This study was aimed at investigating the effects of demethylase fat mass and obesity-associated protein(FTO)and methyltransferase methyltransferase like protein 3(METTL3),key molecules in N6-methyladenosine(m6A)modification,on the replication and proliferation of Japanese encephalitis virus(JEV).Recombinant lentiviruses were generated by packaging the FTO and green fluorescent protein into lentiviral vectors.Neuro2a cells,a mouse neuroblastoma cell line,were infected with the lentivirus,and stable FTO-expressing cell lines were obtained through puromycin selection.Successful overexpression of FTO was confirmed through fluorescence microscopy,real-time quantitative PCR,and western blot analysis.When Neuro2a cells overexpressing FTO were infected with JEV,the overexpression of FTO decreased JEV replication in the cells,and increased the expression of interferon(IFN)and related molecules.Additionally,treatment of JEV-infected Neuro2a cells with the METTL3-specific inhibitor STM2457 resulted in a dose-dependent decrease in JEV replication and viral protein expression.These findings suggested that lowering m6A methylation levels inhibits JEV replication,thus shedding light on the regulatory role of methylation modification in JEV replication.

Objective:To investigate the mechanism of mitochondrial DNA (mtDNA)-reactive oxygen species (ROS)-dynamin-related protein 1 (Drp1) axis in regulating phenotypic transformation of vascular smooth muscle cells (VSMCs).Methods:(1) VSMCs were divided into a control group, a synthetic VSMCs group, and a Drp1 siRNA+synthetic VSMCs group; cells in the Drp1 siRNA+synthetic VSMCs group were transfected with 50 nmol/L Drp1 siRNA for 48 h; cells in the latter two groups were treated with 20 ng/mL platelet-derived growth factor (PDGF)-BB, while cells in the control group were treated with an equal volume of solvent. After another 24 h of culture, Drp1 expression in VSMCs, and mitochondrial Drp1 and mitofusin 2 (Mfn2) expressions were detected by Western blotting, and changes in mitochondrial morphology were detected by mitochondrial fluorescent staining. (2) VSMCs were divided into a control group, a synthetic VSMCs group, and a mitochondrial fission inhibitor 1 (Mdivi-1)+synthetic VSMCs group; cells in the Mdivi-1+synthetic VSMCs group were pretreated with 50 μmol/L Mdivi-1 for 2 h; and cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 hours of continued culture, expressions of α-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), proliferating cell nuclear antigen (PCNA), and Cyclin D1 were detected by Western blotting; invasion and migration abilities of VSMCs were detected by Transwell assay and scratch wound healing assay, respectively. (3) VSMCs were divided into a control group, a synthetic VSMCs group, and a N-acetylcysteine (NAC)+synthetic VSMCs group; cells in the NAC+synthetic VSMCs group were pretreated with 5 mmol/L NAC for 1 h; cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 h of continued culture, expressions of Drp1, phosphorylated (p)-Drp1, α-SMA, SM22-α, PCNA, and Cyclin D1 were detected by Western blotting; changes in mitochondrial morphology were detected by mitochondrial fluorescent staining; intracellular ROS level was detected by 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe; cell invasion and migration abilities were detected by Transwell assay and scratch wound healing assay, respectively. (4) VSMCs were divided into a control group, a synthetic VSMCs group, and a 5-Aza-2'-deoxycytidine (5-Aza-dC)+synthetic VSMCs group; cells in the 5-Aza-dC+synthetic VSMCs group were pretreated with 2 μmol/L 5-Aza-dC for 1 h; and then, cells in the latter two groups were treated with 20 ng/mL PDGF-BB, while cells in the control group were treated with an equal volume of solvent. After 24 h of continued culture, agarose gel electrophoresis was used to analyze the methylation degree in the mitochondrial D-loop region; intracellular ROS level was detected using DCFH-DA fluorescent probe; expressions of mitochondrial DNMT1, α-SMA, SM22-α, PCNA, and Cyclin D1 were detected by Western blotting; invasion and migration abilities were detected by Transwell