Prostaglandin Endoperoxide Research Articles

COX-2/PTGS2-targeted herbal-derived oligonucleotide drug HQi-sRNA-2 was effective in spontaneous mouse lung cancer model.

In 2020, the number of deaths caused by lung cancer worldwide reached 1,796,144, making it the leading cause of cancer-related deaths. Cyclooxygenase-2/prostaglandin endoperoxide synthase 2 (COX-2/PTGS2) is overexpressed in lung cancer, which promotes tumor proliferation, invasion, angiogenesis, and resistance to apoptosis. Here, we report that the oligonucleotide drug HQi-sRNA-2 from Traditional Chinese Medicine Huangqin targeting COX-2/PTGS2 significantly inhibited proliferation, migration, and invasion and induced apoptosis in the human lung cancer cell line NCI-H460. Oral delivery of HQi-sRNA-2 bencaosomes prolonged survival, reduced tumor burden, and maintained weight in a spontaneous mouse lung cancer model. Compared with paclitaxel, HQi-sRNA-2 may be less toxic and have approximately equal efficacy in reducing tumor burden. Our previous studies reported that herbal small RNAs (sRNAs) are functional medical components. Our data suggest that sphingosine (d18:1)-HQi-sRNA-2 bencaosomes, targeting COX-2/PTGS2 and downregulating the PI3K and AKT signaling pathways, may provide novel therapeutics for lung cancer.

Disease Evolution-based Specificity Target Discovery (DESTD) by analyzing Jiawei-Maxing-Shigan Decoctions against COVID-19

IntroductionDiverse disease courses, each corresponding to a unique pathophysiological mechanism, characterize the coronavirus 2019 disease (COVID-19). Intensive research on discovering specificity targets for treating COVID-19 has significant implications for understanding pathogenesis mechanisms in disease evolution. Traditional Chinese medicine (TCM) has shown distinctive advantages in treating COVID-19 in different periods, which may provide new clues for target discovery. In order to accomplish the goal of providing a Disease Evolution-based Specificity Target Discovery (DESTD), the purpose of this paper was to conduct an analysis of the difference in target weighted value during each cycle of COVID-19. MethodsOur study employed a comprehensive methodology combining TCM pharmacology with modern biological techniques. Three Jiawei-Maxing-Shigan Decoctions (MS Decoction), were selected as samples, including Hanshi-Yufei decoction (HY Decoction), Shidu-Yufei decoction (SY Decoction), and Yidu-Bifei decoction (YB Decoction). The core components of these decoctions are the same, while the differences correspond to different periods of COVID-19. The DESTD approach consists of several parts: network pharmacology analysis to identify potential targets, molecular docking simulation to investigate TCM components acting on potential targets, and biological validation. This study employed several cell line models, including RAW264.7 (Mouse Mononuclear Macrophages Cells), 16HBE (Human Bronchial Epithelial Cells), and HUVECC (Human Umbilical Vein Endothelial Cells), to explore new approaches for key target discovery. ResultsDuring the mild phase of COVID-19, HY Decoction exhibited anti-inflammatory effects by specifically down-regulating the expression of heat shock protein HSP 90-alpha (HSP90AA1), transcription factor JUN (JUN) and other five genes in RAW264.7. In the common phrase, inflammation and fibrosis could be specifically alleviated by down-regulating the expression of interleukin-10 (IL10) and mitogen-activated protein kinase 14 (MAPK14) by SY Decoction respectively acting on RAW264.7 and 16HBE cell lines. In severe cases, YB Decoction specifically down-regulated the expression of prostaglandin endoperoxide synthase-2 (PTGS2), caspase-3 (CASP3), and three other genes to protect vascular endothelial cells from oxidative damage. Moreover, YB also played a role in anti-fibrosis by specifically down-regulating the expression of estrogen receptor beta (ESR2), vascular cell adhesion protein 1 (VCAM1), and other three genes, and up-regulating the expression of prostaglandin endoperoxide synthase-1 (PTGS1) in the severe period. These targeted actions align with a holistic treatment approach and provide tailored therapies for different stages of COVID-19. ConclusionThis research proposed a novel strategy for Disease Evolution-based Specificity Target Discovery (DESTD) to identify specific targets of different stages of disease and specific targets and mechanisms throughout the evolution of COVID-19.

Identification of hub genes and molecular pathways in keratoconus by integrating bioinformatics and literature mining at the RNA level.

