Harnessing peroxisome proliferator-activated receptor γ agonists to induce Heme Oxygenase-1: a promising approach for pulmonary inflammatory disorders.

The activation of peroxisome proliferator-activated receptor (PPAR)-γ has been extensively shown to attenuate inflammatory responses in conditions such as asthma, acute lung injury, and acute respiratory distress syndrome, as demonstrated in animal studies. However, the precise molecular mechanisms underlying these inhibitory effects remain largely unknown. The upregulation of heme oxygenase-1 (HO-1) has been shown to confer protective effects, including antioxidant, antiapoptotic, and immunomodulatory effects in vitro and in vivo. PPARγ is highly expressed not only in adipose tissues but also in various other tissues, including the pulmonary system. Thiazolidinediones (TZDs) are highly selective agonists for PPARγ and are used as antihyperglycemic medications. These observations suggest that PPARγ agonists could modulate metabolism and inflammation. Several studies have indicated that PPARγ agonists may serve as potential therapeutic candidates in inflammation-related diseases by upregulating HO-1, which in turn modulates inflammatory responses. In the respiratory system, exposure to external insults triggers the expression of inflammatory molecules, such as cytokines, chemokines, adhesion molecules, matrix metalloproteinases, and reactive oxygen species, leading to the development of pulmonary inflammatory diseases. Previous studies have demonstrated that the upregulation of HO-1 protects tissues and cells from external insults, indicating that the induction of HO-1 by PPARγ agonists could exert protective effects by inhibiting inflammatory signaling pathways and attenuating the development of pulmonary inflammatory diseases. However, the mechanisms underlying TZD-induced HO-1 expression are not well understood. This review aimed to elucidate the molecular mechanisms through which PPARγ agonists induce the expression of HO-1 and explore how they protect against inflammatory and oxidative responses.

Thrombin-Induced COX-2 Expression and PGE2 Synthesis in Human Tracheal Smooth Muscle Cells: Role of PKCδ/Pyk2-Dependent AP-1 Pathway Modulation.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

Thrombin Induces COX-2 and PGE2 Expression via PAR1/PKCalpha/MAPK-Dependent NF-kappaB Activation in Human Tracheal Smooth Muscle Cells.

The inflammation of the airway and lung could be triggered by upregulation cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induced by various proinflammatory factors. COX-2 induction by thrombin has been shown to play a vital role in various inflammatory diseases. However, in human tracheal smooth muscle cells (HTSMCs), how thrombin enhanced the levels of COX-2/PGE2 is not completely characterized. Thus, in this study, the levels of COX-2 expression and PGE2 synthesis induced by thrombin were determined by Western blot, promoter-reporter assay, real-time PCR, and ELISA kit. The various signaling components involved in the thrombin-mediated responses were differentiated by transfection with siRNAs and selective pharmacological inhibitors. The role of NF-κB was assessed by a chromatin immunoprecipitation (ChIP) assay, immunofluorescent staining, as well as Western blot. Our results verified that thrombin markedly triggered PGE2 secretion via COX-2 upregulation which were diminished by the inhibitor of thrombin (PPACK), PAR1 (SCH79797), Gi/o protein (GPA2), Gq protein (GPA2A), PKCα (Gö6976), p38 MAPK (SB202190), JNK1/2 (SP600125), MEK1/2 (U0126), or NF-κB (helenalin) and transfection with siRNA of PAR1, Gq α, Gi α, PKCα, JNK2, p38, p42, or p65. Moreover, thrombin induced PAR1-dependent PKCα phosphorylation in HTSMCs. We also observed that thrombin induced p38 MAPK, JNK1/2, and p42/p44 MAPK activation through a PAR1/PKCα pathway. Thrombin promoted phosphorylation of NF-κB p65, leading to nuclear translocation and binding to the COX-2 promoter element to enhance promoter activity, which was reduced by Gö6976, SP600125, SB202190, or U0126. These findings supported that COX-2/PGE2 expression triggered by thrombin was engaged in PAR1/Gq or Gi/o/PKCα/MAPK-dependent NF-κB activation in HTSMCs.

Anti-Inflammatory Effects of Rhamnetin on Bradykinin-Induced Matrix Metalloproteinase-9 Expression and Cell Migration in Rat Brain Astrocytes.

