Comparison between sodium-glucose cotransporter 2 inhibitors and dipeptidyl peptidase 4 inhibitors on the risk of incident cancer in patients with diabetes mellitus: A real-world evidence study.

AIMS: Sodium-glucose cotransporter 2 inhibitors (SGLT-2is) have been demonstrated to be associated with cancer cell mechanisms. However, whether they increase the risk of cancer remains unclear. Thus, this study aimed to determine the association between SGLT-2i use and the incidence of cancer in patients with diabetes mellitus (DM) in Taiwan. MATERIALS AND METHODS: This retrospective cohort study was based on the Taiwan National Health Insurance database. The study population comprised patients with DM, and those who first used SGLT-2is during 2016-2018 were assigned to the study group. Greedy propensity score matching was performed to select patients who first used dipeptidyl peptidase 4 inhibitors (DPP-4is), and these patients were assigned to the control group. A Cox proportional hazards model was used to estimate the adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs) for cancer risk in the study and control groups; this model was adjusted for demographic characteristics, DM severity, comorbidities and concomitant medication use. RESULTS: After controlling for relevant variables, the SGLT-2i cohort (aHR = 0.90, 95% CI = 0.87-0.93) had a significantly lower risk of developing cancer than the DPP-4i cohort, particularly when the SGLT-2i was dapagliflozin (aHR = 0.91, 95% CI = 0.87-0.95) or empagliflozin (aHR = 0.90, 95% CI = 0.86-0.94). Regarding cancer type, the SGLT-2i cohort's risk of cancer was significantly lower than that of the DPP-4i cohort for leukaemia, oesophageal, colorectal, liver, pancreatic, lung, skin and bladder cancer. CONCLUSIONS: SGLT-2i use was associated with a significantly lower risk of cancer than DPP-4i use.

Use of Transcutaneous Electrical Nerve Stimulation to Alleviate Thirst After Surgery: A Randomized Controlled Trial.

PURPOSE: This prospective study investigated the preventive effect of transcutaneous electrical nerve stimulation (TENS) for postoperative thirst. DESIGN: This experimental study was conducted with the CONSORT checklist. METHODS: A total of 105 surgical patients who received general anesthesia were recruited from a medical center. Each patient was randomly assigned to the experimental group (n = 53; 20 min of TENS) or the control group (n = 52; routine care). In each group, oral moisture wetness was measured at 1 min, 20 min, and 50 min post-surgery. Descriptive and inferential statistics (Chi-square test, t test, one-way ANOVA, and generalized estimating equation (GEE) regression analysis) were performed to assess the proposed relationships. FINDINGS: The two groups showed similar characteristics at baseline. The oral moisture wetness was significantly higher in the experimental group than the control group at each post-surgery assessment time (all P < .001). The GEE results showed that patients in the experimental group reported more oral moisture wetness than patients in the control group. CONCLUSIONS: This study demonstrated that TENS can reduce thirst reported by patients after general anesthesia. Thus, this method may have clinical applications for managing postoperative thirst.

Soil to tobacco component transfer factors for natural radionuclides 40K, 226Ra, and 232Th and the risk assessment of tobacco leaf in smoking.

This study determined the activity concentrations and corresponding transfer factors (TF) of 40K, 226Ra, and 232Th in three tobacco components (root, stem, and leaf). The radiation hazard index parameters were assessed for the tobacco leaf. The activity concentrations in the soil were 589-762, 32-43, and 49-59 Bq kg-dw-1 (dry weight) for 40K, 226Ra, and 232Th, respectively. The average activity concentrations of 40K, 226Ra, and 232Th were 447, 5.41 and 5.69 Bq/kg-dw for the root, 670, 9.64 and 7.61 Bq kg-dw-1 for the stem, and 793, 6.79 and 6.15 Bq kg-dw-1 for the leaf, respectively. The TF values were 0.42-1.42, 0.10-0.49 and 0.06-0.23 for 40K, 226Ra, and 232Th, respectively. The stem and leaf 40K TF values were significantly higher than the root values. The stem 226Ra TF values were significantly higher than the root values. The 226Ra and 232Th activity concentrations and TFs of tobacco components had a significant positive correlation. Based on the activity concentrations of the tobacco leaves, the annual inhalation effective dose to the lungs for an adult smoker was 0.32-0.81 mSv y-1 (average 0.60 mSv y-1). The Excess Lifetime Cancer Risk (ELCR) caused by smoking was an average of 2.39 × 10-3.

