Neuroprotective effect of isovaleraldehyde accompanied with upregulation of BDNF and CREB phosphorylation via the PKA pathway.

Recently, the number of patients diagnosed with dementia has increased. The World Health Organization (WHO) estimates that 50 million patients suffer from dementia. Although several therapeutic strategies have been proposed, currently, there is no curative approach for treating dementia. Neurodegeneration is an irreversible process. As this disease gradually progresses over 15-20 years, a low-cost and sustainable method for preventing these diseases is desired. Cacao nib is consumed in many countries, and a recent clinical study indicated that cocoa intake upregulates brain-derived neurotrophic factor (BDNF), which plays a significant role in memory formation and neuronal cell survival. In the present study, neural cells were treated with cacao nib extract or the 17 characteristic components of cacao nib. Treatment with Cacao nib extract upregulates BDNF mRNA expression. In addition, cacao nib extract elicits the phosphorylation of cAMP-response-element-binding protein (CREB), which regulates the transcription of BDNF. Among the 17 species screened, isovaleraldehyde (IVA), also known as an aroma component of cacao nibs extract, improved BDNF mRNA expression without SH-SY5Y cell toxicity. IVA also promoted CREB phosphorylation through a cAMP-dependent protein kinase (PKA)-dependent mechanism. In conclusion, IVA could be responsible for the BDNF upregulation effect of cacao nib, and IVA upregulated BDNF expression via the PKA-CREB axis.

TET1-mediated epigenetic regulation of tumor necrosis factor-α in trigeminal ganglia contributes to chronic temporomandibular joint pain.

Chronic temporomandibular joint (TMJ) pain profoundly affects patients' quality of life. Trigeminal tumor necrosis factor-α (TNFα) plays a pivotal role in mediating TMJ pain in mice, yet the underlying epigenetic mechanisms remain enigmatic. To unravel these epigenetic intricacies, we employed a multifaceted approach. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and chromatin immunoprecipitation (ChIP) followed by qPCR were employed to investigate the demethylation of TNFα gene (Tnfa) and its regulation by ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in a chronic TMJ pain mouse model. The global levels of 5-hydroxymethylcytosine (5hmc) and percentage of 5hmc at the Tnfa promoter region were measured in the trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) following complete Freund's adjuvant (CFA) or saline treatment. TET1 knockdown and pain behavioral testing were conducted to ascertain the role of TET1-mediated epigenetic regulation of TNFα in the pathogenesis of chronic TMJ pain. Our finding revealed an increase in 5hmc at the Tnfa promoter region in both TG and Sp5C of CFA-treated mice. TET1 was upregulated in the mouse TG, and the ChIP result showed TET1 direct binding to the Tnfa promoter, with higher efficiency in the CFA-treated group. Immunofluorescence revealed the predominant expression of TET1 in trigeminal neurons. TET1 knockdown in the TG significantly reversed CFA-induced TNFα upregulation and alleviated chronic TMJ pain. In conclusion, our study implicates TET1 as a vital epigenetic regulator contributing to chronic inflammatory TMJ pain via trigeminal TNFα signaling. Targeting TET1 holds promise for epigenetic interventions in TMJ pain management.

Metabolic Fingerprints of Effective Fluoxetine Treatment in the Prefrontal Cortex of Chronically Socially Isolated Rats: Marker Candidates and Predictive Metabolites.

The increasing prevalence of depression requires more effective therapy and the understanding of antidepressants' mode of action. We carried out untargeted metabolomics of the prefrontal cortex of rats exposed to chronic social isolation (CSIS), a rat model of depression, and/or fluoxetine treatment using liquid chromatography-high resolution mass spectrometry. The behavioral phenotype was assessed by the forced swim test. To analyze the metabolomics data, we employed univariate and multivariate analysis and biomarker capacity assessment using the receiver operating characteristic (ROC) curve. We also identified the most predictive biomarkers using a support vector machine with linear kernel (SVM-LK). Upregulated myo-inositol following CSIS may represent a potential marker of depressive phenotype. Effective fluoxetine treatment reversed depressive-like behavior and increased sedoheptulose 7-phosphate, hypotaurine, and acetyl-L-carnitine contents, which were identified as marker candidates for fluoxetine efficacy. ROC analysis revealed 4 significant marker candidates for CSIS group discrimination, and 10 for fluoxetine efficacy. SVM-LK with accuracies of 61.50% or 93.30% identified a panel of 7 or 25 predictive metabolites for depressive-like behavior or fluoxetine effectiveness, respectively. Overall, metabolic fingerprints combined with the ROC curve and SVM-LK may represent a new approach to identifying marker candidates or predictive metabolites for ongoing disease or disease risk and treatment outcome.

