Fluorinated hydroxyapatite conditions a favorable osteo-immune microenvironment via triggering metabolic shift from glycolysis to oxidative phosphorylation.

BACKGROUND: Biological-derived hydroxyapatite is widely used as a bone substitute for addressing bone defects, but its limited osteoconductive properties necessitate further improvement. The osteo-immunomodulatory properties hold crucial promise in maintaining bone homeostasis, and precise modulation of macrophage polarization is essential in this process. Metabolism serves as a guiding force for immunity, and fluoride modification represents a promising strategy for modulating the osteoimmunological environment by regulating immunometabolism. In this context, we synthesized fluorinated porcine hydroxyapatite (FPHA), and has demonstrated its enhanced biological properties and osteogenic capacity. However, it remains unknown whether and how FPHA affects the immune microenvironment of the bone defects. METHODS: FPHA was synthesized and its composition and structural properties were confirmed. Macrophages were cultured with FPHA extract to investigate the effects of FPHA on their polarization and the related osteo-immune microenvironment. Furthermore, total RNA of these macrophages was extracted, and RNA-seq analysis was performed to explore the underlying mechanisms associated with the observed changes in macrophages. The metabolic states were evaluated with a Seahorse analyzer. Additionally, immunohistochemical staining was performed to evaluate the macrophages response after implantation of the novel bone substitutes in critical size calvarial defects in SD rats. RESULTS: The incorporation of fluoride ions in FPHA was validated. FPHA promoted macrophage proliferation and enhanced the expression of M2 markers while suppressing the expression of M1 markers. Additionally, FPHA inhibited the expression of inflammatory factors and upregulated the expression of osteogenic factors, thereby enhancing the osteogenic differentiation capacity of the rBMSCs. RNA-seq analysis suggested that the polarization-regulating function of FPHA may be related to changes in cellular metabolism. Further experiments confirmed that FPHA enhanced mitochondrial function and promoted the metabolic shift of macrophages from glycolysis to oxidative phosphorylation. Moreover, in vivo experiments validated the above results in the calvarial defect model in SD rats. CONCLUSION: In summary, our study reveals that FPHA induces a metabolic shift in macrophages from glycolysis to oxidative phosphorylation. This shift leads to an increased tendency toward M2 polarization in macrophages, consequently creating a favorable osteo-immune microenvironment. These findings provide valuable insights into the impact of incorporating an appropriate concentration of fluoride on immunometabolism and macrophage mitochondrial function, which have important implications for the development of fluoride-modified immunometabolism-based bone regenerative biomaterials and the clinical application of FPHA or other fluoride-containing materials.

The Effect Mechanism of N6-adenosine Methylation (m6A) in Melatonin Regulated LPS-induced Colon Inflammation.

Colon inflammation is characterized by disturbances in the intestinal microbiota and inflammation. Melatonin (Mel) can improve colon inflammation. However, the underlying mechanism remains unclear. Recent studies suggest that m6A methylation modification may play an important role in inflammatory responses. This study aimed to explore the effects of melatonin and LPS-mediated m6A methylation on colon inflammation. Our study found that melatonin inhibits M1 macrophages, activates M2 macrophages, inhibit the secretion of pro-inflammatory factors, maintain colon homeostasis and improves colon inflammation through MTNR1B. In addition, the increased methylation level of m6A is associated with the occurrence of colon inflammation, and melatonin can also reduce the level of colon methylation to improve colon inflammation. Among them, the main methylated protein METTL3 can be inhibited by melatonin through MTNR1B. In a word, melatonin regulates m6A methylation by improving abnormal METTL3 protein level to reshape the microflora and activate macrophages to improve colon inflammation, mainly through MTNR1B.

Baicalin nanodelivery system based on functionalized metal-organic framework for targeted therapy of osteoarthritis by modulating macrophage polarization.

Intra-articular drugs used to treat osteoarthritis (OA) often suffer from poor pharmacokinetics and stability. Nano-platforms as drug delivery systems for drug delivery are promising for OA therapy. In this study, we reported an M1 macrophage-targeted delivery system Bai@FA-UIO-66-NH2 based on folic acid (FA) -modified metal-organic framework (MOF) loaded with baicalin (Bai) as antioxidant agent for OA therapy. With outstanding biocompatibility and high drug loading efficiency, Bai@FA-UIO-66-NH2 could be specifically uptaken by LPS-induced macrophages to serve as a potent ROS scavenger, gradually releasing Bai at the subcellular level to reduce ROS production, modulate macrophage polarization to M2, leading to alleviation of synovial inflammation in OA joints. The synergistic effect of Bai@FA-UIO-66-NH2 on macrophage polarization and ROS scavenging significantly improved the therapeutic efficacy of OA, which may provide a new insight into the design of OA precision therapy.