assay and scratch wound healing assay, respectively.Results:(1) Compared with the control group and synthetic VSMCs group, the Drp1 siRNA+synthetic VSMCs group had significantly decreased Drp1 protein expression ( P<0.05). Compared with the control group, the synthetic VSMCs group had significantly increased Drp1 protein expression and decreased Mfn2 protein expression in the mitochondria ( P<0.05); compared with the synthetic VSMCs group, the Drp1 siRNA+synthetic VSMCs group had statistically decreased Drp1 protein expression and increased Mfn2 protein expression in the mitochondria ( P<0.05). Results of mitochondrial fluorescent staining showed that mitochondria in the control group were with filamentous structure, while mitochondrial fission in the synthetic VSMCs group was enhanced, and morphology of mitochondria in the Drp1 siRNA+synthetic VSMCs group tended to be continuous and complete. (2) Compared with the control group, the synthetic VSMCs group had statistically decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the Mdivi-1+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster cell scratch healing; compared with the synthetic VSMCs group, the Mdivi-1+synthetic VSMCs group had smaller number of migrating cells and slower cell scratch healing. (3) Compared with the control group (1.10±0.02), the synthetic VSMCs group (1.53±0.02) had significantly increased p-Drp1 protein expression ( P<0.05). Compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group (0.90±0.02) had statistically decreased p-Drp1 protein expression ( P<0.05). Results of mitochondrial fluorescent staining showed that mitochondria in cells of the control group were in a filamentous structure, while mitochondrial fission in cells of the synthetic VSMCs group was enhanced, and morphology of mitochondria in the NAC+synthetic VSMCs group tended to be continuous and complete. Results of DCFH-DA fluorescent probe showed that ROS level in the synthetic VSMCs group was higher than that in the control group, and ROS level in the NAC+synthetic VSMCs group was lower than that in the synthetic VSMCs group. Compared with the control group, the synthetic VSMCs group had significantly decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster cell scratch healing; compared with the synthetic VSMCs group, the NAC+synthetic VSMCs group had smaller number of migrating cells and slower cell scratch healing. (4) Results of agarose gel electrophoresis showed that compared with the control group, the synthetic VSMCs group had significantly increased methylation rate in the mitochondrial D-loop region ( P<0.05); compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had statistically decreased methylation rate in the mitochondrial D-loop region ( P<0.05). Compared with the control group, the synthetic VSMCs group had statistically increased mitochondrial DNMT1 protein expression (1.03±0.03 vs. 0.55±0.03, P<0.05); and compared with the synthetic VSMCs group, the the 5-Aza-dC+synthetic VSMCs group (0.62±0.03) had significantly decreased mitochondrial DNMT1 protein expression ( P<0.05). Results of DCFH-DA fluorescent probe showed that ROS level in the synthetic VSMCs group was higher than that in the control group; ROS level in the 5-Aza-dC+synthetic VSMCs group was lower than that in the synthetic VSMCs group. Compared with the control group, the synthetic VSMCs group had significantly decreased α-SMA and SM22-α protein expressions and increased PCNA and Cyclin D1 protein expressions ( P<0.05). Compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had significantly increased α-SMA and SM22-α protein expressions and decreased PCNA and Cyclin D1 protein expressions ( P<0.05). Results of Transwell and scratch wound healing assays showed that compared with the control group, the synthetic VSMCs group had larger number of migrating cells and faster scratch healing. Compared with the synthetic VSMCs group, the 5-Aza-dC+synthetic VSMCs group had smaller number of migrating cells and slower scratch healing. Conclusion:The mtDNA-ROS-Drp1 axis may regulate the phenotypic transformation of VSMCs by modulating mitochondrial epigenetic modifications.

