The malondialdehyde levels and apoptosis rate in H2O2+miR-182 group were significantly lower than H2O2 group . The mRNA levels and expression levels of mTOR and PI3K in H2O2+miR-182 group were higher than those in H2O2 group (both P<0.05). The experiment of cerebral ischemic oxidative stress model rats showed that the survival rate, apoptosis rate, malondialdehyde and superoxide dismutase levels in miR-182 group were better than model control group. The positive staining intensity of phosphoinositide 3-kinase (mTOR) and phosphoinositide 3-kinase (PI3K) in model control group were significantly lower than miR-182 group . Increased levels of miR-182 can reduce the damage of H2O2 treatments in RHiN cells. Oxidative stress is decreased in the neuronal cells possibly by activation of the PI3K-AKT-mTOR pathway.To explore protective effects and related mechanisms of microRNA-182 (miR-182) on oxidative stress in rat hippocampal neurons (RHiN), RHiN cells. As the results, the survival rate and superoxide dismutase levels in H These species are generated due to pathological stimulation and promote lipid peroxidation of the cell membrane system, leading to decline in DNA, protein, and other macromolecular activities . The nermicroRNA-182 (miR-182) is a single-stranded noncoding RNA consisting of 23 nucleotides. It was initially discovered in murine retinal tissue, and was found to regulate retinal sensitization in mice . Studies22.12O2 solution (Sangon Biotech (Shanghai) Co., Ltd., China) was added to the culture medium of the hippocampal cells in H2O2 group. A total 200 μmol/L of H2O2 solution and 80 μmol/L of mTOR protein  were added to the culture medium of the hippocampal cells in mTOR+H2O2 group. A total 8 μmol/L of rapamycin solution  was added to the culture medium of the hippocampal cells in mTOR inhibitor group. A total 10 μmol/L of MHY1485 solution  was added to the culture medium of the hippocampal cells in mTOR activator group. Hippocampal neurons in H2O2+miR-182 group were transfected with miR-182 and supplemented with 200 μmol/L H2O2 solution. All cell cultures were placed in an incubator at 37°C, with 5% CO2.RHiN cells were purchased from Shanghai YS Industrial Co., Ltd., China. Dulbeccoʼs Modified Eagle Medium (DMEM) culture media, consisting of 10% fetal bovine serum, penicillin (150 U/mL), and streptomycin (120 U/mL), were all purchased from Shanghai Beinuo Biological Technology Co., Ltd., China. Cell culture groups were defined by their treatments: RHiN in blank control group were routinely cultured with DMEM media culture. Hippocampal neurons in miR-182 group were transfected with miR-182  according to Lipofectamine 2000 Kit instructions . A total of 200 μmol/L of H2.24 cells per well. Cells were cultured for 24 h according to treatment groups, washed 3 times with ice-cold phosphate buffer saline (PBS), followed by addition of 25 μL of MTT (5 g/L)  to each well. Cells were cultured for an additional 4 h. Following the incubation with MTT, the supernatant was discarded and 150 μL dimethylsulfoxide  was added to each well for dissolution. A micro oscillator was used for 10 min to dissolve completely any crystals formed during the incubation. The optical density (OD) at 492 nm was determined using microplate reader . The method for calculating the cell survival rate is as follows: survival rate = (OD in the treatment group / OD in the blank control group) x 100%.Cultured cells at exponential growth phase were seeded into 96-well culture plates, 1 x10Flow cytometry was used to analyze the effect of siRNA transfection. RHiN treatment groups were cultured to logarithmic phase. These cells were digested for 5 min using 0.25% pancreatin , and then centrifuged for 10 min at 2,000 rpm/min. After collecting cells and washing twice with ice-cold PBS, 600 μL ethanol was added to cells, and placed at -4°C overnight. Cells were washed twice more with PBS and 350 μL binding buffer was added to resuspend cells. Annexin V-Enhanced Green Fluorescent Protein (EGFP)  was added for mixing. Propidium iodide (PI) (5 μL) staining solution was added, incubated for 10 min at room temperature, and flow cytometry  was carried out to detect cellular apoptosis levels. The excitation wavelength was set to Ex=488 nm, and the emission wavelength was set to Em=530 nm.2.34 cells per well. Cells were cultured for 24 h according to treatment groups. Following cell collection, radioimmunoprecipitation assay (RIPA) buffer  was added and cells were incubated for 10 min at 4°C, then centrifuged for 5 min at 12,000 rpm. Supernate was taken in low-temperature preservation. The SOD levels of RHiN were detected by enzyme-linked immuno sorbent assay (ELISA) according to the manufacturer’s instructions . The OD value of each well was determined at 450 nm. Thiobarbituric acid (TBA) was utilized to detect the levels of MDA, according to the MDA Assay Kit instructions . TBA was purchased from Beijing Biolab Technology Co., Ltd., China. The OD value of the test sample supernatant was determined at 532 nm.Exponential phase cells were seeded into 96-well culture plates, 1x102.4-ΔΔCt was used to calculate relative expression levels of target genes.RHiN cells were cultured to logarithmic phase after appropriate treatments. Following cell collection, total RNA was extracted by TRIzol reagent . A nanodrop spectrophotometer  was used for verifying the quality of RNA samples. Complementary DNA (cDNA) libraries were synthesized by RNA reverse transcription kit . Reaction conditions were set at 42°C for 15 min and 80°C for 5 min. The product was stored at 4°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene for the purpose of this study. Primer 5.0  was used to design forward and reverse amplification primers. For PI3K, the upstream primer sequence was 5ʼ-CACTGGCATCCTCACTCAC-3ʼ and downstream primer sequence was 5ʼ-CTGACTGACTCACTGC-3ʼ. For mTOR, the upstream primer sequence was 5ʼ-CGTCAACCATCCATGTAC-3ʼ and the downstream primer was 5ʼ-CGTACTGCATGCACTCATGCC-3ʼ. For GAPDH, the upstream primer was 5ʼ-TCACTGCACTGCA-CTGCAC-3ʼ, and the downstream primer was 5ʼ-CGCTGACTGACGTCAC-3ʼ. Reaction conditions were initiated at 95°C for 30 s, 95°C for 5 s, 62°C for 20 s, 72°C for 30 s, and the reaction was run for 32 cycles. The 22.5× protein loading buffer  were mixed. After boiling for 6 min, samples were cooled to room temperature. Denatured protein (15 μg) was added to sample wells of 10% of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and proteins were electrophoresed. Following electrophoresis, total proteins were transferred to polyvinylidene fluoride (PVDF) membrane  by wet-transfer method. Confining liquid with 5% skim milk powder was used to block the membrane with proteins for 1 h. Rabbit anti-rat PI3K and mTOR protein monoclonal antibodies   were added, placed at 4°C for incubation overnight. After tris buffered saline with tween (TBST) washing the membrane, HRP-labeled sheep anti-rabbit IgG secondary antibody   was incubated with the membrane for 1 h. Electrogenerated chemiluminescence (ECL) was used to detect labelled proteins on the membrane. Exposure and photography were performed, followed by film analysis. Image J software was used to measure the gray values of selected protein stripes.RHiN cells in the logarithmic phase were collected. RIPA lysis buffer was used to treat cells for 15 min prior to total protein extraction. Protein concentration was determined using the bicinchoninic acid assay (BCA assay). The protein sample solution and 52.62O2. PBS was used to rinse the samples, and pH 6.0 citrate buffer  was added to the tissue. Tissue samples were boiled for 15 min. After cooling, 5% goat serum (Sangon Biotech (Shanghai) Co., Ltd., China) was dropped and then closed for 1 h. After rinsing with PBS, rabbit anti-rat PI3K and mTOR protein primary antibodies  were added to the tissue, and incubated at room temperature for 2 h. PBS washes were carried out to remove primary antibody from the samples, and sheep anti-rabbit IgG secondary antibody  was added for incubation at room temperature for 1 h. Diaminobenzidine (DAB) (Sangon Biotech (Shanghai) Co., Ltd., China) was added for straining following the secondary antibody washes. Tissue samples were re-strained, dehydrated and hyalinized, followed by