Targeted MDM2 Degradation Reveals a New Vulnerability for p53-Inactivated Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) frequently inactivate p53, increasing their aggressiveness and therapy resistance. We identified an unexpected protein vulnerability in p53-inactivated TNBC and designed a new PROteolysis TArgeting Chimera (PROTAC) to target it. Our PROTAC selectively targets MDM2 for proteasome-mediated degradation with high-affinity binding and VHL recruitment. MDM2 loss in p53 mutant/deleted TNBC cells in two-dimensional/three-dimensional culture and TNBC patient explants, including relapsed tumors, causes apoptosis while sparing normal cells. Our MDM2-PROTAC is stable in vivo, and treatment of TNBC xenograft-bearing mice demonstrates tumor on-target efficacy with no toxicity to normal cells, significantly extending survival. Transcriptomic analyses revealed upregulation of p53 family target genes. Investigations showed activation and a required role for TAp73 to mediate MDM2-PROTAC-induced apoptosis. Our data, challenging the current MDM2/p53 paradigm, show MDM2 is required for p53-inactivated TNBC cell survival, and PROTAC-targeted MDM2 degradation is an innovative potential therapeutic strategy for TNBC and superior to existing MDM2 inhibitors. SIGNIFICANCE: p53-inactivated TNBC is an aggressive, therapy-resistant, and lethal breast cancer subtype. We designed a new compound targeting an unexpected vulnerability we identified in TNBC. Our MDM2-targeted degrader kills p53-inactivated TNBC cells, highlighting the requirement for MDM2 in TNBC cell survival and as a new therapeutic target for this disease. See related commentary by Peuget and Selivanova, p. 1043. This article is highlighted in the In This Issue feature, p. 1027.

Mitochondrial PKM2 deacetylation by procyanidin B2-induced SIRT3 upregulation alleviates lung ischemia/reperfusion injury.

Apoptosis is a critical event in the pathogenesis of lung ischemia/reperfusion (I/R) injury. Sirtuin 3 (SIRT3), an important deacetylase predominantly localized in mitochondria, regulates diverse physiological processes, including apoptosis. However, the detailed mechanisms by which SIRT3 regulates lung I/R injury remain unclear. Many polyphenols strongly regulate the sirtuin family. In this study, we found that a polyphenol compound, procyanidin B2 (PCB2), activated SIRT3 in mouse lungs. Due to this effect, PCB2 administration attenuated histological lesions, relieved pulmonary dysfunction, and improved the survival rate of the murine model of lung I/R injury. Additionally, this treatment inhibited hypoxia/reoxygenation (H/R)-induced A549 cell apoptosis and rescued Bcl-2 expression. Using Sirt3-knockout mice and specific SIRT3 knockdown in vitro, we further found that SIRT3 strongly protects against lung I/R injury. Sirt3 deficiency or enzymatic inactivation substantially aggravated lung I/R-induced pulmonary lesions, promoted apoptosis, and abolished PCB2-mediated protection. Mitochondrial pyruvate kinase M2 (PKM2) inhibits apoptosis by stabilizing Bcl-2. Here, we found that PKM2 accumulates and is hyperacetylated in mitochondria upon lung I/R injury. By screening the potential sites of PKM2 acetylation, we found that SIRT3 deacetylates the K433 residue of PKM2 in A549 cells. Transfection with a deacetylated mimic plasmid of PKM2 noticeably reduced apoptosis, while acetylated mimic transfection abolished the protective effect of PKM2. Furthermore, PKM2 knockdown or inhibition in vivo significantly abrogated the antiapoptotic effects of SIRT3 upregulation. Collectively, this study provides the first evidence that the SIRT3/PKM2 pathway is a protective target for the suppression of apoptosis in lung I/R injury. Moreover, this study identifies K433 deacetylation of PKM2 as a novel modification that regulates its anti-apoptotic activity. In addition, PCB2-mediated modulation of the SIRT3/PKM2 pathway may significantly protect against lung I/R injury, suggesting a novel prophylactic strategy for lung I/R injury.

