iNOS Expression Research Articles

Mesua assamica (King & Prain) kosterm. bark ethanolic extract attenuates rheumatoid arthritis via down-regulating TLR4/NF-κB/COX-2/iNOS and activation of Nrf2/HO-1 pathways: A comprehensive study on in-vitro and in-vivo models

Ethnopharmacological relevanceRheumatoid arthritis (RA) is a multifactorial, polygenic inflammatory disease. Mesua assamica (King & Prain) Kosterm. (MA) is an endangered medicinal plant indigenous to South Asia, primarily to Assam in India. The tree bark is claimed to possess anti-inflammatory, anti-diabetic, anti-cancer, and anti-malarial properties; nevertheless, its role in RA has not been elucidated. Hence, this study aims to investigate the in-vitro and in-vivo anti-arthritic effects of Mesua assamica bark ethanolic extract (MAE). Aim of the studyThis study aims to investigate the anti-rheumatic potential of MAE in-vitro on RAW 264.7 cells for its anti-oxidant and anti-inflammatory activities and in-vivo on the CFA-induced adjuvant arthritis in the rat model. Materials and methodsWe investigated the possible therapeutic effects of MAE in-vitro using RAW 264.7 cells triggered by LPS. Meanwhile, adult Wistar rats were injected intradermally with 100 μl of CFA to induce arthritis, and they were given MAE orally at doses of 100 and 200 mg/kg for up to 28 days. Paw volume analysis, X-ray radiography, anti-oxidant levels analysis, gene and protein expression studies, and histological analysis were carried out to assess the effects of MAE in-vivo. ResultsMAE significantly mitigated the inflammation by reducing ROS levels and dropped the nitrite, PGE2, and COX-2 levels enhanced by LPS in-vitro. At the same time, MAE treatment reduced the paw and joint inflammation and increased the immune organ index in the CFA rats. Histopathology data revealed that MAE mitigated the CFA-induced lesions of the ankle joints and synovial tissues. Similarly, MAE significantly abated the secretion of pro-inflammatory cytokines, inhibited the protein expression of TLR4, NF-кB, COX-2, and iNOS, as well as improved the Nrf2 and HO-1 levels in-vitro and in-vivo. ConclusionAll the results highlighted the anti-rheumatic potential of MAE in RA in-vitro and in-vivo by inhibiting the TLR4/NF-кB/COX-2/iNOS and promoting the Nrf2/HO-1 signaling axis.

Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.

Background: Cardiac fibrosis is a common pathophysiology in a broad variety of heart diseases. GDF11, a member of the TGF-beta superfamily, can reduce cardiac hypertrophy and fibrosis in the pressure overload model of experimental Trans Aortic Constriction (TAC). However, the molecular mechanisms by which GDF11 can reduce cardiac fibrosis are incompletely described. Hypothesis: GDF11 is known to activate both NF-kB as well as SMAD2/3 signaling which are essential in cardiac fibrosis and hypertrophy processes. We hypothesized that exogenous GDF11 can change the dynamics of early inflammatory NF-kB target genes during the response to pressure overload. Methods: We used mice implanted with AngII-osmotic pumps to induce left ventricular fibrosis. The mice were harvested at different time points: 3, 7, 14, and 28 days post-pump implantation (ppi) to follow the evolution of cardiac fibrosis and cardiac hypertrophy, and to identify the role of GDF11 in modulating these processes. Results: GDF11 delivery (1mg/kg every other day) in AngII mice (AngII+GDF11) induced earlier expression of NF-kB target genes (IL-1beta, IL-6, TNF-alpha – p>0.05) compared to AngII+Veh control group at 3d ppi, revealing accelerated activation of NF-kB response pathways in AngII+GDF11 group. This is associated with faster recruitment of the M1 macrophage population observed by an increased expression of CD86 or iNOS at 3d ppi. We also observed decreased markers of fibroblast activation after GDF11 supplementation, with less collagen deposition (F-CHP) and fibrosis (Masson Trichrome) at 28 days. Conclusions: Our data suggest that exogenous GDF11 changes the dynamics of NF-kB inflammatory mechanisms while reducing later fibrosis. This new finding could suggest that the dynamics of the inflammatory response early after pressure overload is a critical factor in cardiac fibrosis.

MiR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes.The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively.HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action.Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.

