Inhibition of Xanthine Oxidase by 4-nitrocinnamic Acid: In Vitro and In Vivo Investigations and Docking Simulations.

Background: Cinnamic acid and its derivatives have gained significant attention in recent medicinal research due to their broad spectrum of pharmacological properties. However, the effects of these compounds on xanthine oxidase (XO) have not been systematically investigated, and the inhibitory mechanism remains unclear.

Objectives: The objective of this study was to screen 18 compounds and identify the XO inhibitor with the strongest inhibitory effect. Furthermore, we aimed to study the inhibitory mechanism of the identified compound.

Methods: The effects of the inhibitors on XO were evaluated using kinetic analysis, docking simulations, and in vivo study. Among the compounds tested, 4-NA was discovered as the first XO inhibitor and exhibited the most potent inhibitory effects, with an IC50 value of 23.02 ± 0.12 µmol/L. The presence of the nitro group in 4-NA was found to be essential for enhancing XO inhibition. The kinetic study revealed that 4-NA inhibited XO in a reversible and noncompetitive manner. Moreover, fluorescence spectra analysis demonstrated that 4-NA could spontaneously form complexes with XO, referred to as 4-NA-XO complexes, with the negative values of △H and ΔS.

Results: This suggests that hydrogen bonds and van der Waals forces play crucial roles in the binding process. Molecular docking studies further supported the kinetic analysis and provided insight into the optimal binding conformation, indicating that 4-NA is located at the bottom outside the catalytic center through the formation of three hydrogen bonds. Furthermore, animal studies confirmed that the inhibitory effects of 4-NA on XO resulted in a significant reduction of serum uric acid level in hyperuricemia mice.

Conclusion: This work elucidates the mechanism of 4-NA inhibiting XO, paving the way for the development of new XO inhibitors.

AgNPs-Modified Polylactic Acid Microneedles: Preparation and In Vivo/In Vitro Antimicrobial Studies.

OBJECTIVE: To prepare polylactic acid microneedles (PLAMNs) with sustained antibacterial effect to avoid skin infection caused by traditional MNs-based biosensors. METHODS: Silver nanoparticles (AgNPs) were synthesized using an in-situ reduction process with polydopamine (PDA). PLAMNs were fabricated using the hot-melt method. A series of pressure tests and puncture experiments were conducted to confirm the physicochemical properties of PLAMNs. Then AgNPs were modified on the surface of PLAMNs through in-situ reduction of PDA, resulting in the formation of PLAMNs@PDA-AgNPs. The in vitro antibacterial efficacy of PLAMNs@PDA-AgNPs was evaluated using agar diffusion assays and bacterial liquid co-culture approach. Wound healing and simulated long-term application were performed to assess the in vivo antibacterial effectiveness of PLAMNs@PDA-AgNPs. RESULTS: The MNs array comprised 169 tiny needle tips in pyramidal rows. Strength and puncture tests confirmed a 100% puncture success rate for PLAMNs on isolated rat skin and tin foil. SEM analysis revealed the integrity of PLAMNs@PDA-AgNPs with the formation of new surface substances. EDS analysis indicated the presence of silver elements on the surface of PLAMNs@PDA-AgNPs, with a content of 14.44%. Transepidermal water loss (TEWL) testing demonstrated the rapid healing of micro-pores created by PLAMNs@PDA-AgNPs, indicating their safety. Both in vitro and in vivo tests confirmed antibacterial efficacy of PLAMNs@PDA-AgNPs. CONCLUSIONS: In conclusion, the sustained antibacterial activity exhibited by PLAMNs@PDA-AgNPs offers a promising solution for addressing skin infections associated with MN applications, especially when compared to traditional MN-based biosensors. This advancement offers significant potential for the field of MN technology.

Effects of theophylline combined with inhaled corticosteroids on patients with moderate and severe asthma and changes of T lymphocyte subsets in peripheral blood.

Introduction: Asthma is a common respiratory disease. Theophylline combined with inhaled corticosteroids (ICS) is a promising therapy for asthma. This study explored the therapeutic effects of ICS combined with theophylline on moderate and severe asthma patients and T lymphocyte subsets (CD3+CD8+ T cells) in peripheral blood. Material and methods: A total of 202 moderate and severe asthma patients were selected, with 101 treated with theophylline combined with ICS and 101 treated with ICS alone as controls. Lung function [forced expiratory volume within 1 second (FEV1), forced vital capacity (FVC), and peak expiratory flow (PEF)] were tested using a spirometer. Asthma symptom control was evaluated by asthma control tests (ACT). The life quality was evaluated using the Asthma Quality of Life Questionnaire (AQLQ). The number and percentage of CD3+ T, CD3+CD4+ T and CD3+CD8+ T cells in peripheral blood mononuclear cells were assessed by flow cytometry. The correlation between CD3+CD8+ T cells and lung function and asthma control of patients after combination therapy was analyzed by Pearson correlation analysis. Results: Compared with moderate and severe patients treated with ICS alone, theophylline improved the efficacy of ICS. Theophylline combined with ICS decreased IL-4 and IL-6 levels, and CD3+ T and CD3+CD8+ T cell number and percentage. After combined treatment, CD3+ CD8+ T cells in peripheral blood of patients were positively correlated with lung function and negatively correlated with asthma control. Conclusions: The additional use of theophylline improved the efficacy of corticosteroids in asthma patient treatment and reduced inflammation level and CD3+ T and CD3+CD8+ T cell contents in peripheral blood.