Upregulation of neuronal progranulin mediates the antinociceptive effect of trimetazidine in paclitaxel-induced peripheral neuropathy: Role of ERK1/2 signaling.

Neuronal progranulin (PGRN) overexpression is an endogenous adaptive pain defense following nerve injury. It allows the survival of injured neurons to block enhanced nociceptive responses. Trimetazidine (TMZ) is widely used by cardiac patients as an anti-anginal drug, reflecting its anti-ischemic property. TMZ promotes axonal regeneration of sciatic nerves after crush injury. This study explored the interplay between PGRN and extracellular signal-regulated kinases (ERK1/2) to address mechanisms underlying neuropathic pain alleviation following paclitaxel (PTX) administration. Rats were given four injections of PTX (2 mg/kg, i.p.) every other day. Two days after the last dose, rats received TMZ (25 mg/kg) with or without the ERK inhibitor, PD98059, daily for 21 days. TMZ preserved the integrity of myelinated nerve fibers, as evidenced by an obvious reduction in axonal damage biomarkers. Accordingly, it alleviated PTX-evoked thermal, cold, and mechanical hyperalgesia/allodynia. TMZ also promoted ERK1/2 phosphorylation with a profound upsurge in PGRN content. These effects were associated with a substantial increase in Notch1 receptor gene expression and a prominent anti-inflammatory effect with a marked increase in mRNA expression of secretory leukocyte protease inhibitor. Further, TMZ decreased oxidative stress and caspase-3 activity in the sciatic nerve. Conversely, co-administration of PD98059 completely abolished these beneficial effects. Thus, the robust antinociceptive effect of TMZ is largely attributed to upregulating PGRN and Notch1 receptors via ERK1/2 activation.

E2F2 enhances the chemoresistance of pancreatic cancer to gemcitabine by regulating the cell cycle and upregulating the expression of RRM2.

Both pro-oncogenic and anti-oncogenic effects of E2F2 have been revealed in different malignancies. However, the precise role of E2F2 in pancreatic cancer, in particular in relation to therapeutic intervention with gemcitabine, remains unclear. In this study, the effect of E2F2 on the proliferation and cell cycle modulation of pancreatic cancer cells, and whether E2F2 plays a role in the treatment of pancreatic cancer cells by gemcitabine, were investigated. The expression of E2F2 in pancreatic cancer was assessed by various methods including bioinformatics prediction, Western blotting, and real-time PCR. The effect of E2F2 on the proliferation and cell cycling of pancreatic cancer cells was analyzed by tissue culture and flow cytometry. In addition, the effect of E2F2 on the intervention of pancreatic cancer by gemcitabine was investigated using both in vitro and in vivo approaches. The expression of E2F2 was found to be significantly increased in pancreatic cancer tissues and cell lines. The pathogenic capacity of E2F2 lied in the fact that this transcription factor promoted the transformation of pancreatic cancer cell cycle from G1-phase to S-phase, thus enhancing the proliferation of pancreatic cancer cells. Furthermore, the expression of E2F2 was increased in pancreatic cancer cells in the presence of gemcitabine, and the augmented expression of E2F2 upregulated the gemcitabine resistance-related gene RRM2 and its downstream signaling molecule deoxycytidine kinase (DCK). The resistance of pancreatic cancer cells to gemcitabine was confirmed using both in vitro and in vivo models. In this study, E2F2 has been demonstrated for the first time to play a pro-oncogenic role in pancreatic cancer by promoting the transition of the cell cycle from G1-phase to S-phase and, therefore, enhancing the proliferation of pancreatic cancer cells. E2F2 has also been demonstrated to enhance the chemotherapy resistance of pancreatic cancer cells to gemcitabine by upregulating the expression of RRM2 and DCK that is downstream of RRM2.

