PTGS2 Expression Research Articles

Diosgenin suppresses COX-2 and mPGES-1 via GR and improves LPS-induced liver injury in mouse

Using a wild yam (Dioscorea japonica), we previously found novel anti-inflammatory and anti-carcinogenic effects via the downregulation of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1. One of the substances in wild yam is a steroidal saponin, diosgenin. We demonstrated that diosgenin suppressed COX-2 in human non–small-cell lung carcinoma A549 cells via nuclear factor-kappa B (NF-κB) translocation and the effects were reversed by a glucocorticoid receptor antagonist, RU486. In lipopolysaccharide (LPS)-induced mouse liver injury, COX-2 and mPGES-1 were induced and localized in sinusoidal macrophages and endothelial cells; however, diosgenin administration significantly suppressed Ptgs2 and Ptges expression and decreased COX-2 and mPGES-1 immunopositive cells in the sinusoids. Multiple immunohistochemical analyses showed that diosgenin had an effect on COX-2 and mPGES-1, particularly in the macrophages. Thus, we showed that diosgenin downregulated COX-2 and mPGES-1 via the glucocorticoid receptor and suppressed COX-2 and mPGES-1 in the macrophages of LPS-induced acute mouse liver injury.

Transcriptional Profile of Cytokines, Regulatory Mediators and TLR in Mesenchymal Stromal Cells after Inflammatory Signaling and Cell-Passaging.

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1β, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFβ. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.

Transfusion of Storage-Damaged Red Blood Cells Induces Ferroptosis in Splenic Macrophages

Red blood cell (RBC) transfusions are a common therapeutic, used in a wide range of settings (e.g., trauma). Therefore, potential outcomes, positive or negative, have special clinical relevance. Preparation of RBC units for transfusion involves refrigerated storage for an extended period of time, during which RBCs undergo multiple metabolic and physical changes, collectively known as the “storage lesion.” As a result, a proportion of the stored RBCs are rapidly cleared from the circulation post-transfusion by macrophages in the liver and spleen. However, the subsequent effects on macrophages of an acute, robust erythrophagocytosis event are still unclear, especially given the substantial amount of iron that is introduced into the cell in a short amount of time. Therefore, we sought to study the effects of substantial erythrophagocytosis on splenic macrophages, which normally play a key role in RBC-derived iron recycling. One possible outcome is macrophage cell death due to ferroptosis, an iron-dependent form of non-apoptotic cell death originally identified in cancer cells, but not yet demonstrated in macrophages.Using a mouse model of storage-damaged RBC transfusion and clearance, wild-type C57BL/6 mice were transfused with fresh (i.e., <24 hr after collection) or 12-14-day stored RBCs, and splenocytes were analyzed at defined time points post-transfusion. By 2 hours post-transfusion, a significant portion of splenic red pulp macrophages (RPMs; VCAM-1+, F4/80hi, CD11blo) had ingested stored RBCs (38.2%; p < 0.0001; Figure A), but not fresh RBCs (2.1%; p < 0.05). Other phagocytes, such as Ly6C+ monocytes (Ly6Chi, CD11b+, CD115+, Ly6G-) and granulocytes (Ly6G+, Ly6Chi, CD11b+) cleared far fewer RBCs: 7.02% (p < 0.0001) and 0.66% respectively. By 2 hours post-transfusion, the number of RPMs dramatically decreased (from 2.4 x 106 to 0.5 x 106 cells per spleen; p < 0.001; Figure B), before returning to steady-state levels by 2 days post-transfusion; thus, transfusion of stored RBCs markedly depletes the available macrophage population in the spleen. Nonetheless, Ki67 staining revealed that RPMs in mice transfused with stored RBCs substantially increased their cell division by 1 day post-transfusion (5.8% to 14.8%; p < 0.01), indicating that these tissue resident cells can self-regenerate locally after transfusion-mediated depletion.Further studies into the mechanism of RPM cell death uncovered ferroptosis-specific characteristics. Thus, by 2 hours after transfusion of old RBCs, increased levels of DCF fluorescence, indicative of reactive oxygen species (16.1% to 24.1%; p < 0.01; Figure C) and C11-Bodipy fluorescence, indicative of lipid peroxidation (2.0% to 19.8%; p < 0.0001; Figure D) were seen. In addition, qPCR analysis of RPMs revealed a 7.9-fold increased expression of PTGS2, which encodes COX-2 (p < 0.001); PTGS2 mRNA is reported to be induced in cells undergoing ferroptosis, downstream of lipid peroxidation. Using bone-marrow-derived macrophages (BMMs) exhibiting a uniform population of F4/80hi and VCAM-1+ cells, which are characteristic of RPMs, we demonstrated that ferrostatin-1 (a ferroptosis inhibitor; 100 µM) rescued cell death in BMMs that had ingested antibody-coated RBCs (30.8% to 13.7%, p < 0.0001; Figure E). Furthermore, ferrostatin-1 decreased the levels of reactive oxygen species (61.4% to 12.6%, p < 0.0001) and lipid peroxides (25.7% to 13.6%; p < 0.001) in BMMs that had ingested RBCs, without decreasing the overall proportion of BMMs that ingested RBCs. In addition, BMMs and J774.1 cells (a murine macrophage cell line) were sensitive to erastin (25% cell death; 1 µM, p < 0.0001), which induces ferroptosis in numerous cancer cell lines.In conclusion, substantial erythrophagocytosis after transfusion of storage-damaged RBCs induces ferroptosis in splenic RPMs. To date, ferroptosis has been described in cancer cells, kidney tubule cells, fibroblasts, neurons, and T cells. However, to our knowledge, this is the first report in macrophages, which play important roles in RBC clearance and iron recycling. This finding sheds light on potential adverse effects on macrophage populations from transfusions of “old” RBCs and also highlights a potential clinical use for ferroptosis inhibitors for preventing substantial losses in an important phagocyte population. [Display omitted] DisclosuresSpitalnik:New Health Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Dimethyl fumarate improves cognitive deficits in chronic cerebral hypoperfusion rats by alleviating inflammation, oxidative stress, and ferroptosis via NRF2/ARE/NF-κB signal pathway