Solanine Inhibits Proliferation and Angiogenesis and Induces Apoptosis through Modulation of EGFR Signaling in KB-ChR-8-5 Multidrug-Resistant Oral Cancer Cells.

Background: The most important factors contributing to multi-drug resistance in oral cancer include overexpression of the EGFR protein and the downstream malignancy regulators that are associated with it. This study investigates the impact of solanine on inflammation, proliferation, and angiogenesis inhibition in multidrug-resistant oral cancer KB-Chr-8-5 cells through inhibition of the EGFR/PI3K/Akt/NF-κB signaling pathway. Methods: Cell viability was assessed using an MTT assay to evaluate cytotoxic effects. Production of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨM), and AO/EtBr staining were analyzed to assess apoptosis and mitochondrial dysfunction. Western blotting was employed to examine protein expression related to angiogenesis, apoptosis, and signaling pathways. Experiments were conducted in triplicate. Results: Solanine treatment at concentrations of 10, 20, and 30 μM significantly increased ROS production, which is indicative of its antioxidant properties. This increase was associated with decreased mitochondrial membrane potential (ΔΨM) with p < 0.05, suggesting mitochondrial dysfunction. Inhibition of EGFR led to reduced activity of PI3K, Akt, and NF-κB, resulting in decreased expression of iNOS, IL-6, Cyclin D1, PCNA, VEGF, Mcl-1, and HIF-1α and increased levels of the apoptotic proteins Bax, caspase-9, and caspase-3. These changes collectively inhibited the growth of multidrug-resistant (MDR) cancer cells. Conclusions: Solanine acts as a potent disruptor of cellular processes by inhibiting the EGFR-mediated PI3K/Akt/NF-κB signaling pathway. These results suggest that solanine holds promise as a potential preventive or therapeutic agent against multidrug-resistant cancers.

Mitigating Acetaminophen-Induced Kidney Injury: The Protective Role of Grape Seed and Peanut Skin Extracts through the iNOS/CYP2E1 Pathway.

The rising number of acute kidney injury cases worldwide due to acetaminophen (APAP) emphasizes the critical need for effective prevention strategies to counteract APAP's detrimental effects. This study examined the kidney-protective capabilities of ethanolic extracts from grape seeds and peanut skins (GSEE and PSEE, respectively) in comparison with silymarin in rats that experienced an APAP overdose. The phenolic compounds in these extracts were measured by using high-performance liquid chromatography (HPLC). In the experiment, Sixty adult male albino rats were divided into five groups of 12. The Control group received 0.5 mL of saline via a gastric tube. Group II received acetaminophen (APAP, 640 mg/kg per day via a gastric tube) to induce renal injury, following Ucar et al. and Islam et al. Groups III, IV, and V received silymarin (50 mg/kg), grape seed extract (200 mg/kg), and peanut skin extract (200 mg/kg), respectively, along with 640 mg of APAP/kg per day for 21 days. Post APAP treatment, significant increases in serum urea and creatinine levels were noted, along with notable decreases in the percentage of body weight gain. Furthermore, there were increases in oxidative stress and inflammatory markers in the kidney tissues, including heightened mRNA expressions of renal iNOS and CYP2E1, which were confirmed through histological studies. The administration of GSEE, PSEE, and silymarin mitigated these adverse effects, likely due to their high phenolic content, which is recognized for its antioxidant and anti-inflammatory effects. GSEE, in particular, showed efficacy comparable to that of silymarin. Molecular docking studies revealed that APAP impeded critical enzymes essential for cellular antioxidant defense, whereas the bioactive compounds in the grape seed and peanut skin extracts effectively inhibited key enzymes and receptors involved in inflammation and oxidative stress. These findings suggest that GSEE and PSEE could serve as viable alternative treatments for kidney damage induced by APAP. Further research to isolate and identify these effective compounds is recommended.

