted from the root of SBG, with potential cardiovascular protective effects. However, the mechanisms underlying the protective effect of Wog on acute myocardial ischemia (AMI) have not yet been clearly elucidated. Aim of the studyTo explore the protective mechanism of Wog on AMI rats by comprehensively integrating traditional pharmacodynamics, metabolomics, and network pharmacology. MethodsThe rat was pretreatment with Wog at a dose of 20 mg/kg/d and 40 mg/kg/d once daily for 10 days and then ligated the left anterior descending coronary artery of rats to establish the AMI rat model. Electrocardiogram (ECG), cardiac enzyme levels, heart weight index (HWI), Triphenyltetrazolium chloride (TTC) staining, and histopathological analyses were adopted to evaluate the protective effect of Wog on AMI rats. Moreover, a serum metabolomic-based UHPLC-Q-Orbitrap MS approach was performed to find metabolic biomarkers and metabolic pathways, and network pharmacology analysis was applied to predict targets and pathways of Wog in treating AMI. Then, the network pharmacology and metabolomic results were integrated to elucidate the mechanism of Wog in treating AMI. Finally, RT- PCR was used to detect the mRNA expression levels of PTGS1, PTGS2, ALOX5, and ALOX15 to validate the result of integrated metabolomics and network analysis. ResultsPharmacodynamic studies suggest that Wog could effectively prevent the ST-segment of electrocardiogram elevation, reduce the myocardial infarct size, heart weight index, and cardiac enzyme levels, and alleviate cardiac histological damage in AMI rats. Metabolomics analysis showed that the disturbances of metabolic profile in AMI rats were partly corrected by Wog and the cardio-protection effects on AMI rats involved 32 differential metabolic biomarkers and 4 metabolic pathways. In addition, the integrated analysis of network pharmacology and metabolomics showed that 7 metabolic biomarkers, 6 targets, and 6 crucial pathways were the main mechanism for the therapeutic application of Wog for AMI. Moreover, the results of RT-PCR showed that PTGS1, PTGS2, ALOX5, and ALOX15 mRNA expression levels were reduced after treatment with Wog. ConclusionWog exerts cardio-protection effects on AMI rats via the regulation of multiple metabolic biomarkers, multiple targets, and multiple pathways, our current study will provide strong scientific evidence supporting the therapeutic application of Wog for AMI.

ERBB2-PTGS2 axis promotes intervertebral disc degeneration by regulating senescence of nucleus pulposus cells

Intervertebral disc degeneration (IDD) is considered one of the main causes of low back pain and lumbar disc herniation. Various studies have shown that disc cell senescence plays a critical role in this process. however, its role in IDD is yet unclear. In this study, we explored the role of senescence-related genes (SR-DEGs) and its underlying mechanism in IDD. A total of 1325 differentially expressed genes (DEGs) were identified using Gene Expression Omnibus (GEO) database GSE41883. 30 SR-DEGs were identified for further functional enrichment and pathway analysis, and two hub SR-DEGs (ERBB2 and PTGS2) were selected to construct transcription factor (TF)–gene interaction and TF-miRNA coregulatory networks, and 10 candidate drugs were screened for the treatment of IDD. Last but not least, in vitro experiments show that ERBB2 expression decreased and PTGS2 expression increased in human nucleus pulposus (NP) cell senescence model treated with TNF-α. After lentivirus-mediated overexpression of ERBB2, the expression of PTGS2 decreased and the senescence level of NP cells decreased. Overexpression of PTGS2 reversed the anti-senescence effects of ERBB2. The findings in this study suggested that ERBB2 overexpression further reduced NP cell senescence by inhibiting PTGS2 levels, which ultimately alleviated IDD. Taken together, our findings provide new insights into the roles of senescence-related genes in IDD and highlight a novel target of ERBB2-PTGS2 axis for therapeutic strategies.