The PDE4 inhibitor tanimilast shows distinct immunomodulatory properties associated with a type 2 endotype and CD141 upregulation.

BACKGROUND: Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanced clinical development for chronic obstructive pulmonary disease (COPD). Tanimilast is known to exert prominent anti-inflammatory activity when tested in preclinical experimental models as well as in human clinical studies. Recently, we have demonstrated that it also finely tunes, rather than suppressing, the cytokine network secreted by activated dendritic cells (DCs). This study was designed to characterize the effects of tanimilast on T-cell polarizing properties of DCs and to investigate additional functional and phenotypical features induced by tanimilast. METHODS: DCs at day 6 of culture were stimulated with LPS in the presence or absence of tanimilast or the control drug budesonide. After 24 h, DCs were analyzed for the expression of surface markers of maturation and activation by flow cytometry and cocultured with T cells to investigate cell proliferation and activation/polarization. The regulation of type 2-skewing mediators was investigated by real-time PCR in DCs and compared to results obtained in vivo in a randomized placebo-controlled trial on COPD patients treated with tanimilast. RESULTS: Our results show that both tanimilast and budesonide reduced the production of the immunostimulatory cytokine IFN-γ by CD4+ T cells. However, the two drugs acted at different levels since budesonide mainly blocked T cell proliferation, while tanimilast skewed T cells towards a Th2 phenotype without affecting cell proliferation. In addition, only DCs matured in the presence of tanimilast displayed increased CD86/CD80 ratio and CD141 expression, which correlated with Th2 T cell induction and dead cell uptake respectively. These cells also upregulated cAMP-dependent immunosuppressive molecules such as IDO1, TSP1, VEGF-A and Amphiregulin. Notably, the translational value of these data was confirmed by the finding that these same genes were upregulated also in sputum cells of COPD patients treated with tanimilast as add-on to inhaled glucocorticoids and bronchodilators. CONCLUSION: Taken together, these findings demonstrate distinct immunomodulatory properties of tanimilast associated with a type 2 endotype and CD141 upregulation in DCs and provide a mechanistic rationale for the administration of tanimilast on top of inhaled corticosteroids.

Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy.

BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

[Estradiol inhibits differentiation of mouse macrophage into a pro-inflammatory phenotype by upregulating the IRE1α-XBP1 signaling axis].

OBJECTIVE: To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway. METHODS: Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 µ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-ß, IL-6, IL-10 and TNF-α were detected with RT-PCR. RESULTS: Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-ß (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 µ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-ß (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist. CONCLUSION: Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.

Exploration of potential mechanism of interleukin-33 up-regulation caused by 1,4-naphthoquinone black carbon in RAW264.7 cells.

BACKGROUND: As air pollution has been paid more attention to by public in recent years, effects and mechanism in particulate matter-triggered health problems become a focus of research. Lysosomes and mitochondria play an important role in regulation of inflammation. Interleukin-33 (IL-33) has been proved to promote inflammation in our previous studies. In this research, macrophage cell line RAW264.7 was used to explore the potential mechanism of upregulation of IL-33 induced by 1,4-naphthoquinone black carbon (1,4-NQ-BC), and to explore changes of lysosomes and mitochondria during the process. RESULTS: 50 µg/mL 1,4-NQ-BC exposure for 24 h dramatically increased expression of IL-33 in RAW264.7 cells. Lysosomal membrane permeability was damaged by 1,4-NQ-BC treatment, and higher mitochondrial membrane potential and ROS level were induced by 1,4-NQ-BC. The results of proteomics suggested that expression of ferritin light chain was increased after cells were challenged with 1,4-NQ-BC, and it was verified by Western blot. Meanwhile, expressions of p62 and LC3B-II were increased by 50 µg/mL 1,4-NQ-BC in RAW264.7 cells. Ultimately, expression of IL-33 could return to same level as control in cells treated with 50 µg/mL 1,4-NQ-BC and 50 µM deferoxamine combined. CONCLUSIONS: 1,4-NQ-BC induces IL-33 upregulation in RAW264.7 cells, and it is responsible for higher lysosomal membrane permeability and ROS level, lower mitochondrial membrane potential, and inhibition of autophagy. Ferritin light chain possibly plays an important role in the upregulation of IL-33 evoked by 1,4-NQ-BC.

