TNF-α Protein Research Articles

Free Electron Density Gradients Enhanced Biosensor for Ultrasensitive and Accurate Affinity Assessment of the Immunotherapy Drugs.

Accurate affinity assessments play an important role in drug discovery, screening, and efficacy evaluation. Label-free affinity biosensors are recognized as a dependable and standard technology for addressing this challenge. This study constructs a free electron density gradient-enhanced meta-surface plasmon resonance (FED-MSPR) biosensor through a finite-difference time-domain simulation model, the biosensor demonstrates superior detection performance in accurately determining affinity and kinetic rate constants. By controlling the dielectric properties of the metal on the surface of the nanocup arrays, the plasmon resonance effects are easily tuned without changing the nanostructure design. Compared with the single-layer gold chip, the triple-layer FED-MSPR chip demonstrated a four-fold improvement in resolution at the optimal resonance peak. Additionally, the sensitivity and figure of merit (FOM) of the multi-layer chip increased by 3.5 and 7.99 times, respectively. Following modification with high- and low-staggered carboxylation, the noise-signal ratio and baseline stability of the real-time kinetic curves based on these chips are significantly enhanced. The developed carboxylation FED-MSPR platform is successfully used to perform affinity assays for Adalimumab and TNF-α protein, resulting in favorable dynamic curves. These findings validate the proposed FED-MSPR biosensor platform as cost-effective, rapid, sensitive, and label-free, facilitating real-time quality control in drug development.

Magnolol Inhibits High Fructose-Induced Podocyte Inflammation via Downregulation of TKFC/Sp1/HDAC4/Notch1 Activation

Background/Objectives: High fructose has been implicated as an important trigger of kidney inflammation in patients and experimental models. Magnolol, isolated from Magnolia officinalis, has an anti-inflammatory effect, but its protective role in podocytes remains underexplored. This study explored the protective effects and underlying mechanism of magnolol against high fructose-induced podocyte inflammation. Methods: The effects of magnolol on high fructose-induced podocyte inflammation were assessed in male Sprague Dawley rats administered 10% (w/v) fructose water for 12 weeks and heat-sensitive human podocyte cell lines (HPCs) exposed to 5 mM fructose. Podocyte foot processes were examined using transmission electron microscopy. The expression levels of nephrin, podocin, tumor necrosis factor-α (TNF-α), Notch1 intracellular domain (NICD1), triokinase/FMN cyclase (TKFC), specificity protein 1 (Sp1) and histone deacetylase 4 (HDAC4) were determined by Western blot, immunofluorescence and real-time quantitative polymerase chain reaction (qRT-PCR). The chromatin immunoprecipitation (ChIP) assay was performed to evaluate the interaction between Sp1 and the promoter region of HDAC4. Results: Magnolol mitigated the impairment of glomerular filtration function in high fructose-fed rats. Besides, it significantly alleviated the inflammatory responses in glomeruli and HPCs, evidenced by decreased protein levels of TNF-α and NICD1. Increased protein levels of TKFC, Sp1 and HDAC4 were observed in high fructose-stimulated HPCs and rat glomeruli. TMP195, an HDAC4 inhibitor, reduced TNF-α and NICD1 protein levels in high fructose-exposed HPCs. The increased Sp1 was shown to associate with the promoter region of HDAC4, promoting HDAC4 protein expression in high fructose-exposed HPCs. The knockdown of TKFC in HPCs by TKFC siRNA decreased Sp1, HDAC4 and NICD1 protein levels, alleviating podocyte inflammatory response. Furthermore, magnolol inhibited TKFC/Sp1/HDAC4/Notch1 activation in vivo and in vitro. Conclusions: Magnolol attenuated high fructose-induced podocyte inflammation possibly through the suppression of TKFC/Sp1/HDAC4/Notch1 activation, providing new evidence for its potential role in podocyte protection.

Histidine-trytophan-ketoglutarate cardioplegia reduces inflammatory response and serum levels of myocardial enzymes in newly developed right-thoracotomy rat model

The objective of this research was to establish a rat model for cardiopulmonary bypass (CPB) with cardiac arrest and resuscitation that is both practical and economical and simulates clinical cardiac surgery. Concurrently, the study aimed to evaluate the myocardial protective effects conferred by histidine–tryptophan–ketoglutarate (HTK) cardioplegia. Thirty rats were randomly assigned to three groups: the histidine–tryptophan–ketoglutarate (HTK), 4:1 blood cardioplegia (BC) and del Nido cardioplegia (DN) groups. The cardiopulmonary bypass (CPB) procedure was implemented and sustained for a duration of one hour. Subsequent to the cessation of CPB, the rats were subjected to monitoring and observation for an additional two hours. Following this observation period, the heart and blood samples were procured for subsequent analysis. During CPB, the average hematocrit level was significantly below the typical physiological range (P < 0.001). Histopathological scores were notably lower in the HTK group in contrast to the BC group (P < 0.001) or the DN group (P < 0.001). At 2 h after weaning off CPB, the levels of CK and CKMB in the DN and BC groups were notably elevated compared to the HTK group (P < 0.001). The levels of IL-6 and TNF-α proteins were notably increased in all three groups (P < 0.001), with the BC and DN groups showing higher increases compared to the HTK group (P < 0.001). This compact animal model of cardiopulmonary bypass (CPB) with cardiac arrest and resuscitation might allow for both the study of myocardial ischemia-reperfusion injury as well as cardioprotective strategies. HTK cardioplegia could reduce inflammatory response and serum levels of myocardial enzymes in this newly developed right thoracotomy rat model.