#3769 MECHANOSENSITIVE ION CHANNEL PIEZO1 MEDIATES BLOOD PRESSURE HOMEOSTASIS BY REGULATING RENIN EXPRESSION IN MICE

Abstract Background and Aims The renin-angiotensin system (RAS) plays a crucial role in the maintenance of fluid-electrolyte and blood pressure homeostasis. Renin, the step-limiting protease of the RAS, is produced, stored, and secreted by the renal juxtaglomerular (JG) cells, which are closely regulated by the blood pressure. Studies have shown that local blood flow-associated shear stress play an important role in renin expression and secretion. To date, however, the molecular identity through which JG cells respond to the blood pressure remains unclear. Method In this study, we determined the expression and subcellular localization of Piezo1 in JG cells by qPCR, Western blot, Immunofluorescence (IF), and multiplex immunohistochemistry. Then, a Fluo-4 AM-based calcium-imaging system was used to detect the dynamic changes of intracellular calcium in response to Piezo1 specific agonist Yoda1 and mechanical stress (MS)-induced by perfusion. Next, CRISPR/Cas9 technology was used to generate Piezo1 knockout JG cells to further confirm its contribution. Then, we used RNA-seq experiment to investigate the downstream signaling of Piezo1 in JG cells. Finally, we generated adeno-associated virus (AAV)-mediated kidney-specific Piezo1 knockdown mice to investigate the in vivo effect of Piezo1. Results We found that the mechanosensitive ion channel Piezo1 was co-expressed with renin in JG cells in mouse kidney slides as well as As4.1 cells, a commonly used JG cell line. Activation of Piezo1 by its agonist Yoda1 induced an intracellular calcium increase and reduced the expression of renin in these cells, while knockout of Piezo1 in JG cells abolished the effect of Yoda1. Meanwhile, fluid induced mechanical stress also led to an intracellular calcium increase and renin downregulation in wildtype but not Piezo1 knockout JG cells. Mechanistically, we demonstrated that activation of Piezo1 upregulated the Ptgs2 expression via the calcineurin-NFAT pathway and increased the production of Ptgs2 downstream molecule COX-2 and PGE2 in JG cells, which inhibited cAMP production and reduced renin expression in JG cells. In mice, we demonstrated that activation of Piezo1 significantly downregulated the blood pressure in wildtype but not kidney-specific Piezo1 knockdown mice. Conclusion In summary, these results revealed that activation of Piezo1 could lead to downregulation of the renin expression in vitro and in vivo, subsequently reduction of blood pressure, highlighting its therapeutic potential as a drug target of the renin-angiotensin system.

Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment.

Epigenetic mechanisms are implicated in transcriptional programs driving chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain and extraterminal (BET) proteins, which are epigenetic readers that modulate gene expression. In the phase 3 BETonMACE trial, apabetalone reduced risk of major adverse cardiac events (MACE) by 50% in the CKD subpopulation, indicating favorable effects along the kidney-heart axis. Activation of human renal mesangial cells (HRMCs) to a contractile phenotype that overproduces extracellular matrix (ECM) and inflammatory cytokines, and promotes calcification, frequently accompanies CKD to drive pathology. Here, we show apabetalone downregulated HRMC activation with TGF-β1 stimulation by suppressing TGF-β1-induced α-smooth muscle actin (α-SMA) expression, α-SMA assembly into stress fibers, enhanced contraction, collagen overproduction, and expression of key drivers of fibrosis, inflammation, or calcification including thrombospondin, fibronectin, periostin, SPARC, interleukin 6, and alkaline phosphatase. Lipopolysaccharide-stimulated expression of inflammatory genes IL6, IL1B, and PTGS2 was also suppressed. Transcriptomics confirmed apabetalone affected gene sets of ECM remodeling and integrins. Clinical translation of in vitro results was indicated in CKD patients where a single dose of apabetalone reduced plasma levels of key pro-fibrotic and inflammatory markers, and indicated inhibition of TGF-β1 signaling. While plasma proteins cannot be traced to the kidney alone, anti-fibrotic and anti-inflammatory effects of apabetalone identified in this study are consistent with the observed decrease in cardiovascular risk in CKD patients.