Keratoconus (KC) is a condition characterized by progressive corneal steepening and thinning. However, its pathophysiological mechanism remains vague. We mainly performed literature mining to extract bioinformatic and related data on KC at the RNA level. The objective of this study was to explore the potential pathological mechanisms of KC by identifying hub genes and key molecular pathways at the RNA level. We performed an exhaustive search of the PubMed database and identified studies that pertained to gene transcripts derived from diverse corneal layers in patients with KC. The identified differentially expressed genes were intersected, and overlapping genes were extracted for further analyses. Significantly enriched genes were screened using "Gene Ontology" (GO) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG) analysis with the "Database for Annotation, Visualization, and Integrated Discovery" (DAVID) database. A protein-protein interaction (PPI) network was constructed for the significantly enriched genes using the STRING database. The PPI network was visualized using the Cytoscape software, and hub genes were screened via betweenness centrality values. Pathways that play a critical role in the pathophysiology of KC were discovered using the GO and KEGG analyses of the hub genes. 68 overlapping genes were obtained. Fifty genes were significantly enriched in 67 biological processes, and 16 genes were identified in 7 KEGG pathways. Moreover, 14 nodes and 32 edges were identified via the PPI network constructed using the STRING database. Multiple analyses identified 4 hub genes, 12 enriched biological processes, and 6 KEGG pathways. GO enrichment analysis showed that the hub genes are mainly involved in the positive regulation of apoptotic process, and KEGG analysis showed that the hub genes are primarily associated with the interleukin-17 (IL-17) and tumor necrosis factor (TNF) pathways. Overall, the matrix metalloproteinase 9, IL-6, estrogen receptor 1, and prostaglandin-endoperoxide synthase 2 were the potential important genes associated with KC. Four genes, matrix metalloproteinase 9, IL-6, estrogen receptor 1, and prostaglandin endoperoxide synthase 2, as well as IL-17 and TNF pathways, are critical in the development of KC. Inflammation and apoptosis may contribute to the pathogenesis of KC.

Effect of PEAR1, PTGS1 gene polymorphisms on the recurrence of aspirin-treated patients with ischemic stroke in the Han population of China: A 4-year follow-up study.

Platelet endothelial aggregation receptor 1 (PEAR1) and prostaglandin endoperoxide synthase 1 (PTGS1) polymorphisms can affect laboratory aspirin resistance. However, the impact of genetic polymorphisms on the recurrence of ischemic stroke (IS) patients treated with aspirin is not fully understood. This study aimed to examine the relationship between gene polymorphisms of PEAR1 and PTGS1 and IS recurrence in patients treated with aspirin. Peripheral blood samples were collected from 174 patients with nonrecurrent IS and 34 with recurrent IS after aspirin treatment. Follow-up was performed on all patients. PEAR1 rs12041331 and PTGS1 rs10306114 polymorphisms were determined using the PCR fluorescence probe method. And the correlations of them with the clinical characteristics were examined by multivariable logistic regression analysis. The distribution frequencies of PEAR1 rs12041331 and PTGS1 rs10306114 genotypes were in Hardy-Weinberg equilibrium, and there was no significant difference in the distribution of PEAR1 rs12041331 polymorphism. Compared to the nonrecurrent group, the AA genotype of the PTGS1 polymorphism was more frequent in the recurrent group (59.77% vs 35.29%, P = .003), and the A allele also showed a higher frequency than the G allele in the recurrent group (P = .001). Multivariable logistic regression analysis showed that smoking (OR = 5.228, 95% CI: 1.938-14.102, P = .001), coronary heart disease (OR = 4.754, 95% CI: 1.498-15.089, P = .008), and the polymorphism at PTGS1(A>G) AA/AG + GG (OR = 2.955, 95% CI: 1.320-6.616, P = .008) were independently associated with IS recurrence in Chinese patients. Our findings suggested that PTGS rs10306114 polymorphisms should receive more attention in the use of aspirin in patients with IS.

Inhibitory effect of downregulating G protein-coupled receptor class C group 5 member A expression on lipopolysaccharide-induced inflammatory response in human gingival fibroblasts

Objective: To clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A (GPRC5A) on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (GFs), thus to provide a foundation for delving into the role of G protein coupled receptor (GPCR) in periodontitis. Methods: Gingival tissue samples were collected from 3 individuals periodontally healthy (health group) and 3 patients with periodontitis (periodontitis group) in Shandong Stomatological Hospital from December 2022 to February 2023. The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining. GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 μmol/L small interfering RNA-GPRC5A (siGPRC5A) or small interfering RNA-negative control (siNC), regarded as the experimental group and the negative control one, respectively. The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR (RT-qPCR). Experiments were then conducted using these cells which were divided into four groups of negative control (NC), LPS, siGPRC5A+LPS and siGPRC5A. The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown. RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS, namely, interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandin endoperoxide synthase 2 (PTGS2) after GPRC5A knockdown. Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B (NF-κB) signaling pathway. Results: Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) (t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 μmol/L group displayed the most significant decrease in siGPRC5A expression (31.16±3.29) compared with that of the siNC group (100.00±4.88) (F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) (F=208.38, P<0.001; F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1β, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions: GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.

