Inducible Nitric Oxide Synthase Expression Research Articles

The Role of Hydrogen Sulfide in iNOS and APP Localization and Expression in Neurons and Glial Cells Under Traumatic Effects: An Experimental Study with Bioinformatics Analysis and Biomodeling

Hydrogen sulfide (H2S) donors are emerging as promising candidates for neuroprotective agents. However, H2S-dependent neuroprotective mechanisms are not yet fully understood. We have demonstrated that an H2S donor (sodium sulfide, Na2S) reduces the expression of inducible NO synthase (iNOS) and amyloid-beta precursor protein (APP) in damaged neural tissue at 24 h and 7 days following traumatic brain injury (TBI). The application of aminooxyacetic acid (AOAA), an inhibitor of cystathionine β-synthase (CBS), produced the opposite effect. Seven days after TBI, iNOS expression was observed not only in the cytoplasm but also in some neuronal nuclei, while APP was exclusively localized in the cytoplasm and axons of damaged neurons. It was also shown that iNOS and APP were present in the cytoplasm of mechanoreceptor neurons (MRNs) in the crayfish, in axons, as well as in certain glial cells 8 h after axotomy. Na2S and AOAA had opposing effects on axotomized MRNs and ganglia in the ventral nerve cord (VNC). Multiple sequence alignments revealed a high degree of identity among iNOS and APP amino acid residues in various vertebrate and invertebrate species. In the final stage of this study, biomodeling identified unique binding sites for H2S, hydrosulfide anion (HS−), and thiosulfate (S2O32−) with iNOS and APP.

Cyclooxygenase-2/prostaglandin E2 pathway orchestrates the replication of infectious bronchitis virus in chicken tracheal explants

ABSTRACT In this study, we investigated the localized pathogenesis of infectious bronchitis virus (IBV) in chicken tracheal organ cultures (TOCs), focusing on the role of inducible cyclooxygenase (COX-2). Two divergent IBV strains, respiratory Connecticut (Conn) A5968 and nephropathogenic Delmarva (DMV)/1639, were studied at 6, 12, 24, and 48 hours post-infection (hpi). Various treatments including exogenous prostaglandin (PGE)2, a selective COX-2 antagonist (SC-236), and inhibitors of PGE2 receptors and Janus kinase (JAK) were administered. IBV genome load and antigen expression were quantified using real-time quantitative PCR and immunohistochemistry. COX-2, interferon (IFN)-α, IFN-β, interleukin (IL)−1β, IL-6, and inducible nitric oxide synthase (iNOS) expressions were measured, along with PGE2 and COX-2 concentrations. IBV genome load and protein expression peaked at 12 and 24 hpi, respectively. Conn A5968-infected TOCs exhibited continuous COX-2 expression for up to 24 hpi, extended PGE2 production up to 48 hpi, and reduced inflammatory cytokine expression. In contrast, DMV/1639-infected TOCs displayed heightened inflammatory cytokine expression, brief COX-2 expression, and PGE2 production. Treatment with IFN-γ, SC-236, PGE2 receptor inhibitors, or JAK inhibitors reduced IBV infection and lesion scores, whereas exogenous PGE2 or IFN-γ pretreatment with a JAK-2 inhibitor augmented infection. These findings shed light on the innate immune regulation of IBV infection in the trachea, highlighting the involvement of the COX-2/PGE2 pathway. IMPORTANCE Understanding the localized pathogenesis of infectious bronchitis virus (IBV) within the trachea of chickens is crucial for developing effective control strategies against this prevalent poultry pathogen. This study sheds light on the role of inducible cyclooxygenase (COX-2) and prostaglandin (PGE)2 in IBV pathogenesis using chicken tracheal organ culture (TOC) models. The findings reveal distinct patterns of COX-2 expression, PGE2 production, and immune responses associated with different IBV strains, highlighting the complexity of host-virus interactions. Furthermore, the identification of specific inhibitors targeting the COX-2/PGE2 pathway and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway provides potential therapeutic avenues for mitigating IBV infection in poultry. Overall, this study contributes to our understanding of the innate immune regulation of IBV infection within the trachea, laying the groundwork for the development of targeted interventions to control IBV outbreaks in poultry populations.