Natural medicine HLXL targets multiple pathways of amyloid-mediated neuroinflammation and immune response in treating alzheimer's disease.

BACKGROUND: Based on the complex pathology of AD, a single chemical approach may not be sufficient to deal simultaneously with multiple pathways of amyloid-tau neuroinflammation. A polydrug approach which contains multiple bioactive components targeting multiple pathways in AD would be more appropriate. Here we focused on a Chinese medicine (HLXL), which contains 56 bioactive natural products identified in 11 medicinal plants and displays potent anti-inflammatory and immuno-modulatory activity. HYPOTHESIS/PURPOSE: We investigated the neuroimmune and neuroinflammation mechanisms by which HLXL may attenuate AD neuropathology. Specifically, we investigated the effects of HLXL on the neuropathology of AD using both transgenic mouse models as well as microglial cell-based models. STUDY DESIGN: The 5XFAD transgenic animals and microglial cell models were respectively treated with HLXL and Aß42, and/or lipopolysaccharide (LPS), and then analyzed focusing on microglia mediated Aß uptake and clearance, as well as pathway changes. METHODS: We showed that HLXL significantly reduced amyloid neuropathology by upregulation of microglia-mediated phagocytosis of Aß both in vivo and in vitro. HLXL displayed multi-modal mechanisms regulating pathways of phagocytosis and energy metabolism. RESULTS: Our results may not only open a new avenue to support pharmacologic modulation of neuroinflammation and the neuroimmune system for AD intervention, but also identify HLXL as a promising natural medicine for AD. CONCLUSION: It is conceivable that the traditional wisdom of natural medicine in combination with modern science and technology would be the best strategy in developing effective therapeutics for AD.

4-Acetylantroquinonol B Suppresses Prostate Cancer Growth and Angiogenesis via a VEGF/PI3K/ERK/mTOR-Dependent Signaling Pathway in Subcutaneous Xenograft and In Vivo Angiogenesis Models.

Prostate cancer is a major cause of cancer-related mortality in men in developed countries. The compound, 4-acetylantroquinonol B (4AAQB), is isolated from Antrodia cinnamomea (commonly known as Niu-Chang-Chih), which has been shown to inhibit cancer growth. However, the anticancer activity of 4AAQB has not previously been examined in prostate cancer. This study aimed to investigate the effect of 4AAQB on cancer and angiogenesis, as well as to explore its mechanism of action. Human prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC) were used in cell viability, cell migration, and cell cycle functional assays to evaluate the anticancer and antiangiogenic efficacy of 4AAQB in vitro. The effects of 4AAQB in vivo were determined using xenograft and angiogenesis models. The signaling events downstream of 4AAQB were also examined. The 4AAQB compound inhibited PC3 cell growth and migration, and reduced in vivo cancer growth, as shown in a subcutaneous xenograft model. Furthermore, 4AAQB inhibited HUVEC migration, tube formation, and aortic ring sprouting; it also reduced neovascularization in a Matrigel implant angiogenesis assay in vivo. The 4AAQB compound also decreased metastasis in the PC3 prostate cancer model in vivo. Serum or vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2), phosphoinositide 3-kinase (PI3K)/Ak strain transforming (Akt), and extracellular signal-regulated kinase ½ (ERK ½) phosphorylation were attenuated by 4AAQB in both PC3 and HUVEC. In conclusion, 4AAQB is a potential candidate for prostate cancer therapy.

Glycemic control was associated with nonprostate cancer and overall mortalities in diabetic patients with prostate cancer.

BACKGROUND: Diabetes mellitus (DM) can worsen the prognosis or survival in prostate cancer (PC) patients. We investigated whether glycemic control impacts mortality in PC patients with existing diabetes. METHODS: All PC patients with or without preexisting DM were enrolled from 2006 to 2017. Mean hemoglobin A1c (HbA1c) values (<7%, 7%-9%, ≥9%) were used to represent glycemic control. Major outcomes included all-cause, PC-specific, and non-PC mortalities. Statistical analyses were performed using Cox regression models with adjusted mean HbA1c and other related confounders. RESULTS: A total of 831 PC patients were enrolled (non-DM group, n = 690; DM group with a record of mean HbA1c values, n = 141). Results showed that the DM group with mean HbA1c level ≥ 9% (n = 14) had significantly increased risk for all-cause and non-PC mortality (hazard ratio [HR], 3.09; 95% CIs, 1.15-8.32; p=0.025 and HR, 5.49; 95% CIs, 1.66-18.16; p = 0.005, respectively), but not for PC-specific mortality (HR, 1.03; 95% CIs, 0.13-8.44; p = 0.975), compared with the non-DM group. CONCLUSION: Our findings indicate that PC patients with DM who had a mean HbA1c level ≥ 9% had higher risks of all-cause and non-PC mortality compared with non-DM subjects. Further large and long-term studies are needed to verify the effect of glycemic control in PC patients with DM.