Unraveling the Mechanism of Xiaochaihu Granules in Alleviating Yeast-Induced Fever Based on Network Analysis and Experimental Validation.

Xiaochaihu granules (XCHG) are extensively used to treat fever. Nevertheless, the underlying mechanism remains elusive. This study aimed to explore the potential of XCHG in mitigating yeast-induced fever and the underlying metabolic pathways. The chemical composition of XCHG was ascertained using ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS), followed by integrated network analysis to predict potential targets. We then conducted experimental validation using pharmacological assays and metabolomics analysis in a yeast-induced mouse fever model. The study identified 133 compounds in XCHG, resulting in the development of a comprehensive network of herb-compound-biological functional modules. Subsequently, molecular dynamic (MD) simulations confirmed the stability of the complexes, including γ-aminobutyric acid B receptor 2 (GABBR2)-saikosaponin C, prostaglandin endoperoxide synthases (PTGS2)-lobetyolin, and NF-κB inhibitor IκBα (NFKBIA)-glycyrrhizic acid. Animal experiments demonstrated that XCHG reduced yeast-induced elevation in NFKBIA's downstream regulators [interleukin (IL)-1β and IL-8], inhibited PTGS2 activity, and consequently decreased prostaglandin E2 (PGE2) levels. XCHG also downregulated the levels of 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), corticotropin releasing hormone (CRH), and adrenocorticotrophin (ACTH). These corroborated the network analysis results indicating XCHG's effectiveness against fever in targeting NFKBIA, PTGS2, and GABBR2. The hypothalamus metabolomics analysis identified 14 distinct metabolites as potential antipyretic biomarkers of XCHG. In conclusion, our findings suggest that XCHG alleviates yeast-induced fever by regulating inflammation/immune responses, neuromodulation, and metabolism modules, providing a scientific basis for the anti-inflammatory and antipyretic properties of XCHG.

Mechanical Stress Induces Sodium Entry and Osmoprotective Responses in Murine Synovial Fibroblasts.

Osteoarthritis (OA) is a multifactorial disease depending on molecular, genetic, and environmental factors like mechanical strain. Next to the cartilage and the subchondral bone, OA also affects the synovium, which is critically involved in the maintenance of joint homeostasis. As there is a correlation between the extracellular sodium content in the knee joint and OA, this study investigates the impact of sodium on OA-associated processes like inflammation and bone remodeling without and with mechanical loading in synovial fibroblasts. For that purpose, murine synovial fibroblasts from the knee joint were exposed to three different extracellular sodium chloride concentrations (-20 mM, ±0 mM and +50 mM NaCl) in the absence or presence of compressive or intermittent tensile strain. In addition to the intracellular Na+ content and gene expression of the osmoprotective transcription factor nuclear factor of activated T cells 5 (Nfat5), the gene and protein expression of inflammatory mediators (interleukin-6 (IL6), prostaglandin endoperoxide synthase-2 (Ptgs2)/prostaglandin E2 (PGE2)), and factors involved in bone metabolism (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG)) were analyzed by qPCR and ELISA. Mechanical strain already increased intracellular Na+ and Nfat5 gene expression at standard salt conditions to levels obtained by exposure to increased extracellular Na+ content. Both high salt and compressive strain resulted in elevated IL6 and PGE2 release. Intermittent tensile strain did not increase Il6 mRNA expression or IL6 protein secretion but triggered Ptgs2 expression and PGE2 production. Increased extracellular Na+ levels and compressive strain increased RANKL expression. In contrast, intermittent tension suppressed RANKL expression without this response being subject to modification by extracellular sodium availability. OPG expression was only induced by compressive strain. Changes in extracellular Na+ levels modified the inflammatory response and altered the expression of mediators involved in bone metabolism in cells exposed to mechanical strain. These findings indicate that Na+ balance and Nfat5 are important players in synovial fibroblast responses to mechanical stress. The integration of Na+ and Na+-dependent signaling will help to improve the understanding of the pathogenesis of osteoarthritis and could lead to the establishment of new therapeutic targets.

Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors

Originally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of PTSG2 and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors.