Fabrication of Antibacterial Sponge Microneedles for Sampling Skin Interstitial Fluid.

Microneedles (MNs) have recently garnered extensive interest concerning direct interstitial fluid (ISF) extraction or their integration into medical devices for continuous biomarker monitoring, owing to their advantages of painlessness, minimal invasiveness, and ease of use. However, micropores created by MN insertion may provide pathways for bacterial infiltration into the skin, causing local or systemic infection, especially with long-term in situ monitoring. To address this, we developed a novel antibacterial sponge MNs (SMNs@PDA-AgNPs) by depositing silver nanoparticles (AgNPs) on polydopamine (PDA)-coated SMNs. The physicochemical properties of SMNs@PDA-AgNPs were characterized regarding morphology, composition, mechanical strength, and liquid absorption capacity. The antibacterial effects were evaluated and optimized through agar diffusion assays in vitro. Wound healing and bacterial inhibition were further examined in vivo during MN application. Finally, the ISF sampling ability and biosafety of SMNs@PDA-AgNPs were assessed in vivo. The results demonstrate that antibacterial SMNs enable direct ISF extraction while preventing infection risks. SMNs@PDA-AgNPs could potentially be used for direct sampling or combined with medical devices for real-time diagnosis and management of chronic diseases.

Xanthine oxidase inhibitory kinetics and mechanism of ellagic acid: In vitro, in silico and in vivo studies.

Ellagic acid (EA), which is widely distributed in many foods, has been found to possess inhibitory activity against xanthine oxidase (XO). However, there is ongoing debate about the difference in XO inhibitory activity between EA and allopurinol. Additionally, the inhibitory kinetics and mechanism of EA on XO are still unclear. Herein, the authors systematically studied the inhibitory effects of EA on XO. The authors' findings showed that EA is a reversible inhibitor with mixed-type inhibition, and its inhibitory activity is weaker than allopurinol. Fluorescence quenching experiments suggested that the generation of EA-XO complex was exothermic and spontaneous. In silico analysis further confirmed that EA entered the XO catalytic centre. Furthermore, the authors verified the anti-hyperuricemia effect of EA in vivo. This study elucidates the inhibition kinetics and mechanism of EA on XO, and lays a theoretical foundation for the further development of drugs and functional foods containing EA for the treatment of hyperuricemia.

High expression of BTLA and B7-H4 on the surface of myeloid dendritic cells has a negative regulatory effect on their anti-tuberculosis immunity activity in pleural tuberculosis patients.

To investigate the effects of the surface markers B- and T-lymphocyte attenuator (BTLA) and B7 homologous body 4 (B7-H4) on expression of CD83, and Human Leukocyte Antigen-DR isotype (HLA-DR) that can activate dendritic cells (DCs). Flow cytometry was used to detect the co-expression of BTLA and B7-H4 on myeloid DCs (mDCs) in peripheral blood (PB) and pleural effusions (PE) in 15 volunteers and 20 tuberculous pleurisy (TP) patients. Co-expression of BTLA and B7-H4 (double positive (DP)) mDCs in PB and PE of TP patients were enhanced. The proportion of DP mDC in PB decreased markedly after 2 weeks treatment, but was still greater than in controls. Expression of CD83 and HLA-DR on DP mDCs was higher than on BTLA and B7-H4 double negative (DN) expressing mDCs in PB of different TP groups. Expression of CD83 on DP mDCs in PB and PE of TP patients was greater than that of controls. Expression of HLA-DR on DP mDCs in TP patient PB was lower than in TP PE and controls. In pleural tuberculosis (TB) patients, high expression of BTLA and B7-H4 promoted a high level of CD83 and HLA-DR, which had a negative regulatory effect on mDCs on anti-TB immunity.

The circular RNA of peripheral blood mononuclear cells: Hsa_circ_0005836 as a new diagnostic biomarker and therapeutic target of active pulmonary tuberculosis.