application of a neutral gum seal before being placed under the microscope  for observation and documentation.Thirty 10-week-old clean-grade male Wistar rats  were purchased with a body weight of 250-300 g, feeding temperature of 22-25 degrees and relative humidity of 45%-55% (license number: SCXK2004-2005). Rats were divided into blank control group, model control group and miR-182 group, with 10 rats in each group. The rats of miR-182 group were injected with miR-182 (15 ng/d) via tail vein injection continuously for 7 days, and the rats of the other two groups were given free diet. On the 8th day, a model of global cerebral ischemia was established by modified Longa method in model control group and miR-182 group. After 90 min of ischemia in rats, reperfusion was performed for 24 h. After anesthesia, blood was collected from the abdominal aorta and rats were killed by cervical dislocation. Hippocampal sections were collected and hippocampal neuron cells were isolated to detect cell viability, apoptosis levels, serum SOD and MDA levels. Hippocampal sections were prepared following paraffin-embedded treatment. Sections were cut to a thickness of 5 μm, and then placed in a 60°C incubator for 2 h. Xylene  was used for dewaxing and hydration, then incubated for 10 min in a 3% solution of HEthical approval The research related to animal use has complied with all the relevant national regulations and institutional policies for the care and use of animals.2.7SPSS19.0 software was used to analyze data. The measurement data were expressed as mean ± standard deviation 33.12O2 group and mTOR inhibitor group were 57.62±3.94% and 64.39±4.95%, significantly lower than that in blank control group (both P<0.05). The survival rates of RHiN cells in H2O2+miR-182 group and H2O2+mTOR group were 73.64±5.37% and 78.13±6.51% respectively, significantly higher than that observed in H2O2 group (both P<0.05). Flow cytometry analysis indicated that the apoptosis rate of RHiN cells in H2O2 group was 22.36±3.15%, significantly higher than blank control group (P<0.05). The apoptosis rate of RHiN cells in miR-182 group was 4.27±1.12%, significantly lower than blank control group (P<0.05). The apoptosis rates of RHiN cells in H2O2+miR-182 group and H2O2+mTOR group were 8.14±2.18% and 9.32±2.43% respectively, significantly lower than that in H2O2 group (both P<0.05), as shown in The survival rates of RHiN cells in miR-182 group and mTOR activator group were 117.56±6.75% and 116.48±7.28%, significantly higher than blank control group (both P<0.05). The survival rates of RHiN cells in H3.22O2 group and mTOR inhibitor group were 0.75±0.08 μmol/mL and 0.79±0.07 μmol/mL, significantly higher than blank control group (both P<0.05). MDA levels in RHiN cells from H2O2+miR-182 group and H2O2+mTOR group were 0.47±0.05 μmol/mL and 0.43±0.04 μmol/mL, significantly lower than H2O2 group (both P<0.05). SOD levels in RHiN cells from miR-182 group and mTOR activator group were 17.75±0.49 U/mL and 18.14±0.74 U/mL, significantly higher than blank control group (both P<0.05). SOD levels in RHiN cells from H2O2 group and mTOR inhibitor group were 5.34±0.12 U/mL and 6.04±0.16 U/mL, significantly lower than blank control group (both P<0.05). SOD levels in RHiN cells from H2O2+miR-182 group and H2O2+mTOR group were 11.42±0.42 U/mL and 12.37±0.62 U/mL, significantly higher than those in H2O2 group (both P<0.05), as illustrated in MDA levels in RHiN cells from miR-182 group and mTOR activator group were 0.34±0.03 μmol/mL and 0.31±0.05 μmol/mL, significantly lower than blank control group (both P<0.05). MDA levels in RHiN cells from H3.32O2 group and mTOR inhibitor group were 0.64±0.08 and 0.43±0.06, lower than blank control group (both P<0.05). The mTOR mRNA levels in RHiN cells from H2O2+miR-182 group and H2O2+mTOR group were 0.82±0.13 and 0.85+0.14, significantly higher than H2O2 group (both P<0.05). The PI3K mRNA level in miR-182 group was 1.13±0.03, significantly higher than H2O2 group (P<0.05). The PI3K mRNA level in H2O2 group was 0.48±0.04, significantly lower than blank control group (P<0.05). The PI3K mRNA levels in H2O2+miR-182 group and H2O2+mTOR group were 0.87±0.06 and 0.89±0.09, which were significantly higher than those in H2O2 group (both P<0.05). Comparisons of the mRNA expression levels are shown in RT-PCR experiments showed that the mTOR mRNA level in RHiN cells from mTOR activator group was 1.17±0.19, higher than blank control group (P<0.05). The mTOR mRNA levels in RHiN cells from H3.42O2 group and mTOR inhibitor group were 0.18±0.03 and 0.23±0.04, lower than blank control group (both P<0.05). The expression levels of mTOR protein in H2O2+miR-182 group and H2O2+mTOR group were 0.45±0.06 and 0.42±0.04, significantly higher than H2O2 group (both P<0.05). The expression level of PI3K protein in miR-182 group was 0.57±0.08, significantly higher than H2O2 group (P<0.05). The expression level of PI3K protein in H2O2 group was 0.23±0.06, significantly lower than blank control group (P<0.05). The expression levels of PI3K protein in H2O2+miR-182 group and H2O2+mTOR group were 0.36±0.04 and 0.38±0.05, significantly higher than H2O2 group (both P<0.05). Comparisons of the protein expression levels are shown in Western Blot evaluation of protein expressions determined that the expression level of mTOR protein in mTOR activator group was 0.71±0.18, higher than blank control group (P<0.05). The expression levels of mTOR protein in H3.5The survival rate and SOD level of rat hippocampal neuronal cells in miR-182 group were 62.15±4.28% and 11.36±2.41 U/mL respectively, significantly higher than model control group (both P<0.05). The apoptosis rate and MDA level of rat hippocampal neuronal cells in miR-182 group were 13.12±2.16% and 0.41±0.05 μmol/mL, significantly lower than model control group (both P<0.05). Immunohistochemical staining showed that the intensities of mTOR and PI3K in model group were 124.57±18.43 and 87.26±18.43 respectively, significantly lower than blank control group (P<0.05). Positive staining intensities of mTOR and PI3K were 141.25±16.52 and 124.18±19.49 respectively in miR-182 group, which were significantly higher than that in model blank group (both P<0.05), as shown in 4The hippocampus is central to information storage and memory functions in the brain. Oxidative stress injury to RHiN cells can lead to neurological deficits in the brain. Furthermore, oxidative injuries may cause various neurodegenerative diseases such as Alzheimerʼs disease and Parkinsonʼs disease . Atalay 2O2+miR-182 group was increased after miR-182 transfection, as demonstrated by effective reduction of cellular apoptosis. Apoptosis results indicate that miR-182 is involved in regulating the oxidative stress response of rat neurons, which effectively reduces the damage incurred from oxidative stress. SOD is an enzyme that can effectively remove free radicals in vivo, thereby influencing cellular antioxidant levels. When the levels of SOD are reduced, there are excess free radicals without an effective removal system. Oxidative damage of brain neurons from this excess of radicals has been reported [2O2 treatment and MDA levels were increased, suggesting higher levels of oxidative stress compared to controls. After transfection of miR-182 in rat neurons, the levels of SOD increased and MDA levels decreased. This affect indicates that miR-182 transfection alleviates oxidative stress in neuronal cells, which is consistent with previous studies [In this study, cell viability of RHiN cells in the Hreported , 14. MDAreported , 16, 17. studies .2O2. The expression levels of mTOR and PI3K proteins were decreased as a result of the peroxide challenge. Transfection of miR-182 increased the expression of mTOR and PI3K proteins in rat neurons and activated the PI3K-AKT-mTOR signaling pathway, with its concomitant positive effects on relieving neuronal oxidative stress. Our results suggest that miR-182 increases the antioxidant capacity of neuronal cells. At the same time, our study showed that miR-182 could alleviate the damage of oxidative stress on rat hippocampal neuronal cells in cerebral ischemic oxidative stress model rats. The mechanism of action of miR-182’s influence on the expression of mTOR and PI3K in rat neurons is beyond the scope of this study, however our initial indications warrant further investigations of related mechanisms.The PI3K-AKT-mTOR signaling pathway plays an important role in regulating cell growth, apoptosis, autophagy and cellular antioxidant capacity. PI3K is a key protein in the signaling pathway, as it can be phosphorylated to form phospholipid creatine phosphate, thereby prompting the phosphorylation of AKT kinase . mTOR is2O2. The mechanism of miR-182’s efficacy involves activation of the PI3K-AKT-mTOR signaling pathway as a means for alleviating the damage from free radicals within the neuronal cells.In conclusion, miR-182 can effectively alleviate oxidative damage to rat hippocampal neurons induced by H