Mesenchymal stromal cell conditioned media is immunomodulatory in the equine endometrium

Endometritis is a common and costly disorder in horses that results in poor reproductive performance. Current therapies are supportive, and the industry suffers from a paucity of immunomodulatory treatment modalities for this disease that is characterized by inflammatory dysregulation. Recently, mesenchymal stromal cell conditioned media (MSC-CM) is being explored as a treatment option for various inflammatory conditions due to its immunoregulatory, antimicrobial, biofilm disruption, and tissue repair properties. Our objective was to evaluate changes in the endometrial inflammatory response after treatment with MSC-CM in vitro. We hypothesized that endometrial cells in culture would have altered cytokine expression and production. Five estrous mares were biopsied to establish primary endometrial cell cultures. After cell dissociation, endometrial cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution (Sigma Aldrich) at 37°C with 5% CO2. At 80% confluence, cultures were hormonally supplemented with 1nM Estradiol-17β, 100 nM Progesterone, or no supplementation; and 24 hours later, treated with MSC-CM or DMSO as control. Supernatants from the cell cultures were analyzed for cytokine expression using an equine cytokine Multiplex assay (Luminex Corp. Austin, TX) quantifying inflammatory biomarkers including IFN-α, IFN-γ, IL-4, IL-17, IL-10, IL1-β, CCL2, CCL3, CCL5, CCL11, TNF-α, and sCD14. In parallel, cells were lysed and RNA extracted for Real-time PCR using the ΔCT method with ACTB as the reference gene for geneexpression of IL-6, IL-8, PTGS2, PTGES, PTGIS, TNFα, TGFB, and NFKb. Data analysis was performed with JMP Pro 16 with level of significant p<0.05. A restricted maximum likelihood model with treatment, supplementation and their interaction and mare as a random factor was used. There was no effect of hormone supplementation on supernatant cytokine concentrations, but treatment with MSC-CM altered their concentration. MSC-CM treatment increased the concentration of CCL2 pg/ml [median (interquartile)] [1862554 (32482, 240080) (p≤0.02); and decreased CCL3 [66 (0.1, 99)] (p≤ 0.01) and IL-10 [6.5 (0.1, 9.5)] (p≤0.007) compared to DMSO control [3114 (2398.5, 7483); 109 (0.1, 154); and 13 (0.1, 17)], respectively. Gene expression analysis showed that MSC-CM-treated cells had increased expression of IL-6 (p≤0.001), IL-8 (p≤0.001), PTGS2 (p≤0.002), PTGES (p≤0.0012), and NFKb (p≤0.01). Additionally, gene expression analysis revealed an effect of hormone supplementation, with increased expression of IL-8 (p≤0.027) and PTGS2 (p≤0.0443) in estrogen or progesterone supplemented cultures compared to no supplementation. Taken together, treatment with MSC-CM resulted in increased inflammatory marker production and greater gene expression of inflammatory cytokines and prostaglandin-related genes. These results are supportive of the potential role of MSC-CM in equine endometrial immunomodulation. Further studies in vivo are warranted to investigate the clinical outcome of this immunomodulatory effect after intrauterine infusion.

Interleukin-1 beta treatment alters prostaglandin-related mRNA expression and cytokine abundance in equine endometrial culture