Objective To construct a hybrid decision-making model that integrates knowledge-driven and data-driven approaches,and to apply it to the etiological diagnosis of ventricular tachycardia(VT).Methods Clinical practice guidelines,expert consensus documents,and medical literature in the field of ar-rhythmia diseases from 2018 to 2023 were retrieved as knowledge sources.Retrospective electronic medical re-cord data of VT patients from Fuwai Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,from 2013 to 2023 were collected as the dataset.A knowledge-driven model was constructed using a knowledge-rule-based approach to establish clinical pathways.A three-class machine learning model for VT eti-ology diagnosis was developed based on real-world data,and the best-performing model was selected as the rep-resentative of the data-driven approach.The machine learning model was embedded into the decision nodes of the clinical pathway in the form of custom operators,forming the hybrid model.The precision,recall,and F1 score of the three models were evaluated.Results Three clinical practice guidelines were included as knowl-edge sources for the knowledge-driven model.A total of 1305 patient records were collected as the dataset,and five machine learning models were constructed,with the XGBoost model performing the best.The hybrid model adopted a knowledge-driven decision-making framework,embedding the XGBoost model into the decision nodes of a two-level classification.The precision,recall,and F1 scores of the three models were as follows:the knowledge-driven model achieved 80.4％,79.1％,and 79.7％;the data-driven model achieved 88.4％,88.5％,and 88.4％;and the hybrid model achieved 90.4％,90.2％,and 90.3％.Conclusions The hybrid model integrating knowledge-driven and data-driven approaches demonstrated higher accuracy,and all its deci-sion outcomes were based on evidence-based practices,aligning more closely with the actual diagnostic reason-ing of clinicians.Further rigorous validation is needed to assess the feasibility of widely applying the hybrid model in the medical field.

Poly(lactic-co-glycolide)(PLGA)is a key excipient in long-acting sustained-release preparations,and its degradation properties directly affect the drug release behavior.In this study,PLGA microspheres were prepared by microfluidic techniques,and the morphology changes of the microspheres were observed by scanning electron microscopy(SEM).In alkaline environment,due to the accelerated hydrolysis of ester bonds,the surface of the microspheres was rapidly dissolved and eroded,and the degradation rate was significantly higher than that in acidic environment.High temperature accelerated the degradation of PLGA microspheres.Under neutral and alkaline conditions,the microspheres showed aggregation and adhesion.Under acidic conditions,the microspheres gradually decomposed into irregular fragments.The high ionic strength further promoted the surface corrosion of the microspheres,especially under extreme pH conditions.Simultaneously,PLGA microspheres encapsulating coumarin were prepared to simulate the microsphere formulation.The release rate of coumarin after degradation of the microspheres under different conditions was observed by measuring the absorbance with ultraviolet-visible spectrophotometry.The results were consistent with those of the blank microspheres.This study revealed that the degradation of PLGA microspheres was significantly pH-dependent,temperature sensitive and ion strength responsive.These findings not only helped to understand and optimize the long-term stability and controlled release performance of drug-carrying microspheres,but also provided a theoretical basis for further improvement of PLGA-based drug carrier design.

OBJECTIVES: To report the development, validation, and findings of the Multi-dimensional Attention Rating Scale (MARS), a self-report tool crafted to evaluate six-dimension attention levels. METHODS: The MARS was developed based on Classical Test Theory (CTT). Totally 202 highly educated healthy adult participants were recruited for reliability and validity tests. Reliability was measured using Cronbach's alpha and test-retest reliability. Structural validity was explored using principal component analysis. Criterion validity was analyzed by correlating MARS scores with the Toronto Hospital Alertness Test (THAT), the Attentional Control Scale (ACS), and the Attention Network Test (ANT). RESULTS: The MARS comprises 12 items spanning six distinct dimensions of attention: focused attention, sustained attention, shifting attention, selective attention, divided attention, and response inhibition.As assessed by six experts, the content validation index (CVI) was 0.95, the Cronbach's alpha for the MARS was 0.78, and the test-retest reliability was 0.81. Four factors were identified (cumulative variance contribution rate 68.79%). The total score of MARS was correlated positively with THAT (r = 0.60, P < 0.01) and ACS (r = 0.78, P < 0.01) and negatively with ANT's reaction time for alerting (r = -0.31, P = 0.049). CONCLUSIONS The MARS can reliably and validly assess six-dimension attention levels in real-world settings and is expected to be a new tool for assessing multi-dimensional attention impairments in different mental disorders.