Fucoidan refined from Saccharina japonica ameliorates ambient particulate matter-induced inflammation in keratinocytes, underlying fibroblasts, and 12-O-tetradecanoylphorbol 13-acetate-induced ear edema in mice

Fucoidan from Saccharina japonica (SJF) was isolated and characterized, and its anti-inflammatory effects on fine dust/ambient particulate matter (PM)-stimulated HaCaT keratinocytes were investigated. SJF increased cell viability by reducing intracellular ROS production in PM-stimulated HaCaT keratinocytes. Moreover, SJF downregulated the expression/production of inflammatory cytokines (IL-6, IL-8, IL-13, IL-25, IL-33, TNF-α, IFN-γ, and TSLP) and chemokines (MDC and TARC) through modulating NF-κB/MAPK signaling in PM-stimulated HaCaT keratinocytes. Extended studies investigated the impact of SJF-treated HaCaT keratinocyte culture media on HDFs. Interestingly, media from SJF-treated HaCaT keratinocytes on HDFs demonstrated a notable downregulation of the production of inflammatory mediators such as TSLP, IL-6, IL-8, IL-13, and TNF-α, as well as TARC and MDC. Furthermore, the study examined the impact of SJF on 12-O-tetradecanoylphorbol 13-acetate (TPA) induced ear edema in BALB/c mice and results indicated the reduced ear thickness and decreased iNOS and COX-2 expression. Our study confirmed the effectiveness of SJF in ameliorating PM-induced skin inflammation in in vitro experiments, along with the TPA-induced in vivo inflammatory model.

Diosmetin ameliorates osteoarthritic inflammation in vivo and ECM macromolecules degradation in interleukin-1β-stimulated murine chondrocytes through the Nrf2/NF-κB pathway.

Osteoarthritis (OA) is a prevailing degenerative disease in elderly population and can lead to severe joint dysfunction. Studies have revealed various pharmacological activities of diosmetin, including the anti-OA efficacy. The present study further investigated its effect on interleukin (IL)-1β-induced OA in chondrocytes. Primary chondrocytes were isolated from young mice, stimulated with IL-1β (10 ng/mL), and pretreated with diosmetin (10 and 20 μM) to conduct the in vitro assays. CCK-8 assay assessed the cytotoxicity of diosmetin whereas the levels of inflammatory factors (PGE2, nitrite, TNF-α, and IL-6) in homogenized cells were evaluated by ELISA. The levels of inflammatory cytokines, content of extracellular matrix (ECM), and signaling-related proteins (Nrf2, HO-1, and NF-κB p65) were assessed by western blotting. Expression of collagen II, p65, and Nrf2 in the chondrocytes was confirmed by immunofluorescence staining. The chondrocytes treated with IL-1β and diosmetin were transfected with Nrf2 knockdown plasmid (si-Nrf2) to investigate the role of Nrf2. In vivo OA mouse model was induced by surgically destabilizing the medial meniscus (DMM). Safranin O staining was conducted to assess the OA severity in the knee-joint tissue. Diosmetin suppressed the expression of iNOS, COX-2, PGE2, nitrite, TNF-α, IL-6, MMP-13, and ADAMTS-5 induced by IL-1β in chondrocytes. The expression of p-p65, p-IκBα, and nuclear p65 was decreased whereas that of Nrf2 and HO-1 increased by diosmetin treatment in IL-1β-treated chondrocytes. Nrf2 knockdown by siRNA reversed the inhibitory effect of diosmetin on IL-1β-induced degradation of ECM proteins and inflammatory factors in cultured chondrocytes. In the DMM-induced model of OA, diosmetin alleviated cartilage degeneration and decreased the Osteoarthritis Research Society International score. Diosmetin ameliorates expression of inflammation biomarkers and ECM macromolecules degradation in cultured murine chondrocytes via inactivation of NF-κB signaling by activating Nrf2/HO-1 signaling pathway.

Discordant effects of Janus kinases inhibition ex-vivo on inflammatory responses in colonic compared to ileal mucosa.

Janus kinase (JAK) inhibitors are used for treating inflammatory bowel diseases (IBD). We aimed to identify molecular effects of JAK inhibition in human intestinal mucosa, considering IBD location and phenotype. Colonic and ileal explants from patients with ulcerative colitis (UC), Crohn's disease (CD), and non-IBD controls (NC) were assessed for phosphorylated signal transducers and activators of transcription (p-STAT) levels and Inflammatory genes expression panel in response to ex-vivo JAK inhibitor (tofacitinib). Cytokine production by lamina propria lymphocytes in response to tofacitinib was assessed. Human intestinal organoids were used to investigate JAK inhibitors' effects on iNOS expression. Explants were collected from 68 patients (UC=20; CD=20; NC=28). p-STAT1\3\5 inhibition rates varied, being higher in colonic compared to ileal explants. p-STAT1\3 inhibition rates negatively correlated with CRP levels. While significant alterations in 120 of 255 inflammatory genes were observed in colonic explants, only 30 were observed in ileal NC explants. In colonic explants from UC, significant alterations were observed in 5 genes, including NOS2. JAK inhibition significantly decreased Th1\Th2\Th17-related cytokine production from lamina propria lymphocytes. Various JAK inhibitors reduced IFN-γ-induced increase in iNOS expression in organoids. Site-specific anti-inflammatory effect of JAK inhibition by tofacitinib was noticed, whereby the colon was more robustly affected than the ileum. Ex-vivo response to tofacitinib is individual. JAK inhibition may attenuate inflammation by decreasing iNOS expression. Ex-vivo mucosal platforms may be a valuable resource for studying personalized drug effects in patients with IBD.