ECM-mimetic, NSAIDs loaded thermo-responsive, immunomodulatory hydrogel for rheumatoid arthritis treatment.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, and it leads to irreversible inflammation in intra-articular joints. Current treatment approaches for RA include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), corticosteroids, and biological agents. To overcome the drug-associated toxicity of conventional therapy and transdermal tissue barrier, an injectable NSAID-loaded hydrogel system was developed and explored its efficacy. RESULTS: The surface morphology and porosity of the hydrogels indicate that they mimic the natural ECM, which is greatly beneficial for tissue healing. Further, NSAIDs, i.e., diclofenac sodium, were loaded into the hydrogel, and the in vitro drug release pattern was found to be burst release for 24 h and subsequently sustainable release of 50% drug up to 10 days. The DPPH assay revealed that the hydrogels have good radical scavenging activity. The biocompatibility study carried out by MTT assay proved good biocompatibility and anti-inflammatory activity of the hydrogels was carried out by gene expression study in RAW 264.7 cells, which indicate the downregulation of several key inflammatory genes such as COX-2, TNF-α & 18s. CONCLUSION: In summary, the proposed ECM-mimetic, thermo-sensitive in situ hydrogels may be utilized for intra-articular inflammation modulation and can be beneficial by reducing the frequency of medication and providing optimum lubrication at intra-articular joints.

Columbianadin ameliorates experimental acute reflux esophagitis in rats via suppression of NF-κB pathway.

PURPOSE: Reflux esophagitis is a condition characterized by inflammation and irritation of the esophagus, resulting from the backflow of stomach acid and other gastric contents into the esophagus. Columbianadin is a coumarin derivative that exhibits anti-inflammatory and antioxidant effects. In this study, we tried to scrutinize the protective effect of Columbianadin against acute reflux esophagitis in rats. METHODS: RAW 264.7 cells were utilized to assess cell viability and measure the production of inflammatory parameters. The rats received anesthesia, and reflux esophagitis was induced via ligation of pylorus and fore stomach and corpus junction. Rats received the oral administration of Columbianadin (25, 50 and 100 mg/kg) and omeprazole (20 mg/kg). The gastric secretion volume, acidity, and pH were measured. Additionally, the levels of oxidative stress parameters, cytokines, and inflammatory markers were determined. At the end of the study, mRNA expression was assessed. RESULTS: Columbianadin remarkably suppressed the cell viability and production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and prostaglandin (PGE2). Columbianadin treatment remarkably suppressed the secretion of gastric volume, total acidity and enhanced the pH level in the stomach. Columbianadin remarkably altered the level of hydrogen peroxidase, free iron, calcium, and plasma scavenging activity, sulfhydryl group; oxidative stress parameters like malonaldehyde, glutathione, superoxide dismutase, catalase, glutathione peroxidase; inflammatory cytokines viz., TNF-α, IL-6, IL-1ß, IL-10, IL-17, and monocyte chemoattractant protein-1; inflammatory parameters including PGE2, iNOS, COX-2, and nuclear kappa B factor (NF-κB). Columbianadin remarkably (P < 0.001) suppressed the mRNA expression TNF-α, IL-6, IL-1ß and plasminogen activator inhibitor-1. CONCLUSIONS: Columbianadin demonstrated a protective effect against acute reflux esophagitis via NF-κB pathway.

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways.

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

Helicobacter pylori disrupts gastric mucosal homeostasis by stimulating macrophages to secrete CCL3.

BACKGROUND: Helicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis. METHODS: The expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry. RESULTS: After H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased. CONCLUSIONS: In summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.

Vikil 20 Suppresses the Proliferation of Prostate Cancer (PC-3) Cells and Quenches Free Radicals In Vitro.