Ilex asprella-derived polysaccharide activates macrophage via TLR4-mediated NF-κB and MAPK signaling pathways

Ilex asprella, a valued herb in China, is recognized for its medicinal and dietary benefits, yet its active components and mechanisms of action have been largely unknown. This study focused on the polysaccharide extracted from Ilex asprella (IAP) and to investigate its structural characteristics and immunomodulatory effects. The results indicated IAP had a molecular weight of 3324 Da and was mainly composed of arabinose, galactose, glucose, and mannose. In vitro study showed 10, 50, and 100 μg/mL of IAP significantly increased the mRNA expression of inducible nitric-oxide synthase, cyclooxygenase-2, reactive oxygen, and cytokines in RAW264.7 macrophages. According to the results, IAP also facilitated the nucleus translocation of NF-κB p65 and activated the phosphorylation of p38, JNK, and ERK, revealing IAP activated RAW264.7 cells via NF-κB and MAPK signaling pathways. Furthermore, the results of membranes receptor inhibitor administration suggested toll-like receptor 4 might be the action target of IAP.

LysoPE mediated by respiratory microorganism Aeromicrobium camelliae alleviates H9N2 challenge in mice

Influenza remains a severe respiratory illness that poses significant global health threats. Recent studies have identified distinct microbial communities within the respiratory tract, from nostrils to alveoli. This research explores specific anti-influenza respiratory microbes using a mouse model supported by 16S rDNA sequencing and untargeted metabolomics. The study found that transferring respiratory microbes from mice that survived H9N2 influenza to antibiotic-treated mice enhanced infection resistance. Notably, the levels of Aeromicrobium were significantly higher in the surviving mice. Mice pre-treated with antibiotics and then inoculated with Aeromicrobium camelliae showed reduced infection severity, as evidenced by decreased weight loss, higher survival rates, and lower lung viral titres. Metabolomic analysis revealed elevated LysoPE (16:0) levels in mildly infected mice. In vivo and in vitro experiments indicated that LysoPE (16:0) suppresses inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX2) expression, enhancing anti-influenza defences. Our findings suggest that Aeromicrobium camelliae could serve as a potential agent for influenza prevention and a prognostic marker for influenza outcomes.

Anti-inflammatory effects of phytosphingosine-regulated cytokines and NF-kB and MAPK mechanism.

Phytosphingosine (PHS) is a major component of the skin barrier and a multifunctional physiologically active substance. This study aimed to investigate the types of cytokines regulated by PHS, their anti-skin inflammatory effects, and their anti-inflammatory mechanisms. RAW264.7 cells stimulated with Lipopolysaccharides (LPS) were treated with PHS to measure inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2), and gene expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX2) were confirmed by q-PCR. Cytokines regulated by PHS against LPS-induced inflammation were found through cytokine array, and each factor was reconfirmed through ELISA. Western blot was performed to confirm anti-inflammatory mechanism of Iκbα and MAPK. To confirm anti-skin inflammatory efficacy, HaCaT cells stimulated with TNF-α/IFN-γ were treated with PHS, and TARC, IL-6, and IL-8 were detected by ELISA. PHS suppressed the gene expression of iNOS and COX2, which were increased by LPS, and suppressed NO and PGE2 production. Through cytokine array, it was confirmed that IL-6, IL-10, IL-27 p28/IL-30, IP-10, I-TAC, MCP-5, and TIMP-1 increased by LPS were decreased by PHS. PHS inhibited NF-κB signaling by inhibiting LPS-induced NF-κB nuclear migration and p-Iκbα-mediated Iκbα degradation, and inhibited p38, ERK, and JNK signaling pathways. PHS reduced the production of TARC, IL-6, and IL-8 increased by TNF-α/IFN-γ. These results indicate PHS has anti-inflammatory effects via the suppression of inflammatory factors and pro-inflammatory cytokines through the NF-κB and MAPK pathways. Moreover, these results may explain beneficial effects of PHS in the treatment of skin inflammatory conditions induced by TNF-α/IFN-γ.

NIR stimulated epigallocatechin gallate loaded polydopamine with enhanced antibacterial and ROS scavenging abilities for improved infectious wound healing