PM2.5-induced airway inflammation may decrease innate immune defenses against bacterial infection. However, there is currently limited information available regarding the effect of PM2.5 exposure on molecular interactions between pneumococcus and macrophages.Pneumococcus is one of the most common human airway pathogens that causes life-threatening infections. Ambient fine particulate matter (PM) with aerodynamic diameter ≤ 2.5 μm (PM2.5 exposure hampered macrophage functions, including phagocytosis and proinflammatory cytokine production, in response to pneumococcal infection. In a PM2.5-exposed pneumococcus-infected mouse model, PM2.5 subverted the pulmonary immune response and caused leukocyte infiltration. Further, PM2.5 exposure suppressed the levels of CXCL10 and its receptor, CXCR3, by inhibiting the PI3K/Akt and MAPK pathways.PM2.5 exposure on macrophage activity enhances pneumococcal infectivity and aggravates pulmonary pathogenesis.The effect of PM PM withammation . Epidemiortality . Low PM2th risks .Pneumococcus, a Gram-positive coccus, is the most common cause of global pneumonia mortality . AlveolaPseudomonas aeruginosa growth [Mounting evidence shows that exposure to ambient pollution particles impairs pulmonary functions and favors infectious diseases –15. For a growth . Recentla growth . These l2.5 exposure in the respiratory tract, particularly with respect to macrophages against bacterial infection, remains unclear. In this study, we aimed to investigate the effects of long-term PM2.5 exposure on macrophage activation against pneumococcal infection. We established a PM2.5-loaded murine model that was pneumococcus-infected to investigate macrophage functions and pulmonary pathogenesis. Further, molecular studies were performed to determine the effect of PM2.5 on signaling pathways and whether it acts as an immune suppressor in response to bacterial challenge, leading to the exacerbation of pneumococcus-induced lung pathogenesis.Ambient particles have been found in airway macrophages in the septum of healthy individuals , and theAntibodies against PI3K, p-Akt, t-Akt, p-p38, t-p38, p-JNK, t-JNK, p-Erk, t-Erk, and p-p65 were purchased from Cell Signaling Technology . Antibody specific to β-actin was obtained from Santa Cruz Biotechnology . Antibodies against inducible nitric oxide synthase (iNOS) and high mobility group box 1 (HMGB1) were purchased from Abcam . Antibodies specific to t-p65 and CXCR3 were purchased from GeneTex  and Novus Biologicals , respectively. Inhibitors specific to Erk (PD98059), p38 (SB203580), JNK (SP600125), and NF-κB (JSH-23) were purchased from Sigma-Aldrich .2 [Streptococcus pneumoniae strain TIGR4  was cultured on blood agar plates  and incubated at 37 °C under 5% CO2 [Macrophage cell line RAW264.7 (ATCC TIB-71) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  containing 10% complement-inactivated fetal bovine serum  and incubated at 37 °C in a humid atmosphere containing 5% CO2 . Streptor 5% CO2 . The bac2.5) (RM8785) was purchased from National Institute of Standards and Technology  [Particulate matter less than 2.5 μm diameter  . The par5) were seeded in 96-well plates and treated with low (5 μg/ml) or high (20 μg/ml) doses of PM2.5 for 24 h. Cells were incubated with 0.5 mg/ml 3--2,5-diphenyltetrazolium bromide (MTT) solution for 2 h. Formazan crystals were dissolved in isopropanol, and the absorbance at 570 nm was determined by a spectrophotometer  [2.5-untreated group.RAW264.7 cells (1 × 102.5 affects the phagocytic activity of macrophages [6) were treated with low (5 μg/ml) or high (20 μg/ml) doses of PM2.5 for 24 h. Latex beads coated with fluorescent-labeled rabbit IgG were incubated with cells at 37 °C for 3 h. The cells were fixed with 4% paraformaldehyde followed by staining with Hoechst 33342 . The signals of fluorescein isothiocyanate (FITC) and Hoechst 33342 were analyzed under a Laser Scanning Confocal Microscope .The Phagocytosis Assay Kit (IgG FITC)  was employed to analyze whether PMrophages . RAW264.2.5 for 24 h prior to infection with pneumococcus (MOI = 10) for 6 h. The infected cells were treated with gentamicin (100 μg/ml) for 1.5 h to kill extracellular bacteria. The cells were lysed with sterilized water and cell lysates were seeded on blood agar plates by serial dilution. Visible colony-forming units (CFU) were calculated to determine the bacterial internalization activity.A gentamicin protection assay was used to analyze the bacterial internalization by macrophages . Briefly6) were untreated or treated with PM2.5 (20 μg/ml) for 24 h followed by infection with pneumococcus (MOI = 10) for an additional 6 h. Cells were washed and lysed with 100 μl RIPA containing protease and phosphatase inhibitors , and then subjected to western blot assay. The samples were resolved by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes . The membranes were blocked by 5% skim milk and incubated with the primary antibodies followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Millipore). The proteins of interests were detected using ECL Western Blotting Detection Reagent  and analyzed by Azure C400  and AzureSpot Analysis Software (Azure Biosystems) [RAW264.7 cells (2 × 10systems) . To dete5) were untreated or treated with 20 μg/ml PM2.5. After incubation for 24 h, the cells were infected with pneumococcus (MOI = 10) for 6 h. The culture medium was collected and the nitric oxide production was assessed by using Griess reagent (Sigma-Aldrich) [RAW264.7 cells (1 × 10Aldrich) .−ΔΔCt method. The method was used to calculate fold changes in each treatment group.To explore the mRNA levels of iNOS, CD80, CD86, CD163, CD206, and F4/80 in macrophages, we performed qRT-PCR analysis in this study. The oligonucleotide primers for qRT-PCR quantification are shown in Table 2.5 for 24 h, the cells were uninfected or infected with pneumococcus (MOI = 10) for 6 h. The supernatant was collected from cell culture, and the expression levels of sHMGB1, IL-1α, IL-1β, TNF-α, CXCL9, CXCL10, and CXCL11 were analyzed using sandwich enzyme-linked immunosorbent assay  [RAW264.7 cells were untreated or treated with 20 μg/ml PMlis, MN) .2.5, pneumococcus, and PM2.5 + pneumococcus  and 4 mice for histopathological examination). PM2.5 was administered by intratracheal (i.t.) instillation twice per week for 3 weeks . Amongst, two mice in PM2.5 + pneumococcus group died during PM2.5 exposure period and were excluded in the following studies. Mice were placed in the chambers, allowed to rest for 4 days, and then infected with pneumococcus by intranasal (i.n.) injection (1 × 108 CFU/10 μl). After infection for 48 h, the mice were euthanized, and the BALF and lungs were isolated as described previously [Male BALB/c mice (aged 6 weeks) were obtained from the National Laboratory Animal Center . The mouse experiments were performed in accordance with the Animal Care and Use Guidelines for Chang Gung University under a protocol approved by the Institutional Animal Care Use Committee . Mice were divided into four groups for the treatments with PBS (mock), PMeviously . Bacterieviously . The inf2. The expression levels of cytokines were expressed as the average signal intensity of duplicate spots subtracted from signal background and normalized to total protein concentration.BALF prepared from each mouse in the same group were pooled into one sample and analyzed by Proteome Profiler Array (R&D Systems). Images were captured using an Azure C400 . The quantifications of each dot were measured by Image J, and the fold changes were calculated