Vikil 20 is an herbal formula produced in Ghana and is widely marketed as a product to boost immunity as well as for general well-being. However, the pharmacological effect of this herbal preparation has not been proven scientifically. Therefore, this study was aimed at investigating the antioxidative as well as the anti-prostate cancer effects of the product. To assess the antioxidative effect of Vikil 20, the DPPH and ABTS activities were investigated. The total phenolic content was investigated using the Folin-Ciocalteu method. The cytotoxic effect of Vikil 20 against prostate cancer (PC-3) cells as well as normal (RAW 264.7) cells was investigated using the MTT assay whereas its anti-metastatic effect was analyzed using the cell migration assay. The effect of Vikil 20 on cell adhesion was analyzed via the cell adhesion assay whereas its effect on TNF-α secretion was investigated using a TNF-α detection kit. Vikil 20 demonstrated significant antioxidant effects by suppressing 57.61% and 92.88% respectively of DPPH and ABTS radicals at 1000 µg/mL with total phenolic contents of 140.45 mg GAE/g. Vikil 20 suppressed the proliferation of PC-3 cells by reducing the number of viable cells to 49.5% while sparing the RAW, 264.7 cells. Further, Vikil 20 significantly suppressed both cellular migration and adhesion of prostate cancer cells. Finally, suppression of cellular migration and adhesion is associated with a reduction in TNF-α secretion by PC-3 cells. Taken together, Vikil 20 was found to possess significant antioxidant and anti-prostate cancer effects in vitro.

Neuroprotective and immunomodulatory effects of superoxide dismutase on SH-SY5Y neuroblastoma cells and RAW264.7 macrophages.

Superoxide dismutase (SOD) is an antioxidant enzyme that protects the body from free radicals. It has both antioxidant and immunomodulatory properties, inducing macrophage polarization from M1 to M2. Macrophages, key mediators of the innate immune response, are divided into the M1 (pro-inflammatory) and M2 (anti-inflammatory) subtypes. In this study, we aimed to assess the antioxidant and neuroprotective effects of SOD on nerve cells and its immunomodulatory effects on macrophages. We observed that SOD inhibited the accumulation of reactive oxygen species and enhanced the viability of H2O2-treated nerve cells. Furthermore, SOD reduced the degree of necrosis in nerve cells treated with the conditioned medium from macrophages, which induced inflammation. In addition, SOD promoted the M1 to M2 transition of macrophages. Our findings suggest that SOD protects nerve cells and regulates immune responses.

Anti-inflammatory properties and characterization of water extracts obtained from Callicarpa kwangtungensis Chun using in vitro and in vivo rat models.

Callicarpa kwangtungensis Chun (CK) is a common remedy exhibits anti-inflammatory properties and has been used in Chinese herbal formulations, such as KangGongYan tablets. It is the main component of KangGongYan tablets, which has been used to treat chronic cervicitis caused by damp heat, red and white bands, cervical erosion, and bleeding. However, the anti-inflammatory effects of CK water extract remains unknown. This study assessed the anti-inflammatory effects of CK in vivo and in vitro, characterized its main components in the serum of rats and verified the anti-inflammatory effects of serum containing CK. Nitric oxide (NO), tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6) release by RAW264.7 cells was examined by ELISA and Griess reagents. Inflammation-related protein expression in LPS-stimulated RAW264.7 cells was measured by western blotting. Furthermore, rat model of foot swelling induced by λ-carrageenan and a collagen-induced arthritis (CIA) rat model were used to explore the anti-inflammatory effects of CK. The components of CK were characterized by LC-MS, and the effects of CK-containing serum on proinflammatory factors levels and the expression of inflammation-related proteins were examined by ELISA, Griess reagents and Western blotting. CK suppressed IL-6, TNF-α, and NO production, and iNOS protein expression in LPS-stimulated RAW264.7 cells. Mechanistic studies showed that CK inhibited the phosphorylation of ERK, P38 and JNK in the MAPK signaling pathway, promoted the expression of IκBα in the NF-κB signaling pathway, and subsequently inhibited the expression of iNOS, thereby exerting anti-inflammatory effects. Moreover, CK reduced the swelling rates with λ-carrageenan induced foot swelling, and reduced the arthritis score and incidence in the collagen-induced arthritis (CIA) rat model. A total of 68 compounds in CK water extract and 31 components in rat serum after intragastric administration of CK were characterized. Serum pharmacological analysis showed that CK-containing serum suppressed iNOS protein expression and NO, TNF-α, and IL-6 release. CK may be an anti-inflammatory agent with therapeutic potential for acute and chronic inflammatory diseases, especially inflammatory diseases associated with MAPK activation.