Infectious wound healing is complicated with and limited by infection and oxidative stress at the wound site. In recent years, various evidences suggest that nanozymes with multiple enzymatic activities have enabled the development of novel strategies for infectious wound healing. In this study, epigallocatechin gallate loaded polydopamine (P@E) was developed to act as a potent reactive oxygen species (ROS) scavenger for scavenging ROS, alleviating inflammatory responses, and promoting infectious wound healing. Combining with near infrared (NIR) irradiation, P@E presented excellent antibacterial ability of Escherichia coli (E. coli, 93.6%) and methicillin-resistant Staphylococcus aureus (MRSA, 87.6%). Specifically, P@E+NIR exhibited the most potent antioxidant, anti-inflammatory and cell proliferation behaviors through down-regulating intracellular ROS levels (81.9% and 94.3% for NIH3T3 and RAW264.7 respectively) and inducible nitric oxide synthase (iNOS) expression level (55.7%), and up-regulating the expression levels of arginase-1 (Arg-1, 71.4%), heat shock protein 70 (HSP70, 48.6%) and platelet endothelial cell adhesion molecule (CD31, 35.3%) compared to control group. Meanwhile, it also efficiently induced M2 directional polarization of lipopolysaccharide induced murine macrophages to achieve anti-inflammation, indicated by the down-regulation of CD86 (86.2%), and up-regulation of CD206 (85.6%). Significantly, it was also observed that P@E+NIR presented the excellent behaviors of inhibiting wound infection, alleviating wound inflammation, as well as promoting skin tissue repairing. Altogether, it has developed the strategy of using P@E combining with NIR irradiation for the synergistic enhanced healing of infectious skin wound, which can serve as a promising therapeutic strategy for its clinical treatment.

Promotion of mandibular distraction osteogenesis by parathyroid hormone via macrophage polarization induced through iNOS downregulation

ObjectiveTo investigate whether Parathyroid hormone (PTH) can promote mandibular distraction osteogenesis by regulating macrophage polarization and the underlying mechanisms of this phenomenon. MethodsForty-eight Rabbits were used to establish the mandibular distraction osteogenesis experimental model, randomly divided into 2 groups. Intermittent post-operative injections of 20 μg/kg PTH and normal saline were administered to the experimental and control groups, respectively. Regenerated new bone was examined using HE staining, osteoclast numbers were determined through tartrate-resistant acid phosphatase (TRAP) staining, and macrophage polarization markers arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) expressions were elucidated using immunohistochemistry (IHC), the mRNA expression of CD206, CD11C, Arg1 and iNOS were detected using qPCR. ResultsThe bone trabeculae in the experimental group were thicker, with a more homogeneous structure and more new osteoid than in the control group. In the area of distraction osteogenesis, the osteoclast count in the experimental group was higher than in the control group (P < 0.05). IHC results indicated differential expressions of Arg1 and iNOS in the experimental group compared to the control group (P < 0.05). Relative mRNA expressions of CD11c and iNOS were lower in the experimental group than in the control group (P < 0.05), whereas the expressions of CD206 and Arg1 mRNA were higher in the experimental group compared to the control group (P < 0.05). ConclusionIntermittent PTH injections increased macrophage quantity in the mandible generated by distraction osteogenesis, downregulated iNOS, upregulated Arg1, and promoted macrophage polarization from M1 to M2 phenotype, thereby promoting mandibular distraction osteogenesis.

Phytochemical profiling of Ananas comosus fruit via UPLC-MS and its anti-inflammatory and anti-arthritic activities: In Silico, In Vitro and In Vivo Approach

Ananas comosus [L.] is one of the most appreciated sources of metabolites for nutraceuticals and therapeutics. Traditional use of A. comosus fruit as anti-inflammatory and anti-arthritic agents warrants scientific validation. A. comosus fruit is well-known for its polyphenolic content and antioxidant activity that pose it good candidate in alleviating arthritis. This study aims to investigate the potential of A. comosus fruit as anti-arthritic and anti-inflammatory agent using different approaches in an attempt to reveal the underlying mechanism of action in accordance to its phytoconstituents. Results revealed that total ethanol extract of A. comosus (TEA) has potent in vitro antioxidant, anti-inflammatory and anti-arthritic activities. Also, TEA (500 and 1000 mg/kg) manifested promising in vivo anti-arthritic activity by alleviating paw edema to only 20.2 and 16.4% increase, respectively. High dose of TEA showed significant reduction in inflammatory biomarkers reverting IL- 6 to near normal value (46.93 pg/mL). TEA reversed the induced histopathological damage caused by the adjuvant and inhibit inducible nitric oxide synthase expression. UPLC/QTOF-MS-MS was carried out for metabolite profiling with a total of 53 metabolites identified including hydroxycinnamic acids (HCA), their depsides, glycerides and glycosides derivatives together with hydroxybenzoic acid glycosides, and amino acids. In silico studies displayed inhibitory potential against TNF-α for most of studied HCA derivatives especially dicinnamoyl glycerides and methoxylated HCA. This study is considered the first pharmacological validation of pineapple fruit traditional use. These observed results are attributed to TEA metabolite profile that exhibited favorable pharmacokinetics and drug likeness properties, suggesting their potential as lead drugs.