It has been reported that circular RNA (circRNA) is associated with human cancer. However, few studies have been reported in active pulmonary tuberculosis (APTB). The global circRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) of APTB patients (n=5) and health controls (HC) (n=5) by using high-throughput sequencing. According to the systematical bioinformatics analysis, the basic content of circRNAs and their fold changes in the two groups were calculated. We selected 6 significant differentially expressed circRNAs, hsa_circ_0005836, hsa_circ_0009128, hsa_circ_0003519, hsa_circ_0023956, hsa_circ_0078768, and hsa_circ_0088452 and validated the expression in PBMCs from APTB (n=10) and HC (n=10) by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Further, the verification of these specific circRNAs (hsa_circ_0005836 and hsa_circ_0009128) between APTB (n=34) and HC (n=30) in PBMCs was also conducted by qRT-PCRs. The RNA-seq data showed the significant differential expression of the 523 circRNAs between the APTB and HC groups (199 circRNAs were significantly up-regulated and 324 circRNAs were down-regulated). Hsa_circ_0005836 and hsa_circ_0009128 expression was significantly down-regulated in the PBMCs of APTB (P<0.05) in the samples of APTB compared to HC in our study. The gene ontology based enrichment analysis of the circRNA-miRNA-mRNAs network showed that cellular catabolic process (P=7.10E-08), regulation of metabolic process (P=2.10E-06), catalytic activity (P=3.67E-08), protein binding (P=1.71E-07), cell part (P=3.46E-06), intracellular part (P=1.71E-07), and intracellular (P=3.67E-08) were recognized in the comparisons between APTB and HC. Based on KEGG analysis, HTLV-I infection, regulation of actin cytoskeleton, neurotrophin signaling pathway and mTOR signaling pathway were relevant during tuberculosis bacillus infection. We found for the first time that hsa_circ_0005836 and hsa_circ_0009128 were significantly down-regulated in the PBMCs of APTB compared with HC. Our findings indicate hsa_circ_0005836 might serve as a novel potential biomarker for TB infection.

Tuberculosis-sensitized monocytes sustain immune response of interleukin-37.

Roles of human IL-37 in infections remain poorly characterized. Although plasma IL-37 is elevated in patients with tuberculosis (TB), IL-37 source and immune correlate in TB have not been investigated. It is also unknown whether and how TB can influence the ability of immune cells to mount innate responses of IL-37 and pre-inflammatory cytokines. Here, we demonstrated that IL-37b-producing monocytes coincided with a source of elevated plasma IL-37b in TB patients. While IL-37b production in TB was associated with prolonged/complicated TB, TB burdens and inflammatory reactions, it negatively correlated with immune responses of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α or IL-10. Interestingly, mycobacterial re-infection of monocytes from TB patients, but not healthy BCG-vaccinated controls, enhanced or sustained IL-37b production by cultured monocytes. TB-sensitized monocytes from TB patients mounted more robust immune responses of IL-37b than those of pre-inflammatory cytokines during mycobacterial re-infection in culture. Our data represent new findings in terms of IL-37b responses, immune correlates and potential mechanisms in TB patients.

Tuberculous pleurisy drives marked effector responses of γδ, CD4+, and CD8+ T cell subpopulations in humans.

Although tuberculous pleurisy (TP) presumably involves a hypersensitivity reaction, there is limited evidence indicating overreactive effector responses of γδ T cells and αß T cells and their interrelation with Foxp3(+) Tregs in pleural and other compartments. We found that TP induced reciprocal representations of Foxp3(+) Tregs and Mtb phosphoantigen-specific Vγ2Vδ2 T cells in different anatomic compartments. Patients with TP exhibited appreciable numbers of "proliferating" Ki-67(+) Vγ2Vδ2 T cells in the airway where Foxp3(+) Tregs were not dominant, whereas striking increases in Foxp3(+) Tregs in the blood and pleural compartments coincided with low frequencies of Vγ2Vδ2 T cells. Interestingly, anti-tuberculosis chemotherapy control of Mtb infection in patients with TP reversed reciprocal representations of Foxp3(+) Tregs and proliferating Vγ2Vδ2 T cells. Surprisingly, despite high-level Foxp3(+) Tregs, TP appeared to drive overreactive responses of IFN-γ-producing Vγ2Vδ2, CD4(+)CD25(+), and CD8(+)CD25(+) T effector subpopulations, whereas IL-22-producing Vγ2Vδ2 T cells increased subtly. Th1 effector responses were sustained despite remarkable declines in Foxp3(+) Tregs at 1 mo after the treatment. Overreactive T effector responses of Mtb-reactive γδ T cells, αß CD25(+)CD4(+), and CD25(+)CD8(+) T cell subpopulations appear to be immune features for TP. Increased Foxp3(+) Tregs might be responsive to overreactive TP but unable to influence T effector responses despite having an inverse relation with proliferating Vγ2Vδ2 T cells.