LCN2 depletion aggravates sepsis-induced liver injury by regulating PTGS2-dependent ferroptosis.

Background: Sepsis-induced liver injury (SILI) is an independent risk factor for organ dysfunction and mortality in critical care units. Methods: In this study, the roles of lipocalin 2 (LCN2) in SILI were investigated because LCN2 expression was increased in liver tissues of the septic mice induced by caecal ligation and puncture (CLP), as well as in hepatocytes treated with lipopolysaccharide (LPS). To evaluate liver injury in mice, the levels of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were measured in both serum and liver tissues. Oxidative stress was evaluated by measuring the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) in serum and liver samples. Additionally, ferroptosis was assessed by examining the expression of prostaglandin endoperoxide synthase 2 (PTGS2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in liver tissue. Results: The results demonstrated that LCN2 depletion significantly exacerbated SILI, oxidative stress, and ferroptosis. Moreover, in in vitro sepsis model, LCN2 overexpression notably ameliorated LPS-induced cell injury, oxidative stress, and ferroptosis by inhibiting PTGS2 expression. Conclusion: In conclusion, our study provides evidence that LCN2 depletion aggravates SILI by regulating PTGS2-mediated ferroptosis.

Effect of "Tongdu Qishen" acupuncture on expressions of ferroptosis-related factors in the hippocampus of APPswe/PS1dE9 Mice.

To investigate the effect of "Tongdu Qishen" acupuncture［dredging the Govern Vessel and normalizing mental activities, electroacupuncture (EA) of "Baihui" (GV20), "Yintang" (EX-HN3) and "Shuigou" (GV26) needling］on the learning-memory ability and the mechanism of ferroptosis in Alzheimer's disease (AD) mice. Twenty-four male APPswe/PS1dE9 mice were randomly and equally divided into model group and EA group, and 12 normal C57BL/6 mice were used as the control group. In the EA group, EA (2 Hz/100 Hz, 20 min) was applied to GV20 and EX-HN3 in combination with manual acupuncture stimulation of GV26. The treatment was performed once a day, for a total of 28 days. The mice in the three groups were given the same fixation and grasping operation. Morris water maze swimming tests were used to assess the mice's learning-memory ability. Nissl staining and transmission electron microscopy were used to observe the morphological changes of neurons in the hippocampus. The activity of superoxide dismutase (SOD) in the hippocampus tissue was detected by superoxide anionic colorimetric assay kit (WST-1), and malondialdehyde (MDA) contents were detected by thiobarbituric acid (TBA) method. The expression levels of prostaglandin endoperoxide synthase 2 (ptgs2) and glutathione peroxidase 4 (GPX4) mRNA in the hippocampus were detected by fluorescent quantitative real-time PCR. Behavioral results showed that compared with the control group, the escape latencies at the 2nd, 3rd, 4th and 5th day of Morris water maze swimming test were significantly increased (P<0.05), and the swimming time in the original platform quadrant and the times of cros-sing the original platform were considerably decreased (P<0.05) in the model group. In comparison with the model group, the escape latencies at the 4th and 5th day were strikingly decreased (P<0.05), and the swimming time in the original platform quadrant and times of crossing the original platform significantly increased (P<0.05) in the EA group. Following modeling, the SOD activity and the expression of GPX4 mRNA were obviously down-regulated (P<0.05), and the content of MDA and the expression of ptgs2 mRNA significantly up-regulated (P<0.05) in the model group rele-vant to the control group. The SOD activity and the expression of GPX4 mRNA were apparently increased (P<0.05), and the content of MDA and the expression of ptgs2 mRNA remarkably down-regulated (P<0.05) in the EA group rele-vant to the model group. Histopathological and ultrastructural results showed scattered arrangement of cells, widened space among cells, reduction in the number of cells, and many shrunk of dissolved nucleoli, shrunking and incomplete mitochondria, and high membrane electron density in the hippocampus of the model group, which was relatively milder in the EA group. "Tongdu Qishen" acupuncture can improve the learning-memory ability of AD mice, which may be related to its functions in up-regulating SOD activity and GPX4 mRNA expression, and down-regulating MDA content and ptgs2 mRNA expression to reduce the lipid peroxidation in the process of ferroptosis.