Bradykinin (BK) has been shown to induce matrix metalloproteinase (MMP)-9 expression and participate in neuroinflammation. The BK/MMP-9 axis can be a target for managing neuroinflammation. Our previous reports have indicated that reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NF-κB) activity is involved in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1). Rhamnetin (RNT), a flavonoid compound, possesses antioxidant and anti-inflammatory effects. Thus, we proposed RNT could attenuate BK-induced response in RBA-1. This study aims to approach mechanisms underlying RNT regulating BK-stimulated MMP-9 expression, especially ROS and NF-κB. We used pharmacological inhibitors and siRNAs to dissect molecular mechanisms. Western blotting and gelatin zymography were used to evaluate protein and MMP-9 expression. Real-time PCR was used for gene expression. Wound healing assay was applied for cell migration. 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) were used for ROS generation and NOX activity, respectively. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were applied to detect gene transcription. Our results showed that RNT inhibits BK-induced MMP-9 protein and mRNA expression, promoter activity, and cell migration in RBA-1 cells. Besides, the levels of phospho-PKCδ, NOX activity, ROS, phospho-ERK1/2, phospho-p65, and NF-κB p65 binding to MMP-9 promoter were attenuated by RNT. In summary, RNT attenuates BK-enhanced MMP-9 upregulation through inhibiting PKCδ/NOX/ROS/ERK1/2-dependent NF-κB activity in RBA-1.

Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) (p38 inhibitor VIII, p38i VIII), c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) (helenalin), or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders.

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.

RTA 408 Inhibits Interleukin-1ß-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent NF-κB and AP-1 Activation in Rat Brain Astrocytes.

Neuroinflammation is characterized by the elevated expression of various inflammatory proteins, including matrix metalloproteinases (MMPs), induced by various pro-inflammatory mediators, which play a critical role in neurodegenerative disorders. Interleukin-1ß (IL-1ß) has been shown to induce the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-reactive oxygen species (ROS)-dependent signaling pathways. N-(2-cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2-2-difluoropropanamide (RTA 408), a novel synthetic triterpenoid, has been shown to possess anti-oxidant and anti-inflammatory properties in various types of cells. Here, we evaluated the effects of RTA 408 on IL-1ß-induced inflammatory responses by suppressing MMP-9 expression in a rat brain astrocyte (RBA-1) line. IL-1ß-induced MMP-9 protein and mRNA expression, and promoter activity were attenuated by RTA 408. The increased level of ROS generation in RBA-1 cells exposed to IL-1ß was attenuated by RTA 408, as determined by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and CellROX. In addition, the inhibitory effects of RTA 408 on MMP-9 expression resulted from the suppression of the IL-1ß-stimulated activation of Pyk2 (proline-rich tyrosine kinase), platelet-derived growth factor receptor ß (PDGFRß), Akt, ROS, and mitogen-activated protein kinases (MAPKs). Pretreatment with RTA 408 attenuated the IL-1ß-induced c-Jun phosphorylation, mRNA expression, and promoter activity. IL-1ß-stimulated nuclear factor-κB (NF-κB) p65 phosphorylation, translocation, and promoter activity were also attenuated by RTA 408. Furthermore, IL-1ß-induced glial fibrillary acidic protein (GFAP) protein and mRNA expression, and cell migration were attenuated by pretreatment with RTA 408. These results provide new insights into the mechanisms by which RTA 408 attenuates IL-1ß-mediated inflammatory responses and exerts beneficial effects for the management of brain 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.

[Exploring Resilience and Related Factors Among Patients With Stroke in the Recovery Stage].

BACKGROUND: Resilience is known to affect the degree to which individuals adapt to the impact of stroke and its sequelae. However, few studies have examined resilience and related factors among stroke patients in Taiwan. PURPOSE: To explore resilience and related factors among stroke patients in the recovery stage. METHODS: A cross-sectional and correlational study design was adopted. Convenience sampling was employed to recruit participants from the rehabilitation inpatient wards of a regional teaching hospital in northern Taiwan. A structured questionnaire, including the social support scale and the Chinese version of the resilience scale, was used for data collection. Data were analyzed using descriptive and inferential statistics and stepwise regression analysis. RESULTS: A total of 128 stroke recovery in-patients who averaged 57.2 ± 11.6 years of age and were predominantly male were recruited. The results of this study showed that the global resilience of participants was moderate and that a significantly positive correlation existed between global social support and resilience. Age, marital status, and global tangible social support accounted for 25.0% of the total variation in resilience. CONCLUSIONS / IMPLICATIONS FOR PRACTICE: Age, marital status and global tangible social support were identified as the crucial predictive factors of resilience in stroke patients. The results support the recommendation that healthcare providers should acquire advanced knowledge and skills through in-service education, proactive caring, and encouraging patients to learn self-care in order to enhance rehabilitation motivation and confidence levels and subsequently promote disease recovery and the ability to adapt to life through cross-disciplinary medical team cooperation and supportive relationships.

