Sjögren’s syndrome (SS) is characterized by extensive lymphocytic infiltration of the salivary and lacrimal gland (LG), resulting in acinar cell destruction and organ dysfunction. The underlying pathogenesis of SS remains largely unknown, and studies historically focus on defining late-stage disease. Here, we identify tissue programs associated with disease onset using transcriptomic and immunohistological analysis of LGs from 5- and 7-week-old mice deficient in autoimmune response element (Aire). At 5 weeks of age (wk), Aire-/- mice show minimal tissue dysfunction and destruction compared to 7 wk Aire-/-, which exhibit severe dry eye, poor tear secretion, extensive lymphocytic infiltration, reduced functional innervation, and increased vascularization. Despite this mild phenotype, 5 wk Aire-/- LGs were highly enriched for signaling pathways previously associated with SS, including interferon gamma (IFNγ), interleukin 1 beta (IL1β), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), toll-like receptor (TLR) signaling, and interleukin-6/signal transducer and activator of transcription 3 (IL6/STAT3) signaling. Novel signaling pathways such as the semaphorin–plexin pathway were also noted. Intriguingly, we found an expansion of the ductal network with increasing disease. Activated STAT3, a blocker of apoptosis, was restricted to the ductal system and also increased with damage, highlighting its potential as a promoter of ductal cell survival. These data demonstrate the early activation of signaling pathways regulating inflammation, innervation, and cell survival before the onset of clinical disease indicators, suggesting their potential value as diagnostic biomarkers. Sjögren’s syndrome (SS) is a complex autoimmune exocrinopathy that has been extensively reported in the clinical literature. Although a systemic disorder, it predominately affects the salivary and lacrimal glands (LG) with lymphocytic infiltration of these tissues resulting in clinical manifestations that include xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes) . While sAlthough some progress has been made in the phenotypic and genomic characterization of SS, including a recent genome-wide association study ,3, causaTo gain a better understanding of global gene expression and how it is altered in the early stages of SS exocrinopathy, we used a well-characterized, spontaneous mouse model of SS and state-of-the-art genetic screening methods to conduct an unbiased exploration of global gene expression. In comparison to the majority of SS mouse models, mice deficient in autoimmune regulator gene (Aire) exhibit classic signs of aqueous-deficient dry eye in a swift fashion, allowing for an understanding of disease processes in the absence of the effects of aging. By 7–8 weeks the lacrimal glands of Aire-/- mice exhibit extensive lymphocytic infiltration combined with corneal pathologies and severe dry eye. However, it is not known when these disease outcomes are initiated in the Aire-/- mouse. Aire-/- mice on the BALB/c background were selected over Aire-/- mice on the NOD background for this study due to their less rapid development of severe disease. NOD. Aire-/- mice develop multi-organ autoimmune disease that results in an average life span of ~8 weeks. In contrast, BALB/c. Aire-/- mice develop SS-associated dry eye and exocrinopathy that is nearly identical in appearance to NOD. Aire-/-, but the time course of disease development is slower, the extent of multi-organ involvement is less, and the average life span extends up to ~16 weeks. These characteristics make BALB/c. Aire-/- mice an ideal model for studies of disease progression.Here, we used RNA sequencing (RNAseq), the gold standard for generating quantitative estimates of RNA abundance and disease-specific alterations in gene expression, to examine the entire transcriptome of CD4+-T-cell-mediated exocrinopathy in 5-week-old (wk) Aire-/- mice. While dry eye researchers have used RNAseq technology to explore the transcriptome of ocular tissues, none have focused on the specific alterations in SS-associated exocrinopathy during the early stages of disease or gone on to validate differences in gene expression using traditional readouts, such as qPCR or protein expression . Our findings provide a holistic overview of genetic changes associated with early disease processes, as well as specific genetic biomarkers that can be used to define the mechanism of disease development and progression.p = 0.0013; 7 wk: 1.00 ± 0.37 vs. 8.13 ± 0.30, p < 0.0001, p = 0.34; 7 wk: 1.00 ± 0.055 vs. 0.20 ± 0.065, p < 0.001, p = 0.03, p = 0.002, p = 0.004, p = 0.22, p < 0.0001, p = 0.015, p = 0.07; 7 wk: 1.00 ± 0.71 vs. 2.00 ± 0.55, p = 0.31 p = 0.33, In order to begin to define pathways activated during early disease onset we utilized the Aire-/- mice which exhibit epithelial barrier disruption, poor tear secretion, extensive lymphocytic infiltration, and vascularization of both lacrimal glands and cornea, as well as severe corneal pathologies and loss of lacrimal acini by 8 weeks of age (wk) as compared to age-matched wild types (WT)  17]. To. To17]. p = 0.0013; 7 wk: 1.00 ± 0.37 vs. 8.13 ± 0.30, p < 0.0001, p = 0.34; 7 wk: 1.00 ± 0.055 vs. 0.20 ± 0.065, p < 0.001, p = 0.03, p = 0.002, p = 0.004, p = 0.22, p < 0.0001, p = 0.015, p = 0.07; 7 wk: 1.00 ± 0.71 vs. 2.00 ± 0.55, p = 0.31 p = 0.33, In order to begin to define pathways activated during early disease onset we utilized the Aire-/- mice which exhibit epithelial barrier disruption, poor tear secretion, extensive lymphocytic infiltration, and vascularization of both lacrimal glands and cornea, as well as severe corneal pathologies and loss of lacrimal acini by 8 weeks of age (wk) as compared to age-matched wild types (WT)  17]. To. To17]. p < 0.01) compared to 694 being downregulated  = −2.3, p = 0.00032), secretoglobulins , and exocrine gland secreted peptides , as well as the acinar marker and master regulator of secretion Basic Helix-Loop-Helix Family Member A15 ; this suggests that, despite tear secretion being similar to in wild-type mice, LG function was being reduced. Concurrent with the loss of acinar genes was an upregulation of the ductal markers mast/stem cell growth factor receptor  and solute carrier 12 member 4 . These results suggest that remodeling of the secretory tissue and alterations in the proportion of acinar and duct cells in LG begins at or before 5 weeks.Current studies have pointed to a number of specific pathways being active in late stage disease, yet whether these are also present at early stages is unclear. To address this issue, we compared gene expression in lacrimal glands from 5 wk Aire-/- with very mild disease, i.e., those with lymphocytic infiltrates ≤10% C to theip = 0.02; 7 wk: 4.64 ± 0.86 vs. 0.70 ± 0.09, p = 0.02, p = 0.04; 7 wk: 0.33% ± 0.33 vs. 9.90% ± 2.33, p = 0.02, p = 0.28; 7 wk: 0.19% ± 0.11 vs. 0.92% ± 0.16, p = 0.01, p < 0.001; 7 wk: 0.50% ± 0.20 vs. 4.41% ± 1.23, p = 0.03, To