Diacerein ameliorates amiodarone-induced pulmonary fibrosis via targeting the TGFβ1/α-SMA/Smad3 pathway.

This study is aimed at investigating the possible protective effect of diacerein (DIA) against AMD-induced pulmonary fibrosis in rats. Rats were classified into 4 groups: a normal group that received distilled water, control group that received AMD (100 mg/kg, p.o.) for 21 days to induce pulmonary fibrosis, and 2 treatment groups that received diacerein, in 2 dose levels (50 and 100 mg/kg, p.o., respectively) in addition to AMD (100 mg/kg, p.o.), for 21 days. Lung function test was assessed using a spirometer; serum and tissue were collected. Biochemical, real-time PCR, histopathological, and immunohistopathological analyses were carried out. AMD reduced tidal volume (TV), peripheral expiratory rate (PER), forced vital capacity (FVC), serum reduced glutathione (GSH) levels, Beclin, and LCII, while it elevated transform growth factor (TGF-β1) gene expression, serum malondialdehyde (MDA) level, alpha-smooth muscle actin (α-SMA), Smad3, phosphorylated signal transducer and activator of transcription (p-STAT3), and p62 lung content. Also, AMD elevated tumor necrosis factor-alpha (TNF-α) and caspase-3 protein expression. DIA elevated TV, PER, FVC, serum GSH level, Beclin, and LCII, while it reduced TGF-β1 gene expression, serum MDA level, α-SMA, Smad3, p-STAT-3, and p62 lung content. Moreover, DIA reduced TNF-α and caspase-3 protein expression. DIA attenuated AMD-induced pulmonary fibrosis via alleviating the TGF1/α-SMA/Smad3 pathway, reducing STAT-3 activation, and combating oxidative stress and inflammation in addition to promoting autophagy and abrogating apoptosis.

The mechanism of wen jing tang in the treatment of endometriosis: Insights from network pharmacology and experimental validation

BackgroundEndometriosis (EM) is a hormone-dependent condition marked by progressively severe secondary dysmenorrhea, significantly impacting patients' quality of life and overall health. Wen Jing Tang (WJT), a traditional Chinese medicinal formulation derived from the Synopsis of the Golden Chamber, has proven to be an effective therapeutic agent for EM. However, the precise molecular mechanisms underlying its efficacy remain unclear. ObjectiveThis study aims to elucidate the underlying mechanisms of WJT in the treatment of EM by integrating network pharmacology analysis with experimental validation. MethodsThe chemical constituents and target sites of WJT were obtained from the TCMSP database, while EM-related target genes were sourced from OMIM, TTD, GeneCards, and the DrugBank databases. A "herbs-components-targets" network was constructed using Cytoscape 3.9.1. The intersecting target genes of WJT and EM were then uploaded to the STRING database for protein-protein interaction (PPI) analysis. Subsequently, the common target genes were subjected to GO and KEGG enrichment analysis via the DAVID database. Molecular docking were employed to analyze the binding affinities between the top five core components and their respective targets. Additionally, ELISA were used to quantify the serum levels of IL-6, IL-1β, E2, and P in EM model rats. The expression levels of TNF-α, HIF1A, STAT3, and EGFR mRNA and proteins in ectopic endometrial tissue were assessed using q-PCR and Western blotting. ResultsA total of 250 chemical components and 553 targets were identified in WJT, while 3491 EM-related targets were screened from multiple databases. Among these, 187 common targets between WJT and EM were found, with quercetin, kaempferol, and beta-sitosterol emerging as the core chemical components, and AKT1, IL6, TNF, and IL1B identified as the key targets. These core components demonstrated strong binding affinities to the targets. GO and KEGG enrichment analyses revealed that the shared targets were primarily involved in the HIF1 signaling pathway. Furthermore, compared to the control group, the EM model rats exhibited an increased ectopic endometrial area, disordered glandular and stromal cells, and notable inflammatory infiltration. Serum levels of IL-6, IL-1β, E2, and P were significantly elevated (P < 0.01), and the expression of TNF-α, HIF1A, STAT3, and EGFR in the ectopic endometrium was markedly increased (P < 0.01). Following WJT intervention, the ectopic endometrial area in model rats was reduced, the morphology and structure of the endometrial cells showed improvement, and serum levels of IL-6, IL-1β, E2, and P were significantly decreased (P < 0.05). WJT also inhibited the expression of HIF1 pathway-related proteins TNF-α, HIF1A, STAT3, and EGFR (P < 0.05). ConclusionThe mechanism by which WJT prevents and treats EM may involve the reduction of inflammation through the inhibition of the HIF1 signaling pathway.

MTORC2 inhibition by JR-AB2-011 improves IL-1β-induced inflammation, catabolic response, and apoptosis in human chondrocytes through IκB-α/NF-κB p65.