Cerebrovascular disease and its risk factors cause persistent decrease of cerebral blood flow, chronic cerebral hypoperfusion (CCH) is the major foundation of vascular cognitive impairment (VCI). The hippocampus is extremely vulnerable to cerebral ischemia and hypoxia. Oxidative stress and neuroinflammation injury are important pathophysiological mechanisms of this process, which is closely related to hippocampal neurons damage and loss. Dimethyl fumarate (DMF), an FDA-approved therapeutic for multiple sclerosis (MS), plays a protective role in multiple neurological disorders. Studies have shown that DMF exerts anti-inflammatory and antioxidant effects via the NRF2/ARE/NF-κB signaling pathway. Thus, this study aimed to evaluate the neuroprotective effect of DMF in the CCH rat model. Ferroptosis, a novel defined iron-dependent cell death form, were found to be strongly associated with the pathophysiology of CCH. Emerging evidences have shown that inhibition of ferroptosis by targeting NRF2 exerted neuroprotective effect in neurodegeneration diseases. We also investigated whether DMF can alleviate cognitive deficits through inhibition of ferroptosis by the NRF2 signaling pathway in this study. DMF was intragastric for consecutive five weeks (100 mg/kg/day). Then behavior test and histological, molecular, and biochemical analysis were performed. We found that DMF treatment significantly improved cognitive deficits and partially reversed hippocampus neuronal damage and loss caused by CCH. And DMF treatment decreased hippocampus IL-1β, TNF-α, and IL-6 pro-inflammatory cytokines concentration, and mediated the NF-κB signaling pathway. And DMF also alleviated hippocampus oxidative stress through reducing MDA, and increasing GSH and SOD levels, which are also closely associated with ferroptosis. Besides, DMF treatment reduced the expression of PTGS2, and increased the expression of FTH1 and xCT, and the iron content is also reduced, which were the important features related to ferroptosis. Furthermore, DMF activated the NRF2/ARE signaling pathway and upregulated the expression of HO-1, NQO1 and GPX4. These outcomes indicated that DMF can improve cognitive impairment in rats with CCH, possibly through alleviating neuroinflammation, oxidative stress damage and inhibiting ferroptosis of hippocampal neurons. Overall, our results provide new evidence for the neuroprotective role of DMF.

Peripheral leucocyte molecular indicators of inflammation and oxidative stress are altered in dairy cows with embryonic loss

Objective of experiment was to determine whether oxidative stress (OS) and inflammation altered embryonic loss in dairy cows. Blood samples were collected at days 0, 16, 32 and 60 after timed (AI) from 200 Holstein cows to determine embryonic loss based on interferon-stimulated gene-15 (ISG15) mRNA expression (day 16) and ultrasound at day 32 and day 60. Leucocyte expressions of mRNA TLR2, TLR4, TNF-α, IL1B, IL10, STAT3 (inflammation), PTGS2, PTGES (prostaglandin synthesis), and PLA2G4A and ALOX5AP (eicosanoid metabolism) at days 0 and 16 were determined. Plasma redox status for antioxidant enzymatic activities of glutathione peroxidase (GPX), superoxide dismutase (SOD), total antioxidant capacity (TAC), and concentrations of malondialdehyde (MDA) were determined at days 0, 16, 32 and 60. All antioxidant-redox responses were beneficially significant in pregnant cows diagnosed pregnant at day16 and sustained pregnancy to day 60 compared to non-pregnant cows at day16 or pregnant at day16 and lost embryos by days 32 or 60. The leucocyte mRNA expressions of TLR2, TLR4, STAT 3, IL1B, PTGS2, PLA2G4A and ALOX5AP were greater and PTGES was lower at day16 in pregnant cows that lost embryos early (P < 0.05). In conclusion peripheral leucocyte molecular indicators of inflammation and plasma indicators of OS were altered in pregnant cows undergoing embryonic losses compared to cows with a sustained pregnancy.

Molecular Mechanism of Xixin-Ganjiang Herb Pair Treating Chronic Obstructive Pulmonary Disease-Integrated Network Pharmacology and Molecular Docking.

Background Chronic obstructive pulmonary disease (COPD) is characterized by high morbidity, disability, and mortality, which seriously threatens human life and health. Xixin and Ganjiang are classic herb pairs of Zhongjing Zhang, which are often used to treat COPD in China. However, the substance basis and mechanism of action of Xixin-Ganjiang herb pair (XGHP) in the treatment of COPD remain unclear. Methods On the website of TCMSP and the DrugBank, effective compounds and targets of XGHP were found. COPD targets were obtained from GeneCards, DisGeNET, and GEO gene chips. Intersecting these databases resulted in a library of drug targets for COPD. Then, intersection targets were used for protein-protein interaction (PPI) and pathway enrichment analysis. Finally, the binding activity between compounds and core genes was evaluated by molecular docking to verify the expression level of PTGS2 and PPARG in rats. Results Twelve effective compounds and 104 core genes were found in the intersection library, and kaempferol, sesamin, β-sitosterol, PTGS2, and PPARG were particularly prominent in the network analysis. A total of 113 pathways were obtained and enrichment of the TNF signaling pathway, IL-17 signaling pathway, and C-type lectin receptor signaling pathway was particularly obvious. Molecular docking indicated that kaempferol, sesamin, and β-sitosterol were closely related to PTGS2 and PPARG and were superior to aminophylline. Key compounds in XGHP could restrict the expression of PTGS2 in the lung tissues of COPD rats and promote the expression of PPARG. Conclusion Inhibition of the expression of inflammatory factor PTGS2 and promotion of the expression of PPARG may be an effective target of XGHP in the treatment of COPD.