validate the alterations in gene expression/pathways/processes measured via transcriptomic analysis, as well as to define their spatiotemporal distribution and abundance with increasing disease, we performed a combination of Western blot and immunofluorescence in 5 and 7 wk Aire-/- and WT LG. Immunofluorescent analysis confirmed the reduction in the acinar cell compartment and acquisition of a more ductal phenotype during disease progression A and thep = 0.02, p = 0.3, p < 0.0001, p < 0.05, Stat1 was also significantly upregulated during early disease  pathways in the Aire-/- LG at 5 wk C and theSema4c , Sema4d , Sema6d , and their receptors Plxnb1  and PlxnD1 . However, by 7 wk, Western blot showed upregulation of glycosylated (100 kDa), nonglycoslyated (75 kDa), and cleaved forms (50 kDa) during disease progression , suggesting that the remaining nerves showed a compensatory increase in function at the early disease stages to maintain tear secretion. However, with disease progression and further destruction of LG tissue, acetylcholine production was significantly reduced in 7 wk Aire-/- mice  were the gift of Mark Anderson, University of California, San Francisco. Genomic DNA isolated from tail clippings was genotyped for the Aire mutations by PCR with manufacturer-recommended specific primers and their optimized PCR protocols. Female mice were used in the study and were between 5 and 7 weeks of age when sacrificed.Mice were handled in strict accordance with the University of California, San Francisco animal welfare guidelines for laboratory and animal care. The protocol was approved on 3 May 2018 by the Institutional Animal Care and Use Committee at the University of California San Francisco . To visualize immune cell subtypes, immunohistochemistry was performed with antibodies specific for CD4  and CD45R , a donkey anti-rat secondary antibody conjugated to horseradish peroxidase and a DAB  staining kit. OCT Tissue Tek embedded lacrimal glands and eyes were sectioned on the cryostat  at 7 μm and mounted on SuperFront Plus slides. To fix the tissue, sections were incubated with ice-cold acetone at −20 °C for 10min and then washed for 5 min with PBS 3 times. Sections were blocked with 5% normal goat serum for 1hr at RT, and then incubated in 3% hydrogen peroxide to inactivate endogenous peroxidases. Primary CD4 (1:50) or CD45R (1:200) antibodies were prepared in 5% normal goat serum and incubated at 4 °C overnight. Sections were washed for 5 min with PBS 3 times, secondary antibody diluted 1:500 in 5% normal goat serum was applied for 30 min at room temperature (RT), and then sections were washed again for 5 min with PBS 3 times. Hematoxylin counterstain was utilized to stain the nuclei. For immunofluorescence staining, OCT Tissue Tek 7 or 20 μm sections were mounted and subsequently fixed with 4% paraformaldehyde for 20 min at RT, followed by 1 min acetone–methanol, and incubated overnight at 4 °C with one or a combinaton of the following primary antibodies: goat anti-COLIV , rat anti-PECAM (1:300 Millipore), goat anti-NKCC1 , rabbit anti-cKit , rabbit anti-MIST1 , rabbit anti-cleaved Caspase 3 , rat anti-Ki67 , rabbit anti-TUBB3 , rat anti-Ecadherin , rabbit anti-pStat3 , rat anti-IL6Ra, Rat anti-CD68 . Antibodies were detected using Cy2-, Cy3-, or Cy5-conjugated secondary Fab fragment antibodies , and nuclei were stained with Hoechst. Fluorescence was analyzed using a Zen Spinning disk confocal , Zeiss Epifluorescence microscope , and NIH ImageJ software .CD4+ T cells were quantified in the central corneal region by counting the total number of DAB+ brown cells with an Hemotoxylin & Eosin counterstain associated nucleus. To quantify CD4+ T cells and CD45R+ B cells in the lacrimal gland, the area of DAB+ signal was measured and divided by the total area of the lacrimal gland. This value was expressed as a percentage of CD4+ T cell or CD45R+ B cell infiltration in the tissue.In the lacrimal gland, MIST1 was utilized to label the acinar population while NKCC1 was utilized to label the ductal population. Lacrimal glands were imaged by confocal microscopy at a magnification of 20× with a stack of 20 1 μm slices. To determine the acinar to ductal cell ratio, the total number of MIST1+ cells associated with Hoeschst-stained nuclei was divided by the total number of NKCC1+ cells associated with Hoeschst-stained nuclei.Blood vessels were labeled for PECAM and imaged by confocal microscopy at 20× using 1 μm confocal sections that were combined to produce a 20 μm projection. For assessing blood vessel dilation, blood vessel area was approximated with an ellipse. The blood vessel diameter was measured as the length of the approximated width for an approximated ellipse, expressed in micrometers. Blood vessels with a diameter greater than 6 μm were labeled as dilated. Blood vessel diameter in three 200 μm × 200 μm Ecadherin (ECAD)-positive areas of the tissue was quantified to determine total number of blood vessels 1–5 μm in diameter and >6 μm in diameter in intact gland. Twenty-micron lacrimal gland sections were labeled for beta3 tubulin (TUBB3) and ECAD, and imaged by confocal microscopy using a 20× objective with 1 μm confocal sections that were combined to give a projected thickness of 20 μm. Nerve density was measured as the area of TUBB3 immunofluorescent signal, as quantified using Tsai’s thresholding method (Moments) in ImageJ . IntegraDuctal cell proliferation was quantified by counting the number of Ki67+ cells in the NKCC1+ ductal cells. To obtain the percentage of proliferating cells per respective region, total cell counts were divided by the number of NKCC1+ Hoechst-stained nuclei. Cell death was labeled using an antibody directed against Cleaved Caspase 3 (CC3). Apoptosis was calculated by counting the number of CC3+ cells in the NKCC1+ ducts and dividing by the total number of NKCC1+ ductal cells to obtain the percentage of apoptotic ductal cells. To obtain the percentage of acinar cell death, the number of CC3+ ECAD+ NKCC1- acinar cells was counted and divided by the total number of ECAD+NKCC1- cells. The result was used to quantitatively assess the loss of acinar cells and the expansion of the ductal cell compartment. Lastly, the percentage of pSTAT3+ ductal cells was obtained by counting the number of pSTAT3+ NKCC1+ cells and dividing by the total number of NKCC1+ ductal cells.Tissues were lysed in radioimmunoprecipitation assay buffer (RIPA buffer) containing a cocktail of protease inhibitors. Protein concentration was measured by bicinchoninic acid (BCA) assay . A quantity of 10 μg of protein per samples was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% Bis Tris gels  and transferred to PVDF membranes. Membranes were blocked in 5% milk and incubated in primary antibodies overnight at 4 °C: rabbit anti-Sema7a , Rabbit anti-Stat1 , and mouse anti-Beta actin . After washing three times in TBST, the blots were incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies and visualized with the Clarity Max Western ECL chemiluminescence detection system . Densitometric quantification of bands was performed using ImageJ software. To correct for sample-to-sample variability, the density of the target protein band was normalized to the density of the loading control beta actin band, for comparison of relative protein levels.Five microliters of lissamine green dye (1%)were applied to the lower conjunctival cul-de-sac of mice anesthetized with isoflurane, and images of the cornea were taken using an Olympus Zoom Stereo Microscope . Corneas were divided into four quadrants and scored independently for the extent of staining in each quadrant. Scores were classified as Grade 0, no staining; Grade 1, sporadic (<25%); Grade 2, diffuse punctate (25–75%), or Grade 3, coalesced punctate staining (75% or more). The sum of the quadrants scored by three masked observers for each eye was calculated on a scale ranging from 0 (no staining) to 12 (most severe staining) and plotted as the fold change relative to the average WT score from the same age group.Pilocarpine (4.5 mg/kg) was injected into the peritoneum (i.p), and after ten minutes, the mice were anesthetized with isoflurane. Tear secretion was measured using a Zone-Quick phenol red thread , to indicate mm of tears absorbed in 15 s.® E/M , which disregarded any reads that aligned to more than one location or more than one gene at a single location. Data was normalized by two procedures: 1. total count normalization, 2. addition of a small offset (0.0001). After normalizing, differential gene expression was determined using the Differential gene expression (GSA) algorithm (Partek). This algorithm generates p values using limma-trend method 4. Genes were considered differentially expressed if the log2 Fold Change between samples was at least 1, with the adjusted p-value held to 0.01. Additionally, we considered only genes with values of at least ten fragments per kilobase of transcript per million mapped reads (FPKM) in at least one biological replicate. A gene list of all significantly modulated genes (p < 0.01) was used as input for gene ontology (GO) analysis using Partek software. The resulting output was ranked by adjusted p-value (Fisher’s exact test on the underlying contingency table). We consider an attribute to be significant if its Expression Analysis Systematic Explorer (EASE) score  is less than 0.05 relative to an appropriate background gene set. Gene sets were selected to highlight unique features of the enrichment analysis. A principal component analysis (PCA) was performed on normalized log2-transformed read counts using seven principal components. The dataset is available on the GEO database.Total RNA was collected and purified using RNAaqueous and DNase reagents according to the manufacturer’s instructions . For each RNA sample (RNA Integrity Number (RIN) >6), cDNA libraries were prepared using the TruSeq mRNA library prep kit  using 1 µg RNA according to the manufacturer’s instructions and were then 50 bp single-end sequenced on an Illumina HiSeq 4000. Quality control metrics were performed on raw sequencing reads using the FASTQC v0.11.6 application . Reads wTo assess acinar cell function, the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217) , was used to measure acetylcholinesterase production in three 20 μm fresh frozen OCT sections of the lacrimal gland.Aire-/- mice. Data are presented as mean ± SEM. Student’s t-test was used for two-group comparisons. One-way ANOVA was used for comparison across multiple groups; p ≤ 0.05 was considered statistically significant.A minimum of three independent repeats were conducted in all experiments. Bar graphs are used to summarize the means and standard errors of each outcome obtained using all data collected from WT and


Staphylococcus aureus. Thirty male Balb/C mice were divided into six groups: normal control, negative control, and treatment groups . Crude polysaccharides were administrated for fourteen days. Furthermore, mice were exposed to S. aureus at the fifteenth day. Two weeks after the end of treatment, the parameters were measured. This study showed that crude polysaccharides at a dose of 75 and 100 mg/kg improved phagocytic activity, spleen index, and splenocytes proliferation. Rising of TNF-α levels was shown in groups treated with crude polysaccharides at doses of 25, 50, and 100 mg/kg. All treatment groups showed a decreasing level of IL-17. Crude okra polysaccharides also showed a slight increase in NK cells activity and IFN-γ level. Thus, crude okra polysaccharides could act as an effective material to enhance immune response including phagocytic activity, spleen index, splenocytes proliferation, and control immune responses through cytokine production.Okra pods were widely consumed by Indonesians to maintain health. The aim of this study was at investigating the potential of crude polysaccharides from okra pods on immune response in mice infected with  S. aureus. These bacteria are able to cause nosocomial infection which can result in serious infections [S. aureus transmission but the bacteria also release components against the immune system. Therefore, the body needs a particular compound to enhance the immune response.The human body is surrounded by environment-contained microbes, including extracellular bacteria, fections . NormallDietary phytochemicals from plants may play important roles in the prevention of many diseases . Plant pA. esculentus) is vegetable crop used as food and traditional medicine for many diseases such as dysentery and diarrhea [Okra  was purchased from Balai Besar Laboratorium Kesehatan, Surabaya, Indonesia. RPMI-1640 was purchased from Gibco . Lipopolysaccharide (LPS) from Escherichia coli 055 : B5, L2880, and lyophilized powder were purchased from Listlab . 3--2, 5-diphenyltetrazolium bromide (MTT) and human hepatoma cell line (huh7it) were acquired from Institute of Tropical Disease, Airlangga University . Interleukin-17 (IL-17), interleukin-23 (IL-23), interferon-γ (IFN-γ), and tumor necrosis factor (TNF)-α, enzyme-linked immunosorbent assay (ELISA) kit were purchased from BioLegend . All other chemicals and solvent used were of analytical reagent grade.Okra pods were collected from the traditional market in Surabaya, Indonesia, in May 2017. The okra pods were packaged 500 g per polyethylene bag and then stored at −20°C until use. 2O and dialyzed through cellulose membrane  for 24 h. The aqueous solution was then collected from the dialysis bag and freeze-dried to obtain the crude okra polysaccharides.According to Ramesh et al.  and ChenµL) and aquadest (90 µL). Then, 50 µL of phenol 5% was added. After being homogenized for 1 min, 2 mL of sulphuric acid was added to the solution and incubated for 10 min in room temperature. The blank solution was made from 50 µL of phenol 5% and 100 µL of aquadest. The absorbance was measured at 490 nm.Polysaccharides content in okra pods was determined using phenol