Lipopolysaccharide induces ICAM-1 expression via a c-Src/NADPH oxidase/ROS-dependent NF-κB pathway in human pulmonary alveolar epithelial cells.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via adhesion molecule induction and then causes lung injury. However, the mechanisms underlying LPS-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. We showed that LPS induced ICAM-1 expression in HPAEpiCs, revealed by Western blotting, RT-PCR, real-time PCR, and promoter assay. Pretreatment with the inhibitor of c-Src (protein phosphatase-1, PP1), reactive oxygen species (ROS) (Edaravone), NADPH oxidase (apocynin and diphenyleneiodonium chloride), EGFR (AG1478), PDGFR (AG1296), phosphatidylinositol-3-kinase (PI3K) (LY294002), MEK1/2 (U0126), or NF-κB (Bay11-7082) and transfection with siRNAs of c-Src, EGFR, PDGFR, Akt, p47(phox), Nox2, Nox4, p42, and p65 markedly reduced LPS-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with LPS. In addition, we established that LPS stimulated phosphorylation of c-Src, EGFR, PDGFR, Akt, or p65, which was inhibited by pretreatment with their respective inhibitors. LPS induced Toll-like receptor 4 (TLR4), MyD88, TNF receptor-associated factor 6 (TRAF6), c-Src, p47(phox), and Rac1 complex formation 2, which was attenuated by transfection with c-Src or TRAF6 siRNA. Furthermore, LPS markedly enhanced NADPH oxidase activation and intracellular ROS generation, which were inhibited by PP1. We established that LPS induced p42/p44 MAPK activation via a c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt-dependent pathway in these cells. Finally, we observed that LPS significantly enhanced NF-κB and IκBα phosphorylation, NF-κB translocation, and NF-κB promoter activity, which were inhibited by PP1, Edaravone, apocynin, diphenyleneiodonium chloride, AG1478, AG1296, LY294002, or U0126. These results demonstrated that LPS induces p42/p44 MAPK activation mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt pathway, which in turn initiates the activation of NF-κB and ultimately induces ICAM-1 expression in HPAEpiCs.

Effect of Acupuncture on Recovery of Consciousness in Patients with Acute Traumatic Brain Injury: A Multi-Institutional Cohort Study.

Traumatic brain injury (TBI) causes cognitive dysfunction and long-term impairments. This study aims to examine the effectiveness of acupuncture on the recovery of consciousness in TBI patients. This is a retrospective, multi-institutional cohort study. We enrolled patients with newly diagnosed TBI from 1 January 2007 to 3 August 2021, aged 20 years and older, from the Chang Gung Research Database (CGRD). The outcome was defined based on the difference between the first and last Glasgow Coma Scale (GCS). A total of 2163 TBI patients were analyzed, and 237 (11%) received acupuncture in the treatment period. Generally, the initial GCS was lower in the acupuncture users (11 vs. 14). For the results of our study, a higher proportion of acupuncture patients achieved significant improvement (GCS differences ≥ 3) compared to non-acupuncture users (46.0% vs. 22.4%, p-value < 0.001). The acupuncture users had a 2.11 times higher chance of achieving a significant improvement when considering all assessable covariates (adjusted odds ratio (aOR) 2, 11, 95% confidence interval [CI]: 1.31-3.40; p-value = 0.002). Using 1:1 propensity score matching (PSM), the acupuncture users still had better outcomes than the non-acupuncture users (45.3% vs. 32.9%, p-value = 0.020). In conclusion, this study suggests that acupuncture treatment may be beneficial for TBI patients.

Rhamnetin Prevents Bradykinin-Induced Expression of Matrix Metalloproteinase-9 in Rat Brain Astrocytes by Suppressing Protein Kinase-Dependent AP-1 Activation.