Aberrant Expression of PAFAH1B3 Affects Proliferation and Apoptosis in Osteosarcoma.

Osteosarcoma is a major malignant tumor of bone and soft tissue, which is presenting with early metastasis and a high mortality rate. Platelet activating factor acetylhydrolase 1B3 (PAFAH1B3), a cancer-relevant molecular, was found to play a vital role in tumorigenesis and aggressiveness in several cancer types. However, the roles and the regulating mechanisms of PAFAH1B3 in osteosarcoma progression remain unclear. PAFAH1B3 expression was detected by immunohistochemistry in 83 osteosarcoma tissues and 44 paired adjacent normal bone tissues. In vitro, loss-of-function assay was performed to explore the role of PAFAH1B3 in osteosarcoma cells. Tumor xenograft growth assay was used to verify the effect of PAFAH1B3 knockdown on osteosarcoma growth in vivo. Chip assay was carried out to investigate the mechanism in osteosarcoma proliferation regulated by PAFAH1B3. PAFAH1B3 was overexpressed in osteosarcoma tissues and cell lines. Moreover, PAFAH1B3 knockdown inhibited osteosarcoma cell proliferation and promoted apoptosis in vitro, and also suppressed osteosarcoma growth in vivo. Furthermore, the proliferative effect of PAFAH1B3 in osteosarcoma was related to the regulation of the expression of EIF4EBP1, MYC, PTGS2 and RPS6KB1. This study demonstrated the biological function of PAFAH1B3 on osteosarcoma proliferation. This research suggested that PAFAH1B3 could be a novel therapeutic target for osteosarcoma patients.

Doxorubicin-paclitaxel sequential treatment: insights of DNA methylation and gene expression changes of luminal A and triple negative breast cancer cell lines.

Breast cancer is one of the significant causes of death among women diagnosed with cancer worldwide. Even though several chemotherapy combinations are still the primary treatment of breast cancer, unsuccessful treatments, and poor prognostic outcomes are still being reported. DNA methylation and gene expression changes among two breast cancer cell lines representing luminal A (MCF-7) and triple-negative (MDA-MB-231) cancers were determined after sequential combination treatment of doxorubicin and paclitaxel and analyzed using Ingenuity Pathway Analysis. Promoter methylation changes were seen in different treated MCF-7 cells and accompanied by changes in the gene expression of CCNA1 and PTGS2. In MDA-MB-231 cells, the hypomethylation of ESR1 was not accompanied by an increase in its gene expression in any treated cells. The hypomethylation of GSTP1 and MGMT was accompanied by an increase in gene expression levels in the group treated with doxorubicin only. Also, significant downregulation of several genes like MUC1 and MKI67 in MCF-7 cells treated with doxorubicin showed much lower gene expression (- 37.63, - 10.88 folds) when compared with cells treated with paclitaxel (- 2.47, - 2.05 folds) or the combination treatment (- 18.99, - 2.81 folds), respectively. On the other hand, a synergistic effect on MMP9 gene expression was significantly seen in MDA-MB-231 cells treated with the combination (- 9.99 folds) in comparison with the cells treated with doxorubicin (- 3.62 folds) or paclitaxel (1.75 folds) alone. Chemotherapy combinations do not always augment the molecular changes seen in each drug alone, and these changes could be utilized as treatment response markers.

The Effects of Naphthenic Acids on Tryptophan Metabolism and Peripheral Serotonin Signalling

Introduction: Serotonin produced in the periphery has been shown to affect glucose and lipid homeostasis. The availability of the amino acid tryptophan, the precursor of serotonin, affects serotonin availability. In addition, the metabolism of tryptophan via the kynurenine pathway produces physiologically active metabolites which have been shown to be altered under conditions of increased adiposity and dysglycemia. There is now evidence demonstrating some environmental xenobiotics, known to affect glucose and lipid homeostasis, can also alter serotonin production and key components of the kynurenine pathway. Recent evidence suggests that exposure to compounds present in petroleum and wastewaters from oil and gas extraction sites can impact endocrine signaling and result in aberrant lipid accumulation and altered glycemic control. However, whether any of these changes can be causally ascribed to altered serotonin synthesis/signaling or tryptophan metabolism remains unknown. The goal of this study was to determine the effects of exposure to naphthenic acid (NA), a key toxicant found in wastewater from bitumen (thick crude oil present in oil sands deposits) extraction on the enzymes involved in tryptophan metabolism and serotonin production. Methods: McA-RH7777 rat hepatoma cells, were exposed to a technical NA mixture for 48 hours at concentrations within the reported range of NA found in wastewaters from oil extraction. We assessed mRNA expression for key rate-limiting enzymes involved in tryptophan metabolism that lead to either serotonin [Tph1] and/or kynurenine [Ido2 and Tdo2] production, as well as downstream enzymes in the kynurenine pathway [Afmid, Kyat1, Aadat, Kyat3, Kmo, Haao, Acmsd, Qprt]. We also examined the effects of NA on prostaglandin synthesis [Ptgs1, Ptgs2, Ptges] and signalling [Ptger2, Ptger4] as prostaglandins have been shown to be induced by serotonin and are linked to hepatic fat accumulation. Results: NA treatment significantly increased Tph1 and Ido2 expression; this occurred in association with a significant increase in the expression of the inducible prostaglandin synthase Ptgs2 (COX-2), prostaglandin E synthase Ptges, and prostaglandin receptors Ptger2 and Ptger4. Acmsd was the only downstream enzyme in the kynurenine pathway that was significantly altered by NA treatment. Conclusion: These results provide proof-of-concept that compounds associated with oil sands extraction have the potential to perturb key components of serotonin synthesis (Tph1) and tryptophan metabolism (Ido2, Acmsd). Furthermore, we found that the increase in Tph1 expression paralleled expression of Ptgs2. As increased prostaglandin production has been reported in association with nonalcoholic steatohepatitis, these data provide a potential mechanism by which exposure to NA and other petroleum-based compounds may increase the risk of metabolic disease.