Carbon Monoxide Releasing Molecule-3 Enhances Heme Oxygenase-1 Induction via ROS-Dependent FoxO1 and Nrf2 in Brain Astrocytes.

Carbon monoxide releasing molecule-3 (CORM-3) has been shown to protect inflammatory diseases via the upregulation of heme oxygenases-1 (HO-1). However, in rat brain astrocytes (RBA-1), the mechanisms underlying CORM-3-induced HO-1 remain poorly defined. This study used western blot, real-time PCR, and promoter activity assays to determine the levels of HO-1 expression and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE) to measure reactive oxygen species (ROS). We found that CORM-3-induced HO-1 expression was mediated through ROS generation by Nox or mitochondria. The signaling components were differentiated by pharmacological inhibitors and small interfering RNA (siRNA). Subcellular fractions, immunofluorescent staining, and chromatin immunoprecipitation assay were used to evaluate the nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The roles of mTOR and FoxO1 in CORM-3-stimulated responses are still unknown in RBA-1 cells. Our results demonstrated that transfection with siRNAs or pretreatment with pharmacological inhibitors attenuated the levels of HO-1 and phosphorylation of signaling components including Akt, mTOR, FoxO1, and Nrf2 stimulated by CORM-3. Moreover, pretreatment with N-acetyl-L-cysteine, diphenyleneiodonium chloride, apocynin, or rotenone blocked nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The present study concluded that in RBA-1 cells, CORM-3-induced HO-1 expression is, at least partially, mediated through Nox and mitochondria/ROS-dependent PI3K/Akt/mTOR cascade to activate FoxO1 or ROS leading to activation of Nrf2 activity.

Mevastatin-Induced AP-1-Dependent HO-1 Expression Suppresses Vascular Cell Adhesion Molecule-1 Expression and Monocyte Adhesion on Human Pulmonary Alveolar Epithelial Cells Challenged with TNF-α.

Mevastatin (MVS) has been previously shown to induce heme oxygenase (HO)-1 expression through Nox/ROS-dependent PDGFRα/PI3K/Akt/Nrf2/ARE axis in human pulmonary alveolar epithelial cells (HPAEpiCs). However, alternative signaling pathways might involve in MVS-induced HO-1 expression. We found that tumor necrosis factor α (TNFα) induced vascular cell adhesion protein 1 (VCAM-1) expression and NF-κB p65 phosphorylation which were attenuated by pretreatment with MVS via up-regulation of HO-1, determined by Western blot and real-time qPCR. TNFα-induced VCAM-1 expression was attenuated by an NF-κB inhibitor, Bay117082. The inhibitory effects of MVS were reversed by tin protoporphyrin (SnPP)IX (an inhibitor of HO-1 activity). In addition, pretreatment with the inhibitor of pan-Protein kinase C (PKC) (GF109203X), PKCα (Gö6983), Pyk2 (PF431396), p38α MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA), and transfection with their respective siRNAs abolished MVS-induced HO-1 expression in HPAEpiCs. c-Jun (one of AP-1 subunits) was activated by PKCα, Pyk2, p38α MAPK, and JNK1/2, which turned on the transcription of the homx1 gene. The interaction between c-Jun and HO-1 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay, which was attenuated by these pharmacological inhibitors. These results suggested that MVS induces AP-1/HO-1 expression via PKCα/Pyk2/p38α MAPK- or JNK1/2-dependent c-Jun activation, which further binds with AP-1-binding site on HO-1 promoter and suppresses the TNFα-mediated inflammatory responses in HPAEpiCs. Thus, upregulation of the AP-1/HO-1 system by MVS exerts a potentially therapeutic strategy to protect against pulmonary inflammation.

Induction of HO-1 by Mevastatin Mediated via a Nox/ROS-Dependent c-Src/PDGFRα/PI3K/Akt/Nrf2/ARE Cascade Suppresses TNF-α-Induced Lung Inflammation.