T-2 toxin-induced testicular impairment by triggering oxidative stress and ferroptosis

T-2 toxin is a trichothecene mycotoxin of significant danger to humans and animals. Its impact on reproductive toxicity is attributed to oxidative stress, which ultimately leads to cell death. Ferroptosis is a programmed cell death that characterized by lipid peroxidation. This study aimed to investigate the toxic effects of T-2 toxin on mouse testis and the potential mechanism of T-2 toxin-induced ferroptosis. T-2 toxin significantly altered the morphology of the testis and decreased testosterone level, sperm concentration, and increased sperm malformation rate, as well as induced oxidative damage with reactive oxygen species and malondialdehyde accumulated, and activity of superoxide dismutase, glutathione peroxidase decreased. Additionally, T-2 toxin induced ferroptosis by accumulating iron ions, increasing prostaglandin endoperoxide synthase 2, downregulating glutathione peroxidase 4 and ferritin heavy chain 1, as well as manifesting ferroptotic morphological alterations, ultimately leading to testicular impairment. Administration of ferroptosis inhibitor liproxstatin-1 or antioxidant resveratrol effectively mitigated the T-2 toxin-induced ferroptosis and testicular injury. These findings provided novel insights into the fundamental mechanism of T-2 toxin-induced cell death and furnished further proof of the potential therapeutic effect in addressing T-2 toxin-induced testicular impairment.

Protective mechanism of Paeoniae Radix Alba against chemical liver injury based on network pharmacology, molecular docking, and in vitro experiments

ObjectiveTo explore and validate the potential targets of Paeoniae Radix Alba (P. Radix, Bai Shao) in protecting against chemical liver injury through network pharmacology, molecular docking technology, and in vitro cell experiments. MethodsNetwork pharmacology was used to identify the common potential targets of P. Radix and chemical liver injury. Molecular docking was used to fit the components, which were subsequently verified in vitro. A cell model of hepatic fibrosis was established by activating hepatic stellate cell (HSC)-LX2 cells with 10 ng/mL transforming growth factor-β1. The cells were exposed to different concentrations of total glucosides of paeony (TGP), the active substance of P. Radix, and then evaluated using the cell counting kit-8 assay, enzyme-linked immunosorbent assay, and western blot. ResultsAnalysis through network pharmacology revealed 13 key compounds of P. Radix, and the potential targets for preventing chemical liver injury were IL-6, AKT serine/threonine kinase 1, jun proto-oncogene, heat shock protein 90 alpha family class A member 1 (HSP90AA1), peroxisome proliferator activated receptor gamma (PPARG), PTGS2, and CASP3. Gene Ontology (GO) enrichment analysis indicated the involvement of response to drugs, membrane rafts, and peptide binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the main pathways involved lipid and atherosclerosis and chemical carcinogenesis-receptor activation. Paeoniflorin and albiflorin exhibited strong affinity for HSP90AA1, PTGS2, PPARG, and CASP3. Different concentrations of TGP can inhibit the expression of COL-Ⅰ, COL-Ⅲ, IL-6, TNF-α, IL-1β, HSP-90α, and PTGS2 while increasing the expression of PPAR-γ and CASP3 in activated HSC-LX2 cells. ConclusionP. Radix primarily can regulate targets such as HSP90AA1, PTGS2, PPARG, CASP3. TGP, the main active compound of P. Radix, protects against chemical liver injury by reducing the inflammatory response, activating apoptotic proteins, and promoting the apoptosis of activated HSCs.

Abstract 12750: Three Dimensional Environment for Efficient and Large Scale Generation of Smooth Muscle Cells From HiPSCs

Introduction: Smooth muscle cells (SMCs) play an important role in maintaining vascular homeostasis and the development of diseases. We assess feasibility and efficacy of differentiation human induced pluripotent stem cells (hiPSCs) to SMCs in a 3-dimension (3D). Methods: hiPSCs were cultured in shaker flasks or PBS-0.1 Vessels for 48 h. Differentiation initiated (as day 0) by culturing hiPSC spheroids in RPMI+B27 without insulin (B27-) and CHIR99021 (CHIR) for 24 h. Day 1, medium was replaced with RPMI+B27-, platelet derived growth factor β (PDGF-β), and transforming growth factor β1 (TGFβ1) for 48 h, refreshed medium on day 3 for 48 h. Day 5, differentiating hiPSC-spheroids were dissociated to single cells and cultured as monolayer in RPMI +4% FBS, PDGF-β, and TGFβ1 for 72 h. Refreshed medium day 8 for 72 h. Differentiation efficiency was determined by flow cytometry and fluorescence immunostaining. The biological function of 3D differentiated SMCs was assessed in vitro and in a mouse model of ischemic limb disease in vivo . Results: Both 3D differentiation methods efficiently differentiated hiPSCs into SMCs. Flow cytometry showed that 99.4% cells expressed smooth muscle actin (SMA), 97.9% expressed myosin heavy chain 11 (MYH11), 95.1% expressed SM22, 97.5% expressed calponin, and 97.7% expressed calveolin-3 using shaker flasks. They were 99.6% for SMA, 97.9% for MYH11, 97% for SM22, 89.6% for calponin, and 87.2% for calveolin-3, respectively, using PBS-0.1 Vessels. Mean SMC cell number obtained was 4.56 x 10 7 for shaker flasks and 5.69 x 10 7 for PBS-0.1 Vessels, respectively, which were significantly higher than hiPSCs differentiated in monolayer (5.7x10 6 ). Fluorescence immunostaining confirmed that SMCs differentiated from both methods expressed SMA, MYH11, SM22, Calponin, and calveolin-3. Differentiated SMCs migrated in scratch assay, contracted in response to U-46619, an analog of the endoperoxide prostaglandin, and formed tubular structure on Matrigel in vitro. Fluorescence angiogram imaging showed that SMC transplantation effectively induced perfusion in mouse ischemic limbs. Conclusions: Both 3D differentiation methods, using shaker flasks or PBS-0.1 vessels, efficiently differentiated hiPSCs into large scales of functional SMCs.