Assessment of therapeutic efficacy of adipose tissue-derived mesenchymal stem cells administration in hyperlipidemia-induced aortic atherosclerosis in adult male albino rats

Atherosclerosis (AS) is a common disease seriously detrimental to human health. AS is a chronic progressive disease related to inflammatory reactions. The present study aimed to characterize and evaluate the effects of adipose tissue stem cells (ADSCs) in high-fat diet-induced atherosclerosis in a rat model. The present study comprises thirty-six rats and they were divided into three groups: the control group, the high-fat diet (HFD) group; which received a high-fat diet, and the high-fat diet + stem cells (HFD+SC) group; which was fed with a high-fat diet along with the administration of intravenous ADSCs. Food was given to the animals for 20 weeks to establish dyslipidemia models. After 20 weeks, animals were sacrificed by cervical dislocation; blood was collected to measure total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL); aortae were collected to detect morphologic changes. Rats of the HFD group showed a significant increase in body weight (B.Wt), altered lipid profile increased expression of inducible nitric oxide synthase (iNOS), and decreased expression of endothelial nitric oxide synthase (eNOS). However, in HFD+SC there was a significant decrease in body weight gain and an improvement in lipid profile. Histopathological and ultrastructural variations observed in the aorta of the HFD group when treated with ADSCs showed preserved normal histological architecture and reduced atherosclerosis compared with the HFD group. This was evidenced by laboratory, histological, immunohistochemical, and morphometric studies. Thus, ADSCs reduced TC, TG, and LDL, reduced the expression of iNOS, and increased the expression of eNOS. The high-fat diet was likely to cause damage to the wall of blood vessels. Systemically transplanted ADSCs could home to the aorta, and further protect the aorta from HFD-induced damage.

Biological Properties of Boletus edulis Extract on Caco-2 Cells: Antioxidant, Anticancer, and Anti-Inflammatory Effects.

Boletus edulis (BE) is a mushroom well known for its taste, nutritional value, and medicinal properties. The objective of this work was to study the biological effects of BE extracts on human colon carcinoma cells (Caco-2), evaluating parameters related to oxidative stress and inflammation. In this study, a hydroethanolic extract of BE was obtained by ohmic heating green technology. The obtained BE extracts are mainly composed of sugars (mainly trehalose), phenolic compounds (taxifolin, rutin, and ellagic acid), and minerals (K, P, Mg, Na, Ca, Zn, Se, etc.). The results showed that BE extracts were able to reduce cancer cell proliferation by the induction of cell cycle arrest at the G0/G1 stage, as well as cell death by autophagy and apoptosis, the alteration of mitochondrial membrane potential, and caspase-3 activation. The extracts modified the redox balance of the cell by increasing the ROS levels associated with a decrease in the thioredoxin reductase activity. Similarly, BE extracts attenuated Caco-2 inflammation by reducing both iNOS and COX-2 mRNA expression and COX-2 protein expression. In addition, BE extracts protected the intestine from the oxidative stress induced by H2O2. Therefore, this study provides information on the potential use of BE bioactive compounds as anticancer therapeutic agents and as functional ingredients to prevent oxidative stress in the intestinal barrier.