Humans ; Adult ; Male ; Attention/physiology* ; Female ; Middle Aged ; Reproducibility of Results ; Young Adult ; Psychometrics

PURPOSE: To investigate the protective effect of sub-hypothermic mechanical perfusion combined with membrane lung oxygenation on ischemic hypoxic injury of yorkshire brain tissue caused by traumatic blood loss. METHODS: This article performed a random controlled trial. Brain tissue of 7 yorkshire was selected and divided into the sub-low temperature anterograde machine perfusion group (n = 4) and the blank control group (n = 3) using the random number table method. A yorkshire model of brain tissue injury induced by traumatic blood loss was established. Firstly, the perfusion temperature and blood oxygen saturation were monitored in real-time during the perfusion process. The number of red blood cells, hemoglobin content, NA⁺, K⁺, and Ca²⁺ ions concentrations and pH of the perfusate were detected. Following perfusion, we specifically examined the parietal lobe to assess its water content. The prefrontal cortex and hippocampus were then dissected for histological evaluation, allowing us to investigate potential regional differences in tissue injury. The blank control group was sampled directly before perfusion. All statistical analyses and graphs were performed using GraphPad Prism 8.0 Student t-test. All tests were two-sided, and p value of less than 0.05 was considered to indicate statistical significance. RESULTS: The contents of red blood cells and hemoglobin during perfusion were maintained at normal levels but more red blood cells were destroyed 3 h after the perfusion. The blood oxygen saturation of the perfusion group was maintained at 95% - 98%. NA⁺ and K⁺ concentrations were normal most of the time during perfusion but increased significantly at about 4 h. The Ca²⁺ concentration remained within the normal range at each period. Glucose levels were slightly higher than the baseline level. The pH of the perfusion solution was slightly lower at the beginning of perfusion, and then gradually increased to the normal level. The water content of brain tissue in the sub-low and docile perfusion group was 78.95% ± 0.39%, which was significantly higher than that in the control group (75.27% ± 0.55%, t = 10.49, p < 0.001), and the difference was statistically significant. Compared with the blank control group, the structure and morphology of pyramidal neurons in the prefrontal cortex and CA1 region of the hippocampal gyrus were similar, and their integrity was better. The structural integrity of granulosa neurons was destroyed and cell edema increased in the perfusion group compared with the blank control group. Immunofluorescence staining for glail fibrillary acidic protein and Iba1, markers of glial cells, revealed well-preserved cell structures in the perfusion group. While there were indications of abnormal cellular activity, the analysis showed no significant difference in axon thickness or integrity compared to the 1-h blank control group. CONCLUSIONS Mild hypothermic machine perfusion can improve ischemia and hypoxia injury of yorkshire brain tissue caused by traumatic blood loss and delay the necrosis and apoptosis of yorkshire brain tissue by continuous oxygen supply, maintaining ion homeostasis and reducing tissue metabolism level.