Osteoarthritis (OA) is a very common chronic joint condition marked by inflammation and cartilage loss. mTOR is a well-known mediator of inflammation, cell survival, and aging; however, its role in OA has not been determined. To explore the role of mTORC2 in OA-and associated pathological changes, we examined the contribution of mTORC2-mediated Akt, rictor and IκB-α/NF-κB p65 pathway in interleukin (IL)-1β-treated human chondrocytes. We focused on the protein expression of proinflammatory cytokines and catabolic and apoptotic factors, including TNF-α, IL-6, iNOS, MMP13, Bax, and caspase3, which may occur through this signalling pathway in IL-1β-treated chondrocytes. Chondrocytes were cultured and treated with either 2 ng/mL IL‑1β alone or in combination with increasing concentrations of JR-AB2-011 (50, 100, or 250 µM), a selective mTORC2 inhibitor. The protein levels of phosphorylated (p)‑Akt, Akt, rictor, p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, iNOS, MMP13, Bax, and caspase3 were evaluated by Western blotting. In IL-1β-stimulated chondrocytes, mTORC2 activity was increased with increased phosphorylation of Akt and expression of rictor. IL-1β increased the expression of p-IκBα, p-NF-κB p65, NF-κB p65, IL-6, TNF-α, iNOS, Bax, and caspase3 proteins and decreased the expression of IκB-α. All of these IL-1β-induced alterations were prevented by JR-AB2-011. The main novel finding in the present study is that selective mTORC2 inhibition by JR-AB2-011 prevents the inflammatory, catabolic, and apoptotic responses induced by IL-1β via modulation of IκB-α/NF-κB activity. Therefore, we demonstrated a previously unknown function of mTORC2 inhibition that seems to be a potential therapeutic target for OA.

Chromone components of Saposhnikovia divaricate attenuate rheumatoid arthritis development by inhibiting the inflammatory response

Ethnopharmacological relevanceSaposhnikovia divaricata (Turcz.) Schisck., a traditional Chinese medicine (TCM), has historically been utilized in the clinical treatment of RA. It was initially documented in the 'Shennong Ben Cao Jing' as a superior quality, with the text stating: 'The herb is widely renowned for its efficacy in alleviating whole-body discomfort, bone pain, malaise, and promoting long-lasting vitality. Chromones (CHR) were identified as the primary active components in Saposhnikovia divaricata. However, the specific molecular mechanisms by which CHR impacts RA remain incompletely understood. Aim of the studyTo explore the therapeutic efficacy of CHR, a class of compound derived from Saposhnikovia divaricata, in alleviating arthropathy and immune hyperactivity in a collagen-induced arthritis (CIA) mouse model. Material and methodsSurface plasmon resonance (SPR) molecular fishing and UHPLC-QTOF/MS technology were used to identify CHR in Saposhnikovia divaricata as an active ingredient for treating RA. A CIA mouse model was used to verify the anti-RA effect of CHR in vivo. The anti-RA efficacy of CHR in vivo was evaluated by body weight change, joint swelling, arthritis index, immune organ index, ankle joint disease, and immunoglobulin G (IgG) content. The mechanism of improving RA was further analyzed by a protein chip assay and verified by Western blotting. ResultsCHR treatment reduced swelling, arthritis index, and IgG, IgG1, IgG2a, and IgG2b levels in CIA mice. Protein microarray indicated that CHR mitigated CIA-induced joint inflammation by inhibiting immune cell activation, reducing the expression of inflammatory factors and chemokines, potentially by modulating the rheumatoid arthritis pathway involving tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), and chemokines. This hypothesis was supported by the upregulation of bone morphogenetic proteins 3 (BMP3) and phospho-Smad2 (p-Smad2) proteins, coupled with the downregulation of interleukin-6 (IL-6), interleukin-1β (IL-1β), TNF-α, and IL-17A proteins in the joints of CHR-treated mice. ConclusionCHR shows promise as a potential therapeutic agent for RA, exerting its effects through anti-inflammatory mechanisms.

Clarifying therapeutic mechanisms of Guanxinshutong capsule for cardiac fibrosis treatment by network pharmacology, molecular docking, molecular dynamics simulation, and in vivo experiments

PurposeGuanxinshutong capsule (GXST) is a Chinese patent medicine used for the treatment of cardiac fibrosis (CF). Nevertheless, the precise mechanism of action of GXST remains unclear. In this study, we applied network pharmacology, molecular docking, molecular dynamics simulation, and in vivo experiments to assess the effects and mechanisms of GXST in the treatment of CF. MethodsThe TCMSP database provided the GXST targets and active substances. The GeneCards, OMIM, and TTD databases were searched for targets relevant to cardiac fibrosis. To filter core targets, a PPI network was set up. Metascape was utilized to conduct enrichment analysis for the KEGG and GO pathways. To evaluate the interaction between putative targets and active drugs, molecular docking was utilized. MDS was performed for the optimal complex of the core ligand protein identified through molecular docking. Induction of cardiac fibrosis in SD rats using isoproterenol, and the protective effects of GXST on the heart were evaluated using echocardiography, H&E staining, and Masson staining. ResultsThrough network pharmacology analysis, we have identified 83 active components and 73 key target proteins related to cardiac fibrosis from GXST. Subsequent molecular docking and molecular dynamics simulation results suggest that quercetin, luteolin, and kaempferol can inhibit cardiac fibrosis by regulating the expression of TNF-α, IL6, and IL1B proteins. These compounds and proteins may be key factors in the treatment of CF with GXST. Further, in vivo experiments demonstrate that GXST can alleviate myocardial injury and improve cardiac function in CF rats. ConclusionThis study successfully predicted the effective components, potential targets, and related pathways involved in treating CF by GXST. GXST can improve cardiac function and alleviate pathological damage in the hearts of SD rats. This provides a new strategy for future investigations into the mechanisms of GXST in the treatment of CF.