Differential Expression Changes in Human Decidua With Term and Preterm Labor: Role for Upstream Targets in the Prostaglandin Pathway

Introduction: Prostaglandins (PGs) are paracrine mediators thought to be important during pregnancy and labor. Synthesis of PGs is complex with three rate limiting steps, the second of which (PTGS2/cyclooxygenase) is a target for managing preterm labor, but this treatment is not always effective. It is suggested that alternate steps in the PG pathway may play distinct roles during labor and hence the potential as alternate targets for labor management. The first step of PG synthesis involves the action of phospholipase A2 (PLA2) enzymes, of which over a dozen isotypes are known. However, PLA2 expression and role in the decidua (maternal-fetal interface) is unknown. We hypothesize that distinct PLA2 isotypes are present within the decidua and play a role in term and preterm labor. To this end, we conducted an expression profile of PLA2 subtypes and related PG genes within human decidua and determined differential expression patterns with labor at term and preterm. Methods: Decidual samples from term (40wks) and preterm (26-37wks) Cesarean deliveries, were used to assess PG gene expression by RNAseq and qRT-PCR (n=7 preterm non-labor, n=11 preterm labor, n=31 term non-labor, n=31 term labor). Analyses were conducted using student’s t-test and one-way ANOVA to compare labor (non-labor vs labor) or with gestation (preterm vs term) and adjusted for multiple testing using the Benjamini-Hochberg method. Results: RNAseq analysis identified 30 highly expressed PG genes in decidua, of which 12 had not been previously reported in the uterus, including 7 PLA2 isotypes. Confirming previous work, expression of PTGS2 was higher with labor (p=0.011). In contrast, we demonstrate that PLA2 isotype expression is lower with labor, with further differences between term and preterm samples. With term labor, expression of PLA2 isotype 2D was lower compared to non-labour (p=0.047). Meanwhile, preterm labor was associated with lower expression of PLA2 isotype 16 (p=0.025) and 4C (p=0.026), compared to preterm non-labour. Epression of PLA2 subtypes 2A, 15 and 4A late was highest in late labor (p=0.016, 0.004, 0.03, respectively). Conclusion: The presence of multiple PLA2 subtypes within the decidua suggests the potential for fine tuning PG synthesis during pregnancy. Expression of PLA2 isotypes 16 and 4C was uniquely associated with preterm labor, suggesting these isotypes may play a role in the pathogenesis of preterm labor. Further investigation of the functional role of PLA2 isotypes may provide insight to a novel mechanism for preterm labor and identify potential targets for labor management.

Effects of maternal calorie restriction on inflammatory mediators in male offspring of preeclamptic‐like BPH/5 mice

Preeclampsia (PE) is characterized by maternal hypertension after 20 weeks of gestation coupled with clinical signs such as proteinuria, thrombocytopenia, hepatic dysfunction, and/or pulmonary edema. Women with pre-existing obesity are 3 times more likely to develop PE. Even though PE is a leading cause of maternal and fetal morbidity and mortality, the long-term impact of the maternal obesogenic environment on the offspring is not completely understood. To investigate this, we utilized the obese BPH/5 mouse, which spontaneously develops hypertension and excessive gestational weight gain early in pregnancy. We have previously shown that calorie restricted pregnant BPH/5 females have improved outcomes when pair-feeding is imposed beginning at the time of conception, embryonic day (e) 0.5, including increased live birth weight and litter sizes, and attenuation of pro-inflammatory cytokines at the maternal-fetal interface compared to BPH/5 ad libitum fed pregnancies. We hypothesized that pair-feeding BPH/5 mice during pregnancy will improve cardiometabolic outcomes in adult BPH/5 males. To test our hypothesis, we first phenotyped adult BPH/5 and control C57 male mice that gestated in ad libitum fed dams. Unlike BPH/5 females, BPH/5 adult male mice have similar body weights, daily food intake, and circulating leptin levels as compared to age-matched control mice (n=5-30; p>0.05). However, as seen in adult BPH/5 females, adult BPH/5 males have increased subcutaneous white adipose tissue (WAT) (BPH/5: 294 vs C57:133 mg; n=5-19; p<0.05) and peri-renal WAT mass (BPH/5: 188 vs C57:59 mg; n=11-22; p<0.05). BPH/5 males demonstrated cardiomegaly (190.2 mg; n=22; p<0.05) as compared to age-matched C57 adult males (168.4 mg; n=8) and on histological evaluation, evidence for cardiomyopathy was present. To investigate the BPH/5 male adipose tissue milieu, real time PCR was used to determine the inflammatory cytokine mRNA expression. Adult BPH5 male showed an increase in tumor necrosis factor alpha (TNFa) relative mRNA in the peri-renal WAT (6-fold; n=3-4; p<0.05). In subcutaneous WAT, there was a significant increase in prostaglandin synthase 2 (Ptgs-2) mRNA expression (6.5-fold; n=3; p<0.05). Pair-feeding BPH/5 dams beginning at day e0.5 and continuing through the first 9 days of pregnancy significantly decreased peri-renal WAT mass and TNFa expression (4.5-fold; n=3-6; p<0.05) as well as decreased subcutaneous WAT mass and Ptgs-2 expression compared to ad libitum fed dams’ BPH5 male offspring (3-fold; n=3-5; p<0.05). In conclusion, we believe that reduction in the maternal obesogenic environment may play an important role in BPH/5 offspring pregnancy outcomes in a sex-dependent manner. Thus, it is possible that fetal programming from an adverse maternal environment in this model could promote a pro-inflammatory response in the WAT, which may promote the development of cardiovascular disease in this mouse model. Future investigations are necessary to understand all the differences observed in BPH/5 offspring into adulthood as well as the transgenerational impact of attenuated maternal obesity in pregnancy.