Study on the Protective Effect and Mechanism of Umbilicaria esculenta Polysaccharide in DSS-Induced Mice Colitis and Secondary Liver Injury.

This study aimed to explore the ameliorative effects and potential mechanisms of Huangshan Umbilicaria esculenta polysaccharide (UEP) in dextran sulfate sodium-induced acute ulcerative colitis (UC) and UC secondary liver injury (SLI). Results showed that UEP could ameliorate both colon and liver pathologic injuries, upregulate mouse intestinal tight junction proteins (TJs) and MUC2 expression, and reduce LPS exposure, thereby attenuating the effects of the gut-liver axis. Importantly, UEP significantly downregulated the secretion levels of TNF-α, IL-1ß, and IL-6 through inhibition of the NF-κB pathway and activated the Nrf2 signaling pathway to increase the expression levels of SOD and GSH-Px. In vitro, UEP inhibited the LPS-induced phosphorylation of NF-κB P65 and promoted nuclear translocation of Nrf2 in RAW264.7 cells. These results revealed that UEP ameliorated UC and SLI through NF-κB and Nrf2-mediated inflammation and oxidative stress. The study first investigated the anticolitis effect of UEP, suggesting its potential for the treatment of colitis and colitis-associated liver disease.

Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration.

Biomaterials can modulate the local immune microenvironments to promote peripheral nerve regeneration. Inspired by the spatial orderly distribution and endogenous electric field of nerve fibers, we aimed to investigate the synergistic effects of electrical and topological cues on immune microenvironments of peripheral nerve regeneration. Nerve guidance conduits (NGCs) with aligned electrospun nanofibers were fabricated using a polyurethane copolymer containing a conductive aniline trimer and degradable L-lysine (PUAT). In vitro experiments showed that the aligned PUAT (A-PUAT) membranes promoted the recruitment of macrophages and induced their polarization towards the pro-healing M2 phenotype, which subsequently facilitated the migration and myelination of Schwann cells. Furthermore, NGCs fabricated from A-PUAT increased the proportion of pro-healing macrophages and improved peripheral nerve regeneration in a rat model of sciatic nerve injury. In conclusion, this study demonstrated the potential application of NGCs in peripheral nerve regeneration from an immunomodulatory perspective and revealed A-PUAT as a clinically-actionable strategy for peripheral nerve injury.

Mesoporous MOFs with ROS scavenging capacity for the alleviation of inflammation through inhibiting stimulator of interferon genes to promote diabetic wound healing.

Excessive production of reactive oxygen species (ROS) and inflammation are the key problems that impede diabetic wound healing. In particular, dressings with ROS scavenging capacity play a crucial role in the process of chronic wound healing. Herein, Zr-based large-pore mesoporous metal-organic frameworks (mesoMOFs) were successfully developed for the construction of spatially organized cascade bioreactors. Natural superoxide dismutase (SOD) and an artificial enzyme were spatially organized in these hierarchical mesoMOFs, forming a cascade antioxidant defense system, and presenting efficient intracellular and extracellular ROS scavenging performance. In vivo experiments demonstrated that the SOD@HMUiO-MnTCPP nanoparticles (S@M@H NPs) significantly accelerated diabetic wound healing. Transcriptomic and western blot results further indicated that the nanocomposite could inhibit fibroblast senescence and ferroptosis as well as the stimulator of interferon genes (STING) signaling pathway activation in macrophages mediated by mitochondrial oxidative stress through ROS elimination. Thus, the biomimetic multi-enzyme cascade catalytic system with spatial ordering demonstrated a high potential for diabetic wound healing, where senescence, ferroptosis, and STING signaling pathways may be potential targets.

GC-MS based antioxidants characterization in Saussurea heteromalla (D. Don) Hand-Mazz by inhibition of nitric oxide generation in macrophages.

For centuries, medicinal plants have served as the cornerstone for traditional health care systems and same practice is still prevalent today. In the Himalayan region, Saussurea heteromalla holds a significant place in traditional medicine and is used to address various health issues. Despite its historical use, little exploration has focused on its potential for scavenging free radicals and reducing inflammation. Hence, our current study aims to investigate the free radical scavenging capabilities of S. heteromalla extracts. The n-hexane extract of entire plant revealed promising activity. This extract underwent extensive extraction on a larger scale. Subsequent purification, employing column chromatography, HPLC-DAD techniques, led to the identification of active compounds, confirmed via GC-MS and the NIST database as 1-O-butyl 2-O-octyl benzene-1,2-dicarboxylate and 2,4-ditert-butylphenol. Assessing the free radical scavenging properties involved utilizing RAW-264.7 macrophages activated by lipopolysaccharides. Notably, the compound 2,4-di-tert-butylphenol exhibited remarkable scavenging abilities, demonstrating over 80% inhibition of Nitric oxide. This study stands as the inaugural report on the isolation of these compounds from S. heteromalla.