Quantitative Proteomics Analysis Expands the Roles of Lysine ß-Hydroxybutyrylation Pathway in Response to Environmental ß-Hydroxybutyrate.

Lysine ß-hydroxybutyrylation (Kbhb) is a newly identified protein posttranslational modification (PTM) derived from ß-hydroxybutyrate (BHB), a product of ketone body metabolism in liver. BHB could serve as an energy source and play a role in the suppression of oxidative stress. The plasma concentration of BHB could increase up to 20 mM during starvation and in pathological conditions. Despite the progress, how the cells derived from extrahepatic tissues respond to elevated environmental BHB remains largely unknown. Given that BHB can significantly drive Kbhb, we characterized the BHB-induced lysine ß-hydroxybutyrylome and acetylome by quantitative proteomics. A total of 840 unique Kbhb sites on 429 proteins were identified, with 42 sites on 39 proteins increased by more than 50% in response to BHB. The results showed that the upregulated Kbhb induced by BHB was involved in aminoacyl-tRNA biosynthesis, 2-oxocarboxylic acid metabolism, citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism pathways. Moreover, some BHB-induced Kbhb substrates were significantly involved in diseases such as cancer. Taken together, we investigate the dynamics of lysine ß-hydroxybutyrylome and acetylome induced by environmental BHB, which reveals the roles of Kbhb in regulating various biological processes and expands the biological functions of BHB.

Sevoflurane Suppresses the Growth, Metastasis, and Invasion of Endometrial Carcinoma Cells via miR-195-5p/JAK2 Axis.

BACKGROUND: Highly invasive and destructive endometrioma is one of the most familiar primary malignant tumors among women. It has been studied that sevoflurane can influence the development of various malignancies. But whatever sevoflurane could influence endometrial tumors is unknown. MATERIALS AND METHODS: Through CCK8 and transwell analysis, we investigated the influence of sevoflurane on the development of endometrial tumors in vitro. Then, we studied the function of miRNA-195-5p to promote sevoflurane to inhibit the development of endometrial tumors. Then, we predicted the target genes of miRNA-195-5p by online software and focused on JAK2. Through luciferase assay, we proved the direct binding and regulation of miRNA-195-5p to JAK2. RESULTS: We showed that sevoflurane could inhibit the growth, metastasis, and invasion of endometrial tumors via miRNA-195-5p/JAK2 axis. CONCLUSIONS: Our research shows the function of sevoflurane in inhibiting the development of endometrial tumors via miRNA-195-5p/JAK2 axis. Our findings proved that sevoflurane is potentially beneficial for endometrial carcinoma patients with surgery and may be helpful for the choice of anesthetics in endometrial carcinoma operations.

Sphingosine 1-Phosphate-Upregulated COX-2/PGE2 System Contributes to Human Cardiac Fibroblast Apoptosis: Involvement of MMP-9-Dependent Transactivation of EGFR Cascade.