How Aconiti Radix Cocta can Treat Gouty Arthritis Based on Systematic Pharmacology and UPLC-QTOF-MS/MS.

Background: Gouty arthritis (GA) is a common metabolic disease caused by a long-term disorder of purine metabolism and increased serum levels of uric acid. The processed product of dried root of Aconitum carmichaeli Debeaux (Aconiti Radix cocta, ARC) is used often in traditional Chinese medicine (TCM) to treat GA, but its specific active components and mechanism of action are not clear. Methods: First, we used ultra-performance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry to identify the chemical spectrum of ARC. Based on this result, we explored the active components of ARC in GA treatment and their potential targets and pathways. Simultaneously, we used computer simulations, in vitro cell experiments and animal experiments to verify the prediction results of systems pharmacology. In vitro, we used aurantiamide acetate (AA) to treat monosodium urate (MSU)-stimulated THP-1 cells and demonstrated the reliability of the prediction by western blotting and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). ELISAs kit were used to measure changes in levels of proinflammatory factors in rats with GA induced by MSU to demonstrate the efficacy of ARC in GA treatment. Results: Forty-three chemical constituents in ARC were identified. ARC could regulate 65 targets through 29 active components, and then treat GA, which involved 1427 Gene Ontology (GO) terms and 146 signaling pathways. Signaling pathways such as proteoglycans in cancer, C-type lectin receptor signaling pathway, and TNF signaling pathway may have an important role in GA treatment with ARC. In silico results showed that the active components songoramine and ignavine had high binding to mitogen-activated protein kinase p38 alpha (MAPK14) and matrix metallopeptidase (MMP)9, indicating that ARC treatment of GA was through multiple components and multiple targets. In vitro experiments showed that AA in ARC could effectively reduce expression of MAPK14, MMP9, and cyclooxygenase2 (PTGS2) in THP-1 cells stimulated by MSU, whereas it could significantly inhibit the mRNA expression of Caspase-1, spleen tyrosine kinase (SYK), and PTGS2. Animal experiments showed that a ARC aqueous extract could significantly reduce expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and intereleukin (IL)-18 in the serum of GA rats stimulated by MSU. Hence, ARC may inhibit inflammation by regulating the proteoglycans in cancer-associated signaling pathways. Conclusion: ARC treatment of GA may have the following mechanisms, ARC can reduce MSU crystal-induced joint swelling, reduce synovial tissue damage, and reduce the expression of inflammatory factors in serum. AA in ARC may inhibit inflammation by regulating the protein expression of MAPK14, MMP9, and PTGS2 and the mRNA expression of caspase-1, SYK, and PTGS2.

A comprehensive strategy to clarify the pharmacodynamic constituents and mechanism of Wu-tou decoction based on the constituents migrating to blood and their in vivo process under pathological state

Ethnopharmacological relevanceAs a traditional Chinese medicine (TCM) formula, Wu-tou decoction has been used for treating rheumatoid arthritis (RA) for more than a thousand years. Identifying pharmacodynamic constituents (PCs) of WTD and exploring their in vivo process are very meaningful for promoting the modernization of TCM. However, the pathological state might change this process. Aim of the studyHence, it is necessary and significant to compare the process in vivo of drugs both in normal and disease state and clarify their action mechanism. Materials and methodsTaking Wu-tou decoction (WTD) as the research object, a comprehensive strategy based on liquid chromatography coupled with mass spectrometry (LC-MS) was developed to identify PCs, clarify and compare their absorption and distribution in normal and model rats, and then explore the potential mechanism of TCM. Firstly, the PCs in WTD were identified. Then, the pharmacokinetics (PK) and tissue distribution of these ingredients were studied. Finally, the constituents with the difference between normal and model rats were selected for target network pharmacological analysis to clarify the mechanism. ResultsA total of 27 PCs of WTD were identified. The absorption and distribution of 20 PCs were successfully analyzed. In the disease state, the absorption and distribution of all these components were improved to have better treatment effects. The results of target network pharmacological analysis indicated that PTGS1, PTGS2, ABCB1, SLC6A4, CHRM2, ESR1, ESR2, CDK2, TNF and IL-6 are 10 key targets for WTD against RA. The regulatory effects of WTD on the expression of PTGS2 and TNF were further verified. Pathway enrichment analysis showed that the key mechanism of WTD against RA is to reduce inflammation and regulate the immune response. ConclusionThese results indicated that this strategy could better understand the in vivo process and mechanism of WTD under the pathological state. Furthermore, this strategy is also appropriate for other TCM.