by LogLung tissues isolated from mice were prepared for hematoxylin-eosin (H&E) or immunohistochemistry (IHC) staining as described previously . The lunt-test for two groups and one-way ANOVA with Tukey post-hoc test for more than two groups. P-value of less than 0.05 was considered statistically significant.Statistical analysis was performed using the SPSS program , and all data are shown as mean ± standard deviation (SD). Statistical significance was determined by Student’s 2.5)  or high (20 μg/ml) PM2.5 doses for 24 h. Cell viability was minimally affected by both doses  Fig. a. RAW264ses Fig. b. Additival Fig. c. Theref2.5 exposure affects macrophage phagocytic activity, we analyzed phagocytosis using antibody-coated latex beads and fluorescence. Low PM2.5 dose (5 μg/ml) slightly reduced macrophage internalization of the latex beads  treatment dramatically reduced latex bead internalization, indicating that PM2.5 hinders macrophage phagocytic activity. We further determined bacterial internalization by macrophages using the gentamicin protection assay. As shown in Fig. 2.5. These results indicate that PM2.5 exposure impairs macrophage phagocytic activity and may reduce pneumococcal clearance.To examine whether PM2.5 on NO production and pneumococcal clearance by macrophages were investigated. Our results showed that iNOS expression was higher in both PM2.5-exposed and pneumococcus-infected cells than in untreated mock cells  generated by inducible nitric oxide synthase (iNOS) in macrophages is known to kill microorganisms . Therefolls Fig. a. In concus Fig. b. These 2.5 on the inflammatory response, we measured the production of several proinflammatory cytokines. HMGB1 exerts proinflammatory activities when released from macrophages [2.5 exposure , followed by pneumococcal infection for 48 h. The body weights and temperatures of the mice were measured every 3 days, and were not observed to be different among the four groups , which was followed by PM2.5 and pneumococcus treatment. As shown in Fig. 2.5 exposure decreased the level of phosphorylated Erk and the expression of iNOS, HMGB1, and CXCR3 in pneumococcus-infected macrophages. In parallel, pneumococcus-induced iNOS, HMGB1, p-Erk, and CXCR3 were significantly suppressed in cells pretreated with PD98059. Interestingly, the suppressive effect of PM2.5 on pneumococcus-induced inflammatory molecules was augmented by PD98059. This trend was also observed during the analysis of NO production and HMGB1 secretion , or SP600125 (JNK inhibitor) . These results demonstrate that PM2.5 inhibits pneumococcus-stimulated macrophage activation through the MAPK and NF-κB signaling pathways.We then conducted an inhibition assay to analyze the involvement of the MAPK and NF-κB signaling pathways in the macrophage function-impairing effect of PMbitor PD9059, whicbitor PD9059, whic2.5 has a larger surface and can adhere to transient metals, toxic substances, and pathogens, and can be inhaled into the respiratory system [2.5 exposure is associated with several respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma [2.5 can penetrate the respiratory barrier and enter the circulatory system, therefore spreading throughout the body, leading to cardiovascular diseases, hypertension, diabetes mellitus, and systemic inflammation [2.5 [2.5 poses adverse health risks, the current study comprehensively explored the mechanism underlying the PM2.5-mediated impairment of the primary immune defense against bacterial infection.Air pollution has become a serious public health concern worldwide. PMy system –38. A grd asthma , 40. Notammation –44. Alveion [2.5  that impion [2.5 , 18. Giv2.5 subverts macrophage phagocytic activity, and decreases proinflammatory cytokine production in response to pneumococcal infection. Furthermore, the nitric oxide level, which exhibits antimicrobial activity, was suppressed by PM2.5-mediated inhibition of iNOS expression. Collectively, these findings provide information on how particulate matter subverts macrophage activity to enhance bacterial infectivity.Phagocytosis is a strategy by which macrophages trigger lysosomes to degrade internalized bacteria . The act2.5 exposure levels (≥ 100 μg/ml) are known to enhance proinflammatory cytokine production, resulting in lung toxicity [2.5 levels (20 μg/ml). In contrast, pneumococcal infection markedly enhanced proinflammatory cytokine secretion; however, this trend was in turn remarkably reduced by PM2.5 exposure. The low dose of PM2.5 administered to macrophages before pneumococcal challenge may have substantially suppressed proinflammatory cytokine production rather than being cytotoxic. The association between PM2.5 exposure at different concentrations and impaired macrophage function following pneumococcal infection merits further investigation.High PMtoxicity –50. Howe2 particles [It has been reported that PM induces inflammatory response through the TLR2 and TLR4 pathways , 52. TLRarticles . Collect2.5-exposure or bacterial infection but decreased by PM2.5 + pneumococcus co-treatment. A similar trend was also observed for the expression level of phosphorylated p65. PM2.5-treatment plus bacterial infection inhibited the production of proinflammatory cytokines including IL-1α, IL-1β, and TNF-α, thereby enhancing bacterial infectivity. HMGB1 appears to significantly manipulate macrophage proinflammatory cytokine production during bacterial infection. However, the mechanism underlying PM2.5 suppression of HMGB1-mediated immune defense against pneumococcus remains to be elucidated.HMGB1 is one of the danger-associated molecular pattern (DAMP) proteins, which are endogenous danger signals . HMGB1 i2.5 exposure significantly induced the inflammatory M1 polarization, which contributed to lung disorders [2.5 activated M2-polarization to exacerbate lung eosinophilia and allergic responses [2.5 on macrophage phenotypic transition. The obtained ELISA and qRT-PCR results demonstrated that pneumococcal infection activated M1 macrophages  and M2 (anti-inflammatory phenotype) . Previouisorders , 63. In esponses , 65. How2.5 treatment. Furthermore, we identified that pneumococcus-induced CXCL10/CXCR3, MAPK, and NF-κB signaling pathway activation was suppressed by PM2.5  untreated (mock) or (B) treated with 20 μg/ml PM2.5 for 24 h and subjected to light microscopic analysis. Arrows represent macrophages with phagocytosed PM2.5. Scale bars, 5 μm. Figure S2. The body weight and temperature of mice during the experiment. Mice were divided into four groups (10 per group) for the treatments with PBS (mock), PM2.5, pneumococcus (Sp), and PM2.5 + pneumococcus, respectively. The body weights and temperatures of the mice were measured every three days during the experiment. Figure S3. Long-term PM2.5-exposure decreases the expression of CXCR3 and IL-1β in pneumococcus-infected lung tissues. Mice were administered PM2.5 and pneumococcus-infected as described in Fig. Figure S4. Involvement of the MAPK and NF-κB signaling pathways in the suppression of pneumococcus-induced nitric oxide production by PM2.5. RAW264.7 cells were pretreated with SB203580 (10 μM), SP000125 (10 μM), or JSH-23 (20 μM) for 1 h, and exposed to PM2.5 (20 μg/ml) for 24 h, followed by pneumococcal infection for 6 h. The nitric oxide concentration was determined using Griess reagent. Statistical significance was evaluated using one-way ANOVA followed by a post-hoc test  to compare with pneumococcus infection alone group. Figure S5. PM2.5 subverts the macrophage polarization. RAW264.7 cells were unexposed or exposed to 20 μg/ml of PM2.5 for 24 h, followed by pneumococcal challenge for 6 h. mRNA levels of (A) iNOS, (B) CD80, (C) CD86, (D) CD163, (E) CD206, and (F) F4/80 in macrophages were determined by qRT-PCR analysis. Data are presented as means ± standard deviations from triplicate independent experiments. Statistical significance was analyzed using one-way ANOVA, followed by a post-hoc test . (G) Mouse lung tissues were subjected to immunohistochemical (IHC) and stained with anti-F4/80 antibody . Magnified images of each cropped area are shown in the lower panel. Arrows in red indicated F4/80-positive cells. Scale bars, 100 μm. Table S1. Primers used for qRT-PCR. Table S2. Differentiated cell counts in BALF.