Animals ; Perfusion/methods* ; Disease Models, Animal ; Brain Injuries/etiology* ; Swine ; Male ; Hypothermia, Induced/methods*

OBJECTIVES: To determine the pharmacological impact of hesperidin, the main component of Citri Reticulatae Pericarpium, on depressive behavior and elucidate the mechanism by which hesperidin treats depression, focusing on the gut-brain axis. METHODS: Fifty-four Sprague Dawley male rats were randomly allocated to 6 groups using a random number table, including control, model, hesperidin, probiotics, fluoxetine, and Citri Reticulatae Pericarpium groups. Except for the control group, rats in the remaining 5 groups were challenged with chronic unpredictable mild stress (CUMS) for 21 days and housed in single cages. The sucrose preference test (SPT), immobility time in the forced swim test (FST), and number in the open field test (OFT) were performed to measure the behavioral changes in the rats. Enzyme-linked immunosorbent assay was used to determine the levels of 5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) in brain tissue, and the histopathology was performed to evaluate the changes of colon tissue, together with sequencing of the V3-V4 regions of 16S rRNA gene on feces to explore the changes of intestinal flora in the rats. RESULTS: Compared to the control group, the rats in the model group showed notable reductions in body weight, SPF, and number in OFT (P<0.01). Hesperidin was found to ameliorate depression induced by CUMS, as seen by improvements in body weight, SPT, immobility time in FST, and number in OFT (P<0.05 or P<0.01). Regarding neurotransmitters, it was found that at a dose of 50 mg/kg hesperidin treatment upregulated the levels of 5-HT and BDNF in depressed rats (P<0.05). Compared to the control group, the colon tissue of the model group exhibited greater inflammatory cell infiltration, with markedly reduced numbers of goblet cells and crypts and were significantly improved following treatment with hesperidin. Simultaneously, the administration of hesperidin demonstrated a positive impact on the gut microbiome of rats treated with CUMS, such as Shannon index increased and Simpson index decreased (P<0.01), while the abundance of Pseudomonadota and Bacteroidota increased in the hesperidin-treated group (P<0.05). CONCLUSION The mechanism responsible for the beneficial effects of hesperidin on depressive behavior in rats may be related to inhibition of the expressions of BDNF and 5-HT and preservation of the gut microbiota.
Animals ; Hesperidin/therapeutic use* ; Rats, Sprague-Dawley ; Depression/drug therapy* ; Male ; Stress, Psychological/drug therapy* ; Brain/metabolism* ; Brain-Derived Neurotrophic Factor/metabolism* ; Serotonin/metabolism* ; Gastrointestinal Microbiome/drug effects* ; Behavior, Animal/drug effects* ; Rats ; Brain-Gut Axis/drug effects* ; Chronic Disease ; Colon/drug effects*

ObjectiveThis study proposes a novel single-cell protein localization method based on a class perception graph convolutional network (CP-GCN) to overcome several critical challenges in protein microscopic image analysis, including the scarcity of cell-level annotations, inadequate feature extraction, and the difficulty in achieving precise protein localization within individual cells. The methodology involves multiple innovative components designed to enhance both feature extraction and localization accuracy. MethodsFirst, a class perception module (CPM) is developed to effectively capture and distinguish semantic features across different subcellular categories, enabling more discriminative feature representation. Building upon this, the CP-GCN network is designed to explore global features of subcellular proteins in multicellular environments. This network incorporates a category feature-aware module to extract protein semantic features aligned with label dimensions and establishes a subcellular relationship mining module to model correlations between different subcellular structures. By doing so, it generates co-occurrence embedding features that encode spatial and contextual relationships among subcellular locations, thereby improving feature representation. To further refine localization, a multi-scale feature analysis approach is employed using the K-means clustering algorithm, which classifies multi-scale features within each subcellular category and generates multi-cell class activation maps (CAMs). These CAMs highlight discriminative regions associated with specific subcellular locations, facilitating more accurate protein localization. Additionally, a pseudo-label generation strategy is introduced to address the lack of annotated single-cell data. This strategy segments multicellular images into single-cell images and assigns reliable pseudo-labels based on the CAM-predicted regions, ensuring high-quality training data for single-cell analysis. Under a transfer learning framework, the model is trained to achieve precise single-cell-level protein localization, leveraging both the extracted features and pseudo-labels for robust performance. ResultsExperimental validation on multiple single-cell test datasets demonstrates that the proposed method significantly outperforms existing approaches in terms of robustness and localization accuracy. Specifically, on the Kaggle 2021 dataset, the method achieves superior mean average precision (mAP) metrics across 18 subcellular categories, highlighting its effectiveness in diverse protein localization tasks. Visualization of the generated CAM results further confirms the model’s capability to accurately localize subcellular proteins within individual cells, even in complex multicellular environments. ConclusionThe integration of the CP-GCN network with a pseudo-labeling strategy enables the proposed method to effectively capture heterogeneous cellular features in protein images and achieve precise single-cell protein localization. This advancement not only addresses key limitations in current protein image analysis but also provides a scalable and accurate solution for subcellular protein studies, with potential applications in biomedical research and diagnostic imaging. The success of this method underscores the importance of combining advanced deep learning architectures with innovative training strategies to overcome data scarcity and improve localization performance in biological image analysis. Future work could explore the extension of this framework to other types of microscopic imaging and its application in large-scale protein interaction studies.