Bradykinin (BK) has been recognized as a stimulant for matrix metalloproteinase (MMP)-9 expression, contributing to neuroinflammation. Modulating the BK/MMP-9 pathway offers potential in the treatment of neuroinflammatory disorders. Rhamnetin (RNT), a flavonoid compound known for its antioxidant and anti-inflammatory effects, has shown promise. However, the specific mechanisms through which RNT inhibits BK-induced MMP-9 expression remain unclear. Therefore, this study aims to delve into the intricate mechanisms underlying this process. Here, we initially demonstrated that RNT effectively attenuated BK-induced MMP-9 expression and its associated cell migration in rat brain astrocyte-1 (RBA-1) cells. Further investigation revealed that BK-driven MMP-9 protein, mRNA, and promoter activity linked to cell migration relied on c-Src, Pyk2, EGFR, PDGFR, PI3K/Akt, JNK1/2, and c-Jun. This was validated by the inhibition of these effects through specific inhibitors, a finding substantiated by the introduction of siRNAs targeting these signaling molecules. Notably, the phosphorylated levels of these signaling components induced by BK were significantly reduced by their respective inhibitors and RNT, underscoring the inhibitory role of RNT in this process. These findings indicate that, in RBA-1 cells, RNT diminishes the heightened induction of MMP-9 triggered by BK through the inhibition of c-Src/Pyk2/PDGFR and EGFR/PI3K/Akt/JNK1/2-dependent AP-1 activation. This suggests that RNT holds promise as a potential therapeutic approach for addressing neuroinflammation in the brain.

Reactive Oxygen Species-Dependent Activation of EGFR/Akt/p38 Mitogen-Activated Protein Kinase and JNK1/2/FoxO1 and AP-1 Pathways in Human Pulmonary Alveolar Epithelial Cells Leads to Up-Regulation of COX-2/PGE2 Induced by Silica Nanoparticles.

The risk of lung exposure to silica nanoparticles (SiNPs) and related lung inflammatory injury is increasing with the wide application of SiNPs in a variety of industries. A growing body of research has revealed that cyclooxygenase (COX)-2/prostaglandin E2 (PGE2) up-regulated by SiNP toxicity has a role during pulmonary inflammation. The detailed mechanisms underlying SiNP-induced COX-2 expression and PGE2 synthesis remain unknown. The present study aims to dissect the molecular components involved in COX-2/PGE2 up-regulated by SiNPs in human pulmonary alveolar epithelial cells (HPAEpiCs) which are one of the major targets while SiNPs are inhaled. In the present study, we demonstrated that SiNPs induced COX-2 expression and PGE2 release, which were inhibited by pretreatment with a reactive oxygen species (ROS) scavenger (edaravone) or the inhibitors of proline-rich tyrosine kinase 2 (Pyk2, PF-431396), epidermal growth factor receptor (EGFR, AG1478), phosphatidylinositol 3-kinase (PI3K, LY294002), protein kinase B (Akt, Akt inhibitor VIII), p38 mitogen-activated protein kinase (MAPK) (p38 MAPK inhibitor VIII), c-Jun N-terminal kinases (JNK)1/2 (SP600125), Forkhead Box O1 (FoxO1, AS1842856), and activator protein 1 (AP-1, Tanshinone IIA). In addition, we also found that SiNPs induced ROS-dependent Pyk2, EGFR, Akt, p38 MAPK, and JNK1/2 activation in these cells. These signaling pathways induced by SiNPs could further cause c-Jun and FoxO1 activation and translocation from the cytosol to the nucleus. AP-1 and FoxO1 activation could increase COX-2 and PGE2 levels induced by SiNPs. Finally, the COX-2/PGE2 axis might promote the inflammatory responses in HPAEpiCs. In conclusion, we suggested that SiNPs induced COX-2 expression accompanied by PGE2 synthesis mediated via ROS/Pyk2/EGFR/PI3K/Akt/p38 MAPK- and JNK1/2-dependent FoxO1 and AP-1 activation in HPAEpiCs.

Induction of Heme Oxygenase-1 by 15d-Prostaglandin J2 Mediated via a ROS-Dependent Sp1 and AP-1 Cascade Suppresses Lipopolysaccharide-Triggered Interleukin-6 Expression in Mouse Brain Microvascular Endothelial Cells.