BACKGROUND: Mevastatin (MVS), a 3-hydroxy-3-methylglutaryl coenzyme, a reductase (HMG-CoA) inhibitor, has anti-inflammatory effects potentially via up-regulation of heme oxygenase-1 (HO-1). However, the mechanisms underlying MVS-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). METHODS: HO-1 and intercellular adhesion molecule (ICAM)-1 expression were determined using real-time PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated using pharmacological inhibitors or specific small interfering RNA (siRNA)s. Interaction between Nrf2 and the antioxidant response element (ARE) binding site for the HO-1 promoter was determined by chromatin immunoprecipitation (ChIP) assay. RESULTS: Upregulation of HO-1 by MVS attenuated the tumor necrosis factor (TNF)-α-stimulated ICAM-1 expression associated with THP-1 adhesion to HPAEpiCs. These inhibitory effects of HO-1 were reversed by tin protoporphyrin (SnPP)IX or by transfection with HO-1 siRNA. MVS-induced HO-1 expression was mediated via NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation. Activation of Nox2/ROS further stimulated the phosphorylation of p47phox, proto-oncogene tyrosine-protein kinase (c-Src), platelet-derived growth factor receptor (PDFGR)α, protein kinase B (Akt), and Nrf2, which were inhibited by siRNAs. Pretreatment with pharmacological inhibitors, including diphenyleneiodonium (DPI), apocynin (APO), N-acetyl-L-cysteine (NAC), PP1, AG1296, or LY294002, reduced the MVS-activated Nrf2 nuclear-translocation binding to the ARE on the HO-1 promoter. CONCLUSIONS: MVS-induced HO-1 is, at least in part, mediated through a p47phox/Nox2/ROS-dependent activation of c-Src/PDGFRα/PI3K/Akt-regulated Nrf2/ARE axis and suppresses the TNF-α-mediated inflammatory responses in HPAEpiCs.

Lysophosphatidylcholine-induced mitochondrial fission contributes to collagen production in human cardiac fibroblasts.

Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation. Here, we evaluated the mechanisms underlying LPC-mediated collagen induction via mitochondrial events in human cardiac fibroblasts (HCFs), coupling application of the pharmacologic cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and genetic mutations in FOXO1 on the fibrosis pathway. In HCFs, LPC caused prostaglandin E2 (PGE2)/PGE2 receptor 4 (EP4)-dependent collagen induction via activation of transcriptional activity of forkhead box protein O1 (FoxO1) on COX-2 gene expression. These responses were mediated through LPC-induced generation of mitochondrial reactive oxygen species (mitoROS), as confirmed by ex vivo studies, which indicated that LPC increased COX-2 expression and oxidative stress. LPC-induced mitoROS mediated the activation of protein kinase C (PKC)α, which interacted with and phosphorylated dynamin-related protein 1 (Drp1) at Ser616, thereby increasing Drp1-mediated mitochondrial fission and mitochondrial depolarization. Furthermore, inhibition of PKCα and Drp1 reduced FoxO1-mediated phosphorylation at Ser256 and nuclear accumulation, which suppressed COX-2/PGE2 expression and collagen production. Moreover, pretreatment with celecoxib or COX-2 siRNA suppressed WT FoxO1; mutated Ser256-to-Asp256 FoxO1-enhanced collagen induction, which was reversed by addition of PGE2 Our results demonstrate that LPC-induced generation of mitoROS regulates PKCα-mediated Drp1-dependent mitochondrial fission and COX-2 expression via a PKCα/Drp1/FoxO1 cascade, leading to PGE2/EP4-mediated collagen induction. These findings provide new insights about the role of LPC in the pathway of fibrotic injury in HCFs.

Carbon Monoxide Releasing Molecule-2-Upregulated ROS-Dependent Heme Oxygenase-1 Axis Suppresses Lipopolysaccharide-Induced Airway Inflammation.