New sesquineolignan glycoside isomers from the aerial parts of Leonurus japonicus and their absolute configurations

The acid water-soluble fraction of the 95% EtOH extract of Leonurus japonicus exhibited significant anti-inflammatory activity by suppressing the p-ERK/ERK ratio and iNOS expression. A further phytochemical investigation of this acid water-soluble fraction led to the isolation of three previously undescribed sesquineolignan glycosides, leolignosides A–C (1–3), and their planar structures were elucidated using HR-ESI-MS and 1D and 2D NMR. To determine the complicated absolute configuration of sesquineolignan glycosides, coupling constants, NOESY data, empirical rules, enzymatic hydrolysis, and ECD data were comprehensively used. These isolated compounds were also screened for anti-inflammatory activity. Encouragingly, leolignosides A–C all suppressed LPS-induced NO overproduction in RAW 264.7 macrophages. The aglycone (3a) of leolignoside C was chosen for further investigation for its activity and the available amount. Similar to the acid water-soluble fraction, compound 3a inhibited NO overproduction and decreased the p-ERK/ERK and p-NF-κB/NF-κB ratios, indicating that 3a may exert anti-inflammatory effects through the ERK/NF-κB signaling pathway.

Ameliorative effect of nodakenin in combating TNBS-induced ulcerative colitis by suppressing NFƙB-mediated NLRP3 inflammasome pathway.

Ulcerative colitis (UC) is an enduring and complex inflammatory bowel disease that is clinically prevalent, progressive, and debilitating. As of now, the few effective medical treatments for UC have unacceptably high side effects. It is crucial to find safer and more effective UC treatments. Nodakenin possesses anti-inflammatory and antioxidant activity by suppressing several pro-inflammatory mediators. In the present study, we aimed to evaluate the colonoprotective effect of nodakenin in combating colitis through the NFƙB-mediated NLRP3 inflammasome pathway. In mice, UC was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Nodakenin (10, 20, and 40mg/kg) was introduced intragastrically, and disease activity index (DAI) score was calculated. Malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), nitric oxide (NO) levels, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentration were evaluated in colon homogenate. Colon samples were used for histopathological investigation and mRNA expression studies involving nuclear factor kappa B (NFƙB), cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), nucleotide-binding receptor domain 3 (NLRP3), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Nodakenin treatment was found effective in lowering the DAI score, histological score, MPO, MDA, and NO levels while elevating SOD levels as compared to the model control group, showcasing its anti-inflammatory and antioxidant properties. Nodakenin (40mg/kg) significantly downregulated the expression of TNF-α, IL-6, NFƙB (1.24-fold), iNOS (1.2-fold), COX-2 (1.98-fold), NLRP3 (1.78-fold), IL-1β (1.29-fold), and IL-18 (1.17-fold) conferring its great anti-inflammatory potential in combating colitis. Taking together, nodakenin presumably alleviated TNBS-induced colitis by NFƙB-mediated NLRP3 inflammasome pathway and reduced colon damage by downregulating various transcriptional genes and pro-inflammatory mediators.

In vivo anti-gastric ulcer activity of 7-O-methyl aromadendrin and sakuranetin via mitigating inflammatory and oxidative stress trails

Ethnopharmacological relevanceEucalyptus genus has been used for a very long time in conventional treatment as an anti-ulcer remedy. Aim of the studyThe study aimed to explore the gastroprotective potential of 7-O-methyl aromadendrin (7-OMA), and sakuranetin (SKN) in comparison with omeprazole. The study tackled the contribution of their anti-inflammatory, antioxidant, and antiapoptotic capabilities to their anti-gastric ulcer effects. Materials and methodsAn ethanol-induced gastric ulcer model in rats was adopted and the consequences were confirmed by a molecular docking study. ResultsThe oral pretreatment of rats 1 h before ethanol using omeprazole (20 mg/kg) or 7-OMA (20 or 40 mg/kg) or SKN (20 or 40 mg/kg) exhibited gastroprotective and anti-inflammatory properties to different extents. These amendments witnessed as restorations in the stomach histological architecture in H and E-stained sections, mucus content in periodic acid-Schiff (PAS) stained sections with increased cellular proliferation, as demonstrated by increased immunohistochemical staining of PCNA, and increments in stomach COX-1 activity and eNOS. The highest dose of SKN showed the best corrections to reach 4.8, 1.8, and 2.1 folds increase in PAS, COX-1 and eNOS, respectively as compared to the untreated ethanol-induced gastric ulcer group; effects that were comparable to that of omeprazole. Moreover, reductions in COX-2 activity, and the protein expression of NF-κB, IL-6, TNF-α and NOx, in addition to the gene expression of inducible iNOS were also noted. Moreover, the antioxidant and antiapoptotic capabilities of omeprazole, 7-OMA, and SKN were perceived. SKN (40 mg/kg) succeeded to show the unsurpassed results to reach 293.6%, 237.1%, 274.7%, 248.2%, and 175.4% in total and reduced GSH, catalase, SOD, and Bcl2, respectively, as well as 50.0%, 46.8%, and 52.1 % in oxidized GSSG, TBARS and caspase-3, respectively. The gastroprotective potential of the tested compounds can be assigned to their anti-inflammatory, antioxidant and antiapoptotic properties.7-OMA and SKN were studied using molecular docking into the binding sites of the most significant inflammatory targets, including COX-2, TNF-α, iNOS, and NF-κB. Pharmacokinetic and physicochemical parameters in silico were appropriate. ConclusionThe prophylactic use of 7-OMA and SKN could be considered as an add-on to recurrent gastric ulcers and might influence its therapeutic approaches.