Exploration of Pharmacological Mechanism of Cinnamomum tamala Essential Oil in Treating Inflammation based on Network Pharmacology, Molecular Modelling, and Experimental Validation.

Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm., also known as Indian Bay leaf, holds a distinctive position in complementary and alternative medicinal systems due to its anti-inflammatory properties. However, the active constituents and key molecular targets by which C. tamala essential oil (CTEO) exerts its anti-inflammatory action remain unclear. The present study used network pharmacology and experimental validation to investigate the mechanism of CTEO in the treatment of inflammation. GC-MS analysis was used to identify the constituents of CTEO. The key constituents and core targets of CTEO against inflammation were obtained by network pharmacology. The binding mechanism between the active compounds and inflammatory genes was ascertained by molecular docking and molecular dynamics simulation analysis. The pharmacological mechanism predicted by network pharmacology was verified in lipopolysaccharide-stimulated murine macrophage (RAW 264.7) cell lines. Forty-nine constituents were identified by GC-MS analysis, with 44 constituents being drug-like candidates. A total of 549 compounds and 213 inflammation-related genes were obtained, revealing 68 overlapping genes between them. Compound target network analysis revealed cinnamaldehyde as the core bioactive compound with the highest degree score. PPI network analysis demonstrated Il-1β, TNF-α, IL8, IL6 and TLR4 as key hub anti-inflammatory targets. KEGG enrichment analysis revealed a Toll-like receptor signalling pathway as the principally regulated pathway associated with inflammation. A molecular docking study showed that cinnamaldehyde strongly interacted with the Il-1β, TNF-α and TLR-4 proteins. Molecular dynamics simulations and MMPBSA analysis revealed that these complexes are stable without much deviation and have better free energy values. In cellular experiments, CTEO showed no cytotoxic effects on RAW 264.7 murine macrophages. The cells treated with LPS exhibited significant reductions in NO, PGE2, IL-6, TNF-α, and IL-1β levels following treatment with CTEO. Additionally, CTEO treatment reduced the ROS levels and increased the antioxidant enzymes such as SOD, GSH, GPx and CAT. Immunofluorescence analysis revealed that CTEO inhibited LPS-stimulated NF-κB nuclear translocation. The mRNA expression of TLR4, MyD88 and TRAF6 in the CTEO group decreased significantly compared to the LPS-treated group. The current findings suggest that CTEO attenuates inflammation by regulating TLR4/MyD88/NF- κB signalling pathway.

BML-111 Modulates and Alleviates p38/MAPK Signaling Pathway and Th1/Th2/Th17 Cytokine Response in Murine Psoriasis-Like Dermatitis.

Psoriasis is a prevalent cutaneous inflammatory disorder characterized by elevated keratinocyte inflammation. 5(S)-6(R)-7-trihydroxyheptanoic-acid-methyl-ester (BML-111), an established analogue of lipoxin A4, is known for its potent anti-inflammatory properties. However, the precise role of BML-111 within a murine psoriasis-like dermatitis model requires further clarification. This research aims to investigate the modulatory effects of BML-111 on inflammatory responses, the p38/mitogen-activated protein kinase (MAPK) signaling cascade, and T helper type 1 (Th1), Th2, and Th17 cell responses within the context of a murine psoriasis-like dermatitis model. A psoriasis-like dermatitis model was established by applying 5% imiquimod (IMQ) cream to the backs of C57BL/6 mice, which were pretreated intraperitoneally with or without BML-111 prior to IMQ application. Hematoxylin-eosin staining was utilized to detect the pathological alterations of the murine dorsal skin tissue. Furthermore, the psoriasis area and severity index (PASI) scoring system was used to assess the dynamic cutaneous alterations in the mice. The levels of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin (IL)-1β, IL-6, IL-4, and IL-17A in the murine serum samples were quantified by means of enzyme-linked immunosorbent assays (ELISA). Western blotting was conducted to detect the proteins of TNF-α, IL-1β, IL-6, phospho-p38 (p-p38), and p38 in murine skin tissues. Lastly, a flow cytometry analysis was executed to evaluate the expression of peripheral blood Th1/Th2/Th17 cell subsets. BML-111 attenuated IMQ-induced pathological changes in skin tissue of psoriasis-like dermatitis mice. BML-111 treatment substantially reduced TNF-α, IL-1β, IL-6, IFN-γ and IL-17A levels and elevated IL-4 levels in serum and skin lesion tissues of IMQ-induced mice (p < 0.01, p < 0.01, p < 0.01, p < 0.05, p < 0.05, p < 0.05, respectively). The ratio of Th1/Th17 cells in the peripheral blood of BML-111-treated mice was substantially diminished and the ratio of Th2 cells was substantially augmented (p < 0.05, p < 0.01, p < 0.001, respectively). Mechanistically, p-p38 protein level was substantially reduced in the skin tissues of BML-111-treated mice (p < 0.05). While, dehydrocorydaline (DHC, a p38/MAPK pathway agonists) reversed the reduction of p-p38 protein level induced by BML-111 treatment in psoriasis-like mice (p < 0.05). BML-111 modulates the p38/MAPK signaling pathway and Th1/Th2/Th17 cytokine response, and alleviates psoriasis-like dermatitis in mice.