New insights on Prostaglandin endoperoxide H synthase 2 (PGHS2) inhibition mechanism by nitroarachidonic acid (NO2AA)

New insights on Prostaglandin endoperoxide H synthase 2 (PGHS2) inhibition mechanism by nitroarachidonic acid (NO2AA)

Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1.

Tanshinone IIA (TSN) extracted from danshen (Salviamiltiorrhiza) could protect cardiomyocytes against myocardial ischemia/reperfusion injury (IRI), however the underlying molecular mechanisms of action remain unclear. The aim of the present study was to identify the protective effects of TSN and its mechanisms of action through invitro studies. An anoxia/reoxygenation (A/R) injury model was established using H9c2 cells to simulate myocardial IRI invitro. Before A/R, H9c2 cardiomyoblasts were pretreated with 8 µM TSN or 10µM ferrostatin‑1 (Fer‑1) or erastin. The cell counting kit 8 (CCK‑8) and lactate dehydrogenase (LDH) assay kit were used to detect the cell viability and cytotoxicity. The levels of total iron, glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde (MDA), ferrous iron, caspase‑3 activity, and reactive oxygen species (ROS) were assessed using commercial kit. The levels of mitochondrial membrane potential (MMP), lipid ROS, cell apoptosis, and mitochondrial permeability transition pore (mPTP) opening were detected by flow cytometry. Transmission electron microscopy (TEM) was used to observed the mitochondrial damage. Protein levels were detected by western blot analysis. The interaction between TSN and voltage‑dependent anion channel 1 (VDAC1) was evaluated by molecular docking simulation. The results showed that pretreatment with TSN and Fer‑1 significantly decreased cell viability, glutathione peroxidase4 (GPX4) protein and GSH expression and GSH/GSSG ratio and inhibited upregulation of LDH activity, prostaglandin endoperoxide synthase 2 and VDAC1 protein expression, ROS levels, mitochondrial injury and GSSG induced by A/R. TSN also effectively inhibited the damaging effects of erastin treatment. Additionally, TSN increased MMP and Bcl‑2/Bax ratio, while decreasing levels of apoptotic cells, activating Caspase‑3 and closing the mPTP. These effects were blocked by VDAC1 overexpression and the results of molecular docking simulation studies revealed a direct interaction between TSN and VDAC1. In conclusion, TSN pretreatment effectively attenuated H9c2 cardiomyocyte damage in an A/R injury model and VDAC1‑mediated ferroptosis and apoptosis served a vital role in the protective effects of TSN.

Elucidating the molecular mechanisms underlying anti-inflammatory effects of Morchella esculenta in the arachidonic acid metabolic pathway by network pharmacology and molecular docking