sulphuric acid assay. Sample solution of crude okra polysaccharides was made from stock of crude okra polysaccharides  were provided by the Laboratory Animal from Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia. The animals were maintained in cages made of a plastic with a lid made of woven wire cage at 20°C, with 12 h light/12 h dark cycle, fed and watered by ad libitum. All procedures involving animal care were approved by Animal Care and Use Committee (ACUC) of Veterinary Faculty, Airlangga University, Surabaya, Indonesia, no. 714-KE.S. aureus without okra crude polysaccharides administration; P1, P2, P3: okra crude polysaccharides doses 25, 50, 75, and 100 mg/kg BW, respectively). Okra crude polysaccharides were administrated by gavage in fourteen days. Furthermore, mice were exposed to S. aureus (0.5 Mc. Farland) once through intraperitoneal at the fifteenth days. Two weeks after the last administration, the animals were weighed, blood samples were collected to obtain serum, and then the animals were killed. The intraperitoneal fluid was collected. The spleen was excised from the animal and weighed immediately and placed in cold PBS-penicillin-streptomycin. The relative was calculated according to the following formula: spleen index (mg/g) = (weight of spleen/body weight).After 10 days of acclimatization, BALB/c mice were randomly divided into six groups  kits  according to the manufacturer's protocol. The absorbance was measured using ELISA reader at 450 nm.Whole blood was collected and centrifuged at 3000 rpm and 4°C for 10 min, while the upper layer contained the serum. The levels of IFN-µg/ml streptomycin and resuspended in RPMI 1640-FBS 10% medium. Then, splenocytes were used in splenocytes proliferation assay and natural killer cell activity assay.The spleens were gently homogenized and passed through a sterilized copper sieve (200-mesh) to obtain single cell suspensions. Splenocytes suspension was centrifuged at 1000 rpm for 5 minutes. Pellet containing red blood cells was resuspended in tris-buffered NH4Cl pH 7.2 and centrifuged at 1000 rpm for 5 minutes until white pellet was obtained. Splenocytes were washed with 5 ml of PBS-100 unit/ml penicillin-100 µl of splenocytes (3 × 105 cells/well) and 5 µl of LPS (200 µg/ml) were seeded in 96-well plates. After incubation at 37°C in an incubator with 5% CO2 for 48 hours, MTT assay was used. The absorbance was measured at 560 nm. Splenocytes proliferation (%) = /) × 100.Cell numbers of splenocytes were counted by haemocytometer. 195 2 for 48 hours. The activity of NK cell was evaluated by the MTT assay. The absorbance was measured at 560 nm. NK cell activity (%) = /) × 100.The splenocytes as the effector (3 × 105 cells/well) was added to human hepatocyte cell line as the target cells (6 × 103 cells/well) to give E/T ratio 50 : 1. They were cultured in 96-well plate and incubated at 37°C in an incubator with 5% COP value of <0.05 was considered statistically significant.Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan's post hoc test. All analysis was performed using IBM SPSS Statistics 24 software. The results were reported as the mean ± standard deviation (SD) of five repeats. Using the polysaccharide standard regression equation, the polysaccharides content in the stock solution with dose of 100 mg/kg BW was 22.87 mg/mL.P < 0.05). The highest increase on phagocytic activity was shown by P3 group. Meanwhile, P1 group and P2 group increased phagocytic activity but did not show significant difference compared with normal and negative control groups , P2 group (460.40 ± 79.28 pg/ml), and P4 group (282.40 ± 80.38 pg/ml) compared to normal control and negative control groups (P < 0.05). P3 group (175.50 ± 79.76) also showed slight increase of TNF-α level compared to normal control and negative control groups (P > 0.05) (Serum levels of TNF- > 0.05) .P < 0.05). Although the difference was not significance, P1 showed decrease level of IL-17. There was slight increase in the serum level of IFN-γ in P4 group (174.60 ± 64.55 pg/ml) compared to normal control and negative control groups but did not show a significant difference. Meanwhile, there was no difference in the result of IL-23 . P4 group at a dose of 100 mg/kg showed highest spleen index (16.72 ± 3.60%) and increased spleen index significantly compared to normal control and negative control groups (P < 0.05) (Spleen index was significantly increased in P2 group (14.84 ± 2.76%) and P3 group (14.43 ± 3.31%) compared to normal control group ( < 0.05) .P < 0.05). The highest increase of splenocytes proliferation was demonstrated in P3 group with 157.77 ± 11.06%  did not show significant increase of natural killer cell activity compared with normal control group. P3 and P4 groups showed slight increase in NK cell activity (104.67 ± 15.32% and 106.75 ± 15.32%) compared to normal control, which was similar to the results of the splenocytes proliferation .Immune system gives protection to organism against bacterial infection through layered defense, nonspecific immunity, and specific immunity. When bacteria pass through nonspecific defense, the body forms a more complex immune system . These dNonspecific immunity component that firstly recognizes bacteria is macrophage . Activatα, IFN-γ, IL-17, and IL-23 and improves host defense against bacteria. TNF-α and IFN-γ are cytokines produced by macrophage and Th cell due to all kinds of antigen infections. Macrophage also produced IL-23 to induce activation of Th17. Meanwhile, IL-17 is produced by Th17 specifically. Staphylococcus aureus is extracellular bacteria. Extracellular bacteria specifically induce Th naïve to differentiate as Th17 [α, IFN-γ, IL-17, and IL-23.Polysaccharides regulate the host immune system by activating immune cell-related to lymphocytes, macrophages, and NK cells . The pre as Th17 . Based oS. aureus presented by active macrophage induces activation of Th-17. T helper-17 produced cytokine such as IL-17, IL-22, and TNF-α [α is one of the most important proinflammatory cytokines against microbe. The present result showed that serum level of TNF-α significantly increased in groups with administration of crude okra polysaccharide at doses of 25, 50, and 100 mg/kg BW. The previous study also reported that crude okra polysaccharides significantly increased TNF-α level in RAW264.7 macrophage [Cytokines are small proteins produced by cells such as T helper, NK cells, and macrophages that regulate the immune response and inflammation . S. aurend TNF-α . Among tα. This present study of TNF-α demonstrated that administration of crude okra polysaccharides at a dose of 75 mg/kg did not significantly increase the level of TNF-α.From the previous study, okra polysaccharide-induced cytokine production from macrophages through activation of the transcription factor NFкB . The traα level in this study. This result indicated that TNF-α was not dominantly produced by macrophages. Crude polysaccharides from okra pods may