Ligand-Based Pharmacophoric Design and Anti-inflammatory Evaluation of Triazole Linked Semisynthetic Labdane Conjugates.

This study employed a ligand-based pharmacophoric approach to design and synthesize 33 novel semisynthetic labdane-appended triazolyl isatins to discover potential anti-inflammatory agents. The anti-inflammatory efficacy of the derivatives was evaluated by their ability to inhibit the production of NO, TNF-α, and IL-6, in lipopolysaccharide-induced RAW264.7 macrophages. The initial screening revealed that compound 7a ((1-(2-(2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl (E)-3-formyl-5-((1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl)pent-3-enoate) exhibited an anti-inflammatory effect (NO inhibition, IC50 = 3.13 μΜ), surpassing both the positive control indomethacin (NO inhibition, IC50 = 7.31 μΜ) and the parent compound labdane dialdehyde. Notably, 7a reduced the levels of pro-inflammatory cytokines TNF-α and IL-6 while increasing the levels of the anti-inflammatory cytokine IL-10. Mechanistic studies revealed that 7a downregulated the expression of COX-2 and iNOS by inhibiting the NF-κB signaling pathway. In silico molecular modeling studies on NF-κB proteins support these findings, suggesting that 7a is a promising candidate for developing into a potent anti-inflammatory clinical agent.

Retraction Note: ICB3E induces iNOS expression by ROS-dependent JNK and ERK activation for apoptosis of leukemic cells.

Retraction Note: ICB3E induces iNOS expression by ROS-dependent JNK and ERK activation for apoptosis of leukemic cells.

Synthesis, characterization, and mechanistic insights into the enhanced anti-inflammatory activity of baicalin butyl ester via the PI3K-AKT pathway.

To investigate the anti-inflammatory activity and mechanism of Baicalin derivative (Baicalin butyl ester, BE). BE was synthesized and identified using UV-Vis spectroscopy, FT-IR spectroscopy, mass spectrometry (MS) and high-performance liquid chromatography (HPLC) methods. Its anti-inflammatory potential was explored by an in vitro inflammation model. Network pharmacology was employed to predict the anti-inflammatory targets of BE, construct protein-protein interaction (PPI) networks, and analysis topological features and KEGG pathway enrichment. Additionally, molecular docking was conducted to evaluate the binding affinity between BE and its core targets. qRT-PCR analysis was conducted to validate the network pharmacology results. The organizational efficiency was further evaluated through octanol-water partition coefficient and transmembrane activity analysis. UV-Vis, FT-IR, MS, and HPLC analyses confirmed the successfully synthesis of BE with a high purity of 93.75%. In vitro anti-inflammatory research showed that BE could more effectively suppress the expression of NO, COX-2, IL-6, IL-1β, and iNOS. Network pharmacology and in vitro experiments validated that BE's anti-inflammatory effects was mediated through the suppression of SRC, HSP90AA1, PIK3CA, JAK2, AKT1, and NF-κB via PI3K-AKT pathway. Molecular docking results revealed that the binding affinities of BA to the core targets were lower than those of BE. The Log p-value of BE (1.7) was markedly higher than that of BA (-0.5). Furthermore, BE accumulated in cells at a level approximately 200 times greater than BA. BE exhibits stronger anti-inflammatory activity relative to BA, possibly attributed to its better lipid solubility and cellular penetration capabilities. The anti-inflammatory mechanism of BE may be mediated through the PI3K-AKT pathway.