Heme oxygenase-1 (HO-1) has been shown to exert antioxidant, anti-inflammatory, and anti-apoptotic effects in various types of cells. Therefore, the induction of HO-1 is an excellent rationale for the development of protective drugs. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) can modulate the expression of antioxidant defense proteins and be beneficial for neuroinflammation. Brain endothelial cells play an important role in the pathophysiology of brain disorders. Whether 15d-PGJ2 can induce HO-1 expression and protect against the inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells remains unclear. Here, we reveal that 15d-PGJ2 stimulated HO-1 protein and mRNA expression in a time- and concentration-dependent manner in bEnd.3 cells, which was attenuated by diphenyleneiodonium chloride (DPI) and MitoTempo. Thus, activation of NADPH oxidase (NOX)- and mitochondria-derived reactive oxygen species (ROS) mediated 15d-PGJ2-induced HO-1 expression. ROS generation could cause phosphorylation of protein kinase C (PKC)δ, leading to HO-1 expression, which was suppressed by Rottlerin (selective inhibitor PKCδ), DPI, and MitoTempo. We further demonstrated that phosphorylation of c-Jun N-terminal kinase (JNK)1/2 participated in 15d-PGJ2-upregulated HO-1 expression, which was blocked by SP600125 or Rottlerin. Moreover, 15d-PGJ2-induced HO-1 expression was mediated through the activation of c-Jun (a subunit of activator protein 1 (AP-1)) and specificity protein 1 (Sp1), leading to their interaction with the HO-1 promoter, revealed by chromatin immunoprecipitation assay, which was attenuated by SP600125, Mithramycin A, or Tanshinone II A. We further verified the anti-inflammatory effect of HO-1 expression. Our results showed that 15d-PGJ2-induced HO-1 could mitigate the lipopolysaccharide-triggered interleukin-6 expression and secretion, as measured by an ELISA assay kit. These results suggest that 15d-PGJ2-induced HO-1 expression is mediated through the activation of NOX- and mitochondria-derived ROS-dependent PKCδ/JNK1/2/Sp1 and the AP-1 signaling pathway and protects against inflammatory responses in bEnd.3 cells.

Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ2 Attenuates H2O2-Induced IL-6 Expression in Rat Brain Astrocytes.

Excessive production of reactive oxygen species (ROS) by NADPH oxidase (Nox) resulted in inflammation. The negative regulator of ROS (NRROS) dampens ROS generation during inflammatory responses. 15-Deoxy-∆12,14-prostaglandin J2 (15d-PGJ2) exhibits neuroprotective effects on central nervous system (CNS). However, whether 15d-PGJ2-induced NRROS expression was unknown in rat brain astrocytes (RBA-1). NRROS expression was determined by Western blot, RT/real-time PCR, and promoter activity assays. The signaling components were investigated using pharmacological inhibitors or specific siRNAs. The interaction between transcription factors and the NRROS promoter was investigated by chromatin immunoprecipitation assay. Upregulation of NRROS on the hydrogen peroxide (H2O2)-mediated ROS generation and interleukin 6 (IL-6) secretion was measured. 15d-PGJ2-induced NRROS expression was mediated through PI3K/Akt-dependent activation of Sp1 and FoxO1 and established the essential promoter regions. We demonstrated that 15d-PGJ2 activated PI3K/Akt and following by cooperation between phosphorylated nuclear FoxO1 and Sp1 to initiate the NRROS transcription. In addition, Nrf2 played a key role in NRROS expression induced by 15d-PGJ2 which was mediated through its phosphorylation. Finally, the NRROS stable clones attenuated the H2O2-induced ROS generation and expression of IL-6 through suppressing the Nox-2 activity. These results suggested that 15d-PGJ2-induced NRROS expression is mediated through a PI3K/Akt-dependent FoxO1 and Sp1 phosphorylation, and Nrf2 cascade, which suppresses ROS generation through attenuating the p47phox phosphorylation and gp91phox formation and IL-6 expression in RBA-1 cells. These results confirmed the mechanisms underlying 15d-PGJ2-induced NRROS expression which might be a potential strategy for prevention and management of brain inflammatory and neurodegenerative diseases.

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.

5,8-Dihydroxy-4 ', 7-dimethoxyflavone Attenuates TNF-α-Induced Expression of Vascular Cell Adhesion Molecule-1 through EGFR/PKCα/PI3K/Akt/Sp1-Dependent Induction of Heme Oxygenase-1 in Human Cardiac Fibroblasts.