induce other cells to produce TNF-α. Zhou et al. [T. wilfordii strongly inhibits TNF-α and IL-1 production. The overproduction of TNF-α is related with development of various diseases [α is suppressed in higher doses. Insignificant level of TNF-α in P3 with dose of 75 mg/kg BW could be due to its optimal doses. Based on this result, optimal doses of polysaccharides to increase TNF-α were lower doses (25 and 50 mg/kg BW). Although P4 showed significant result, the level of TNF-α did not raise as high as P1 and P2 groups.Contrast with this result, the highest increase of phagocytic activity was found in the group with at a dose of 75 mg/kg BW. There is no relation between active macrophages that undergo phagocytosis with production of TNF-u et al.  reporteddiseases . Thus, wγ but the difference was insignificant. Meanwhile, serum level of IL-23 did not show any difference. Contrast with serum level of TNF-α, all of the treatment groups had decreased serum level of IL-17 compared with normal control group and negative control group. IL-17 is one of the proinflammatory cytokines [γ and controlled serum level of IL-17 and IL-23 possibly to prevent healthy cells from becoming damaged. Both IFN-γ and IL-17 are proinflammatory cytokines, and overexpression of proinflammatory cytokines will induce excessive inflammation.This study also showed increase of serum level of IFN-ytokines . These rLymphocytes circulated in the blood and the lymph are also found in large numbers in lymphoid tissues or lymphoid organs. The spleen is a secondary lymphoid organ, a place for maintaining mature naive lymphocytes . Spleen Dendrobium huoshanense [Splenocytes are immune cells in the spleen. Splenocytes consist of T cell, B cell, macrophages, and dendritic cells . This reshanense . Recent shanense . As we rAs a member of lymphocyte class, NK cells are best known for their nonspecific killing of tumor cells, and there is evidence for their role in controlling infection in the earliest phases of the body's immune response . They caTherefore, an NK cell activity assay is a routine method for analysis of a patient's cellular immune response in vitro and can also be used to test the antitumor activities of possible drugs . In thisImmunomodulatory activities of polysaccharides may be due to direct or indirect interaction with immune system components. Complement proteins and monocytes, macrophages, dendritic cells, neutrophils, and lymphocytes have been reported as target responding to polysaccharides –29. Bindα level. Contrast with other results, treatment groups with higher doses of crude okra polysaccharides showed decrease level of IL-17 as response to prevent overexpression of proinflammatory cytokines.The results of the present study all together showed that crude polysaccharides of the okra pods had the potential to enhance the immune response of some immune components. Treatment groups with higher doses of crude okra polysaccharides increased phagocytic activity, spleen index, and splenocytes proliferation. Meanwhile, treatment groups with lower doses of crude polysaccharides from okra pods showed the highest significant rising of TNF-α level. Crude okra polysaccharides could suppress immune response (immunosuppression), showed with decrease of IL-17 level.Based on this study, crude okra polysaccharides could act as an immunomodulator. Crude okra polysaccharides had both immunostimulator activity and immunosuppression activity. Most of the immune components examined in this study showed significant increase and decrease at the doses of 75–100 mg/kg. Crude okra polysaccharide could enhance immune response, showed with rising of phagocytic activity, spleen index, splenocytes proliferation, and TNF-α level, but decrease IL-17 level as a response to prevent overexpression of proinflammatory cytokines. This study suggests that crude polysaccharides from okra pods grown in Indonesia could act as an effective compound to improve immune response.We concluded that crude polysaccharides from okra pods could enhance phagocytic activity, spleen index, splenocyte proliferation, and TNF-


Phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinase (MAPK) dual activation was induced by ZnO NPs in a dose-dependent manner. Additionally, the Akt (protein kinase B) inhibitor BML257 and the mTOR  inhibitor rapamycin contributed to the survival of astrocytes. Inhibitors of cyclooxygenase-2 and lipoxygenase attenuated ZnO NP-induced toxicity. Calcium-modulating compounds, antioxidants, and zinc/iron chelators also decreased ZnO NP-induced toxicity. Together, these results suggest that ZnO NP-induced autophagy and apoptosis may be associated with oxidative stress and the inflammatory process in primary astrocyte cultures.The present study examined the potential toxic concentrations of zinc oxide nanoparticles (ZnO NPs) and associated autophagy and apoptosis-related injuries in primary neocortical astrocyte cultures. Concentrations of ZnO NPs ≥3 μg/mL induced significant toxicity in the astrocytes. At 24 h after exposure to the ZnO NPs, transmission electron microscopy revealed swelling of the endoplasmic reticulum (ER) and increased numbers of autophagolysosomes in the cultured astrocytes, and increased levels of LC3 (microtubule-associated protein 1 light chain 3)-mediated autophagy were identified by flow cytometry. Apoptosis induced by ZnO NP exposure was confirmed by the elevation of caspase-3/7 activity and 4′,6′-diamidino-2-phenylindole (DAPI) staining. Significant ( Zinc oxide nanoparticles (ZnO NPs) have been widely used for medicinal purposes, such as photodynamic therapy  and piezZinc is one of several essential endogenous metallic ions, and is thought to be a modulator of critical physiological functions such as systemic growth, bone metabolism, wound healing, endocrine function, neuronal excitability, and survival in the brain ,16. HoweA recent study employing an ischemic stroke model found that zinc overload-induced activation and/or the cell death of astrocytes is correlated with autophagy ,22 and aPreviously, we reported that lipoxygenase (LOX), not COX-2, is the predominant arachidonate enzyme in ZnO NP-related toxicity in human dopaminergic neuroblastoma SH-SY5Y cells . However®) were obtained from Alfa Aesar Co. ; the average particle size of the ZnO NPs in the powder was 67 nm (40–100 nm), and the range of the specific surface area (based on the Brunauer–Emmett–Teller [BET] theory) was 16 m2/g (10–25 m2/g). Horse serum (HS), fetal bovine serum (FBS), penicillin-streptomycin, and Minimal Essential Eagle’s Medium (MEM) were purchased from Gibco Co. ; N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), LY294002, and rapamycin were purchased from Enzo Life Sciences ; and esculetin, meloxicam, phenidone, N-acetylcysteine (NAC), deferoxamine mesylate, 4′,6′-diamidino-2-phenylindole (DAPI), and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. . The ZnO NP suspensions were prepared with Millipore water at a