Cyclic adenosine monophosphate critically modulates cardiac GLP-1 receptor's anti-inflammatory effects.

Glucagon-like peptide (GLP)-1 receptor (GLP1R) agonists exert a multitude of beneficial cardiovascular effects beyond control of blood glucose levels and obesity reduction. They also have anti-inflammatory actions through both central and peripheral mechanisms. GLP1R is a G protein-coupled receptor (GPCR), coupling to adenylyl cyclase (AC)-stimulatory Gs proteins to raise cyclic 3`-5`-adenosine monophosphate (cAMP) levels in cells. cAMP exerts various anti-apoptotic and anti-inflammatory effects via its effectors protein kinase A (PKA) and Exchange protein directly activated by cAMP (Epac). However, the precise role and importance of cAMP in mediating GLP1R`s anti-inflammatory actions, at least in the heart, remains to be determined. To this end, we tested the effects of the GLP1R agonist liraglutide on lipopolysaccharide (LPS)-induced acute inflammatory injury in H9c2 cardiac cells, either in the absence of cAMP production (AC inhibition) or upon enhancement of cAMP levels via phosphodiesterase (PDE)-4 inhibition with roflumilast. Liraglutide dose-dependently inhibited LPS-induced apoptosis and increased cAMP levels in H9c2 cells, with roflumilast but also PDE8 inhibition further enhancing cAMP production by liraglutide. GLP1R-stimulated cAMP markedly suppressed the LPS-dependent induction of pro-inflammatory tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-6 cytokine expression, of inducible nitric oxide synthase (iNOS) expression and nuclear factor (NF)-kB activity, of matrix metalloproteinases (MMP)-2 and MMP-9 levels and activities, and of myocardial injury markers in H9c2 cardiac cells. The effects of liraglutide were mediated by the GLP1R since they were abolished by the GLP1R antagonist exendin(9-39). Importantly, AC inhibition completely abrogated liraglutide`s suppression of LPS-dependent inflammatory injury, whereas roflumilast significantly enhanced the protective effects of liraglutide against LPS-induced inflammation. Finally, PKA inhibition or Epac1/2 inhibition alone only partially blocked liraglutide`s suppression of LPS-induced inflammation in H9c2 cardiac cells, but, together, PKA and Epac1/2 inhibition fully prevented liraglutide from reducing LPS-dependent inflammation. cAMP, via activation of both PKA and Epac, is essential for GLP1R`s anti-inflammatory signaling in cardiac cells and that cAMP levels crucially regulate the anti-inflammatory efficacy of GLP1R agonists in the heart. Strategies that elevate cardiac cAMP levels, such as PDE4 inhibition, may potentiate the cardiovascular, including anti-inflammatory, benefits of GLP1R agonist drugs.

Anti-inflammation Mechanisms of Flavones Are Highly Sensitive to the Position Isomers of Flavonoids: Acacetin vs Biochanin A.

Acacetin (ACA) and biochanin A (BCA) are isomeric monomethoxyflavones with different structural positions of the 4'-methoxy-phenyl group. Both of them are present in many commonly consumed foods, such as citrus fruits and vegetables, and have been discovered with anti-inflammatory activities, but their mechanisms of action are not clearly elucidated at the molecular level. Herein, we reported the structure-activity relationship of ACA and BCA regarding their potency in inhibiting nitric oxide (NO) production, proinflammatory enzyme expression, and mRNA expression of proinflammatory cytokines in the lipopolysaccharide (LPS)-induced RAW 264.7 cells. Furthermore, transcriptome analysis was conducted to map out the overall pathways impacted by these two isomers. Our results showed that ACA possessed higher suppressive activity in nitric oxide (NO) production (IC50 = 23.93 ± 1.74 μM for ACA and IC50 = 71.41 ± 8.07 μM for BCA), inducible nitric oxide synthase (iNOS) expression, and proinflammatory interleukin (IL)-1β mRNA expression, but lower inhibitory activity in IL-6 mRNA expression as compared with BCA. Although two compounds were revealed to bind to c-Src protein according to drug affinity responsive target stability (DARTS) and western blotting results, molecular docking and molecular dynamics (MD) simulation showed that they had different binding sites, which subsequently resulted in different signaling transduction. ACA primarily exerted anti-inflammatory activity through the mitogen-activated protein kinase (MAPK) signaling pathway, the tumor necrosis factor (TNF) signaling pathway, and the hypoxia-inducible factor (HIF)-1 signaling pathway, while BCA was particularly involved in the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway, the nuclear factor kappa B (NF-κB) signaling pathway, the TNF signaling pathway, and the toll-like receptor (TLR) signaling pathway. Moreover, two isomers remained stable in the cell culture medium and did not encounter the biotransformation process in RAW 264.7 cells. However, BCA exhibited insufficient cellular uptake ability and transport efficiency in macrophages compared to ACA. Taken together, ACA is more promising for controlling inflammation and worthy of further study for human use.