Berberine Inhibits the Inflammatory Response Induced by Staphylococcus aureus Isolated from Atopic Eczema Patients via the TNF-α/Inflammation/RAGE Pathways.

Atopic eczema patients exhibit high levels of Staphylococcus aureus (S. aureus) skin colonization. S. aureus can stimulate macrophages and the expression of proinflammatory cytokines. Berberine (BBR), an alkaloid, attenuates S. aureus toxin production. This study investigated if BBR suppressed bacterial growth and inflammatory response induced by eczema-patient-derived S. aureus using murine macrophage (RAW 264.7) and human monocyte cell lines (U937). RAW 264.7 and U937 were treated with BBR at different concentrations and stimulated with heat-killed S. aureus (ATCC #33591) or S. aureus derived from severe eczema patients (EC01-EC10), who were undergoing topical steroid withdrawal, for 24 h. TNF-α protein levels were determined by ELISA, gene expression by qRT-PCR, cell cytotoxicity by trypan blue excursion, and reactive oxygen species (ROS) levels by fluorometric assay. BBR showed a bacteriostatic effect in S. aureus (ATCC strain #33591 and clinical isolates (EC01-EC10) and suppressed TNF-α production in RAW 264.7 and U937 cells exposed to heat-killed S. aureus (ATCC and clinical isolates) dose-dependently without any cell cytotoxicity. BBR (20 µg/mL) suppressed >90% of TNF-α production (p < 0.001), downregulated genes involved in inflammatory pathways, and inhibited S. aureus ROS production in U937 and RAW 264.7 cells (p < 0.01). BBR suppresses S. aureus-induced inflammation via inhibition of TNF-α release, ROS production, and expression of key genes involved in the inflammatory pathway.

Extract of Angelica keiskei Leaves Attenuates Spatial Memory Impairment on the D-galactose Model of Brain Aging in Mice

Aging is a complex and inexorable phenomenon, entailing several physiological changes. Impaired memory skills are associated with cell death in the hippocampus as a result of the age-related buildup of free radicals. D-galactose can artificially accelerate brain aging, causing memory impairment in mice due to neuroinflammation and oxidative stress. Numerous phytoconstituents found in Angelica keiskei possess anti-oxidant, anti-inflammatory, and memory-enhancing properties. In this study, we sought to determine the effects of an ethanol extract from the leaves of A. keiskei (EELAK) on spatial memory in mice with impaired memory functions due to D-gal. Here, we showed that administering 300 mg/kg BW/day of D-gal orally for a duration of 28 days significantly decreased spatial memory as quantified by the Morris Water Maze, and this was linked to a marked increase in hippocampal acetylcholinesterase (AChE), inflammation, and oxidative stress. The spatial memory of D-gal-induced mice was markedly enhanced by 20 mg/kg BW/day of EELAK, demonstrating its potent memory-boosting properties. The mice treated with EELAK also showed a notable decrease in hippocampal neuroinflammation (p65 NF-kB, NO, and TNF-α protein) and an increase in antioxidant activity (elevated SOD activity and reduced MDA levels), suggesting its potent neuroprotective activity. In conclusion, our results establish for the first time that by reducing oxidative stress and neuroinflammation, EELAK enhances spatial memory. Hence, for the prevention and treatment of age-related neurodegenerative illnesses like Alzheimer's disease, EELAK may be a useful therapeutic approach.

The Effect of Metformin Therapy on Serum IL-33 and ST-2 Receptor Levels in Patients With Type 2 Diabetes Mellitus

Introduction: Patients with type 2 had elevated levels of inflammatory biomarkers, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF-α). Significant elevations in serum levels of TNF-α and C-reactive protein were seen in individuals with type 2 diabetes mellitus (T2DM). The IL-1 superfamily (IL-1β, IL-18, and IL-33) plays a crucial role in the development of diabetes mellitus by modifying immunological and inflammatory immune responses. Objectives: Little is known about the role of metformin in type 2 diabetes mellitus with respect to serum levels of IL-33 and ST-2 receptors. Therefore, in the present study, we sought toexplore such role. Patients and Methods: This single arm study included 45 type 2 diabetes patients with an age range of 30 to less than 60 years. Patients were given metformin (500 mg orally; for two months then, serum level of interleukin- 33 (IL-33) and ST-2 receptors were evaluated. Results: Treatment resulted in significant reduction of serum the suppression of tumorigenicity suppressor 2 (ST2) from 0.71 ±0.157 µg/mlto 0.59 ±0.11 µg/ml (p&lt;0.001), across with significant increase in serum IL-33 from 0.60 ±0.18 pg/ml to 0.75 ±0.13 pg/ml (p &lt; 0.001). Conclusion: Treatment of type 2 diabetes mellitus using metformin revealed a crucial role for IL-33 and ST-2 receptors in glycemic control since increment in IL-33 and reduction in ST-2 receptors were associated with good glycemic control suggesting an inflammatory role in disease pathogenesis and response to treatment.