Klebsiella pneumoniae, an environmental pathogen causing mastitis in dairy cattle, is often resistant to antibiotics. K. pneumoniae was used as the host bacteria to support bacteriophage replication; 2 bacteriophages, CM8-1 and SJT-2 were isolated and considered to have therapeutic potential. In the present study, we determined the ability of these 2 bacteriophages to mitigate cytotoxicity, pathomorphological changes, inflammatory responses and apoptosis induced by K. pneumoniae (bacteriophage to K. pneumoniae MOI 1:10) in bovine mammary epithelial cells (bMECs) cultured in vitro.K. pneumoniae, but each bacteriophage significantly suppressed damage and decreased TNF-α and IL-1β concentrations. K. pneumoniae enhanced mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, IL-6, IL-8, caspase-3, caspase-9 and cyt-c in bMECs and increased apoptosis of bMECs, although these effects were mitigated by treatment with either bacteriophage for 8 h.Bacteriophages reduced bacterial adhesion and invasion and cytotoxicity (lactate dehydrogenase release). Morphological changes in bMECs, including swelling, shrinkage, necrosis and hematoxylin and eosin staining of cytoplasm, were apparent 4 to 8 h after infection with K. pneumoniae-induced inflammation in bMECs cultured in vitro. Therefore, the potential of these bacteriophages for treating mastitis in cows should be determined in clinical trials.Bacteriophages CM8-1 and SJT-2 mitigated  Klebsiella pneumoniae, a common environmental pathogen, causes mastitis in dairy cows [E. coli [K. pneumoniae, it often has single or multidrug resistance [Bovine mastitis caused by infection with pathogenic microorganisms reduces milk production and quality, inflicting huge economic losses in the dairy industry , 2. Klebiry cows , 4, acco[E. coli , 6. AlthK. pneumoniae can multiply rapidly in bovine mammary tissues, causing a severe inflammatory response and tissue damage [K. pneumoniae, infection of bMECs is an important component. Antibiotics are commonly used to treat bovine mastitis and consequently, drug resistance in gram-negative bacteria, including K. pneumoniae, is rapidly increasing [K. pneumoniae.Bovine mammary epithelial cells (bMECs) are the first line of defense against invading pathogens that enter the udder through the streak canal, at the distal end of the bovine teat , 7. Thuse damage , 15. In creasing , 16, 17.in vivo and in vitro without harmful effects on host cells and have been widely used in various fields [Staphylococcus aureus infections [K. pneumoniae has apparently not been reported. Therefore, our objective was to investigate the role of bacteriophages for controlling K. pneumoniae in an in vitro infection model of bMECs.Bacteriophages are small viruses, nearly ubiquitous in the natural environment . Bacteris fields , 20. Forfections . Furtherfections . HoweverK. pneumoniae bacteriophages CM8-1 and SJT-2 are shown  of K. pneumoniae to bMECs in the K. pneumoniae + CM8-1 and K. pneumoniae + SJT-2 groups as compared to the K. pneumoniae group at 1.5, 2, 2.5 and 3 h post infection (hpi)  compared to the Control group  than that of the K. pneumoniae group at 2, 2.5 and 3 hpi  than in the Control group  than in the Control group  at 4, 6 and 8 hpi with K. pneumoniae  in bMECs were determined by real-time PCR. At 2 hpi, mRNA expression levels of TLR4, NF-κB, TNF-α, IL-1β, IL-6 and IL-8 in bMECs were significantly higher in the K. pneumoniae, mRNA expression levels of apoptosis factors caspase-3 and caspase-9 were higher (P < 0.05) in the K. pneumoniae groups than in the Control group  than in the Control group at 2 hpi  compared to the K. pneumoniae group at 4, 6 and 8 hpi  is an important marker of bacterial cytotoxicity , 29. Infl damage , 30. In K. pneumoniae, reducing its virulence and mitigating damage to bMECs, manifested by less morphological damage and lower LDH concentrations.A lack of toxic effects on host cells is characteristic of bacteriophages , 31. TreK. pneumoniae, E. coli, S. aureus and S. agalactiae, cause inflammation by producing inflammatory factors [K. pneumoniae; furthermore, there was a positive association with duration of infection time, confirming K. pneumoniae-induced inflammation of bMECs. Gram negative bacteria such as K. pneumoniae and E. coli can activate TLR4 mediated NF-κB pathway to induce inflammation of bMECs [K. pneumoniae, mRNA levels of TLR4 and NF-κB, in the pathway of regulating inflammatory response in bMECs, were significantly up-regulated and positively associated with duration of infection. In addition, downstream genes in TLR4 and NF-κB pathways, namely mRNA expression levels of TNF-α, IL-1β, IL-6 and IL-8 in bMECs were also up-regulated, confirming that K. pneumoniae induced an inflammatory response in bMECs. However, bacteriophages CM8-1 and SJT-2 were able to lyse K. pneumoniae and mitigate the inflammatory response induced by K. pneumoniae, manifested by decreased concentrations of IL-1β and TNF-α in bMECs culture medium, as well as reduced mRNA expression levels of TLR4, NF-κB, TNF-α, IL-1β, IL-6 and IL-8.Infection of bMECs by pathogens in development of bovine mastitis is usually accompanied by a severe inflammatory response . Various factors , 33–35. of bMECs , 36, 37.K. pneumoniae induced apoptosis of bMECs. Mitochondria are involved in the common apoptotic pathway, which mainly depends on the caspase families and cyt-c for regulation [K. pneumoniae infection of bMECs presumably enhanced release of cyt-c from mitochondria, triggered the caspase-9 and caspase-3 cascade reaction and accelerated cell apoptosis. The mRNA expression of caspase-3, caspase-9 and cyt-c in bMECs significantly increased with duration of infection time, implying apoptosis was triggered by K. pneumoniae. Based on flow cytometry, the apoptosis rate of bMECs increased gradually after K. pneumoniae treatment, which further confirmed that K. pneumoniae induced apoptosis of bMECs. However, bacteriophages CM8-1 and SJT-2 decreased expression of caspase-3, caspase-9 and cyt-c, as well as apoptosis rate of bMECs. Therefore, bacteriophages CM8-1 and SJT-2 mitigated apoptosis of bMECs induced by K. pneumoniae.In this study, gulation –40. In tK. pneumoniae-induced inflammation in bovine mammary epithelial cells cultured in vitro. Therefore, the potential of these bacteriophages for treating mastitis in cows should be determined in clinical trials.Bacteriophages CM8-1 and SJT-2 mitigated K. pneumoniae (CM-KP8) with K57 capsule serotype [oC for 18 h. Thereafter, bacteria were sub-cultured on brain heart infusion broth for 18 h to mid-log phase and bacterial concentration was determined by the plate counting method for the following experiments.The bacterial strain used in this study, isolated from clinical mastitis in a dairy cow, was serotype . This isoC. Then, the bacteriophage suspension was centrifuged at 10,000 × g for 10 min to obtain bacteriophage precipitation. The above process was repeated twice to obtain pure bacteriophage. The bacteriophage precipitation was resuspended with DMEM for the following experiments.Bacteriophages CM8-1 and SJT-2 were isolated from dairy farm wastewater. Prior to each experiment, a bacterial solution in mid-log phase was mixed with bacteriophage stock solution and spread over a double-layer plate (96.6% LB liquid medium and 3.4% agar powder), with sodium magnesium (SM) buffer added after the bacteriophage had grown on the entire plate. Thereafter, this plate was placed on a shaker at 120 rpm/min for 2 h to liberate the bacteriophage from the plate and into the SM solution. The latter was collected and passed through a 0.22 µm filter to obtain the bacteriophage stock solution. Thereafter, PEG8000 (10%) was add into the bacteriophage suspension and left overnight at 4 oC in a humidified atmosphere of 5% CO2. Cells from 3 to 8 passages were used for experiments, with the same passages of cells used in each experiment. The bMECs were treated with or without K. pneumoniae  and bacteriophages CM8-1 or SJT-2 , to form the following groups: Control; K. pneumoniae infection group (K. pneumoniae), K. pneumoniae plus bacteriophage CM8-1 group (K. pneumoniae + CM8-1) and K. pneumoniae plus bacteriophage SJT-2 group (K. pneumoniae + SJT-2).Bovine mammary epithelial cells (bMECs), line MAC-T , were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) Gibco® fetal bovine serum at 37 9 PFU/mL) was dropped on a copper mesh and precipitated for 2 min at room temperature, with excess liquid absorbed by filter paper. Bacteriophages were then stained with 2% uranium acetate for 1 min, excess liquid absorbed, naturally dried for 15 min at room temperature and then observed under a transmission electron microscope  at 80 kV.A suspension (20 µL) of