Interleukin-1β (IL-1β) is a proinflammatory cytokine that modulates the immunoinflammatory response and induces prostaglandin synthesis. IL-1β has a variety of roles in the endometrium during inflammation, decidualization, pregnancy, and post-partum. It can cause changes in endometrial gene expression and may affect cytokine, chemokine, and prostaglandin-related pathways. The relationship between IL-1β, prostaglandins, and downstream inflammatory responses in the equine uterine environment is not fully known. Our objective was to determine the effect that IL-1β has on prostaglandin-related gene expression, as well as cytokine and chemokine abundance, in endometrial cell culture. Light breed mares (n=8) were examined daily by transrectal ultrasound until ovulation. An endometrial biopsy was collected at day 11 post ovulation, and used for primary endometrial cell culture. Cells were cultured in DMEM with 10% FBS. At 80% confluence, cells were either not supplemented or supplemented with: 100 nM Progesterone (Prog); 1 nM Estradiol-17β (Est); or 100 nM Prog and 1 nM Est (P+E). After 24 hours, cells were treated with recombinant equine IL-1β at either 10 ng/mL (Low) or 1000 ng/mL (High), or 0.2% DMSO as an untreated control. Twenty-four hours post-treatment, supernatant was collected, and a bead-based multiplex assay was used to quantify concentration of twelve cytokines and chemokines. Total RNA was extracted from the cells and mRNA expression was evaluatedfor 10 prostaglandin-related genes, using the ΔCT method with ACTB as the reference gene. Data analysis was performed using JMP Pro 16, using a restricted maximum likelihood model with treatment and supplementation as factors, and mare as a random factor. Treatment with IL-1β High resulted in increased levels of CCL2, CCL5, and CCL11 (p<0.001) in cell culture supernatant, as well as increased mRNA expression of PTGS2 (p<0.02), regardless of supplementation. Cells supplemented with Prog or P+E and treated with IL-1β High had increased expression of IL1R1, IL1RAP, PTGS1, and PTGES compared to non-supplemented cells (p<0.05). IL-1β High treatment increased LiF expression in cells supplemented with Prog or P+E compared to untreated cells (p<0.05). IL-1β likely acts to modulate inflammatory pathways in the endometrium, altering chemokine levels and activating expression of PTGS2, leading to changes in downstream prostaglandin synthesis and other related pathways. Progesterone appears to increase expression of several target genes when compared to non-supplemented cells. Progesterone has been shown to be involved in the suppression of several inflammatory pathways, and investigation into the ovarian steroid regulation of genes and proteins in the equine endometrium is warranted. Further studies on the effect of IL-1β on molecular pathways and prostaglandin regulation could lead to a greater understanding of the uterine environment in the pregnant and non-pregnant mare.

Differential gene expression in the equine endometrium and conceptus from Day 11 to 16 post-ovulation

Mobility of the equine conceptus is essential for maternal recognition of pregnancy (MRP) and is regulated by the conceptus's own derived prostaglandin secretion. Our objective was to compare prostaglandin-related genes and other genes of interest during the expected MRP time. Light breed mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation. Mares in the pregnant (P) group were bred by artificial insemination with >500 million progressively motile normal sperm from one fertile stallion until D0. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Endometrium was collected via a transcervical biopsy punch following conceptus retrieval. Endometrium of non-pregnant (NP) mares was collected at: D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium from the pregnant horn and conceptuses was collected at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). Total RNA from trophoblast and endometrial samples was isolated and evaluated for expression of PTGS2, PTGES, mPGES2, PTGER4, PTGER2, PTGIS, PTGIR, HPGD, SLCO2A1, CRTC2, NR3C1, CBR1, CREB, NF-Kβ, SPP1, IGFBP1, ESR1 and DNTTIP2 using Real-time PCR. Mean threshold cycle was determined and normalized to the reference gene (GAPDH and ACTB) (ΔCT). Statistical analyses were performed using JMP Pro 16, and data was ranked, transformed if necessary, followed by a restricted maximum likelihood model with mare as arandom factor to test differences between Groups and Day. Regarding endometrial mRNA, NP11 had higher expression for NF-Kβ, PTGIS, PTGIR and CBR1 compared to NP13 (p<0.007). ESR1, mPGES2, PTGIS, and CBR1 mRNA were higher in P11 compared to P16 (p<0.05), while CRTC2 was higher in P16 compared to P11 (p<0.06). Lastly, NF-Kβ, PTGIR and CBR1 were higher in P11 compared to P13 (p<0.01), but NF-Kβ P13 was lower compared to P16 (p<0.04). Regarding the conceptuses, CREB and PTGFR expressions were higher in D16 compared to D11 (p<0.003), while CRTC2, NR3C1, NF-Kβ and SPP1 were higher in D16 compared to D11 and 13 (p<0.02). D13 conceptuses had lower expression of HPGD compared to D11 and 16 (p<0.02). Lastly, D11 conceptuses had higher expression of mPGES2, PTGIS and PTGER4 compared to D13 and D16 (p<0.03). Higher expression of D11 PTGIS from both tissues may increase nutrient and blood flow to the endometrium in support of the early conceptus. The upregulation of endometrial NF-Kβ at D11 may be a result of endometrial receptivity through the regulation of inflammation, and the up-regulation of HPGD may function to regulate PGE2 during early pregnancy. Understanding embryonic and endometrial prostaglandin regulation during MRP may help to develop future therapies to ameliorate pregnancy loss in the horse.