The up-regulation of heme oxygenase-1 (HO-1) is mediated through nicotinamaide adenine dinucleotide phosphate (NADPH) oxidases (Nox) and reactive oxygen species (ROS) generation, which could provide cytoprotection against inflammation. However, the molecular mechanisms of carbon monoxide-releasing molecule (CORM)-2-induced HO-1 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we found that pretreatment with CORM-2 attenuated the lipopolysaccharide (LPS)-induced intercellular adhesion molecule (ICAM-1) expression and leukocyte count through the up-regulation of HO-1 in mice, which was revealed by immunohistochemistrical staining, Western blot, real-time PCR, and cell count. The inhibitory effects of HO-1 by CORM-2 were reversed by transfection with HO-1 siRNA. Next, Western blot, real-time PCR, and promoter activity assay were performed to examine the HO-1 induction in HTSMCs. We found that CORM-2 induced HO-1 expression via the activation of protein kinase C (PKC)α and proline-rich tyrosine kinase (Pyk2), which was mediated through Nox-derived ROS generation using pharmacological inhibitors or small interfering ribonucleic acids (siRNAs). CORM-2-induced HO-1 expression was mediated through Nox-(1, 2, 4) or p47phox, which was confirmed by transfection with their own siRNAs. The Nox-derived ROS signals promoted the activities of extracellular signal-regulated kinase 1/2 (ERK1/2). Subsequently, c-Fos and c-Jun-activator protein-1 (AP-1) subunits-were up-regulated by activated ERK1/2, which turned on transcription of the HO-1 gene by regulating the HO-1 promoter. These results suggested that in HTSMCs, CORM-2 activates PKCα/Pyk2-dependent Nox/ROS/ERK1/2/AP-1, leading to HO-1 up-regulation, which suppresses the lipopolysaccharide (LPS)-induced airway inflammation.

Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity.

Most tissue-resident macrophage populations develop during embryogenesis, self-renew in the steady state and expand during type 2 immunity. Whether shared mechanisms regulate the proliferation of macrophages in homeostasis and disease is unclear. Here we found that the transcription factor Bhlhe40 was required in a cell-intrinsic manner for the self-renewal and maintenance of large peritoneal macrophages (LPMs), but not that of other tissue-resident macrophages. Bhlhe40 was necessary for the proliferation, but not the polarization, of LPMs in response to the cytokine IL-4. During infection with the helminth Heligmosomoides polygyrus bakeri, Bhlhe40 was required for cell cycling of LPMs. Bhlhe40 repressed the expression of genes encoding the transcription factors c-Maf and Mafb and directly promoted expression of transcripts encoding cell cycle-related proteins to enable the proliferation of LPMs. In LPMs, Bhlhe40 bound to genomic sites co-bound by the macrophage lineage-determining factor PU.1 and to unique sites, including Maf and loci encoding cell-cycle-related proteins. Our findings demonstrate a tissue-specific control mechanism that regulates the proliferation of resident macrophages in homeostasis and type 2 immunity.

CO-Releasing Molecule-2 Induces Nrf2/ARE-Dependent Heme Oxygenase-1 Expression Suppressing TNF-α-Induced Pulmonary Inflammation.

The upregulation of heme oxygenase-1 (HO-1) by the carbon monoxide-releasing molecule (CORM)-2 may be mediated through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [Nox] and reactive oxygen species (ROS) generation, which could provide cytoprotection against various cellular injuries. However, the detailed mechanisms of CORM-2-induced HO-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain largely unknown. Therefore, we dissected the mechanisms underlying CORM-2-induced HO-1 expression in HPAEpiCs. We found that the administration of mice with CORM-2 attenuated the tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte count as revealed by immunohistochemical staining, western blot, real-time polymerase chain reaction (PCR), and cell count. Furthermore, TNF-α-induced ICAM-1 expression associated with monocyte adhesion to HPAEpiCs was attenuated by infection with adenovirus (adv)-HO-1 or incubation with CORM-2. These inhibitory effects of HO-1 were reversed by pretreatment with hemoglobin (Hb). Moreover, CORM-2-induced HO-1 expression was mediated via the phosphorylation of p47phox, c-Src, epidermal growth factor receptor (EGFR), Akt, and NF-E2-related factor 2 (Nrf2), which were inhibited by their pharmacological inhibitors, including diphenyleneiodonium (DPI) or apocynin (APO), ROS [N-acetyl-L-cysteine (NAC)], PP1, AG1478, PI3K (LY294002), or Akt (SH-5), and small interfering RNAs (siRNAs). CORM-2-enhanced Nrf2 expression, and anti-oxidant response element (ARE) promoter activity was also inhibited by these pharmacological inhibitors. The interaction between Nrf2 and AREs was confirmed with a chromatin immunoprecipitation (ChIP) assay. These findings suggest that CORM-2 increases the formation of the Nrf2 and AREs complex and binds with ARE-binding sites via Src, EGFR, and PI3K/Akt, which further induces HO-1 expression in HPAEpiCs. Thus, the HO-1/CO system might suppress TNF-α-mediated inflammatory responses and exert a potential therapeutic strategy in pulmonary diseases.