Human cardiac fibroblasts (HCFs) play key roles in normal physiological functions and pathological processes in the heart. Our recent study has found that, in HCFs, sphingosine 1-phosphate (S1P) can upregulate the expression of cyclooxygenase-2 (COX-2) leading to prostaglandin E2 (PGE2) generation mediated by S1P receptors/PKCα/MAPKs cascade-dependent activation of NF-κB. Alternatively, G protein-coupled receptor- (GPCR-) mediated transactivation of receptor tyrosine kinases (RTKs) has been proved to induce inflammatory responses. However, whether GPCR-mediated transactivation of RTKs participated in the COX-2/PGE2 system induced by S1P is still unclear in HCFs. We hypothesize that GPCR-mediated transactivation of RTKs-dependent signaling cascade is involved in S1P-induced responses. This study is aimed at exploring the comprehensive mechanisms of S1P-promoted COX-2/PGE2 expression and apoptotic effects on HCFs. Here, we used pharmacological inhibitors and transfection with siRNA to evaluate whether matrix metalloprotease (MMP)2/9, heparin-binding- (HB-) epidermal growth factor (EGF), EGF receptor (EGFR), PI3K/Akt, MAPKs, and transcription factor AP-1 participated in the S1P-induced COX-2/PGE2 system determined by Western blotting, real-time polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), and promoter-reporter assays in HCFs. Our results showed that S1PR1/3 activated by S1P coupled to Gq- and Gi-mediated MMP9 activity to stimulate EGFR/PI3K/Akt/MAPKs/AP-1-dependent activity of transcription to upregulate COX-2 accompanied with PGE2 production, leading to stimulation of caspase-3 activity and apoptosis. Moreover, S1P-enhanced c-Jun bound to COX-2 promoters on its corresponding binding sites, which was attenuated by these inhibitors of protein kinases, determined by a ChIP assay. These results concluded that transactivation of MMP9/EGFR-mediated PI3K/Akt/MAPKs-dependent AP-1 activity was involved in the upregulation of the COX-2/PGE2 system induced by S1P, in turn leading to apoptosis in HCFs.

PPARγ Alleviates Sepsis-Induced Liver Injury by Inhibiting Hepatocyte Pyroptosis via Inhibition of the ROS/TXNIP/NLRP3 Signaling Pathway.

Sepsis is a systemic inflammatory response syndrome caused by a dysregulated host response to infection. Peroxisome proliferator-activated receptor gamma (PPARγ) exerts anti-inflammatory and antioxidative properties. To investigate the potential effects of PPARγ on sepsis-induced liver injury and determine the related mechanisms, C57BL/6 male mice were subjected to cecal ligation and puncture (CLP) to create a sepsis model which was treated with GW1929 or GW9662 to upregulate or downregulate the expression of PPARγ. We found that upregulation of PPARγ decreased the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), and liver pathological damage and improved the 5-day survival rate. Increased expression of PPARγ also decreased sepsis-induced reactive oxygen species (ROS) by promoting the expression of Nrf2. In addition, upregulated PPARγ inhibited the expression of the TXNIP/NLRP3 signaling pathway by reducing ROS-induced injury in the liver during sepsis, which further reduced NLRP3-mediated pyroptosis and the inflammatory response. The role of PPARγ was further examined in in vitro experiments, where lipopolysaccharide- (LPS-) treated HepG2 and Hep3B cells were incubated with GW1929 or GW9662 to upregulate or downregulate the expression of PPARγ. We found that upregulated PPARγ ameliorated LDH release and improved cell viability. Our results indicated that increased expression of PPARγ reduced ROS levels and inhibited the TXNIP/NLRP3 signaling pathway, resulting in decreased pyroptosis and reduced liver dysfunction during sepsis.

Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues.

Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1-aged Gclcf/f;Le-CreTg/- (KO) and Gclcf/f;Le-Cre-/- control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclcw/w;Le-CreTg/- (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-l-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), α-SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.

Lipopolysaccharide acting via toll-like receptor 4 transactivates the TGF-ß receptor in vascular smooth muscle cells.