Potential of CME@ZIF-8 MOF Nanoformulation: Smart Delivery of Silymarin for Enhanced Performance and Mechanism in Albino Rats.

Silymarin, an antioxidant, is locally used for kidney and heart ailments. However, its limited water solubility and less oral bioavailability limit its therapeutic efficiency. The present study investigated the enhancement of solubility and bioavailability of silymarin by loading it in Cordia myxa plant extract-coated zeolitic imidazole framework (CME@ZIF-8) against carbon tetrachloride (CCl4)-induced nephrotoxicity and cardiac toxicity in albino rats. The synthesized PEG-coated silymarin drug-loaded CME@ZIF-8 MOFs (PEG-Sily@CME@ZIF-8) were characterized by scanning electron microscopy, transmission electron microscopy, high-resolution transmission electron microscopy, energy dispersive X-ray spectroscopy, UV-visible spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, and zeta potential. The average crystal size of CME@ZIF-8 and PEG-Sily@CME@ZIF-8 was 12.69 and 16.81 nm, respectively. The silymarin drug loading percentage in PEG-Sily@CME@ZIF-8 was 33.05% (w/w). In the animal model with CCl4 treatment, different parameters like serum profile, enzymatic level, genotoxicity, and histopathology were assessed. Treatment with PEG-Sily@CME@ZIF-8 with different doses of 500, 1000, and 1500 μg/kg body weight efficiently ameliorated the alterations in the antioxidant defenses, biochemical parameters, and histopathological alterations and DNA damage in comparison to silymarin drug in a CCl4-induced toxicity rat model via alleviating the cellular abnormalities and attenuation of normal antioxidant enzymes levels. Moreover, the molecular mechanism of drug-silymarin interaction with the target protein was investigated. It involves the binding pockets of silymarin molecules with VEGFR, TNF-α, NLRP3, AT1R, NOX1, RIPK1, Caspase-3, CHOP, and MMP-9 proteins, elucidating the silymarin-protein interactions by the formation of hydrogen bonds and hydrophobic interactions. This study suggests that the nanodrug PEG-Sily@CME@ZIF-8 MOFs protect the kidneys and heart possibly by mitigating oxidative stress more efficiently than the conventional drug silymarin.

IL-37 Inhibits Inflammation of Lacrimal Gland in Dry Eye Mice via the IL-37-PTEN-NFκB Signaling Pathway

ABSTRACT Purpose This study aims to investigate the role of Interleukin-37 (IL-37) in mouse models of dry eye. Methods Two murine models of dry eye were employed in this investigation. The evaluation of the anti-inflammatory impact of IL-37 (200 μl, 10 μg/ml) on dry eye mice involved intraperitoneal injections administered once daily for 7 days. Additionally, intraperitoneal injection of VO-Ohpic trihydrate (VO, 0.25 mg/kg) in dry eye mice was performed to investigate the role of PTEN in the IL-37 anti-inflammatory signaling pathway. Tear production was assessed using phenol red cotton thread, while corneal damage was examined through sodium fluorescein staining using a slit lamp. Histological alterations in the lacrimal gland were observed through H&E staining. PAS staining was used to assess conjunctival goblet cells. The levels of NFκB-P65, p-NFκB-P65, IL-1β, IL-6, TNF-α, CD3, AQP5, α-SMA and PTEN proteins were determined via Western blotting or immunofluorescence. Results Following IL-37 treatment, both dry eye models exhibited reduced corneal fluorescence staining scores and enhanced tear production. In lacrimal gland, the expression of p-NFκB-P65, IL-1β, IL-6, CD3 and TNF-α was diminished, while PTEN, AQP5, α-SMA expression increased after IL-37 treatment in both dry eye mice. However, the intraperitoneal injection of VO significantly attenuated the anti-inflammatory effect of IL-37 on dry eye mice. Conclusion IL-37 emerges as an anti-inflammatory mediator within the lacrimal gland of dry eye mice, exerting its effects through the IL-37-PTEN-NFκB signaling pathway.