Lipopolysaccharide-Induced Matrix Metalloproteinase-9 Expression Associated with Cell Migration in Rat Brain Astrocytes.

Neuroinflammation is a landmark of neuroinflammatory and neurodegenerative diseases. Matrix metalloproteinase (MMP)-9, one member of MMPs, has been shown to contribute to the pathology of these brain diseases. Several experimental models have demonstrated that lipopolysaccharide (LPS) exerts a pathological role through Toll-like receptors (TLRs) in neuroinflammation and neurodegeneration. However, the mechanisms underlying LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1) are not completely understood. Here, we applied pharmacological inhibitors and siRNA transfection to assess the levels of MMP-9 protein, mRNA, and promoter activity, as well as protein kinase phosphorylation in RBA-1 cells triggered by LPS. We found that LPS-induced expression of pro-form MMP-9 and cell migration were mediated through TLR4, proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), p38 mitogen-activated protein kinase (MAPK), and Jun amino-terminal kinase (JNK)1/2 signaling molecules in RBA-1 cells. In addition, LPS-stimulated binding of c-Jun to the MMP-9 promoter was confirmed by chromatin immunoprecipitation (ChIP) assay, which was blocked by pretreatment with c-Src inhibitor II, PF431396, AG1296, LY294002, Akt inhibitor VIII, p38 MAP kinase inhibitor VIII, SP600125, and tanshinone IIA. These results suggest that in RBA-1 cells, LPS activates a TLR4/c-Src/Pyk2/PDGFR/PI3K/Akt/p38 MAPK and JNK1/2 pathway, which in turn triggers activator protein 1 (AP-1) activation and ultimately induces MMP-9 expression and cell migration.

Galangin Inhibits Thrombin-Induced MMP-9 Expression in SK-N-SH Cells via Protein Kinase-Dependent NF-κB Phosphorylation.

Galangin, a member of the flavonol compounds of the flavonoids, could exert anti-inflammatory effects in various cell types. It has been used for the treatment of arthritis, airway inflammation, stroke, and cognitive impairment. Thrombin, one of the regulators of matrix metalloproteinase (MMPs), has been known as a vital factor of physiological and pathological processes, including cell migration, the blood⁻brain barrier breakdown, brain edema formation, neuroinflammation, and neuronal death. MMP-9 especially may contribute to neurodegenerative diseases. However, the effect of galangin in combating thrombin-induced MMP-9 expression is not well understood in neurons. Therefore, we attempted to explore the molecular mechanisms by which galangin inhibited MMP-9 expression and cell migration induced by thrombin in SK-N-SH cells (a human neuroblastoma cell line). Gelatin zymography, western blot, real-time PCR, and cell migration assay were used to elucidate the inhibitory effects of galangin on the thrmbin-mediated responses. The results showed that galangin markedly attenuated the thrombin-stimulated phosphorylation of proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), protein kinase C (PKC)α/ß/δ, protein kinase B (Akt), mammalian target of rapamycin (mTOR), p42/p44 mitogen-activated protein kinase (MAPK), Jun amino-terminal kinases (JNK)1/2, p38 MAPK, forkhead box protein O1 (FoxO1), p65, and c-Jun and suppressed MMP-9 expression and cell migration in SK-N-SH cells. Our results concluded that galangin blocked the thrombin-induced MMP-9 expression in SK-N-SH cells via inhibiting c-Src, Pyk2, PKCα/ßII/δ, Akt, mTOR, p42/p44 MAPK, JNK1/2, p38 MAPK, FoxO1, c-Jun, and p65 phosphorylation and ultimately attenuated cell migration. Therefore, galangin may be a potential candidate for the management of brain inflammatory diseases.

Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells.