concentration of 1 mg/mL. Prior to adding the ZnO NPs to the culture media, each suspension was sonicated for at least 30 min and then vigorously vortexed. Thereafter, the suspension was immediately applied to the cultured astrocytes.ZnO NPs  was added to halt the overgrowth of the astrocyte cultures and dividing cells. The culture medium was exchanged every 3 to 4 days until experiments were performed. The astrocyte cultures were maintained for 3 to 5 weeks, until the grown cells formed a confluent monolayer. The study protocol was approved by Kangwon National University Institutional Review Board (KW-170322-1 and KW-180621-1). Murine astrocyte cultures were prepared as previously described ,29. BrieAfter changing the growing media to MEM media without serum, the cultured astrocytes were treated with either 3 µg/mL or 5 µg/mL of ZnO NPs or co-treated with ZnO NPs and test chemicals for 20 to 24 h. After treatment, cell injury was quantitatively estimated at 20 to 24 h by measuring lactate dehydrogenase (LDH) release from damaged cells into the culture medium, as described previously . LDH act2 incubator. After a monolayer of astrocytes was formed, the cells were exposed to ZnO NPs and/or test chemicals in 200-µL volume, and then incubated for 20 to 24 h. Next, the MTT solution was added, and the cells were incubated at 37 °C for 2 to 3 h. After discarding the culture medium, 100 µL of dimethyl sulfoxide (DMSO) was added, and the cells were incubated for 30 min to dissolve the formazan crystals. The viability of the cells was estimated at 570 nm using a VersaMax microplate reader (Molecular Devices). The astrocytes were cultured in a 96-well culture plate at 37 °C in a 5% COg for 5 min at 4 °C, the supernatant was removed, anti-LC3 Alexa Fluor® 555 and 1× Autophagy Reagent B were added, and the samples were incubated on ice for 30 min in the dark. Next, the samples were centrifuged at 300× g for 5 min at 4 °C, the supernatant was removed, and then each sample was resuspended with 200 µL of 1× assay buffer and assayed using MuseTM flow cytometry. To assess autophagy, cultured astrocytes growing in six-well plates were exposed to ZnO NPs for 12 or 24 h. Autophagy Reagent A was applied to the cultured cells for 3 h; then, the cells were washed once with 1× Hank’s Balanced Sat Solution (HBSS) and accutase for 5 min at 37 °C to detach the cells. The harvested cells were transferred to Muse Assays microtubes, and 100 µL of 1× HBSS was added to each well. Next, the cells were centrifuged at 300× Prior to TEM, the cultured astrocytes were washed with 0.1 M of phosphate-buffered saline (PBS) and fixed with a mixture of 1% paraformaldehyde and 4% glutaraldehyde overnight at 4 °C. Then, the astrocytes were post-fixed in 1% osmium tetroxide in the same buffer and dehydrated with ethanol and propylene oxide. Subsequently, the samples were embedded in Epon-812 resin, and ultra-thin sections were obtained using an ultra-cut microtome . Finally, the sections were stained with uranyl acetate and lead citrate and subjected to TEM visualization .TM RIPA Buffer  and HaltTM Protease Inhibitor Cocktail (Thermo Fisher Scientific), and sonicated in 3-min ON and 30-s OFF cycles. The cycle was repeated three times. After sonication, the cells were centrifuged at 12,000 rpm for 20 min at 4 °C, and 100 μL of supernatant was transferred to a new tube. Next, 100 μL of media  or supernatant  and standard were added to specific antibody-coated 96-well microplates and incubated at 37 °C in a 5% CO2 incubator for 2 h. All of the supernatant was removed, 100 μL 1× biotin antibody was added to each well, and the plates were incubated at 37 °C in a 5% CO2 incubator for 1 h. Then, each well was washed three times with 1× wash buffer, 100 μL 1× HRP (horseradish peroxidase)-avidin was added, and the plates were incubated at 37 °C in a 5% CO2 incubator for 1 h. Each well was washed five times, 90 μL of TMB  substrate was added, and the plates were incubated at 37 °C in a 5% CO2 incubator for 25 min in the dark. Following the addition of 50 μL of stop solution, the activities of IL-6, TNF-α, SOD, and GPx were estimated at 450 nm using a VersaMax microplate reader (Molecular Devices).To measure the activities of IL-6, TNF-α, SOD, and GPx, a mouse enzyme-linked immunoassay (ELISA) assay kit  was used according to the manufacturer’s instructions. Briefly, IL-6 and TNF-α activities were assessed by collecting 100 μL of media from cultured astrocytes. To assess the activities of SOD and GPx, cultured astrocytes were treated with PierceTo measure the amount of protein from cultured astrocytes, a BCA (bicinchoninic acid) Protein Assay Kit  was used according to the manufacturer’s instructions. Supernatant was obtained from sonicated and centrifuged cells, and 100 μL of supernatant, standard, and working solution were added to a 96-well microplate and incubated at 60 °C for 1 h. After 1 h, the amount of protein was measured at 562 nm using a VersaMax microplate reader (Molecular Devices).g for 5 min at 4 °C. The supernatant was discarded, and the cells were resuspended with 50 μL 1× assay buffer. Then, an equal volume of fixation buffer was added to the cell suspension and incubated for 10 min on ice. The cell suspension was centrifuged at 300× g for 5 min, and the supernatant was removed. To permeabilize the cells, 100 μL of ice-cold 1× permeabilization buffer was added and incubated for 10 min on ice. Afterwards, the cells were centrifuged at 300× g for 5 min, and the supernatant was removed. The cells were resuspended by adding a 90 μL 1× assay buffer and 10 μL of working antibody cocktail solution. Following incubation for 30 min in the dark at room temperature, 100 μL 1× assay buffer was added to the cells. Next, the cells were centrifuged at 300× g for 5 min, and the supernatant was removed. Finally, the cells were resuspended in 200 μL 1× Assay Buffer and analyzed using the MuseTM Cell Analyzer.To analyze activation of the PI3K/MAPK dual pathway, the Muse PI3K/MAPK Dual Pathway Activation Kit  was used according to the manufacturer’s instructions. The cultured astrocytes were grown in 24-well plates and exposed to ZnO NPs for 12 h. Then, the cells were deactivated by replacing the media with fresh media. Next, the cells were harvested using a cell scraper and centrifuged at 300× ® Caspase-3/7 Kit (Merck-Millipore) was used according to the manufacturer’s instructions. The cultured astrocytes were grown in 24-well plates and exposed to ZnO NPs for 12 h. Then, the cells were washed once with 1× HBSS and harvested using a cell scraper. The harvested cells were transferred to a Muse Assays microtube and centrifuged at 300× g for 5 min at 4 °C. The supernatant was removed, and each sample was resuspended with 50 μL 1× assay buffer. Next, 5 μL of MuseTM Caspase-3/7 Reagent working solution was added to each sample and incubated at 37 °C in a 5% CO2 incubator for 30 min. After incubation, 150 μL of MuseTM Caspase 7-AAD working solution was