Cerebral Ischemia-Reperfusion Induced Neuronal Damage, Inflammation, miR-374a-5p, MAPK6, NLRP3, and Smad6 Alterations: Rescue Effect of N-acetylcysteine

Background: Ischemic stroke (IS) is still a major cause of neurological disability. This study aimed to ascertain potential markers closely related to IS diagnosis and treatment and then we examined the neuroprotective effect of N-acetylcysteine (NAC) in a transient cerebral ischemia. Methods: Male Wistar rats were randomly divided into three groups (n=6), including sham, IR (ischemia-reperfusion), IR+NAC (150 mg/kg, ip; intraperitoneally, 1 hour prior to ischemia and 5 min before reperfusion). The infarct volume was evaluated by 2,3,5-triphenyl tetrazolium chloride staining. H&amp;E and Nissle staining were performed to evaluate cerebral ischemia-reperfusion injury. miR-374a-5p gene expression, MAPK6, NLRP3, smad6, TNF-α, and IL-1β protein levels were determined by Real-time PCR, Western blot, and Elisa in the cerebral cortex exposed to IR. Results: Herein, we found that IR increased infarct volume and pathological damage to the cerebral cortex after global cerebral artery occlusion/reperfusion. In addition, miR-374a-5p gene expression decreased, while MAPK6, NLRP3, and smad6 protein expressions increased in the IR group. TNF-α and IL-1β protein levels increased in the ischemic cortex. Treatment with NAC significantly attenuated infarct size, inflammation and reversed aforementioned molecule levels. Conclusion: Taken together, these results suggested that ischemic insult can increase infarct size, neuronal damage, and inflammation may in part by modulating miR-374a-5p, MAPK6, NLRP3, and smad6 pathway in the brain cortex after cerebral IR insult and providing new clues to molecular mechanisms and treatment targets in IS. It can be alleviated by NAC as a potential therapy for someone afflicted with ischemia.

Uncovering the mechanisms of diosmin in treating obesity-related kidney injury based on network pharmacology, molecular docking, and in vitro validation.

Obesity increases the risk of kidney injury, involving various pathological events such as inflammation, insulin resistance, lipid metabolism disorders, and hemodynamic changes, making it a significant risk factor for the development and progression of chronic kidney disease. Diosmin, a natural flavonoid glycoside, exhibits anti-inflammatory, antioxidant, anti-lipid, and vasodilatory effects. However, whether diosmin has a protective effect on obesity-related kidney injury remains unclear. The molecular formula of diosmin was obtained, and diosmin and target genes related to obesity-related kidney injury were screened. The interaction between overlapping target genes was analyzed. GO functional enrichment and KEGG pathway enrichment analyses were performed on overlapping target genes. Molecular docking was employed to assess the binding strength between overlapping target genes. Palmitic acid-induced damage to HK-2 cells, which were then treated with diosmin. Subsequently, the expression levels of relevant mRNAs and proteins were measured. Network analysis identified 219 potential diosmin target genes, 6800 potential target genes related to obesity-related kidney injury, and 93 potential overlapping target genes. Protein-protein interaction networks and molecular docking results revealed that AKT1, TNF-α, SRC, EGFR, ESR1, CASP3, MMP9, PPAR-γ, GSK-3β, and MMP2 were identified as key therapeutic targets, and they exhibited stable binding with diosmin. GO analysis indicated that these key targets may participate in inflammation, chemical stress, and protein phosphorylation. KEGG revealed that pathways in cancer, AGE-RAGE signaling pathway, PI3K-AKT signaling pathway, PPAR signaling pathway, and insulin resistance as crucial in treating obesity-related kidney injury. CCK-8 assay showed that diosmin significantly restored the viability of HK-2 cells affected by palmitic acid. Oil Red O staining demonstrated that diosmin significantly improved lipid deposition in HK-2 cells induced by palmitic acid. PCR results showed that diosmin inhibited the mRNA levels of AKT1, TNF-α, EGFR, ESR1, CASP3, MMP9, GSK-3β, and MMP2 while promoting the mRNA level of PPAR-γ. Western blot analysis revealed that diosmin restored PPAR-γ protein expression, inhibited NF-kB p-p65 protein expression, and reduced TNF-α protein expression. Diosmin demonstrated multi-target and multi-pathway effects in the treatment of obesity-associated renal injury, with key targets including AKT1, TNF-α, EGFR, ESR1, CASP3, MMP9, PPAR-γ, GSK-3β, and MMP2. The mechanism may be through the modulation of the PPAR-γ/NF-κB signaling pathway, which can attenuate inflammatory responses and protect the kidney.

Anti-inflammatory Effect of Novel 2-Phenylphthalazin-2-ium Bromides on LPS-induced RAW264.7 Cells and their Mechanism

Background: Inspired by natural anti-inflammatory quaternary benzo[C]phenanthridine alkaloids, novel 2-phenylphthalazin-2-ium bromides were previously designed and synthesized. Objective: The anti-inflammatory effect of 2-phenylphthalazin-2-ium bromides was evaluated based on inflammatory cytokines, and their possible mechanism was explored through the NF-κB, TLR4 and MAPK signaling pathways. Methods: The tested concentrations of two compounds were assessed using MTT assay in vitro. Griess assay was used to determine the changes in nitric oxide (NO) in the cell culture supernatant. qRT‒PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS. The secretion levels of TNF-α and IL-1β were detected by ELISA. Western blot test was used to detect the protein expression of IL-6, IL-10, TLR4, iNOS, NF-κB, p-P38/P38, p- ERK/ERK and p-JNK/JNK. Results: 2-(3,5-Dichlorophenyl)phthalazin-2-ium bromide (2) with a concentration below 1 μg/mL showed no significant effect on the growth inhibition of RAW264.7 cells, so the concentrations of compound 2 used for experiments were set to 0, 0.25, 0.5 and 1 μg/mL. Compared with the blank control group, the model group showed increased release of NO, transcription levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS (p&lt; 0.05), and ratios of p-P38/P38, p-ERK/ERK, p-JNK/JNK (p&lt; 0.05). Compared with the model group, the sample groups displayed decreased NO release and reduced transcriptional levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4, and iNOS and reduced protein expression ratios of IL-6, IL-1ß, IL-10, TNF-α, NF-κB, TLR4, iNOS, p-P38/P38, p-ERK/ERK and p- JNK/JNK (p&lt; 0.05). Conclusion: This study showed that 2-phenylphthalazin-2-ium bromides partially protected macrophages from the LPS-induced inflammatory response by suppressing TLR4-NF-κB/MAPK signaling and reducing NO production.