Toll-like receptors (TLRs) recognise pathogen­associated molecular patterns, which allow the detection of microbial infection by host cells. Bacterial-derived toxin lipopolysaccharide activates TLR4 and leads to the activation of the Smad2 transcription factor. The phosphorylation of the Smad2 transcription factor is the result of the activation of the transforming growth factor-ß receptor 1 (TGFBR1). Therefore, we sought to investigate LPS via TLR4-mediated Smad2 carboxy terminal phosphorylation dependent on the transactivation of the TGFBR1. The in vitro model used human aortic vascular smooth muscle cells to assess the implications of TLR4 transactivation of the TGFBR1 in vascular pathophysiology. We show that LPS-mediated Smad2 carboxy terminal phosphorylation is inhibited in the presence of TGFBR1 inhibitor, SB431542. Treatment with MyD88 and TRIF pathway antagonists does not affect LPS-mediated phosphorylation of Smad2 carboxy terminal; however, LPS-mediated Smad2 phosphorylation was inhibited in the presence of MMP inhibitor, GM6001, and unaffected in the presence of ROCK inhibitor Y27632 or ROS/NOX inhibitor DPI. LPS via transactivation of the TGFBR1 stimulates PAI-1 mRNA expression. TLRs are first in line to respond to exogenous invading substances and endogenous molecules; our findings characterise a novel signalling pathway in the context of cell biology. Identifying TLR transactivation of the TGFBR1 may provide future insight into the detrimental implications of pathogens in pathophysiology.

CCN2 (Cellular Communication Network Factor 2) Deletion Alters Vascular Integrity and Function Predisposing to Aneurysm Formation.

BACKGROUND: CCN2 (cellular communication network factor 2) is a matricellular protein involved in cell communication and microenvironmental signaling responses. CCN2 is known to be overexpressed in several cardiovascular diseases, but its role is not completely understood. METHODS: Here, CCN2 involvement in aortic wall homeostasis and response to vascular injury was investigated in inducible <i>Ccn2</i>-deficient mice, with induction of vascular damage by infusion of Ang II (angiotensin II; 15 days), which is known to upregulate CCN2 expression in the aorta. RESULTS: Ang II infusion in CCN2-silenced mice lead to 60% mortality within 10 days due to rapid development and rupture of aortic aneurysms, as evidenced by magnetic resonance imaging, echography, and histological examination. <i>Ccn2</i> deletion decreased systolic blood pressure and caused aortic structural and functional changes, including elastin layer disruption, smooth muscle cell alterations, augmented distensibility, and increased metalloproteinase activity, which were aggravated by Ang II administration. Gene ontology analysis of RNA sequencing data identified aldosterone biosynthesis as one of the most enriched terms in CCN2-deficient aortas. Consistently, treatment with the mineralocorticoid receptor antagonist spironolactone before and during Ang II infusion reduced aneurysm formation and mortality, underscoring the importance of the aldosterone pathway in Ang II-induced aorta pathology. CONCLUSIONS: CCN2 is critically involved in the functional and structural homeostasis of the aorta and in maintenance of its integrity under Ang II-induced stress, at least, in part, by disruption of the aldosterone pathway. Thus, this study opens new avenues to future studies in disorders associated to vascular pathologies.

BMP7 increases protein synthesis in SW1353 cells and determines rRNA levels in a NKX3-2-dependent manner.

BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.

The ACE genes in Aphelenchoides besseyi isolates and their expression correlation to the fenamiphos treatment.

Aphelenchoides besseyi could cause great yield losses of rice and many economically important crops. Acetylcholinesterase (AChE) inhibitors were commonly used to manage plant-parasitic nematodes. However, nematodes resistant to AChE inhibitors have been increasingly reported due to the extensive use of these chemicals. The current study was aimed to establish the correlation between fenamiphos (an AChE-inhibitor) sensitivities and acetylcholinesterase genes (ace) by analyzing two isolates of A. besseyi (designated Rl and HSF), which displayed differential sensitivities to fenamiphos. The concentrations of fenamiphos that led to the death of 50% (LD50) of Rl and HSF were 572.2 ppm and 129.4 ppm, respectively. Three ace genes were cloned from A. besseyi and sequenced. Sequence searching and phylogenic analyses revealed that AChEs of R1 and HSF shared strong similarities with those of various vertebrate and invertebrate species. Molecular docking analysis indicated that AChEs-HSF had much higher affinities to fenamiphos than AChEs-R1. Quantitative reverse transcriptase-PCR analyses revealed that expression of three ace genes were downregulated in HSF but were upregulated in Rl after exposure to 100 ppm fenamiphos for 12 h. The results indicated that the expression of the ace genes was modulated in response to fenamiphos in different nematode strains. An increased expression of the ace genes might contribute to fenamiphos-insensitivity as seen in the Rl isolate.