Sulfate-Reducing Bacteria Induce Pro-Inflammatory TNF-α and iNOS via PI3K/Akt Pathway in a TLR 2-Dependent Manner.

Desulfovibrio, resident gut sulfate-reducing bacteria (SRB), are found to overgrow in diseases such as inflammatory bowel disease and Parkinson's disease. They activate a pro-inflammatory response, suggesting that Desulfovibrio may play a causal role in inflammation. Class I phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway regulates key events in the inflammatory response to infection. Dysfunctional PI3K/Akt signaling is linked to numerous diseases. Bacterial-induced PI3K/Akt pathway may be activated downstream of toll-like receptor (TLR) signaling. Here, we tested the hypothesis that Desulfovibrio vulgaris (DSV) may induce tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) expression via PI3K/Akt in a TLR 2-dependent manner. RAW 264.7 macrophages were infected with DSV, and protein expression of p-Akt, p-p70S6K, p-NF-κB, p-IkB, TNF-α, and iNOS was measured. We found that DSV induced these proteins in a time-dependent manner. Heat-killed and live DSV, but not bacterial culture supernatant or a probiotic Lactobacillus plantarum, significantly caused PI3K/AKT/TNF/iNOS activation. LY294002, a PI3K/Akt signaling inhibitor, and TL2-C29, a TLR 2 antagonist, inhibited DSV-induced PI3K/AKT pathway. Thus, DSV induces pro-inflammatory TNF-α and iNOS via PI3K/Akt pathway in a TLR 2-dependent manner. Taken together, our study identifies a novel mechanism by which SRB such as Desulfovibrio may trigger inflammation in diseases associated with SRB overgrowth.

Anti-neuroinflammatory effects of the KIOM -patented Polygonum multiflorum maximized root tuber against LPS-stimulated BV2 Cells

The main pathological mechanism of neurodegeneration is neuroinflammation. It is known that the persistent neuroinflammatory response is harmful by causing secondary nerve tissue damage. Meanwhile, P. multiflorum is a traditional oriental medicinal herb. It has been used as a hematopoietic agent and is used to treat a variety of diseases and conditions. The aim of the present study was to compare the anti-inflammatory efficacy between the commonly available P. multiflorum (C1) and the KIOM-patented in vitro-propagated P. multiflorum (K1), which had higher content of active ingredients and biomass, using culture and cultivation conditions of LPS-induced neuroinflammation. After stimulation with LPS and treatment with C1 and K1 in mouse microglial BV-2 cells, nitric oxide (NO) production, pro-inflammatory cytokine secretion, inducible NO synthase (iNOS) expression, MAPK phosphorylation and transcription factor activity were assessed. We examined the antioxidant effect using DPPH and production of nitric oxide (NO). C1 and K1 suppressed the expression of iNOS and COX-2 and the production of pro-inflammatory cytokines. Furthermore, we determined the levels of inflammatory mediators, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and mitogen-activated protein kinases and IκBα via Western blotting to understand the regulating mechanisms. Additionally, C1 and K1 also inhibited the activation of p38 and nuclear factor-kappa B (NF-κB) in LPS-stimulated BV2 cells. In all experimental results, excellent anti-neuroinflammatory effects were confirmed at a lower dose in K1 than in C1, which is believed to be due to the increased biomass. Therefore, K1 is expected to be more effective than C1 and can be applied more broadly in the development of prevention and treatment of various inflammatory-mediated neurodegenerative diseases.

Enhanced eNOS/nitric oxide production by nebivolol interferes with TGF-β1/Smad3 signaling and collagen I deposition in the kidney after prolonged tacrolimus administration