Concentration Effects of Methylprednisolone in Human Vocal Fold Fibroblast-Macrophage Co-Culture.

The diversity of glucocorticoid (GC) properties may underlie variability of clinical efficacy for vocal fold (VF) disease. Optimized therapeutic approaches must account for tissue complexity as well as interactions between cell types. We previously reported that reduced GC concentrations inhibited inflammation without eliciting fibrosis in mono-cultured VF fibroblasts and macrophages. These data suggested that a refined approach to GC concentration may improve outcomes. In the current study, co-culture of VF fibroblasts and macrophages was employed to investigate the effects of different concentrations of methylprednisolone on fibrotic and inflammatory response genes in VF fibroblasts to optimize management paradigms. In vitro. THP-1 monocyte-derived macrophages were stimulated with interferon-γ (IFN-γ), lipopolysaccharide (LPS), or transforming growth factor-β (TGF-β) to induce inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes. Macrophages were then co-cultured with a human VF fibroblast cell line using a 0.4 μm pore membrane with or without 0.1-3000 nM methylprednisolone. Inflammatory (CXCL10, TNF, and PTGS2) and fibrotic (ACTA2, CCN2, and COL1A1) gene expression was quantified in fibroblasts. Incubating VF fibroblasts with M(IFN/LPS) macrophages increased expression of TNF and PTGS2, and this effect was inhibited by methylprednisolone. Incubation of VF fibroblasts with M(TGF) macrophages increased expression of ACTA2, CCN2, and COL1A1, and this effect was enhanced by methylprednisolone. The concentration of methylprednisolone required to downregulate inflammatory genes (TNF and PTGS2) was lower than that to upregulate fibrotic genes (ACTA2, CCN2, and COL1A1). Reduced concentration of methylprednisolone effectively suppressed inflammatory genes without enhancing fibrotic genes, suggesting that a refined approach to GC concentration may improve clinical outcomes. N/A Laryngoscope, 133:3116-3122, 2023.

Quercetin prevents the ferroptosis of OPCs by inhibiting the Id2/transferrin pathway

Spinal cord injury (SCI) is a destructive neurological disorder that causes impaired mobility, sensory, and autonomic dysfunctions. The loss of oligodendrocyte progenitor cells (OPCs), which can differentiate into mature oligodendrocytes and re-myelinate damaged axons, is related to poorer recovery for SCI patients. However, inhibiting OPCs loss has always been a difficult problem to overcome. In this study, we demonstrated the anti-ferroptosis effects of quercetin as a mechanism in erastin-induced OPC ferroptosis. Quercetin ameliorated erastin-induced ferroptosis in OPCs, as indicated by decreased iron concentration, reactive oxygen species (ROS) production, and increased content of glutathione (GSH) as well as more normal mitochondria morphology. Compared with erastin-induced OPCs, the myelin basic protein (MBP)-positive myelin and NF200-positive axonal was remarkably increased in quercetin-treated OPCs. Furthermore, quercetin ameliorated the erastin-induced ferroptosis as well as the myelin and axon loss of OPCs by downregulating transferrin. Transfected OPCs with transferrin overexpression plasmids significantly abrogated the protective role of quercetin in OPC ferroptosis. Using ChIP-qPCR, a direct interaction of transferrin with its upstream gene Id2 was found. The overexpression of Id2 reversed the effect of quercetin on OPC ferroptosis. In vivo study found that quercetin greatly decreased the area of injury, and enhanced the BBB score after SCI. Furthermore, in the SCI model, quercetin significantly downregulated Id2 and transferrin expression, while significantly up-regulated GPX4 and PTGS2 expression. In conclusion, quercetin prevents the ferroptosis of OPCs by inhibiting the Id2/transferrin pathway. These findings highlight quercetin as an anti-ferroptosis agent for the treatment or prevention of spinal cord injury.