Effects of Life-Long Exercise on Age-Related Inflammation, Apoptosis, Oxidative Stress, Ferroptosis Markers, and NRF2/KAEP 1/Klotho Pathway in Rat Kidneys

To investigate the effects of life-long exercise (LLE) on age-related inflammatory cytokines, apoptosis, oxidative stress, ferroptosis markers, and the NRF2/KAEP 1/Klotho pathway in rats. Eight-month-old female Sprague-Dawley rats were divided into four groups: 1) LLE: 18-month LLE training starting at 8 months of age, 2) Old moderate-intensity continuous training (OMICT): 8 months of moderate-intensity continuous training starting at 18 months of age, 3) Adult sedentary (ASED): 8 month-old adult sedentary control group, and 4) Old sedentary (OSED): a 26-month-old sedentary control group. Hematoxylin eosin staining was performed to observe the pathological changes of kidney tissue injury in rats; Masson’s staining to observe the deposition of collagen fibers in rat kidney tissues; and western blotting to detect the expression levels of IL-6, IL 1β, p53, p21, TNF-α, GPX4, KAEP 1, NRF2, SLC7A11, and other proteins in kidney tissues. Results: Compared with the ASED group, the OSED group showed significant morphological changes in renal tubules and glomeruli, which were swollen and deformed, with a small number of inflammatory cells infiltrated in the tubules. Compared with the OSED group, the expression levels of inflammation-related proteins such as IL-1β, IL-6, TNF α, and MMP3 were significantly lower in the LLE group. Quantitative immunofluorescence analysis and western blotting revealed that compared with the ASED group, KAEP 1 protein fluorescence intensity and protein expression levels were significantly enhanced, while Klotho and NRF2 protein fluorescence intensity and protein expression levels were reduced in the OSED group. Compared with the OSED group, KAEP 1 protein fluorescence intensity and protein expression levels were reduced in the LLE and OMICT groups. Klotho and KAEP 1 protein expression levels and immunofluorescence intensity were higher in the LLE group than in the OSED group. The expression levels of GPX4 and SLC7A11, two negative marker proteins associated with ferroptosis, were significantly higher in the LLE group than in the OSED group, while the expression of p53 a cellular senescence-associated protein that negatively regulates SLC7A11, and the downstream protein p21 were significantly decreased. LLE may ameliorated aging-induced oxidative stress, inflammatory response, apoptosis, and ferroptosis by regulating Klotho and synergistically activating the NRF2/KAEP 1 pathway.

Nandrolone decanoate induced kidney injury through miRNA-146a targeting IRAK1 and TRAF6 via activation of the NF-κB pathway: The effect of moderate exercise

Anabolic-androgenic steroids (AAS) abuse is linked to some abnormalities in several tissues including the kidney. However, the precise molecular mediators involved in AAS-induced kidney disorder remain elusive. The main objective of the present study was to investigate the effect of Nandrolone decanoate on kidney injury alone or in combination with moderate exercise and its related mechanisms. Thirty-two male Wistar rats were subdivided randomly into four groups. control (Con), Nandrolone (10 mg/kg)(N), Exercise (Exe), Nandrolone + Exercise (N+Exe). ResultsAfter 6 weeks, nandrolone treatment led to a significant increase in functional parameters such as serum cystatin c, urea, creatinine, albuminuria and Albumin/ creatinine ratio indicating kidney dysfunction. Moreover, nandrolone treatment increased vacuolization, focal inflammation, hemorragia, cast formation fibrosis in the renal tissue of rats. miRNA-146a increased in kidney tissue after nandrolone exposure by using RT-PCR which may be considered idealtheranomiRNAcandidates for diagnosis and treatment.Western blotting indicated that IRAK1, TRAF6, TNF-α, NF-κB, iNOS and TGF-β protein expressions were considerably elevated in the kidneys of nandrolone treated rats. Moderate exercise could alleviate the renal dysfunction, histological abnormalities and aforementioned proteins. Our findings suggested that nandrolone consumption can cause damage to kidney tissue probably through miRNA-146a targeting IRAK1 and TRAF6 via activation of the NF-κB and TGF-β pathway. These results provide future lines of research in the identification of theranoMiRNAs related to nandrolone treatment, which can be ameliorated by moderate exercise.