AimsTacrolimus is an effective immunosuppressant commonly used post-transplantation and in certain autoimmune diseases. However, its long-term administration is associated with renal fibrosis through transforming growth factor-beta/suppressor of mother against decapentaplegic (TGF-β/Smad) signaling that could be partly attributed to endothelial dysfunction alongside decreased nitric oxide (NO) release. Our study aimed to investigate the prospective renal anti-fibrotic effect of enhanced NO production by nebivolol against tacrolimus-stimulated TGF-β1/Smad3 signaling. Materials and methodsTo illustrate the proposed mechanism of nebivolol, Nω-nitro-L-arginine methyl ester (L-NAME); nitric oxide synthase inhibitor; was co-administered with nebivolol. Rats were treated for 30 days as control, tacrolimus, tacrolimus/nebivolol, tacrolimus/L-NAME, and tacrolimus/nebivolol/L-NAME groups. Key findingsOur results revealed that renal NO content was reduced in tacrolimus-treated rats, while treatment with tacrolimus/nebivolol enhanced NO content via up-regulated endothelial nitric oxide synthase (eNOS), but down-regulated inducible nitric oxide synthase (iNOS) expression. That participated in the inhibition of TGF-β1/Smad3 signaling induced by tacrolimus, where the addition of L-NAME abolished the defensive effects of nebivolol. Subsequently, the deposition of collagen I and alpha-smooth muscle actin (α-SMA) was retarded by nebivolol, emphasized by reduced Masson's trichrome staining. In accordance, there was a strong negative correlation between eNOS and both TGF-β1 and collagen I protein expression. The protective effects of nebivolol were further confirmed by the improvement in kidney function biomarkers and histological features. SignificanceIt can be suggested that treatment with nebivolol along with tacrolimus could effectively suppress renal TGF-β1/Smad3 fibrotic signaling via the enhancement of endothelial NO production, thus curbing renal fibrosis development.

Sulindac prevents increased mitochondrial VDAC1 expression and cell surface mistargeting induced by pathological conditions in retinal cells

Diabetic retinopathy (DR) continues to be the primary cause of vision loss in poorly controlled diabetic subjects. The molecular mechanisms underlying retinal pigment epithelium (RPE) cell dysfunction in DR still remain elusive. We investigated the role of mitochondrial volt-age-dependent anion channel 1 (VDAC1) in RPE dysfunction under glucotoxic and inflammatory conditions. Our results demonstrate that both glucotoxicity and cytokine treatment reduces cellular viability accompanied by increased VDAC1 and inducible nitric oxide synthase (iNOS) expression, concomitant with decreased expression of mitochondrial VDAC2 and constitutively ex-pressed endothelial NOS (eNOS). Increased VDAC1 expression during these conditions leads to its mistargeting to the cell surface, leading to ATP loss. Additionally, VDAC1 upregulation by glucotoxicity and inflammatory cytokines induces leakage of mitochondrial DNA (mtDNA) into the cytosol. Sulindac, a nonsteroidal anti-inflammatory agent, mitigates the adverse effects associated with increased VDAC1 level under pathophysiological conditions, by suppressing VDAC1 expression. The effect of sulindac on restoring cell viability could be comparably achieved only with VDAC1 inhibitor (VBIT-4) or VDAC1-specific antibody and not with the iNOS inhibitor aminoguanidine.Our findings suggest that sulindac's beneficial effects on ARPE-19 cell function are mediated by prevention of increased VDAC1 expression under pathological conditions, thus preventing mtDNA leakage and ATP loss, which are the key steps in induction of cellular inflammatory responses involved in the development of DR.

Neuroprotective role of chlorogenic acid against hippocampal neuroinflammation, oxidative stress, and apoptosis following acute seizures induced by pentylenetetrazole.

This study investigated the neuroprotective effect of chlorogenic acid (CGA) on pentylenetetrazole (PTZ)-induced acute epileptic seizures in mice. Epileptic animals received CGA (200mg/kg) or sodium valproate (standard antiepileptic agent, 200mg/kg) for four weeks. Results revealed that pre-administration of CGA significantly reversed the behavioral changes following pentylenetetrazole (PTZ) injection. Further, CGA pre-treatment caused significant increases in acetylcholinesterase (AChE) activity and brain-derived neurotrophic factor (BDNF) levels, along with marked increases in dopamine, norepinephrine, and serotonin levels. Additionally, the increased antioxidant enzymes activities, along with higher glutathione (GSH) contents and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) gene expression, were indicative of a notable improvement in the cellular antioxidant defense in mice treated with CGA. These results were associated with lowered malondialdehyde (MDA) and nitric oxide (NO) levels. Moreover, epileptic mice that received CGA showed significant declines in the content of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and nuclear factor kappa-B (NF-κB), besides downregulating inducible nitric oxide synthase (iNOS) expression. Remarkably, CGA counteracted hippocampal apoptosis by lessening the levels of pro-apoptotic biomarkers [Bcl-2-associated X protein (Bax) and caspase-3] and increasing the anti-apoptogenic marker level of B-cell lymphoma 2 (Bcl-2). The hippocampal histopathological findings corroborated the abovementioned changes. In sum, these findings suggest that CGA could mediate the neuroprotective effect against PTZ-induced epilepsy via modulation of neurotransmitters, oxidative damage, neuroinflammation, and apoptosis. CGA, therefore, could be considered a valuable antiepileptic therapeutic supplement.