added to each sample, and the samples were vortexed at medium speed for 3 to 5 s. Then, the samples were incubated at room temperature for 5 min in the dark. Samples were assayed using the MuseTM Cell Analyzer.To assess caspase-3/7 activation, the MuseFor fluorescent staining of DNA damage (intact and condensed or fragmented DNA), the medium of cultured astrocyte cells was aspirated, and the cells were washed three times with 0.1 M of PBS. Cells were fixed for 10 min in 4% paraformaldehyde and rinsed three times with the same buffer. Cells were permeabilized with 0.2% Triton X-100 for 5 min. After washing three times with PBS, cells were incubated at room temperature in DAPI labeling solution (1 µg/mL in PBS). Stained images were photographed using the fluorescence detector of an Olympus inverted microscope .p values < 0.05 were considered to indicate statistical significance, and all the experimental data are expressed as a mean ± standard error of the mean (SEM). All the statistical analyses were conducted with the SAS 9.4 program using one-way analysis of variance (ANOVA) tests and Tukey’s multiple comparison test. ®) were characterized using field emission TEM  was evaluated using LDH A and MTTThe astrocytes exhibited various types of cell morphology that contained intact organelles such as mitochondria, endoplasmic reticulum (ER), the nucleus, and lysosomes A. HoweveThe exposure of cultured astrocytes to toxic concentrations of ZnO NPs  significantly increased the number of autophagic LC3-positive cells at 12 h and 24 h compared with the control A. The thThe exposure of cultured astrocyte cells with ZnO NPs (3 µg/mL and 10 µg/mL) resulted in significantly increased caspase-3/7 activity at 12 h in a dose-dependent manner A. The toChanges in the inflammatory cytokines IL-6 and TNF-α induced by ZnO NP exposure were evaluated in the media of cultured astrocyte cells. ZnO NPs significantly elevated the levels of IL-6 and TNF-α at 10 µg/mL A,B, whilPI3K/MAPK activity induced by ZnO NP exposure was examined to identify the relationship with apoptosis by flow cytometry in cultured astrocyte cells. ZnO NPs slightly but significantly induced activation of the dual pathway or MAPK in the PI3K/MAPK assay at 3 µg/mL and 10 µg/mL A,B. HoweMeloxicam, which is a selective COX-2 inhibitor, attenuated astrocyte injury in a concentration-dependent manner and exhibited a significant difference at 500 µM A. PhenidThe Akt inhibitor BML-257 significantly decreased ZnO NP-induced toxicity at 50 µM A. RapamyBAPTA/AM, which is an intracellular calcium chelator, significantly blocked ZnO NP-induced toxicity at a concentration range of 0.3 to 3 µM A. NimodiThe increased use of ZnO NPs can result in their consistent accumulation in the body , and the50) of ZnO NPs is 6.6 µg/mL in microglial cells. Although the culture conditions in each of the above-mentioned studies differed somewhat, together, the results suggest that microglia have a twofold lower vulnerability than astrocytes [In this study, we determined that ZnO NPs were toxic at concentrations ≥3 µg/mL. The LDH and MTT assays showed similar results. Above this concentration, morphological cell damage was evident. Interestingly, we found that the mouse primary cultured astrocytes used in this study had 2.6-fold and 20-fold higher vulnerability to ZnO NPs compared to cultured rat primary astrocytes in previous studies by Wang et al.  and Sudhtrocytes . Thus, atrocytes  reportedAutophagy is the elimination of metabolic debris to maintain cell survival. Autophagic functions lead to abnormal deterioration during the acceleration of the aging process and in neurodegenerative states . In the Using TEM, we confirmed that autophagolysosome formation and ER swelling were evident following treatment with ZnO NPs. These results suggest that ZnO NPs may induce the production of pro-inflammatory cytokines, such as IL-6 and TNF-α, via activation of the ER stress–autophagy axis .In primary astrocyte cultures, treatment with ZnO NPs increased apoptosis (twofold and threefold increases compared to controls) dose-dependently at 3 µg/mL and 10 µg/mL at 12 h, respectively. However, NAC reversed this effect to control levels. Sudahkaran et al.  reportedRecently, in vivo studies have shown that levels of the pro-inflammatory cytokines IL-6 and TNF-α are elevated following treatment with ZnO NPs, whereas those of SOD and GPx are reduced . Roy et Recently, PI3K/Akt and MAPK have been shown to be involved in autophagy or apoptosis and cell survival through various cellular stress mechanisms or pro-inflammatory cytokines in ZnO NP toxicity ,43. In tIn a previous study, ZnO NPs were introduced to elevate the expression of COX-2 as well as pro-inflammatory cytokines in macrophages . We wereWe evaluated the effects of the Akt inhibitor BML-257 in ZnO NP-induced astrocyte injury. BML-257 attenuated astrocyte injury induced by ZnO NPs, suggesting that the Akt signaling activation process, rather than the PI3K process, may contribute to astrocyte injury. In contrast with previous studies that used neuroblastomas, the PI3K inhibitor LY294002 did not significantly inhibit ZnO NP-induced astrocyte toxicity in LDH assays (data not shown) . Roy et Maintaining an appropriate balance in intracellular calcium levels is also very important for the survival of astrocytes. In the present study, BAPTA/AM, which is an intracellular calcium chelator and L-type calcium channel blocker, reduced ZnO NP-induced damage to astrocytes. These results suggest that the excessive accumulation of intracellular zinc ions resulting from the permeabilization of dissolved ZnO NPs in cultured astrocytes may trigger calcium dyshomeostasis ,47. Howe


Paeonia, also known as the “King of Flowers” in China, is an important source of traditional Chinese medicine (TCM). Plants of this genus have been used to treat a range of cardiovascular and gynecological diseases. However, the potential pharmacological activity of one particular species, Paeonia rockii, has not been fully investigated. In the first part of the present study, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), reducing power assays, and metal ion chelating assays were used to investigate the in vitro antioxidant activities of Paeonia rockii. In the second portion of the study, a mouse model of d-galactose-induced aging was used to validate the antioxidant effects of the flowers from Paeonia rockii in vivo. Lastly, potential antioxidant constituents were screened and identified by ultra-high pressure liquid chromatography and electrospray ionization coupled with high-resolution mass spectrometry (UHPLC-ESI-HRMSn) combined with the DPPH assay. Results indicated that the flowers and leaves exhibited stronger antioxidant activity than ascorbic acid in vitro. The therapeutic effect of Paeoniarockii was determined in relation to the levels of biochemical indicators, such as 8-iso-prostaglandin F2α (8-iso PGF2α) in the serum, superoxide dismutase (SOD), protein