Triptolide Modulates the Expression of Inflammation-Associated lncRNA-PACER and lincRNA-p21 in Mycobacterium tuberculosis-Infected Monocyte-Derived Macrophages.

Triptolide is a diterpene triepoxide, which performs its biological activities via mechanisms including induction of apoptosis, targeting of pro-inflammatory cytokines, and reshaping of the epigenetic landscape of target cells. However, the targeting of long non-coding RNAs (lncRNAs) by triptolide has not yet been investigated, despite their emerging roles as key epigenetic regulators of inflammation and immune cell function during Mycobacterium tuberculosis (Mtb) infection. Hence, we investigated whether triptolide targets inflammation-associated lncRNA-PACER and lincRNA-p21 and how this targeting associates with Mtb killing within monocyte-derived macrophages (MDMs).Using RT-qPCR, we found that triptolide induced the expression of lincRNA-p21 but inhibited the expression of lncRNA-PACER in resting MDMs in a dose- and time-dependent manner. Moreover, Mtb infection induced the expression of lincRNA-p21 and lncRNA-PACER, and exposure to triptolide before or after Mtb infection led to further increase of Mtb-induced expression of these lncRNAs in MDMs. We further found that contrary to lncRNA-PACER, triptolide time- and dose-dependently upregulated Ptgs-2, which is a proximal gene regulated by lncRNA-PACER. Also, low-concentration triptolide inhibited the expression of cytokine IL-6, a known target of lincRNA-p21. Mtb infection induced the expression of IL-6 and Ptgs-2, and triptolide treatment further increased IL-6 but decreased Ptgs-2 expression in Mtb-infected MDMs. The inverse relation between the expression of these lncRNAs and their target genes is concordant with the conception that these lncRNAs mediate, at least partially, the cytotoxic and/or anti-inflammatory activities of triptolide in both resting and activated MDMs. Using the CFU count method, we found that triptolide decreased the intracellular growth of Mtb HN878. The alamarBlue assay showed that this decreased Mtb HN878 growth was not as a result of direct targeting of Mtb HN878 by triptolide, but rather evoking MDMs’ intracellular killing mechanisms which we speculate could include triptolide-induced enhancement of MDMs’ effector killing functions mediated by lncRNA-PACER and lincRNA-p21. Altogether, these results provide proof of the modulation of lncRNA-PACER and lincRNA-p21 expression by triptolide, and a possible link between these lncRNAs, the enhancement of MDMs’ effector killing functions and the intracellular Mtb-killing activities of triptolide. These findings prompt for further investigation of the precise contribution of these lncRNAs to triptolide-induced activities in MDMs.

Network pharmacology integrated with molecular docking reveals the common experiment-validated antipyretic mechanism of bitter-cold herbs

Ethnopharmacological relevanceBitter-cold herbs have been used to clearing heat and expelling damp in clinical practice in China for thousands of years.Aim of the study: This study aimed to investigate the common molecular mechanism of bitter-cold herbs through network pharmacology analysis, molecular docking and experimental validation in vivo. Materials and methodsNetwork pharmacological analysis integrated with molecular docking was employed to identify the active compounds and core action targets of the bitter-cold herbs. Then, the yeast-induced pathological model was established, and the antipyretic effect of the herbs was evaluated by checking rectal temperatures of the mice hourly. Lastly, the protein expression of core targets was examined to reveal the antipyretic mechanism. ResultsA total of 52 lead compounds from the four bitter-cold herbs, Phellodendri Chinensis Cortex (PCC), Sophorae Flavescentis Radix (SFR), Gentianae Radix Et Rhozima (GRER) and Coptidis Rhizoma (CR), and 248 compounds-related targets were screened out with PTGS2 ranking the first. The results from molecular docking showed that 22 compounds adopted the same orientation as aspirin and had an excellent stability in the active site pocket of PTGS2. Furthermore, these herbs exerted potential therapeutic effects through 38 related pathways. On the other hand, the outcome of animal experiments showed that they could significantly attenuate the yeast-induced mice fever with dose-dependent relationship. Further experimental results demonstrated that administration of yeast suspension raised protein expression of PTGS2 significantly, which was evidently inhibited in the high or low-dose groups of GRER as well as in the low-dose group of SFR (P < 0.01) though a higher expression of PTGS2 was shown in the low-dose group of CR compared with FM group (P < 0.01). ConclusionsThe bitter-cold herbs can alleviate fever response and their antipyretic effect may mainly be attributed to regulating the expression of PTGS2 after the formation of ligand-receptor/PTGS2 complexes, and their active compounds might be nominated as antipyretic lead-ligand candidates.