[Arbutin ameliorates liver fibrosis in mice by inhibiting macrophage recruitment and regulating the Akt/NF-κB and Smad signaling pathways].

OBJECTIVE: To investigate the protective effect of arbutin against CCl4-induced hepatic fibrosis in mice and explore the underlying mechanisms. METHODS: Twenty-four C57BL/6 mice were randomly divided into control group, model group, and low- and high-dose arbutin treatment (25 and 50 mg/kg, respectively) groups. Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4, and arbutin was administered daily via gavage for 6 weeks. After the treatments, serum biochemical parameters of the mice were tested, and liver tissues were taken for HE staining, Sirius Red staining and immunohistochemical staining. RT-qPCR was used to detect the mRNA levels of α-SMA, Pdgfb, Col1α1, Timp-1, Ccl2 and Tnf-a, and Western blotting was performed to detect α-SMA protein expression in the liver tissues. In the cell experiment, the effect of arbutin treatment for 24 h on THP-1 and RAW264.7 cell migration and recruitment was examined using Transwell migration assay and DAPI staining; The changes in protein levels of Akt, p65, Smad3, p-Akt, p-p65, p-Smad3 and α-SMA in arbutintreated LX-2 cells were detected with Western blotting. RESULTS: Arbutin treatment significantly lowered serum alanine aminotransferase and aspartate aminotransferase levels, alleviated liver tissue damage and collagen deposition, and reduced macrophage infiltration and α-SMA protein expression in the liver of the mouse models (P < 0.05 or 0.001). Arbutin treatment also significantly reduced CCl4-induced elevation of a-SMA, Pdgfb, Col1α1, Timp-1, Ccl2 and Tnf-a mRNA levels in mice (P < 0.05). In the cell experiment, arbutin treatment obviously inhibited migration and recruitment of THP-1 and RAW264.7 cells and lowered the phosphorylation levels of Akt, p65 and Smad3 and the protein expression level of α-SMA in LX-2 cells. CONCLUSION: Arbutin ameliorates liver inflammation and fibrosis in mice by inhibiting hepatic stellate cell activation via reducing macrophage recruitment and infiltration and suppressing activation of the Akt/NF-κB and Smad signaling pathways.

Asiatic acid alleviates vascular remodeling in BAPN-induced aortic dissection through inhibiting NF-κB p65/CX3CL1 signaling.

Inflammation assumes a pivotal role in the aortic remodeling of aortic dissection (AD). Asiatic acid (AA), a triterpene compound, is recognized for its strong anti-inflammatory properties. Yet, its effects on ß-aminopropionitrile (BAPN)-triggered AD have not been clearly established. The objective is to determine whether AA attenuates adverse aortic remodeling in BAPN-induced AD and clarify potential molecular mechanisms. In vitro studies, RAW264.7 cells pretreated with AA were challenged with lipopolysaccharide (LPS), and then the vascular smooth muscle cells (VSMCs)-macrophage coculture system was established to explore intercellular interactions. To induce AD, male C57BL/6J mice at three weeks of age were administered BAPN at a dosage of 1 g/kg/d for four weeks. To decipher the mechanism underlying the effects of AA, RNA sequencing analysis was conducted, with subsequent validation of these pathways through cellular experiments. AA exhibited significant suppression of M1 macrophage polarization. In the cell coculture system, AA facilitated the transformation of VSMCs into a contractile phenotype. In the mouse model of AD, AA strikingly prevented the BAPN-induced increases in inflammation cell infiltration and extracellular matrix degradation. Mechanistically, RNA sequencing analysis revealed a substantial upregulation of CX3CL1 expression in BAPN group but downregulation in AA-treated group. Additionally, it was observed that the upregulation of CX3CL1 negated the beneficial impact of AA on the polarization of macrophages and the phenotypic transformation of VSMCs. Crucially, our findings revealed that AA is capable of downregulating CX3CL1 expression, accomplishing this by obstructing the nuclear translocation of NF-κB p65. The findings indicate that AA holds promise as a prospective treatment for adverse aortic remodeling by suppressing the activity of NF-κB p65/CX3CL1 signaling pathway.

hCeO2@ Cu5.4O nanoparticle alleviates inflammatory responses by regulating the CTSB-NLRP3 signaling pathway.