Morchella esculenta is an edible fungus with a uniquely delicious flavor and remarkable benefits for health. Herein, the molecular mechanism underlying the anti-inflammatory effects of Morchella esculenta was elucidated using molecular docking and network pharmacology. NPASS, Super-pred, SEA, Swiss Target Prediction, GeneCards, DisGeNET, Omim database, and STRING platform were used to select anti-inflammatory targets and construct target protein interaction networks using the active ingredients of Morchella esculenta. The OmicShare cloud platform was used to analyze GO functions and KEGG pathways related to the target, and the AutoDock Vina software was used to perform molecular docking and molecular dynamics (MD) simulation on the main target. Based on Cytoscape’s “Network Analysis”, the degree was used to identify potential key targets, and different inflammatory transcriptome data sets were used to evaluate core targets showing clinical significance. The active ingredient of Morchella esculenta identified from the NPASS database was EOYA, which had 43 anti-inflammatory targets, including NR1I2, PTGS1, PTGS2, CYP4F2, CYP3A4, TLR4, MAPK1, PLA2G4A, and PTPN11, and was mainly implicated in arachidonic acid metabolism, vascular endothelial growth factor signal pathway, and sphingomyelin signal transduction pathway, indicating that the anti-inflammatory effects of EOYA were mainly related to these biological processes. The degree was used to select 9 potential effective targets, namely NR1I2, PTGS1, PTGS2, CYP4F2, CYP3A4, TLR4, MAPK1, PLA2G4A, and PTPN11, among which NR1I2, PTGS1, PTGS2, PLA2G4A, MAPK1, CYP3A4, and TLR4 showed clinical significance. Molecular docking results showed that (E)-Octadec-11-En-9-Ynoic Acid (EOYA) could spontaneously bind to the 9 core targets, and the binding fractions of NR1I2, PTGS1, PTGS2, CYP4F2, and CYP3A4 were the highest. The MD simulation results showed that EYOA did indeed bind well NR1I2 to PTGS2, and the complex has high stability. Morchella esculenta can regulate the activity of prostaglandin endoperoxide synthetase, and affect the biosynthesis of prostaglandins, thereby impacting the metabolic pathway of arachidonic acid.

Discovery of (S)-flurbiprofen-based novel azine derivatives as prostaglandin endoperoxide synthase-II inhibitors: Synthesis, in-vivo analgesic, anti-inflammatory activities, and their molecular docking

The anti-inflammatory and analgesic drugs currently used are associated with several adverse effects and found to be highly unsafe for long-term use. Currently, nineteen novel bis-Schiff base derivatives (1–19) of flurbiprofen have been designed, prepared and assessed for in-vivo analgesic, anti-inflammatory and in vivo acute toxicity evaluation. The structures of the acquired compounds were deduced through modern spectroscopic techniques including HR-ESI-MS, 13C-, and 1H NMR. Amongst the series, compounds 7, 9, and 10 attributed potent activities with 93.89, 92.50, and 90.47% decreased edema, respectively compared to flurbiprofen (90.01%), however, compounds 11 and 15 exhibited significant activity of 90.00% decrease. Out of them, fourteen compounds (1–6, 8, 12–14, and 16–19) displayed good activity in the range of 68.96–86.95%. In case of an analgesic study, all the derivatives significantly (p 0.001) increased the pain threshold time particularly compound 7 had the best analgesic effect (24 ± 2.08 s) in comparison with flurbiprofen (21.66 ± 2.02 s) using hot plate test. Similarly, in the acetic acid-induced writhing test, compound 7 determined a potent inhibitory effect (60.47 %) close to flurbiprofen (59.28%). All the synthesized derivatives were found safe up to the dose of 30 mg/kg, in acute toxicity study. On a molecular scale, the synthesized compounds were modeled through a ligand-based pharmacophore study and molecular docking to have insight into the different possible interactions leading to high inhibition levels against the COX-2 enzyme.

T-2 toxin induces cardiotoxicity by activating ferroptosis and inhibiting heme oxygenase-1

T-2 toxin, a natural secondary sesquiterpenoid metabolite produced by numerous strains of Fusarium fungi, is prevalent in both contaminated food and the environment. T-2 toxin is known to be highly toxic to the cardiovascular system, but the precise mechanisms that lead to T-2 toxin-induced cardiotoxicity are not yet fully understood. Recent findings indicate that ferroptosis is a pivotal factor in cardiovascular damage and exhibits a strong correlation with the detrimental impacts of T-2 toxin. The present study was designed to examine the involvement of ferroptosis in T-2 toxin-induced cardiac injury. Male mice and human cardiomyocytes were subjected to T-2 toxin for 24 h to induce acute cardiotoxicity for in vivo and in vitro studies, respectively. Our results demonstrated that T-2 toxin increased reactive oxygen species production, malondialdehyde, and decreased glutathione/oxidized glutathione and adenosine triphosphate levels. Furthermore, T-2 toxin was observed to activate ferroptosis, as evidenced by an increase in iron (Fe2+) concentration and upregulation of prostaglandin endoperoxide synthase 2, downregulation of glutathione peroxidase 4 and ferritin heavy chain 1, as well as ferroptotic morphological alterations. Inhibition of ferroptosis by Liproxstatin-1 reversed T-2 toxin-induced cardiac injury. Additionally, the downregulation of heme oxgenase-1 (HO-1) expression by T-2 toxin exacerbates ferroptosis and oxidative damage, which can be further aggravated by HO-1 inhibition with Sn-protoporphyrin. These findings provide novel insights into the mechanism of T-2 toxin-induced cardiotoxicity and suggest that targeting ferroptosis and HO-1 may represent a promising cardioprotective strategy against T-2 toxin.