Higher PGD2 production by synovial mast cells from rheumatoid arthritis patients compared with osteoarthritis patients via miR-199a-3p/prostaglandin synthetase 2 axis

We previously reported that synovial mast cells (MCs) from patients with rheumatoid arthritis (RA) produced TNF-α in response to immune complexes via FcγRI and FcγRIIA. However, the specific functions of synovial MCs in RA remain unclear. This study aimed to elucidate those functions. Synovial tissues and fluid were obtained from RA and osteoarthritis (OA) patients undergoing joint replacement surgery. Synovium-derived, cultured MCs were generated by culturing dispersed synovial cells with stem cell factor. We performed microarray-based screening of mRNA and microRNA (miRNA), followed by quantitative RT-PCR-based verification. Synovial MCs from RA patients showed significantly higher prostaglandin systhetase (PTGS)1 and PTGS2 expression compared with OA patients’ MCs, and they produced significantly more prostaglandin D2 (PGD2) following aggregation of FcγRI. PGD2 induced IL-8 production by human group 2 innate lymphoid cells, suggesting that PGD2-producing MCs induce neutrophil recruitment into the synovium of RA patients. PTGS2 mRNA expression in RA patients’ MCs correlated inversely with miRNA-199a-3p expression, which down-regulated PTGS2. RA patients’ synovial fluid contained significantly more PGD2 compared with OA patients’ fluid. Synovial MCs might regulate inflammation in RA through hyper-production of PGD2 following FcRγ aggregation. Our findings indicate functional heterogeneity of human MCs among diseases.

Abstract PR008: Tumor-cell-intrinsic transcriptional and epigenetic regulation of EGFR underlies the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer

Abstract Resistance to immunotherapy is a major problem of current clinical care for cancer patients. While T cell abundance is essential for tumor responsiveness to immunotherapy, factors that dictate T cell infiltration in tumor microenvironments are not fully understood. To understand the tumor-cell-intrinsic factors underlying the heterogeneity of tumor immunity and sensitivity to immunotherapy, we established a new experimental system by generating a library of congenic pancreatic tumor cell clones from a genetic mouse model driven by mutant Kras and p53. These tumor cell clones robustly formed implanted tumors that recapitulated T-cell-inflamed and non-T-cell-inflamed tumor microenvironments, associated with distinct patterns of infiltration by T cells and myeloid cells. We found that the non-T-cell-inflamed phenotype was dominant over the T-cell-inflamed phenotype in the local tumor microenvironment. An integrated transcriptomic and epigenetic analysis revealed that tumor-cell-intrinsic expression of CXCL1, EPHA2, PTGS2, and USP22 as determinants of the non-T-cell-inflamed microenvironment, and ablation of tumor-cell-intrinsic CXCL1, EPHA2, PTGS2, or USP22 promoted T cell infiltration and sensitivity to a combination of chemotherapies, CD40 agonist, and checkpoint blockades. Furthermore, we performed an in vivo CRISPR-based genetic screen to identify tumor cell intrinsic epigenetic regulators of anti-tumor immunity and discovered novel therapeutic opportunities to improve the efficacy of currently developed immunotherapy for pancreatic cancer. Specifically, we identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, while treatment of established tumors with an EGFR inhibitor erlotinib prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. Together, these results demonstrated that heterogeneity of tumor immune phenotypes is driven by tumor-cell-intrinsic factors, including epigenetic factors, that can be manipulated to influence the outcome of immunotherapies. Citation Format: Jinyang Li, Salina Yuan, Robert Norgard, Fangxue Yan, Andres Blanco, Ben Stanger. Tumor-cell-intrinsic transcriptional and epigenetic regulation of EGFR underlies the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR008.

Abstract PS18-44: Simvastatin induces ferroptosis in breast cancer cells by inhibiting GPX4 and sensitizes chemotherapy

Abstract Background The incidence of breast cancer ranks the first among female malignant tumors. Although the effect of breast cancer treatment strategies has been very ideal and the mortality rate of breast cancer has decreased, some patients are still not sensitive to these, which eventually leads to rapid recurrence, metastasis and poor prognosis. Ferroptosis is a kind of Regulated cell death, which induced by accumulation of lipid peroxidation products and Reactive Oxygen Species (ROS). It is quite different from apoptosis and autophagy in cell morphological characteristics and biochemical indicators. According to the latest studies, ferroptosis can reduce the activity of GPX4 (Gutathione Peroxidase 4) in cells and accumulate ROS and lipid Peroxidase products through iron metabolism, ROS metabolism, amino acid metabolism and lipid metabolism, etc., thus causing ferroptosis. Ferroptosis is a hotspot in the study of drug resistance and metastasis of breast cancer. Simvastatin is a kind of commonly used oral cholesterol-lowering drugs, belongs to the HMG CoA reductase inhibitors. Some clinical studies have shown that statins can reduce the risk of a variety of cancers, including breast cancer, and the risk of recurrence. Other basic experiment show lipophilic statins have anti-cancer effect. Simvastatin is the most lipophilic of all statins in use. Recent studies have found that simvastatin can be used in combination with docetaxel to enhance the effect of chemotherapy in prostate cancer, but has not been reported in breast cancer. In this study, we investigated whether simvastatin could induce ferroptosis in breast cancer by inhibiting GPX4 and play the role of chemotherapy sensitization in combination with docetaxel. Methods (1) CCK8 assay was used for cell proliferation in vitro by simvastatin and docetaxel in breast cancer cells MDA-MB-231 and MCF-7. (2) The anti-cancer effect of simvastatin in vivo was verified by xenograft experiments in nude mice. (3) GPX4 mRNA and protein levels were detected by qPCR and Western blot. (4) GPXs activity test, ROS level test and MDA level test were used for ferroptosis detection.(5) the expression of ferroptosis-related genes ACSL4, PTGS2 and NOX1 was detected by qPCR.(6) anti-tumor effect of simvastatin combined with docetaxel in breast cancer cells mda-mb-231 and McF-7 was detected in vitro by CCK8 method.(7) By MDA-MB-231 tumor xenograft models surveyed anti-tumor activity of simvastatin and docetaxel. Results CCK8 results showed that simvastatin significantly inhibited cell proliferation in breast cancer cells MDA-MB-231 and MCF-7 (P&amp;lt;0.001), with the IC50 values of MDA-MB-231 was 4.67um and MCF-7 was 81.53um,. In the xenograft experiments of nude mice, it was found that compared with the control group, the tumor volume of the simvastatin group was significantly reduced. In qPCR and Western blot, simvastatin was found to significantly inhibit GPX4 mRNA and protein expression. Compared with the control group, in breast cancer cells MDA-MB-231 and MCF-7, simvastatin can reduce the activity of GPXs and increase the level of ROS and MDA, thus causing ferroptosis in breast cancer cells. qPCR showed that ferroptosis -related gene ACSL4 and PTGS2, which are related to lipid metabolism, and ROS metabolism were significantly increased. In the xenograft experiments of nude mice, it was found that compared with the control group and the single drug group, the tumor volume of simvastatin combined with docetaxel group was significantly reduced. Conclusion Simvastatin can inhibit the proliferation of breast cancer cells both in vivo and in vitro, inhibit the expression of breast cancer cell MDA-MB-231 and MCF-7 GPX4, and induce ferroptosis. Besides, simvastatin sensitizes the effect of docetaxel. Citation Format: Sihong Lu, Nan Shao, Jong Bi, Ying Lin. Simvastatin induces ferroptosis in breast cancer cells by inhibiting GPX4 and sensitizes chemotherapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-44.