Integrating UPLC-Q-Exactive Orbitrap/MS, network pharmacology and experimental validation to reveal the potential mechanism of Tibetan medicine Rhodiola granules in improving myocardial ischemia-reperfusion injury

Ethnopharmacology relevanceRhodiola granules (RG) is a traditional Tibetan medicine prescription that can be used to improve the symptoms of ischemia and hypoxia in cardiovascular and cerebrovascular diseases. However, there is no report on its use to improve myocardial ischemia/reperfusion (I/R) injury, and its potential active ingredients and mechanism against myocardial ischemia/reperfusion (I/R) injury remain unclear. Aim of the studyThis study aimed to reveal the potential bioactive components and underlying pharmacological mechanisms of RG in improving myocardial I/R injury through a comprehensive strategy. Materials and methodsUPLC-Q-Exactive Orbitrap/MS technology was used to analyze the chemical components of RG, the potential bioactive components and targets were tracked and predicted by the SwissADME and SwissTargetPrediction databases, and the core targets were predicted through the PPI network, as well the functions and pathways were determined by GO and KEGG analysis. In addition, the molecular docking and ligation of the anterior descending coronary artery-induced rat I/R models were experimentally validated. ResultsA total of 37 ingredients were detected from RG, including nine flavones, ten flavonoid glycosides, one glycoside, eight organic acids, four amides, two nucleosides, one amino acid, and two other components. Among them, 15 chemical components, such as salidroside, morin, diosmetin, and gallic acid were identified as key active compounds. Ten core targets, including AKT1, VEGF, PTGS2, and STAT3, were discovered through the analysis of the PPI network constructed from 124 common potential targets. These possible targets were involved in the regulation of oxidative stress and HIF-1/VEGF/PI3K-Akt signaling pathways. Furthermore, molecular docking confirmed that the potential bioactive compounds in RG have good potential binding abilities to AKT1, VEGFA, PTGS2, STAT3, and HIF-1α proteins. Then, the animal experiments showed that RG could significantly improve the cardiac function of I/R rats, reduce the size of myocardial infarction, improve the myocardial structure, and reduce the degree of myocardial fibrosis, inflammatory cell infiltration, and myocardial cell apoptosis rate in I/R rats. In addition, we also found that RG could decrease the concentration of AGE, Ox-LDL, MDA, MPO, XOD, SDH, Ca2+, and ROS, and increase the concentration of Trx, TrxR1, SOD, T-AOC, NO, ATP, Na+k+-ATPase, Ca2+-ATPase, and CCO. Moreover, RG could significantly down-regulate the expressions of Bax, Cleaved-caspase3, HIF-1α, and PTGS2, as well up-regulate the expressions of Bcl-2, VEGFA, p-AKT1, and p-STAT3. ConclusionIn summary, we revealed for the first time the potential active ingredients and mechanisms of RG for myocardial I/R injury therapy through a comprehensive research strategy. RG may synergistically improve myocardial I/R injury through anti-inflammatory, regulating energy metabolism, and oxidative stress, improving I/R-induced myocardial apoptosis, which may be related to the HIF-1/VEGF/PI3K-Akt signaling pathway. Our study provides new insights into the clinical application of RG and also provides a reference for the development and mechanism research of other Tibetan medicine compound preparations.