bacteriophages (titer = 10K. pneumoniae to cells) of 10:1, the bMECs were cultured at 37 °C in a humidified atmosphere of 5% CO2 for 0.5 h, and then with addition of bacteriophages CM8-1 or SJT-2  of 1:10 for 0.5, 1, 1.5, 2 and 2.5 h. After incubation, cells were washed with PBS (to remove non-adherent bacteria), and the cell suspension was 10-fold diluted and cultured on LB liquid medium plate for counting colony forming units. Meanwhile, after incubation, cells were washed with PBS and treated with 1 mL of DMEM containing kanamycin (100 µg/mL) and then cultured at 37 °C in a humidified atmosphere of 5% CO2 for 2 h. The culture medium was removed, cells were lysed with 0.5% TritonX-100 and the lysate diluted and cultured on LB liquid medium plate as described above. Colonies were counted and invasion rate of K. pneumoniae into bMECs was calculated.Adhesion and invasion were measured as described . The bME5 cells/well), grown to 80% confluence, then DMEM supplemented with 1% (v/v) Gibco® Fetal Bovine Serum was added. Cells were then treated with K. pneumoniae and bacteriophages CM8-1 or SJT-2, as described above. After culture for 1, 2, 4, 6 or 8 h, supernatants were collected, centrifuged at 11,586 × g for 15 min at 4 oC and LDH release measured by cytotoxicity , according to manufacturer’s instructions. Absorbance of optical density was determined at 492 nm with a microplate reader .The bMECs were seeded in 96-well plates  staining was performed according to manufacturer’s instructions. An increasing alcohol gradient  was used to dehydrate cells, which were subsequently observed under a light microscope at 40×.The bMECs were seeded in 6-well plates with glass slides and grown to 80% confluence. Cells were then treated with oC and supernatant removed. Concentrations of inflammatory cytokines TNF-α and IL-1β in supernatant were measured using ELISA kits , used in accordance with manufacturer’s instructions.Culture medium was collected, centrifuged at 11,586 × g for 10 min at 4 K. pneumoniae and bacteriophages CM8-1 or SJT-2, and cells collected for total RNA extraction. Trizol Reagent (1 mL) was pre-chilled on ice and added to cell samples for 5 min to lyse cells. Mixed liquid was centrifuged at 11,586 × g for 15 min at 4 oC and supernatant collected. Total mRNA of bMECs was extracted with mRNA extraction kit , used according to manufacturer’s instructions. Primers for GAPDH (housekeeping gene), TLR4, NF-κB, TNF-α, IL-1β, IL-6, IL-8, caspase-3, caspase-9 and cyt-c were designed using primer 5.0 software . Cell culture fluid and washing fluid were collected. Then, cells were harvested with trypsin without EDTA and washed with PBS. Cells were centrifuged at 965 × g for 10 min, supernatant removed and cells re-suspended in 195 µL Annexin V-FITC binding solution and then transferred to a sterile flow cytometry glass tube. Annexin V-FITC (5 µL) was add to the cell suspension, followed by 10 µL propidium iodide staining solution. After 20 min at room temperature in dark conditions, apoptotic cells were detected using flow cytometry.After bMECs were treated with P < 0.05 was considered significant.Data were expressed as mean ± standard deviation (SD), followed by post hoc Tukey’s tests using SPSS 22.0 . All measurements were replicated three times and


This study revealed that the antioxidant capacity of the microalgae varied between divisions, strains, and growth phase and was also related to the content of antioxidant compounds present in the cells. Carotenoids and phenolics were found to be the major contributors to the antioxidant capacity, followed by polyunsaturated fatty acids linoleic acid (LA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), and docosahexaenoic acid (DHA) compared to other fatty acids. The antioxidant capacity of the selected bacillariophytes and haptophytes was found to be positively correlated to phenolic  under exponential phase, and to carotenoid fucoxanthin and β-carotene  under stationary phase. Meanwhile, antioxidant capacity of chlorophyte strains was positively correlated with lutein, β-carotene and zeaxanthin under the exponential phase . In the stationary phase, chlorophyte strains were positively correlated with violaxanthin (0.755 with •NO–), neoxanthin , and lutein (0.582 with •NO–). This study showed that antioxidant capacity and related antioxidant compound production of tropical microalgae strains are growth phase-dependent. The results can be used to improve the microalgal antioxidant compound production for application in pharmaceutical, nutraceutical, food, and feed industry.Antioxidants found in microalgae play an essential role in both animals and humans, against various diseases and aging processes by protecting cells from oxidative damage. In this study, 26 indigenous tropical marine microalgae were screened. Out of the 26 screened strains, 10 were selected and were further investigated for their natural antioxidant compounds which include carotenoids, phenolics, and fatty acids collected in their exponential and stationary phases. The antioxidant capacity was also evaluated by a total of four assays, which include ABTS, DPPH, superoxide radical (O Under normal conditions, these radicals are part of the cellular redox signaling and immune function and are readily converted into a safer intermediate  and reactive nitrogen species (RNS), such as hydroxyl radicals (•OH), superoxide anions  Remis Beach, Selangor (N03°12′ E101°18′), (2) Teluk Ketapang Beach, Terengganu (N05°23′ E103°06′), and (3) Shrimp pond, Terengganu (N05°38′ E102°45′). For molecular identification, DNA was extracted from the microalgae cells using the DNeasy Plant Mini Kit . Prior to extraction, microalgal cell disruption was carried out using zirconium beads  to ensure the release of intracellular contents  into the medium. One milliliter of microalgal cells was bead-beaten for 2 min, centrifuged at 14,000 rpm for 1 min, and the supernatant was used for PCR amplification. For amplification of the SSU region of chlorophytes, the primer combinations used were NS1F-1650R, NS1F-1150R, 1170F-1650R, or PRIMER A-PRIMER B as described previously by Chaetoceros gracilis, Isochrysis galbana, and Chlorella vulgaris were obtained from Universiti Putra Malaysia  was obtained from the Provosoli-Guillard National Center for Culture of Marine Phytoplankton, United States, and used as the control strain. This strain is known to be rich in fucoxanthin, a carotenoid known for its antioxidant activity , 0.1 mL L–1 of trace metals 6MO7O2, 2 g L–1 CuSO4.5H2O), 2 mL of silicate solution (15 g L–1 Na2SiO3) and 0.1 mL L–1 of vitamins solution. The pre-inoculum was then inoculated at an initial optical density of 0.001 into 250 mL of fresh medium in 300 mL Erlenmeyer flasks (triplicate). Each algal culture was maintained at 25°C and salinity of 30 ppt under an illumination intensity of 150 μmol m–2 s–1 under a 12:12 h light/dark photoperiod until the exponential phase. Daily growth was monitored by optical density measurement at 750 nm (OD750) using a microplate reader . On day 7, all cells were harvested by centrifugation at 3,500 rpm for 10 min, freeze-dried, and then kept at −20°C before analysis. As a first screening process, the total antioxidant capacity of all strains was spectrophotometrically determined by their scavenging capacity using 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) total antioxidant capacity assay .A pre-inoculum was cultivated for 4 days to ensure that the cells are in the exponential phase by the time of inoculation in 100 mL of Conway medium  containi750) and dry weight (DW). The optical density was measured daily using a 96-well microplate. Briefly, 200 μL algae sample was added into each well, and OD750 was measured using a microplate reader. The total biomass dry weight (g DW L–1) of each strain was obtained according to Based on the first screening of their antioxidant capacity, 10 potential microalgal species which produced high antioxidant were selected. The selected species are comprised of bacillariophytes, chlorophytes, and haptophytes. For further experiments involving antioxidant compound characterization, each potential strain was cultivated in triplicate in a 4L Nalgene polycarbonate bottle until the stationary phase, which varied among strains. Aeration was supplied for the whole culture period to provide mixing and equal light and nutrient exposure of the algal cells. The number of days for the exponential and stationary phase of each algal strains was determined based on a preliminary growth curve experiment conducted prior using similar initial cell density and culture conditions. All strains were inoculated at an initial optical density of 0.010, and the growth of each strain was monitored by determining optical density at 750 nm (OD2•–) scavenging capacity and RNS: Nitric oxide (•NO–) scavenging capacity was also analyzed at different growth phases. Details of the measurements are shown below.The antioxidant capacity was evaluated by a total of four antioxidant assays, including the two radical scavenging assays, ABTS and DPPH, and two other assays that are more specific against the ROS: superoxide radical  was added to a tube of 1 mg of finely ground freeze-dried sample and shaken vigorously for 30 s. The sample was sonicated in an ice bath for 45 min . The samples were then centrifuged at 5,000 g for 10 min at 4°C and the supernatant was separated. The collected supernatant was combined and used immediately in analysis or purged to dryness using nitrogen gas before it was stored at -20°C.