Bmal1 promotes prostaglandin E2 synthesis by upregulating Ptgs2 transcription in response to increasing estradiol levels in day 4 pregnant mice.

Prostaglandin G/H synthase 2 (PTGS2) is a rate-limiting enzyme in prostaglandin synthesis. The present study assessed the role of the uterine circadian clock on Ptgs2 transcription in response to steroid hormones during early pregnancy. We demonstrated that the core clock genes (Bmal1, Per2, Nr1d1, and Dbp), Vegf, and Ptgs2, and their encoded proteins, have rhythmic expression in the mouse uterus from days 3.5 to 4.5 (D3.5-4.5) of pregnancy. Progesterone (P4) treatment of cultured uterus endometrial stromal cells (UESCs) isolated from mPer2Luciferase reporter gene knock-in mice on D4 induced a phase shift in PER2::LUCIFERASE oscillations. This P4-induced phase shift of PER2::LUCIFERASE oscillations was significantly attenuated by the P4 antagonist RU486. Additionally, the amplitude of PER2::LUCIFERASE oscillations was increased by estradiol (E2) treatment in the presence of P4. Consistently, the mRNA levels of clock genes (Bmal1 and Per2), Vegf, and Ptgs2 were markedly increased by E2 treatment of UESCs in the presence of P4. Treatment with E2 also promoted prostaglandin E2 (PGE2) synthesis by UESCs. Depletion of Bmal1 in UESCs by small-interfering RNA (siRNA) decreased the transcript levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2 compared with nonsilencing siRNA treatment. Bmal1 knockdown also inhibited PGE2 synthesis. Moreover, the mRNA expression levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2, and their respective proteins were significantly decreased in the uterus of Bmal1-/- mice. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.NEW & NOTEWORTHY Rhythmic expression of Bmal1 and Ptgs2 was observed in the uterus isolated from D3.5-4.5 of pregnant mice. E2 increased the expression of Bmal1 and Ptg2 in UESCs isolated from mice on D4. The expression of Ptgs2 was significantly decreased in Bmal1-siRNA treated UESCs. Bmal1 knockdown also inhibited PGE2 synthesis. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.

Abstract PO015: Tumor-cell-intrinsic epigenetic factors underlie the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer

Abstract Resistance to immunotherapy is a major problem of current clinical care for cancer patients. While T cell abundance is essential for tumor responsiveness to immunotherapy, factors that dictate T cell infiltration in tumor microenvironments are not fully understood. To understand the tumor-cell-intrinsic factors underlying the heterogeneity of tumor immunity and sensitivity to immunotherapy, we established a new experimental system by generating a library of congenic pancreatic tumor cell clones from a genetic mouse model driven by mutant Kras and p53. These tumor cell clones robustly formed implanted tumors that recapitulated T-cell-inflamed and non-T-cell-inflamed tumor microenvironments, associated with distinct patterns of infiltration by T cells and myeloid cells. We found that the non-T-cell-inflamed phenotype was dominant over the T-cell-inflamed phenotype in the local tumor microenvironment. An integrated transcriptomic and epigenetic analysis revealed that tumor-cell-intrinsic expression of CXCL1, EPHA2, PTGS2, and USP22 as determinants of the non-T-cell-inflamed microenvironment, and ablation of tumor-cell-intrinsic CXCL1, EPHA2, PTGS2, or USP22 promoted T cell infiltration and sensitivity to a combination of chemotherapies, CD40 agonist, and checkpoint blockades. Furthermore, we performed an in vivo CRISPR-based genetic screen to identify tumor cell intrinsic epigenetic regulators of anti-tumor immunity and discovered novel therapeutic opportunities to improve the efficacy of currently developed immunotherapy for pancreatic cancer. These results demonstrated that heterogeneity of tumor immune phenotypes is driven by tumor-cell-intrinsic factors, including epigenetic factors, that can be manipulated to influence the outcome of immunotherapies. Citation Format: Jinyang Li, Salina Yuan, Robert J. Norgard, Fangxue Yan, Robert H. Vonderheide, Andres Blanco, Ben Z. Stanger. Tumor-cell-intrinsic epigenetic factors underlie the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO015.