Myrislignan ameliorates the progression of osteoarthritis: An in vitro and in vivo study

Osteoarthritis (OA) is a prevalent disease of the musculoskeletal system that causes functional deterioration and diminished quality of life. Myrislignan (MRL) has a wide range of pharmacological characteristics, including an anti-inflammatory ability. Although inflammation is a major cause of OA, the role of MRL in OA treatment is still not well-understood. In this study, we analyze the anti-inflammatory and anti-ECM degradation effects of MRL both in vivo and in vitro. Rat primary chondrocytes were treated with interleukin-1β (IL-1β) to simulate inflammatory environmental conditions and OA in vitro. The in vivo OA rat model was established by anterior cruciate ligament transection (ACLT) on rat. Our investigation discovered that MRL lowers the IL-1β-activated tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2) and inducible nitric-oxide synthase (iNOS) expression in chondrocytes. Moreover, MRL effectively alleviates IL-1β-induced extracellular matrix (ECM) degradation and promotes ECM synthesis in chondrocytes by upregulating the mRNA level expression of collagen-II and aggrecan (ACAN), downregulating the expression of matrix metalloproteinases-3,-13 (MMP-3, MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5). Gene expression profiles of different groups identified DEGs that were mainly enriched in functions associated with NF-κB signaling pathway, and other highly enriched in functions related to TNF, IL-17, Rheumatoid arthritis and cytokine-cytokine receptor signaling pathways. Venn interaction of DEGs from the abovementioned five pathways showed that Nfkbia, Il1b, Il6, Nfkb1, Ccl2, Mmp3 were highly enriched DEGs. In addition, our research revealed that MRL suppresses NF-κB and modulates the Nrf2/HO-1/JNK signaling pathway activated by IL-1β in chondrocytes. In vivo research shows that MRL slows the progression of OA in rats. Our findings imply that MRL might be a viable OA therapeutic choice.

Physicochemical properties and immune enhancement activity of oligosaccharide fraction purified from biomass of green macro-algal Ulva prolifera

Ulva prolifera (U. prolifera), as the dominant species of green macro-algal bloom, caused great damage to marine aquaculture and economy. This study was carried out to find its industrial utilization ways by determination the structure characteristics and the immune enhancement effect of oligosaccharide fractions produced from this alga. The result indicated that oligosaccharide fraction of F3 showed the best immune enhancement activity via RAW 264.7 macrophages and zebrafish assays. Chemical and structural analysis indicated that F3 with 11.14%sulfate content was composed of rhamnose, xylose, and glucose in ratio of 77.11:16.33:6.56. NMR spectroscopy showed that F3 contained (1 → 4)-α-l-rhamnopyranose-3-SO3 and α-l-rhamnopyranose-3-SO3-(1 → 4)-β-D-xylopyranose, with sulfate groups located at C-3 of rhamnopyranose. The results of immune effect in vitro demonstrated that F3 increased the production of nitric oxide (NO) and prostaglandin E2 (PGE2) with up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. F3 also promoted the secretion of cytokines (TNF-α, IL-1β and IL-6) and activated the expression of mitogen-activated protein kinases (MAPK) and NF-κB signaling pathways. In vivo assay, F3 boosted the embryo survival rate and heart rate, as well as promoted the generation of NO and reactive oxygen species (ROS) in a dose-dependent manner. The novel structure and excellent activity in this study suggested that F3 could be the potential material for use as a natural immuno-enhancing agent in the biomedical and functional food industries.