•+) assay, as originally described by •+ radical cation was prepared by mixing 7 mol m–3 ABTS  with 2.45 mol m–3 potassium persulfate  for 8 h in the dark at room temperature (21°C). Briefly, 350 μl of the microalgal extract was added to 1 mL of the ABTS•+ solution, and the absorbance was measured at 734 nm using a spectrophotometer  exactly 1 min after initial mixing and up to 6 min. The results were then expressed as micromole Trolox (TE) equivalent per gram of dry algae biomass (μmol TE g–1 DW).The total antioxidant radical-scavenging capacities were evaluated using the ABTS radical cation  stable radical, according to –1 DW.Meanwhile, superoxide anions were generated in a phenazine methosulphate-NADH system and assayed by reduction of nitro blue tetrazolium (NBT) as described by –1 DW.The nitric oxide radical scavenging activity was determined, according to a were estimated from respective peaks calibrated against pure standards .Carotenoids in each extract were tentatively identified and quantified according to using a reverse-phase gradient elution HPLC system, according to –1. EZ Chrom Elite software  was used for recording and integration. Chromatographic grade standards of fatty acids in methyl ester formed from Nu-Check-Prep  containing 20 FAME and from Kitasato University containing 13 FAME were used for tentative peak identification.Total crude lipids were extracted from the freeze-dried samples with a mixture of chloroform: methanol  according to –1 dry weight of microalgae.The total phenolic content was determined using Folin–Ciocalteu reagent described by post-hoc comparison test to measure differences between data. Statistical significance was determined at p < 0.05. Correlations among antioxidant capacities and contents of bioactive compounds were calculated using Pearson’s correlation coefficient (r). Statistical analysis was carried out using the statistical software SPPS, version 23 .The experiments were carried out in triplicate, and all results are expressed as mean ± standard error. Data were then analyzed using one-way variance analysis (ANOVA), followed by Tukey’s Nanochlorum eucaryotum (two strains), Picochlorum maculatum (two strains), Chlorella sorokiniana (two strains), Oocystis heteromucosa, Oocystis marina, and Chlamydomonas uva-maris as chlorophytes, and Amphora montana (two strains), Nitzschia capitellata (two strains), Nitzschia palea, Psammodictyon pustulatum, Pauliella taeniata, Navicula arenaria (three strains), Navicula radiosa (two strains) and Thalassiosira weisflogii (two strains) as bacillariophytes  at 34.16 ± 1.18 μmol TE g–1 DW, followed by Nanochlorum eucaryotum , and Chlorella sorokiniana (SLG4-13), and Tetraselmis suecica. On the other hand, the lowest (p < 0.05) antioxidant capacity among the chlorophyte strains was observed in Chlamydomonas uva-maris (SLG4-14) at 4.32 ± 0.02 μmol TE g–1 DW. The only haptophyte strain evaluated was Isochrysis galbana (33.60 ± 0.67 μmol TE g–1 DW), showed higher (p < 0.05) antioxidant capacity by ABTS than the bacillariophyte strain Phaeodactylum tricornutum (control) at 25.23 ± 0.12 μmol TE g–1 DW. Among the 12 pennate diatoms studied, only Amphora montana (SLG4-03) showed higher (p < 0.05) antioxidant capacity in ABTS than that of the control strain (25.23 ± 0.12 μmol TE g–1 DW) at 30.74 ± 0.05 μmol TE g–1 DW. The lowest (p < 0.05) ABTS scavenging capacity among all diatoms was observed in Navicula arenaria (SLG4-18) at only 5.33 ± 1.14 μmol TE g–1 DW, respectively. All three centric diatom strains, Chaetoceros gracilis, Thalassiosira weissflogii (TRG10-P103), and Thalassiosira weissflogii (TRG10-P105), indicated a higher (p < 0.05) total antioxidant capacity than Phaeodactylum tricornutum.The 26 indigenous microalgae showed that antioxidant capacity varied among division, class, species, and strain . Among t–1 DW (p < 0.05) antioxidant capacity than the control (6.68 ± 1.09 μmol TE g–1 DW) were Isochrysis galbana (19.01 ± 1.34 μmol TE g–1 DW), Thalassiosira weissflogii (TRG10-P105) (16.83 ± 1.03 μmol TE g–1 DW), Chaetoceros gracilis (9.56 ± 0.23 μmol TE g–1 DW), Amphora montana (SLG4-03) (8.50 ± 0.21 μmol TE g–1 DW) and Navicula radiosa (SLG4-02) (6.89 ± 0.61 μmol TE g–1 DW), respectively. Among the chlorophytes, high (p < 0.05) DPPH scavenging capacity compared to control was observed in Tetraselmis suecica, Chlorella vulgaris, Chlorella sorokiniana (SLG4-12), Nanochlorum eucaryotum (SLG4-11), Chlorella sorokiniana (SLG4-13), and Nanochlorum eucaryotum (SLG4-08) ranging from 12.29 ± 0.29 to 8.51 ± 0.28 μmol TE g–1 DW, respectively.Meanwhile, the total antioxidant capacity, according to the DPPH assay, varied from 0.68 ± 0.36 to 19.01 ± 1.34 μmol TE g–1 DW . The bacNanochlorum eucaryotum (SLG4-08), Nanochlorum eucaryotum (SLG4-11), Chlorella sorokiniana (SLG4-12), Chlorella sorokiniana (SLG4-13), and Tetraselmis suecica, 2. Bacillariophyte strains; Amphora montana (SLG4-03), Chaetoceros gracilis, Thalassiosira weissflogii (TRG10-P103) and Thalassiosira weissflogii (TRG10-P105), and 3. Haptophyte strain; Isochrysis galbana.Based on the screening results , 10 strap < 0.05) SGR, OD750 and biomass dry weight among all strains were found in the chlorophyte Tetraselmis suecica at 1.420 ± 0.31 g–1 DW day–1, 0.304 ± 0.009 OD750 and 0.524 ± 0.002 g DW L–1. Meanwhile, under the exponential phase, the highest (p < 0.05) OD750 and biomass dry weight was observed in the Chlorella sorokiniana (SLG4-13) at 0.256 ± 0.025 OD750 and 0.328 ± 0.045 g DW L–1, respectively. Bacillariophyte, chlorophyte, and haptophyte strains showed relatively higher OD750 and biomass dry weight during the stationary phase compared to that during the exponential phase.Growth characteristics in terms of maximum optical density, maximum dry biomass weight, and specific growth rate were different among the 10 selected potential strains as natural antioxidant producers . Under sIsochrysis galbana compared to chlorophytes was mainly due to their high fucoxanthin composition. The highest (p < 0.05) total carotenoid in the stationary phase was observed in Amphora montana (SLG4-03), followed by Isochrysis galbana and the control Phaeodactylum tricornutum at 18.97 ± 3.12, 15.66 ± 2.13, and 14.37 ± 1.84 mg g–1 DW, respectively. Correspondingly, the highest fucoxanthin level under the stationary phase was also observed in Amphora montana (SLG4-03) (16.90 ± 2.75 mg g–1 DW) followed by Isochrysis galbana, Phaeodactylum tricornutum, Chaetoceros gracilis, and Thalassiosira weissflogii (TRG10-P105) at 13.96 ± 1.91, 12.34 ± 1.65, 11.37 ± 2.11, and 10.29 ± 0.17 mg g–1 DW, respectively. Dry weight-specific fucoxanthin content was generally higher during the early stationary phase than in the exponential phase. Meanwhile, total carotenoids of chlorophytes tended to be higher in the exponential phase than in the early stationary phase and can be characterized by the presence of lutein, neoxanthin, violaxanthin, and zeaxanthin. Total carotenoid content (10.68 ± 4.09) was higher in the chlorophyte strain Nanochlorum eucaryotum (SLG4-08), in the exponential phase, as compared to the other chlorophyte strains with relatively rich contents of β-carotene, neoxanthin, violaxanthin, and zeaxanthin.The microalgal biomass showed division and species-specific carotenoid profiles . GeneralIsochrysis galbana under exponential phase at 749.25 μg g–1 DW, followed by Thalassiosira weissflogii (TRG10-P103)  and Chaetoceros gracilis   contained the highest amount of PUFA compared to other strains during the exponential phase at 364.33 ± 73.43 and 343.45 ± 52.69 μg g–1 DW, respectively. High PUFA composition observed in chlorophytes is contributed mainly by their α-linoleic acid (ALA) and linoleic acid (LA) content. Whereas, for bacillariophytes, PUFA was mainly contributed by their eicosapentaenoic acid (EPA) content. Among the bacillariophytes, Chaetoceros gracilis, Phaeodactylum tricornutum, and Thalassiosira weissflogii (TRG10-P103), the high EPA content was observed during the exponential phase at 94.93 ± 39.89, 82.29 ± 8.34, and 78.62 ± 1.06 μg g–1 DW, respectively.The highest fatty acid content was found in l phase) . Fatty aNanochlorum eucaryotum (SLG4-11) (91.02 ± 6.26 μg g–1 DW), followed by Tetraselmis suecica (87.38 ± 15.17 μg g–1 DW). Likewise, Nanochlorum eucaryotum (SLG4-08) under the exponential phase showed the highest LA content at 77.21 ± 7.08 μg g–1 DW compared to other strains. Among the 10 strains analyzed, only Isochrysis galbana contained docosahexaenoic acid (DHA) at 61.76 ± 9.37 μg g–1 DW during the exponential phase and 23.00 ± 4.30 μg g–1 DW during the stationary phase, respectively. Meanwhile, γ-linolenic acid (GLA) was only detected at low concentrations in Tetraselmis suecica , Isochrysis galbana , Amphora montana (SLG4-03) , and Chaetoceros gracilis . The highest ARA was observed in Amphora montana (SLG4-03) at 25.08 ± 7.06 μg g–1 DW at the exponential phase.Meanwhile, the highest ALA was observed during the exponential phase in –1 DW under the exponential phase and from 0.79 to 11.80 mg GAE g–1 DW under the stationary phase (Amphora montana (SLG4-03) and Phaeodactylum tricornutum  at 14.53 ± 2.04 and 12.46 ± 1.55 mg GAE g–1 DW, respectively (The phenolic content of the 10 strains ranged from 0.53 to 15.54 mg GAE gry phase . The phecornutum . The higectively .p > 0.05) total antioxidant capacity among the bacillariophyte and haptophyte strains compared to chlorophyte was found in Thalassiosira weissflogii (TRG10-P105) with ABTS and DPPH scavenging at 73.8 ± 1.94 (stationary phase) and 31.42 ± 0.80  μmol TE g–1 DW, respectively. ABTS scavenging activity was also high in Chaetoceros gracilis (67.79 ± 7.45 μmol TE g–1 DW) and Isochrysis galbana (63.12 ± 6.51 μmol TE g–1 DW) during the stationary phase compared to the chlorophyte strains. Meanwhile, among the chlorophyte strains, the highest (p > 0.05) total antioxidant capacity was found in Nanochlorum eucaryotum (SLG4-08) under the exponential phase with ABTS scavenging at 40.11 ± 1.11 μmol TE g–1 DW. As for DPPH, Isochrysis galbana at the exponential phase showed a scavenging capacity at 32.11 ± 0.89 μmol TE g–1 DW. The highest O2•– scavenging capacity was observed in Chaetoceros gracilis and Thalassiosira weissflogii (TRG10-P105) under the early stationary phase at 30.4 ± 3.66 and 30.94 ± 3.15 μmol TE g–1 DW, respectively  showed a group-specific variation . Higher ectively . Similarry phase .R2-values of the correlation analysis for ABTS (0.623), DPPH (0.714), and •NO– (0.786) compared to total carotenoid, total fatty acid, and PUFA, suggest that their antioxidant capacity was significantly influenced by the phenolic content during the exponential phase (p < 0.05). Meanwhile, during the stationary phase, their antioxidant capacity was significantly contributed by carotenoids, as observed with ABTS (0.565), •NO– (0.466), and O2•– (0.657). As for the chlorophyte strains, significant correlations with antioxidant capacity were observed in carotenoids (ABTS and •NO–) and phenolic (•NO– and O2•–) during both the exponential and stationary phases  was significantly (p < 0.05) influenced by their fucoxanthin, diadinoxanthin, and β-carotene content for bacillariophytes and haptophytes , lutein (0.615), and zeaxanthin (0.507). Similarly, •NO– was also largely contributed by lutein (0.794), β-carotene (0.659), and neoxanthin (0.509). Meanwhile, a significant influence of α-carotene was observed for O2•– (0.534). In the stationary phase, chlorophytes were significantly influenced (p < 0.05) by the presence of violaxanthin (0.755 with •NO–), neoxanthin , and lutein (0.582 with •NO–).Detailed analysis of the correlation with each carotenoid showed that their antioxidant capacity , ALA (0.510), and DHA (0.536) toward antioxidant capacity was observed for DPPH, while they were lower than the R2 value of total phenolic (0.714) (2•–) was observed for ARA (0.923) and EPA (0.947). In addition, LA also showed a significant contribution toward ABTS (0.566) and •NO–(0.781). Significant relationships were also observed under the stationary phases of the chlorophytes for DPPH with ARA (0.503) and EPA (0.517).Some essential fatty acids also showed a significant correlation with antioxidant capacity, especially during the exponential phase. For bacillariophytes and haptophytes , the con (0.714) . No signIn the present study, 26 indigenous tropical marine tropical microalgae from Malaysian waters were screened for their antioxidant capacity by ABTS and DPPH radical scavenging assays. Both assays measure the total antioxidant capacity through HAT and SET mechanisms of the polar and non-polar compounds present in the extract . Due to Nanochlorum eucaryotum (SLG4-08), Nanochlorum eucaryotum (SLG4-11), Chlorella sorokiniana (SLG4-12), Chlorella sorokiniana (SLG4-13), Tetraselmis suecica, Amphora montana (SLG4-03), Chaetoceros gracilis, Thalassiosira weissflogii (TRG10-P103), Thalassiosira weisflogii (TRG10-P105) and Isochrysis galbana. Among them, eight strains, except for Chaetoceros gracilis and Isochrysis galbana, which were previously isolated by Amphora montana (SLG4-03), Thalassiosira weisflogii (TRG10-P105), Chaetoceros gracilis, and Isochrysis galbana, were two to three times better in scavenging ABTS radical in comparison to the value reported by Isochrysis galbana with 15.66 ± 2.13 mg g–1 DW carotenoid content compared to 4.33 ± 0.04 mg g–1 DW and 7.75 ± 0.13 mg g–1 DW as reported by Chaetoceros gracilis also showed 12.66 ± 2.45 mg g–1 DW of total carotenoid compared to the previously reported Chaetoceros calcitrans at 2.33 ± 0.14 (–1 DW (Nanochlorum eucaryotum (SLG4-08), Amphora montana (SLG4-03) and Thalassiosira weissflogii (TRG10-P105) as shown in DPPH scavenging assay has been described as less sensitive for samples with high carotenoids, but more sensitive with high phenolic sample . Meanwhi3 ± 0.14  and 6.134 , except than Picochlorum sp., which has the highest phenolic content, specifically three phenolic acids . Scenedesmus sp. ME02 extract (high in quercetin and rutin). In this study, we showed that phenolic compounds also contribute to the antioxidant activities of bacillariophytes and haptophytes. In addition, the contribution of phenolic content to the antioxidant capacity is particularly significant during the exponential phase. Because the phenolic compound accumulation is known to be growth-dependent, where their content is reduced under nutrient-limiting conditions  (high fucoxanthin), Isochrysis galbana , Amphora montana (SLG4-03) (high fucoxanthin and ARA), Chlorella sorokiniana (SLG4-12) , and Tetraselmis suecica .This study showed that the selected 10 tropical microalgal strains belonging to Bacillariophyta, Chlorophyta, and Haptophyta, have potential as a new source of natural antioxidants, along with a substantial amount of carotenoids, fatty acids, and phenolic compounds. In particular, carotenoid and phenolic content significantly influenced the antioxidant capacity of the selected strains. This study also suggests that the antioxidant capacity and related bioactive compound production of these strains are growth phase-dependent. The higher antioxidant capacity observed in the bacillariophytes and the haptophyte was majorly influenced by their phenolic content  and their dominant carotenoid, fucoxanthin, and β-carotene (under stationary phase), which may have been acting synergistically or antagonistically with other compounds. Meanwhile, the antioxidant capacity of chlorophytes during the exponential phase is largely influenced by the presence of the carotenoid lutein and β-carotene, and during the stationary phase, by violaxanthin, neoxanthin, and lutein. The high carotenoid, phenolic, and fatty acid contents still have the potential to be enhanced further for commercial purposes by cultivation under certain environmental or abiotic stressors. Moreover, our results revealed several high antioxidants producing candidate strains that can be exploited as feedstocks for various other applications, such as pharmaceutical, aquaculture, and biofuels, with their high nutritional value. These potential strains include The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.NR, KT, and TK conceived and designed the experiments. NR collected and analyzed data, prepared the manuscript, and participated in the assembly and editing of the final manuscript. KT and TK supervised and assisted in the interpretation of data, assembly, and editing of the manuscript. TK, MW, NK, and HK assisted in collecting algal material and editing of the manuscript. YY assisted in the fatty acid analysis and the interpretation of the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.