This study aims to comprehensively analyze the material basis of toad visceral oil(hereafter referred to as toad oil), and explore the pharmacological effect of toad oil on atopic dermatitis(AD). Ultra-high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry(UHPLC-LTQ-Orbitrap-MS) and gas chromatography-mass spectrometry(GC-MS) were employed to comprehensively identify the chemical components in toad oil. The animal model of AD was prepared by the hapten stimulation method. The modeled animals were respectively administrated with positive drug(0.1% hydrocortisone butyrate cream) and low-and high-doses(1%, 10%) of toad oil by gavage. The effect of toad oil on AD was evaluated with the AD score, ear swelling rate, spleen index, and pathological section results as indicators. A total of 99 components were identified by UHPLC-LTQ-Orbitrap-MS, including 14 bufadienolides, 7 fatty acids, 6 alkaloids, 10 ketones, 18 amides, and other compounds. After methylation of toad oil samples, a total of 20 compounds were identified by GC-MS. Compared with the model group, the low-and high-dose toad oil groups showed declined AD score, ear swelling rate, and spleen index, alleviated skin lesions, and reduced infiltrating mast cells. This study comprehensively analyzes the chemical composition and clarifies the material basis of toad oil. Meanwhile, this study proves that toad oil has a good therapeutic effect on AD and is a reserve resource of traditional Chinese medicine for external use in the treatment of AD.
Animals ; Dermatitis, Atopic/immunology* ; Disease Models, Animal ; Mice ; Male ; Gas Chromatography-Mass Spectrometry ; Humans ; Bufonidae ; Oils/administration & dosage* ; Chromatography, High Pressure Liquid ; Female ; Mice, Inbred BALB C

Objective To investigate the ameliorative effects and mechanisms of six types of tea(green tea,cyan tea,red tea,white tea,black tea and yellow tea)on metabolic disorders in obesity mice induced by high-fat diet(HFD).Methods Four-week-old male C57BL/6J mice were randomly divided into 8 groups with 7 mice per group.An HFD-induced obese mouse model was established,and the mice in control group maintained on standard diet followed by intragastric administration of different teas for 5 weeks.The body weight,liver weight ratio,fasting blood glucose,and lipid profile of the mice were measured to assess glucose and lipid metabolism.Serum inflammatory factors including IL-6,tumor necrosis factor-alpha(TNF-α)and oxidative stress markers[malondialdehyde(MDA)and superoxide dismutase(SOD)were measured.Additionally,liver histopathology and the expression of key glycolipid metabolism-related genes,adenosine monophosphate-activated protein kinase(AMPK)and carnitine palmitoyltransferase 1(CPT-1),were analyzed to explore underlying mechanisms.Results Cyan tea significantly suppressed weight gain,demonstrating superior weight control.White tea markedly reduced fasting blood glucose levels and decreased the area under the curve of oral glucose tolerance test(OGTT)and insulin tolerance test(ITT),indicating synergistic improvements in glucose metabolism and insulin sensitivity.Yellow tea exhibited exceptional anti-inflammatory and antioxidant effects,reducing hepatic IL-6 and MDA while enhancing SOD activity.Green tea activated the lipid oxidation pathway by upregulating AMPK/CPT-1 expression.All kinds of tea significantly attenuated hepatic lipid droplet accumulation.Conclusion All six types of tea alleviated metabolic disorders by reducing hepatic fat content in obesity mice.However,different types of tea exert their unique effects on improving metabolic disorders through differential mechanisms such as glucose metabolism regulation,lipid oxidation,and anti-inflammatory and antioxidant actions.