Recently, we found that 5,8-dihydroxy-4',7-dimethoxyflavone (DDF) upregulated the expression of heme oxygenase (HO)-1 via p38 mitogen-activated protein kinase/nuclear factor-erythroid factor 2-related factor 2 (MAPK/Nrf2) pathway in human cardiac fibroblasts (HCFs). However, the alternative processes by which DDF induces the upregulation of HO-1 expression are unknown. Activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and protein kinase C (PKC)α may initiate specificity protein (Sp)1 activity, which has been reported to induce expression of antioxidant molecules. Thus, we explored whether these components are engaged in DDF-induced HO-1 upregulation in HCFs. Western blotting, promoter-reporter analyses, and real-time polymerase chain reactions were adopted to measure HO-1 and vascular cell adhesion molecule (VCAM)-1 expressions in HCFs. Respective small interfering (si)RNAs and pharmacological inhibitors were employed to investigate the signaling components engaged in DDF-induced HO-1 upregulation. The chromatin immunoprecipitation assay was conducted to detect the binding interaction of Sp1 and antioxidant response elements (ARE) on the promoter of HO-1. An adhesion assay of THP-1 monocyte was undertaken to examine the functional effect of HO-1 on tumor necrosis factor (TNF)-α-induced VCAM-1 expression. DDF stimulated the EGFR/PKCα/PI3K/Akt pathway leading to activation of Sp1 in HCFs. The roles of these protein kinases in HO-1 induction were ensured by transfection with their respective siRNAs. Chromatin immunoprecipitation assays revealed the interaction between Sp1 and the binding site of proximal ARE on the HO-1 promoter, which was abolished by glutathione, AG1478, Gö6976, LY294002, or mithramycin A. HO-1 expression enhanced by DDF abolished the monocyte adherence to HCFs and VCAM-1 expression induced by TNF-α. Pretreatment with an inhibitor of HO-1: zinc protoporphyrin IX reversed these inhibitory effects of HO-1. We concluded that DDF-induced HO-1 expression was mediated via an EGFR/PKCα/PI3K/Akt-dependent Sp1 pathway and attenuated the responses of inflammation in HCFs.

HO-1 Upregulation by Kaempferol via ROS-Dependent Nrf2-ARE Cascade Attenuates Lipopolysaccharide-Mediated Intercellular Cell Adhesion Molecule-1 Expression in Human Pulmonary Alveolar Epithelial Cells.

Lung inflammation is a pivotal event in the pathogenesis of acute lung injury. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme that could be induced by kaempferol (KPR) and exerts anti-inflammatory effects. However, the molecular mechanisms of KPR-mediated HO-1 expression and its effects on inflammatory responses remain unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). This study aimed to verify the relationship between HO-1 expression and KPR treatment in both in vitro and in vivo models. HO-1 expression was determined by real time-PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated by using pharmacological inhibitors or specific siRNAs. Chromatin immunoprecipitation (ChIP) assay was performed to investigate the interaction between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of HO-1 promoter. The effect of KPR on monocytes (THP-1) binding to HPAEpiCs challenged with lipopolysaccharides (LPS) was determined by adhesion assay. We found that KPR-induced HO-1 level attenuated the LPS-induced intercellular cell adhesion protein 1 (ICAM-1) expression in HPAEpiCs. KPR-induced HO-1 mRNA and protein expression also attenuated ICAM-1 expression in mice. Tin protoporphyrin (SnPP)IX reversed the inhibitory effects of KPR in HPAEpiCs. In addition, in HPAEpiCs, KPR-induced HO-1 expression was abolished by both pretreating with the inhibitor of NADPH oxidase (NOX, apocynin (APO)), reactive oxygen species (ROS) (N-acetyl-L-cysteine (NAC)), Src (Src kinase inhibitor II (Srci II)), Pyk2 (PF431396), protein kinase C (PKC)α (Gö6976), p38 mitogen-activated protein kinase (MAPK) inhibitor (p38i) VIII, or c-Jun N-terminal kinases (JNK)1/2 (SP600125) and transfection with their respective siRNAs. The transcription of the homx1 gene was enhanced by Nrf2 activated by JNK1/2 and p38α MAPK. The binding activity between Nrf2 and HO-1 promoter was attenuated by APO, NAC, Srci II, PF431396, or Gö6983. KPR-mediated NOX/ROS/c-Src/Pyk2/PKCα/p38α MAPK and JNK1/2 activate Nrf2 to bind with ARE on the HO-1 promoter and induce HO-1 expression, which further suppresses the LPS-mediated inflammation in HPAEpiCs. Thus, KPR exerts a potential strategy to protect against pulmonary inflammation via upregulation of the HO-1.