Experimental validation for mechanisms of Qizhiweitong particles against Chronic Non-atrophic gastritis based on metabolomics and network pharmacology

Qizhiweitong particles (QZWT), a classic Chinese herbal prescription derived from the Sinisan decoction in Shang Han Za Bing Lun, has definitive clinical efficacy in treating Chronic Non-atrophic Gastritis (CNG) in China. However, its mechanism of action at the metabolic level remains unclear. The aim of this study was to explore the mechanisms of QZWT against CNG based on non-targeted metabolomics combined with network pharmacology and experimentally validated by enzyme linked immunosorbent assays (ELISA). First, CNG model rats were established by free drinking ammonia water combined with starvation and satiety disorder for 12 weeks. Taking gastric tissue as the object, ultra-high performance liquid chromatography tandem mass spectrometry based metabolomics and network pharmacology were conducted to identify the key compounds, core targets and pathways that mediate the effects of QZWT against CNG. Furthermore, the targets from network pharmacology and the metabolites from metabolomics were jointly analyzed to select crucial metabolism pathways by MetaScape. Finally, the key metabolic enzymes and metabolites were experimentally validated by ELISA. The results indicated that there were 29 differential metabolites were identified and considered to be metabolic biomarkers of QZWT in the treatment of CNG. Among them, 8 of the differential metabolites showed a significant reduction in the content of QZWT groups. Arachidonic acid (AA) metabolic and glycerophospholipid (GP) metabolic are the most crucial metabolic pathways for QZWT to treat CNG. QZWT regulated AA and GP metabolism by synergetic reducing the level of AA, Phospholipid acid and Lysophosphatidic acid and inhibiting the enzyme activity of prostaglandin endoperoxide synthase 1 and prostaglandin endoperoxide synthase 2. And a compound-reaction-enzyme-gene network of mechanism for QZWT against CNG was established. In conclusion, this study reveals the complicated mechanisms of QZWT against CNG. Our work presents a novel strategy to identify the potential mechanisms of pharmacological effects derived from a compound prescription of TCM.

Effects of rumen cannulation combined with different pre-weaning feeding intensities on the intestinal, splenic and thymic immune system in heifer calves several month after surgery.

Fistulation is a helpful procedure in animal nutritional research and also common practise in human medicine. However, there are indications that alterations in the upper gastrointestinal tract contribute to intestinal immune modulations. The present study aimed to investigate effects of a rumen cannulation in week 3 of life on the intestinal and tissue specific immune system of 34-week old heifers. Nutrition influences the development of the neonatal intestinal immune system to a high extent. Therefore, rumen cannulation was investigated in combination with different pre-weaning milk feeding intensities (20% (20MR) vs. 10% milk replacer feeding (10MR). Heifers of 20MR without rumen cannula (NRC) showed higher cluster of differentiation (CD)8+ T cell subsets in mesenteric lymph nodes (MSL) compared to heifers with rumen cannula (RC) and 10MRNRC heifers. CD4+ T cell subsets in jejunal intraepithelial lymphocytes (IELs) were higher in 10MRNRC heifers compared to 10MRRC heifers. CD4+ T cell subsets in ileal IELs were lower and CD21+ B cell subsets were higher in NRC heifers compared to RC heifers. CD8+ T cell subsets in spleen tended to be lower in 20MRNRC heifers compared to all other groups. Splenic CD21+ B cell subsets were higher in 20MRNRC heifers compared to RC heifers. Splenic toll like receptor (TLR) 6 expression was increased and IL4 expression tended to be increased in RC heifers than NRC heifers. Splenic TLR2, 3 and 10 gene expression was higher in 20MR compared to 10MR heifers. Jejunal prostaglandin endoperoxide synthase 2 expression was higher in RC heifers than NRC heifers, and MUC2 expression tended to increase in 20MR heifers compared to 10MR heifers. In conclusion, rumen cannulation modulated T and B cell subsets in the down streaming gastrointestinal tract and spleen. Pre-weaning feeding intensity seemed to affect intestinal mucin secretion and T and B cell subsets in MSL, spleen and thymus until several month later. Interestingly, in MSL, spleen and thymus the 10MR feeding regime evoked similar modulations of T and B cell subsets like rumen cannulation.