Imperatorin inhibits LPS-induced bone marrow-derived macrophages activation by decreased NF-κB p65 phosphorylation

Background Imperatorin (IMP) is a secondary metabolite of plants and is the most abundant in Angelica dahurica. Previous studies showed that IMP exhibited anti-inflammatory activity in RAW264.7 cell line. Here, we aim to investigate the roles and mechanisms of IMP in bone marrow-derived macrophages (BMDMs), in view of the difference between primary macrophages and cell lines. Methods BMDMs were stimulated with LPS for the inflammation model. Flow cytometry was performed with BMDMs treated with different doses of IMP (0–20mg/L) within staining Annexin V-APC for 5 min. The cytokines and inflammatory mediators were detected by RT-PCR or ELISA. RNA-seq was performed in IMP-treated BMDMs or control, stimulated with LPS for 6h. Western blotting is carried out to determine the phosphorylation of p65, ERK1/2, JNK1, p38, and Akt. Results Our results showed that IMP inhibited IL-12p40, IL-6, TNF-α and IL-1β in LPS-stimulated BMDMs. RNA-seq analysis suggested that IMP inhibits Toll-like receptor signaling pathway (KEGG), TNF signaling pathway (KEGG), NF-κB signaling pathway (KEGG), Inflammatory Response (GO). In addition, IMP inhibited myd88, tpl2, cxcl1, ptgs2(COX-2) expression in mRNA level. Finally, we found decreased phosphorylation of NF-κB p65 in IMP-treated BMDMs, after stimulated with LPS. Conclusion IMP inhibits IL-12p40, IL-6, TNF-α, and IL-1β expression in LPS-stimulated BMDMs. IMP inhibits macrophage activation, which maybe resulted in decreased phosphorylation of NF-κB p65. Furthermore, IMP may protect against the progress of inflammatory-related diseases.

Abstract 2694: Blockade of the cyclooxygenase-2 prevents chemotherapy-induced cognitive impairments

Abstract Chemotherapy-induced cognitive impairment (CICI, also termed “chemobrain”) is a major neurotoxic side effect exhibited by a wide range of chemotherapeutic agents. These side effects affect approximately 14 million cancer survivors. More importantly, CICI persists well after cessation of therapy, severely interfering with quality of life. Unfortunately, there is no treatment and novel therapies are urgently needed. Utilizing cisplatin, a platinum-based chemotherapy, to model chemobrain mice in vivo, we have established that cisplatin accelerates the brain aging process thus leading to long-term memory impairment in mice similar to what is clinically reported. We further show that cisplatin causes oxidative DNA damage, mitochondrial defects, impaired neurogenesis, synaptic defects, and increased gliosis in the adult hippocampus, a brain region critical for learning and memory. Mechanistically, our RNA-sequencing and qRT-PCR analysis revealed that cisplatin dramatically increases COX-2 (Ptgs2) expression and its major product, prostaglandin E2 (PGE2) in adult mouse brain and human cortical neurons derived from induced pluripotent stem cells (iPSCs). Similar as cisplatin treatment, the levels of COX-2 expression began to increase at 0.1 µM methotrexate treatment, which significantly increased at 1 µM in human cortical neurons, indicating that COX-2 induction is a common pathogenic mechanism mediating cognitive impairment associated with cisplatin and methotrexate in spite of the fact that these compounds have different mechanisms of action for cancer eradication. Most importantly, NS-398, a selective COX-2 inhibitor, effectively prevents cognitive deficits in mice and reduction in cell viability of human neurons associated with these chemotherapies, without promoting tumor growth or interfering with cisplatin’s anti-tumor activity. Taken together, our findings strongly suggest that COX-2 induction is a main cause of CICI, making COX-2 inhibition a therapeutic target for CICI. Given that COX-2 inhibitors are currently being tested in cancer prevention as well as age-related memory loss in clinical trials, inhibiting COX-2 may have far-reaching therapeutic effects on CICI and cancer treatment, which can be applied quickly, safely, and effectively to clinical trials, in the effort to improve cancer survivor quality of life. Citation Format: Mohammad Abdur Rashid, Jason J. Tang, Ki-Hyun Yoo, Ana Corujo-Ramirez, Sang Hoon Kim, Alfredo Oliveros, Peter Cole, John R. Hawse, Mi-Hyeon Jang. Blockade of the cyclooxygenase-2 prevents chemotherapy-induced cognitive impairments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2694.