Ganospirones A−G, seven undescribed spiro-meroterpenoids from Ganoderma lucidum and their biological activities

Ganoderma lucidum, a medicinal mushroom with a long history in traditional Chinese medicine, is widely used for chronic diseases. Ganospirones A−G (1–7), seven pairs of undescribed spiro-meroterpenoids, were isolated from the fruiting bodies of G. lucidum. Their structures including absolute configurations were characterized by using NMR spectroscopic data, ECD computational and X-ray diffraction methods. The anti-inflammatory and anti-renal fibrosis activities of the meroterpenoids 1–7 were tested, and the results revealed that (−)-2 and (+)-2 could inhibit iNOS expression in lipopolysaccharide-induced RAW264.7 cells at 20 μM.

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.

Hepatoprotective potential of alpha-lipoic acid against gliclazide-induced liver injury in high-glucose-exposed human liver cells and experimental type 2 diabetic rats

Growing clinical evidence shows that sulfonylurea therapy for patients with type 2 diabetic mellitus (T2DM) contributes to progressive worsening of their liver. The present study presents hepatotoxicity induced by gliclazide, a second-generation sulfonylurea, and alpha-lipoic acid (ALA) as a novel and promising drug for T2DM treatment. Normal human liver cells (HL-7702) were incubated with high-glucose DMEM in the presence or absence of gliclazide and ALA for 72 h, and cell viability and death were measured by flow cytometry. Next, Sprague-Dawley rats were subjected to 12 h of fasting, and fasting blood glucose was measured. The rats were randomized into four groups: HC (healthy control; n = 7), T2DM (diabetic rats without treatment; n = 9), GLC (diabetic rats with 15 mg/kg gliclazide treatment; n = 7) and GLC+ALA (diabetic rats with gliclazide and 60 mg/kg ALA treatment; n = 7). T2DM was induced by a bolus administration of 110 mg/kg nicotinamide and 55 mg/kg streptozotocin intraperitoneally. The experimental protocol lasted for 6 weeks after which the animals were sacrificed and pancreas, liver and blood samples were collected for biochemical, histological and molecular analyses. Compared to healthy control (HC) group, exposure of HL-7702 cells to high glucose induced significant cell death by 19 % (p < 0.001), which was exacerbated with gliclazide treatment by 29 % (p < 0.0001) but markedly reduced by 6 % to near HC value following ALA treatment. In vivo, GLC-treated rats had severe liver damage characterized by increased hepatocellular vacuolation, and significant expression of ED-1, iNOS and caspase-3 as well as markedly high levels of liver enzymes (aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase compared to T2DM rats. Interestingly, ALA administration prevented these pathological changes and protected the diabetic liver to levels comparable to HC rats. ALA showed hepatoprotective effect against gliclazide-induced hepatotoxicity by suppressing inflammation and apoptosis while activating antioxidant pathway in the diabetic liver.Abbreviations: ALA, Alpha-lipoic acid; ALT, Alanine aminotransferase; ALP, Alkaline phosphatase; AMPK, Adenosine monophosphate-activated protein kinase; AST, Aspartate aminotransferase; ATP, Adenosine triphosphate; DMEM, Dulbecco’s Modified Eagle Medium; EDTA, ethylenediaminetetraacetic acid; FBG, Fasting blood glucose; FBS, Fetal bovine serum; GLC, Gliclazide; GLUT4, Glucose transporter type 4; GSH, Glutathione; H&E, Hematoxylin/Eosin; HbA1c, Glycosylated haemoglobin A1c; HC, Healthy control; HG, Hyperglycemic group; HOMA-β, Homeostasis model assessment of β-cell function; IL-1β, Interleukin-1β; IL-6, Interleukin-6; iNOS, Inducible nitric oxide synthase; KATP, ATP-dependent potassium channels; MDA, Malondialdehyde; MPTP, Mitochondrial permeability transition pore; NO, Nitric oxide; P/S, Penicillin/streptomycin; PAS, Periodic acid-Schiff; RIA, Radioimmunoassay; ROS, Reactive oxygen species; SOD, Superoxide dismutase; T2DM, Type 2 diabetes mellitus; TBARS, Thiobarbituric acid reactive substances; TNF-α, Tumor necrosis factor-alpha.

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.