Mevalonate kinase of Leishmania donovani promotes its survival and plays a pivotal role in pathogenesis

The infectivity of Leishmania is determined by its ability to invade and evade host and its thriving capacity within the macrophage. Our study revealed the role of Leishmania donovani mevalonate kinase (MVK), an enzyme of mevalonate pathway in visceral leishmaniasis pathogenesis. Peritoneal exudate cells (PEC)-derived macrophages from BALB/c mice were infected with wild type (WT), MVK over expressing (MVK OE) and knockdown (KD) parasites and MVK OE parasites were found to be more infective than WT and MVK KD parasites. Incubation of macrophages with MVK OE parasites declined inducible nitric oxide synthase (iNOS) expression as well as nitric oxide (NO) production, both by 2 times in comparison to WT parasites. Moreover, ∼3 fold increase in Arginase1 expression indicated that MVK might induce polarization of macrophage towards M2, favouring the survival of parasite within the macrophages. Post 24 h infection of the macrophages with mutant strains, the levels of different cytokines (TNF-α, IL-12, IL-10 and IFN-γ) were measured. Infection of macrophages with MVK OE parasites showed an increase in the level of anti-inflammatory cytokine: IL-10 while infection with MVK KD parasites exhibited an increase in the level of pro-inflammatory cytokines: TNF-α, IL-12, and IFN-γ. Hence, Leishmania donovani mevalonate kinase (LdMVK) modulates macrophage functions and has a significant role in pathogenesis.

Porphyromonas gingivalis virulence factors induce toxic effects in SH-SY5Y neuroblastoma cells: GRK5 modulation as a protective strategy

Periodontitis (PDS) is a chronic inflammatory disease initiated by a dysbiosis of oral pathogenic bacterial species, such as Porphyromonas gingivalis (Pg). These bacteria can penetrate the bloodstream, releasing various endo and exotoxins that fuel the infection, and stimulate toxic inflammation in different compartments, including the brain. However, the specific mechanisms by which PDS/Pg contribute to brain disorders, such as Alzheimer’s disease (AD), remain unclear. This study assessed the effects of Pg’s virulence factors - lipopolysaccharide (LPS-Pg) and gingipains (gps) K (Kgp) and Rgp - on SH-SY5Y cells. Our results demonstrated that LPS-Pg activated signaling through the Toll-like receptor (TLR)-2/4 induced a significant downregulation of G protein-coupled receptor kinase 5 (GRK5). Additionally, LPS-Pg stimulation resulted in a robust increase in Tau phosphorylation (pTau) and p53 levels, while causing a marked reduction in Bcl2 and increased cell death compared to unstimulated cells (Ns). LPS-Pg also elevated inducible nitric oxide synthase (iNOS) expression, leading to oxidative damage. In cells overexpressing GRK5 via Adenovirus, LPS-Pg failed to increase iNOS and pTau levels compared to GFP control cells. High GRK5 levels also prevented the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Furthermore, the overexpression of a GRK5 mutant form lacking the nuclear localization signal (ΔNLS) nearly abolished LPS-Pg induced p53 and iNOS upregulation. Finally, we tested whether Kgp and Rgp mediated similar effects and our data showed that both gps caused a marked downregulation of GRK5 leading to increased p53 and pTau levels.In conclusion, this study provides further insight into the toxic effects elicited by Pg in cells and suggests that preventing GRK5 deficiency may be a valid strategy to mitigate Pg-induced toxic effects (i.e. cell death, oxidative damage, and Tau hyperphosphorylation) in SH-SY5Y cells, which are typical molecular hallmarks of neurodegenerative disorders.

ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury.

In this study we investigate the role of Zipper-interacting protein kinase (ZIPK) in high glucose-induced vascular injury, focusing on its interaction with STAT5A and its effects on p53 and inducible nitric oxide synthase (NOS2) expression. Human umbilical vein endothelial cells (HUVECs) are cultured under normal (5 mM) and high (25 mM) glucose conditions. Protein and gene expression levels are assessed by western blot analysis and qPCR respectively, while ROS levels are measured via flow cytometry. ZIPK expression is manipulated using overexpression plasmids, siRNAs, and shRNAs. The effects of the ZIPK inhibitor TC-DAPK6 are evaluated in a diabetic rat model. Our results show that high glucose significantly upregulates ZIPK, STAT5A, p53, and NOS2 expressions in HUVECs, thus increasing oxidative stress. Silencing of STAT5A reduces p53 and NOS2 expressions and reactive oxygen species (ROS) accumulation. ZIPK is essential for high glucose-induced p53 expression and ROS accumulation, while silencing of ZIPK reverses these effects. Overexpression of ZIPK combined with STAT5A silencing attenuates glucose-induced alterations in p53 and NOS2 expression, thereby preventing cell damage. Coimmunoprecipitation reveals a direct interaction between ZIPK and STAT5A in the nucleus under high-glucose condition. In diabetic rats, TC-DAPK6 treatment significantly decreases ZIPK, p53, and NOS2 expressions. Our findings suggest that ZIPK plays a critical role in high glucose-induced vascular injury via STAT5A-mediated pathways, proposing that ZIPK is a potential therapeutic target for diabetic vascular complications.