Gene Expression in Glenoid Articular Cartilage Varies Across Acute Instability, Chronic Instability, and Osteoarthritis.

Shoulder instability is a common pathology associated with an elevated risk of osteoarthritis (OA). Little is known about gene expression in the cartilage of the glenohumeral joint after dislocation events, particularly as it relates to the risk of posttraumatic OA. This study tested the hypothesis that gene expression in glenoid cartilage varies among acute instability (<3 dislocations), chronic instability (≥3 dislocations), and OA. Articular cartilage was collected from the anteroinferior glenoid of consenting patients undergoing shoulder stabilization surgery (n = 17) or total shoulder arthroplasty (n = 16). Digital quantitative polymerase chain reaction was used to assess the relative expression of 57 genes (36 genes from OA risk allele studies, 21 genes from differential expression studies), comparing (1) OA versus instability (acute and chronic combined), (2) acute versus chronic instability, (3) OA versus acute instability, and (4) OA versus chronic instability. The expression of 11 genes from OA risk allele studies and 9 genes from differential expression studies was significantly different between cartilage from patients with instability and those with OA. Pro-inflammatory genes from differential expression studies and genes from OA risk allele studies were more highly expressed in cartilage in the OA group compared with the instability group, which expressed higher levels of extracellular matrix and pro-anabolic genes. The expression of 14 genes from OA risk allele studies and 4 genes from differential expression studies, including pro-inflammatory genes, anti-anabolic genes, and multiple genes from OA risk allele studies, was higher in the acute instability group compared with the chronic instability group. Cartilage in the OA group displayed higher expression of CCL3, CHST11, GPR22, PRKAR2B, and PTGS2 than cartilage in the group with acute or chronic instability. Whereas cartilage in both the acute and chronic instability groups had higher expression of collagen genes, cartilage in the OA group had expression of a subset of genes from OA risk allele studies or from differential expression studies that was lower than in the acute group and higher than in the chronic group. Glenoid cartilage has an inflammatory and catabolic phenotype in shoulders with OA but an anabolic phenotype in shoulders with instability. Cartilage from shoulders with acute instability displayed greater (cellular) metabolic activity compared with shoulders with chronic instability. This exploratory study identified genes of interest, such as CCL3, CHST11, GPR22, PRKAR2B, and PTGS2, that have elevated expression in osteoarthritic glenoid cartilage. These findings provide new biological insight into the relationship between shoulder instability and OA, which could lead to strategies to predict and potentially modify patients' risk of degenerative arthritis due to shoulder instability.

Dynamics of inflammatory cytokine expression in bovine endometrial cells exposed to cow blood plasma small extracellular vesicles (sEV) may reflect high fertility

Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30–200 nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein–Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (FBV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded lower expression of PTGS1 and PTGS2 compared to the control. In bCSC cells exposed to HF-EXO, pro-inflammatory cytokine IL1-α was downregulated compared to the untreated control, IL-12α and IL-8 were downregulated compared to the LF-EXO treatment. Our findings demonstrate that sEVs interact with both endometrial epithelial and stromal cells to initiate differential gene expression, specifically genes relate to inflammation. Therefore, even subtle changes on the inflammatory gene cascade in the endometrium via sEV may affect reproductive performance and/or outcomes. Further, sEV from high-fertile animals acts in a unique direction to deactivate prostaglandin synthases in both bCSC and bEEL cells and deactivate pro-inflammatory cytokines in the endometrial stroma. The results suggest that circulating sEV may serve as a potential biomarker of fertility.