Staphylococcus aureus (S. aureus) is a very common Gram-positive bacterium. It is widely distributed in air, soil, and water. S. aureus often causes septicemia and pneumonia in patients. In addition, it is considered to play a key role in mediating cell adhesion molecules upregulation. Resveratrol is a natural antioxidant with diverse biological effects, including the modulation of immune function, anti-inflammation, and cancer chemoprevention. In this study, we proved that S. aureus-upregulated vascular cell adhesion molecule-1 (VCAM-1) expression in human lung epithelial cells (HPAEpiCs) was inhibited by resveratrol. We also observed that resveratrol downregulated S. aureus-enhanced leukocyte count in bronchoalveolar lavage (BAL) fluid in mice. In HPAEpiCs, S. aureus stimulated c-Src, PDGFR, p38 MAPK, or JNK1/2 phosphorylation, which was inhibited by resveratrol. S. aureus induced the adhesion of THP-1 cells (a human monocytic cell line) to HPAEpiCs, which was also reduced by resveratrol. Finally, we found that S. aureus induced c-Src/PDGFR/p38 MAPK and JNK1/2-dependent c-Jun and ATF2 activation and in vivo binding of c-Jun and ATF2 to the VCAM-1 promoter, which were inhibited by resveratrol. Thus, resveratrol functions as a suppressor of S. aureus-induced inflammatory signaling, not only by inhibiting VCAM-1 expression but also by diminishing c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 activation in HPAEpiCs.

Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.

Lysophosphatidylcholine (LysoPC) has been shown to induce the expression of inflammatory proteins, including cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6), associated with cardiac fibrosis. Here, we demonstrated that LysoPC-induced COX-2 and IL-6 expression was inhibited by silencing NADPH oxidase 1, 2, 4, 5; p65; and FoxO1 in human cardiac fibroblasts (HCFs). LysoPC-induced IL-6 expression was attenuated by a COX-2 inhibitor. LysoPC-induced responses were mediated via the NADPH oxidase-derived reactive oxygen species-dependent JNK1/2 phosphorylation pathway, leading to NF-κB and FoxO1 activation. In addition, we demonstrated that both FoxO1 and p65 regulated COX-2 promoter activity stimulated by LysoPC. Overexpression of wild-type FoxO1 and S256D FoxO1 enhanced COX-2 promoter activity and protein expression in HCFs. These results were confirmed by ex vivo studies, where LysoPC-induced COX-2 and IL-6 expression was attenuated by the inhibitors of NADPH oxidase, NF-κB, and FoxO1. Our findings demonstrate that LysoPC-induced COX-2 expression is mediated via NADPH oxidase-derived reactive oxygen species generation linked to the JNK1/2-dependent pathway leading to FoxO1 and NF-κB activation in HCFs. LysoPC-induced COX-2-dependent IL-6 expression provided novel insights into the therapeutic targets of the cardiac fibrotic responses.

Haem oxygenase-1 up-regulation by rosiglitazone via ROS-dependent Nrf2-antioxidant response elements axis or PPARγ attenuates LPS-mediated lung inflammation.

BACKGROUND AND PURPOSE: Haem oxygenase-1 (HO-1) is induced by thiazolidinediones including rosiglitazone and exerts anti-inflammatory effects in various models. However, the molecular mechanisms underlying rosiglitazone-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). EXPERIMENTAL APPROACH: HO-1 expression was determined by real time-PCR, Western blotting and promoter reporter analyses. Signalling pathways were investigated using pharmacological inhibitors or specific siRNAs. Interactions between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of the HO-1 promoter were investigated with chromatin immunoprecipitation assays. KEY RESULTS: Up-regulation of HO-1 in HPAEpiCs or in mice by rosiglitazone blunted ICAM-1 expression and monocyte adhesion to HPAEpiCs challenged with LPS. Rosiglitazone-induced HO-1 expression was significantly attenuated by NADPH oxidase (NOX) inhibitors (apocynin and diphenyleneiodonium) or ROS scavenger (N-acetyl cysteine). The involvement of NOX activity and ROS generation in rosiglitazone-induced HO-1 expression was confirmed by transfection with p47phox or NOX2 siRNA. Moreover, pretreatment with the inhibitors of c-Src (c-Srci II), proline-rich tyrosine kinase 2 (Pyk2) (PF431396), Akt (Akti VIII) or PPARγ (GW9662) and transfection with siRNA of c-Src, Pyk2, Akt or PPARγ abolished the rosiglitazone-induced HO-1 expression in HPAEpiCs. Subsequently, Nrf2 was activated by phosphorylation of c-Src, Pyk2 and Akt, which turned on transcription of HO-1 gene by binding to AREs binding site and enhancing ARE promoter activity. CONCLUSIONS AND IMPLICATIONS: Rosiglitazone induces HO-1 expression via either NOX/ROS/c-Src/Pyk2/Akt-dependent Nrf2 activation or PPARγ in HPAEpiCs and suppresses LPS-mediated inflammatory responses, suggesting that PPARγ agonists may be useful for protection against pulmonary inflammation.