Inflammatory responses, especially chronic inflammation, are closely associated with many systemic diseases. There are many ways to treat and alleviate inflammation, but how to solve this problem at the molecular level has always been a hot topic in research. The use of nanoparticles (NPs) as anti-inflammatory agents is a potential treatment method. We synthesized new hollow cerium oxide nanomaterials (hCeO2 NPs) doped with different concentrations of Cu5.4O NPs [the molar ratio of Cu/(Ce + Cu) was 50%, 67%, and 83%, respectively], characterized their surface morphology and physicochemical properties, and screened the safe concentration of hCeO2@Cu5.4O using the CCK8 method. Macrophages were cultured, and P.g-lipopolysaccharide-stimulated was used as a model of inflammation and co-cultured with hCeO2@Cu5.4O NPs. We then observe the effect of the transcription levels of CTSB, NLRP3, caspase-1, ASC, IL-18, and IL-1ß by PCR and detect its effect on the expression level of CTSB protein by Western blot. The levels of IL-18 and IL-1ß in the cell supernatant were measured by enzyme-linked immunosorbent assay. Our results indicated that hCeO2@Cu5.4O NPs could reduce the production of reactive oxygen species and inhibit CTSB and NLRP3 to alleviate the damage caused by the inflammatory response to cells. More importantly, hCeO2@Cu5.4O NPs showed stronger anti-inflammatory effects as Cu5.4O NP doping increased. Therefore, the development of the novel nanomaterial hCeO2@Cu5.4O NPs provides a possible new approach for the treatment of inflammatory diseases.

In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

The role of RUNX1/NF-κB in regulating PVAT inflammation in aortic dissection.

The pathogenesis of aortic dissection (AD), an aortic disease associated with high mortality, involves significant vascular inflammatory infiltration. However, the precise relationship between perivascular adipose tissue (PVAT) and aortic dissection remains incompletely understood. The objective of this study is to investigate the role of PVAT inflammation in the pathogenesis of aortic dissection and identify novel therapeutic targets for this disease. The mouse model of aortic dissection was established in this study through intraperitoneal injection of Ang II and administration of BAPN in drinking water. Additionally, control groups were established at different time points including the 2-week group, 3-week group, and 4-week group. qPCR and immunohistochemistry techniques were employed to detect the expression of inflammatory markers and RUNX1 in PVAT surrounding the thoracic aorta in mice. Additionally, an aortic dissection model was established using RUNX1 knockout mice, and the aforementioned indicators were assessed. The 3T3-L1 cells were induced to differentiate into mature adipocytes in vitro, followed by lentivirus transfection for the knockdown or overexpression of RUNX1. The study aimed to investigate the potential cell-to-cell interactions by co-culturing 3T3-L1 cells with A7r5 or RAW264.7 cells. Subsequently, human aortic PVAT samples were obtained through clinical surgery and the aforementioned indicators were detected. In comparison to the control group, the aortic dissection model group exhibited decreased expression of MMP-2 and NF-κB in PVAT, while TNF-α and RUNX1 expression increased. Suppression of RUNX1 expression resulted in increased MMP-2 and NF-κB expression in PVAT, along with decreased TNF-α expression. Overexpression of RUNX1 upregulated the expression levels of NF-Κb, MMP-2, and TNF-α in adipocytes, whereas knockdown of RUNX1 exerted an opposite effect. Macrophages co-cultured with adipocytes overexpressing RUNX1 exhibited enhanced CD86 expression, while vascular smooth muscle cells co-cultured with these adipocytes showed reduced α-SMA expression. In human samples, there was an increase in both RUNX1 and MMP-2 expression levels, accompanied by a decrease in TNF-α and NF-Κb expression. The presence of aortic dissection is accompanied by evident inflammatory alterations in the PVAT, and this phenomenon appears to be associated with the involvement of RUNX1. It is plausible that the regulation of PVAT's inflammatory changes by RUNX1/NF-κB signaling pathway plays a role in the pathogenesis of aortic dissection.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.