Up-regulation of PYK2/PKCα-dependent haem oxygenase-1 by CO-releasing molecule-2 attenuates TNF-α-induced lung inflammation.

BACKGROUND AND PURPOSE: Haem oxygenase-1 (HO-1) could provide cytoprotection against various inflammatory diseases. However, the mechanisms underlying the protective effect of CO-releasing molecule-2 (CORM-2)-induced HO-1 expression against TNF-α-induced inflammatory responses in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unknown. EXPERIMENTAL APPROACH: CORM-2-induced HO-1 protein and mRNA expression, and signalling pathways were determined by Western blot and real-time PCR, coupled with respective pharmacological inhibitors or transfection with siRNAs. The effect of CORM-2 on TNF-α-induced increase in leukocyte counts in BAL fluid and VCAM-1 expression in lung was determined by cell counting and Western blot analysis. KEY RESULTS: CORM-2 attenuated the TNF-α-induced pulmonary haematoma, VCAM-1 expression and increase in leukocytes through an up-regulation of HO-1 in mice; this effect of CORM-2 was reversed by the HO-1 inhibitor zinc protoporphyrin IX. Furthermore, CORM-2 increased HO-1 protein and mRNA expression as well as the phosphorylation of PYK2, PKCα and ERK1/2 (p44/p42 MAPK) in HPAEpiCs; these effects were attenuated by their respective pharmacological inhibitors or transfection with siRNAs. Inhibition of PKCα by Gö6976 or Gö6983 attenuated CORM-2-induced stimulation of PKCα and ERK1/2 phosphorylation but had no effect on PYK2 phosphorylation. Moreover, inhibition of PYK2 by PF431396 reduced the phosphorylation of all three protein kinases. Finally, PYK2/PKCα/ERK1/2-mediated stimulation of activator protein 1 was shown to play a key role in CORM-2-induced HO-1 expression via an up-regulation of c-Fos mRNA. CONCLUSIONS AND IMPLICATIONS: CORM-2 activates a PYK2/PKCα/ERK1/2/AP-1 pathway leading to HO-1 expression in HPAEpiCs. This HO-1/CO system might have potential as a therapeutic target in pulmonary inflammation.

Arachidonic Acid Induces ARE/Nrf2-Dependent Heme Oxygenase-1 Transcription in Rat Brain Astrocytes.

Arachidonic acid (AA) is a major product of phospholipid hydrolysis catalyzed by phospholipase A2 during neurodegenerative diseases. AA exerts as a second messenger to regulate various signaling components which may be involved in different pathophysiological processes. Astrocytes are the main types of CNS resident cells which maintain and support the physiological function of brain. AA has been shown to induce ROS generation through activation of NADPH oxidases (Noxs) which may play a key role in the expression of heme oxygenase-1 (HO-1). Therefore, this study was designed to investigate the mechanisms underlying AA-induced HO-1 expression in rat brain astrocytes (RBA-1). We found that AA induced HO-1 protein and mRNA expression and promoter activity in RBA-1, which was mediated through the synthesis of 15-deoxy-Δ12,14-prostaglandin D2-activated peroxisome proliferator-activated receptor-γ (PPARγ) receptors. This note was confirmed by transfection with PPARγ small interfering RNAs (siRNA) which attenuated the AA-mediated responses. AA-induced HO-1 expression was mediated through Nox/ROS generation, which was inhibited by Nox inhibitors (diphenyleneiodonium and apocynin) and ROS scavengers (N-acetyl cysteine). Moreover, AA-induced HO-1 expression was mediated through phosphorylation of Src, Pyk2, platelet-derived growth factor, PI3K/Akt, and ERK1/2 which were inhibited by the pharmacological inhibitors including PP1, PF431396, AG1296, LY294002, and U0126 or by transfection with respective siRNAs. AA-enhanced Nrf2 expression and HO-1 promoter activity was inhibited by transfection with Nrf2 siRNA or by these pharmacological inhibitors. Furthermore, chromatin immunoprecipitation assay confirmed that Nrf2 and PPARγ were associated with the proximal antioxidant response element (ARE)-binding site on HO-1 promoter, suggesting that Nrf2/PPARγ are key transcription factors modulating HO-1 expression. AA-induced ARE promoter activity was also reduced by these pharmacological inhibitors. These findings suggested that AA increases formation of Nrf2 and PPARγ complex and binding with ARE1 binding site through Src, Pyk2, PI3K/Akt, and ERK1/2, which further induced HO-1 expression in RBA-1 cells.