carbonyl, malondialdehyde (MDA), and glutathione (GSH) in the liver and brain, after daily intra-gastric administration of different concentrations of extracts  for three weeks. The levels of 8-iso PGF2α (p < 0.01) and protein carbonyl groups (p < 0.01) were significantly reduced, whereas those of SOD (p < 0.05) had significantly increased, indicating that components of the flowers of Paeonia rockii had favorable antioxidant activities in vivo. Furthermore, UHPLC-ESI-HRMSn, combined with pre-column DPPH reaction, detected 25 potential antioxidant compounds. Of these, 18 compounds were tentatively identified, including 11 flavonoids, four phenolic acids, two tannins, and one monoterpene glycoside. This study concluded that the leaves and flowers from Paeonia rockii possess excellent antioxidant properties, highlighting their candidacy as “new” antioxidants, which can be utilized therapeutically to protect the body from diseases caused by oxidative stress.The genus  Bletilla striata, Lonicera japonica, and Paeonia have been reported to possess favorable antioxidant activity . After incubation at 50 °C for 30 min, 2 mL of 10% trichloroacetic acid (TCA) was added. A portion of the solution (2 mL) was mixed with 2 mL of distilled water and 1 mL of 0.1% ferric chloride (FeCl3). The absorbance was then measured at 700 nm. Ascorbic acid was used as a reference. Ascorbic acid equivalent values were calculated from linear equations for the samples and reducing power was expressed as ascorbic acid equivalents (AAE) given in μg AAE/100 μg of the extracts.The reducing power of the extracts was determined according to a previously described procedure  with som2+-chelating activity was measured according to a previously described method [4 (1 mM) and 2.3 mL of distilled water. The mixture was shaken vigorously and left at room temperature for 5 min. Afterwards, 1 mL of ferrozine (1 mM in methanol) was added to the mixture, which was mixed and left for another 5 min to react with the residual Fe2+. The absorbance of the Fe2+ ferrozine complex was measured at 562 nm against a blank; EDTA-Na2 was used as a reference. Fe2+ chelating activity was calculated using the same formula as that used for the DPPH assay. IC50 was determined by probit regression in SPSS .Fed method  with somSpecific-pathogen-free (SPF) male mice (20 ± 2 g) were purchased from the Experimental Animal Company of Chendu Dashuo . Mice were allowed to acclimate to room conditions for three days prior to experimentation and were kept in a controlled environment at 25 ± 2 °C under a 12 h light/12 h dark cycle and in 60 ± 2% relative humidity. Mice were fed a standard rodent pellet diet and had access to fresh water at all times.d-galactose (0.1 mL/10 g) once a day for 6 weeks [Mice were randomly divided into six groups, each containing ten mice. Group I was a blank control group that was subcutaneously injected with normal saline (100 mg/kg) once a day for six weeks. Groups II–VI were intraperitoneally injected with 100 mg/kg doses of  6 weeks . Group I2α analysis. Liver and brain homogenates (10% w/v) were prepared in cold saline. The resulting suspension was centrifuged at 3000 rpm for 10 min at 4 °C and frozen at −80 °C until the assay was conducted as described below. The activities of 8-iso PGF2α from blood serum, along with SOD, protein carbonyl, MDA, and GSH from liver and brain, were assayed using commercial reagent kits obtained from the Institute of Biological Engineering of Chengdu Kelong  according to the manufacturer’s instructions and using standard assay procedures [The collected eyeball blood was allowed to naturally sediment for 2 h. The resulting suspension was centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatant was collected for 8-iso PGFocedures ,52.n analysis. A sample (2.5 mg/mL) without DPPH was used as a blank control. Peaks representing antioxidant components reduced or disappeared after reacting with DPPH. UHPLC analyses were performed using an Ultimate 3000 system  equipped with an online vacuum degasser, a quaternary pump, an autosampler, and a temperature-controlled column compartment. Chromatographic separation was performed on a SunFireTM C18  column. The eluent consisted of solvents A (0.1% aqueous formic acid in water) and B (acetonitrile); flow rate was 0.3 mL/min. Gradient elution was programmed as follows: 0–2 min, 5% B; 2–10 min, 5%–15% B; 10–30 min, 15%–40% B; and 30–40 min, 40%–80% B. The injection volume was 2 μL, and the injection temperature was 15 °C.MF and ML extracts (5.0 mg/mL in methanol) were reacted with DPPH (0.2 mM in methanol) at 37 °C for 30 min; the volume ratio of the extracts and DPPH solutions was 1:1. The resulting mixtures were filtered through a 0.22 μm filter prior to UHPLC-ESI-HRMSm/z in both positive and negative ion modes. The operating conditions were as follows: capillary voltage, 2.00 kV; pressure of nebulizer, 30 psi; auxiliary gas pressure, 10 L/min; capillary temp, 320 °C; auxiliary gas heater temp, 350 °C. MS/MS spectra were acquired with collision energy at 35 eV to achieve the maximum number of characteristic fragments for structural elucidation.Tandem mass spectrometry was performed in a Q-Exactive Orbitrap mass spectrometer (MS)  using a heated electrospray ionization source for the ionization of target compounds. MS data were acquired across a range of 80–1200 t-tests, and the means compared using Duncan’s multiple range test. Statistical significance was determined at p < 0.05.All antioxidant experiments were performed in triplicate. SPSS .and Graph Pad Prism were used to analyze the data statistically. Data were expressed as mean ± standard deviation (SD). The statistical evaluations used one-way analysis of variance (ANOVA) with multiple comparisons, followed by Dunnett’s Paeonia rockii and revealed the presence of antioxidant compounds. The flowers and leaves from Paeonia rockii showed stronger antioxidant activity than ascorbic acid in vitro in reducing power, DPPH, and ABTS radical scavenging assay. The antioxidant ability of Paeonia rockii flowers was evidenced by the reduction of 8-iso PGF2α and protein carbonyl and a concurrent increase in SOD levels in vivo. In addition, 25 potential antioxidant compounds in the leaves and flowers were screened, and 18 compounds were identified by DPPH-UHPLC-ESI-HRMSn methodology in vitro. The result demonstrated that the flavonoids and phenolic acids might be the major compounds responsible for the strong antioxidant activity of the flowers and leaves from Paeonia rockii. This study calls attention to the important antioxidant properties of Paeonia rockii and highlights the therapeutic potential of this traditional Chinese herb in preventing and treating diseases caused by oxidative stress.This study first evaluated the antioxidant activities of leaves and flowers from


Consistently, these cells did not activate cAMP synthesis in response to noradrenaline. These data indicate that immortalized adipose-derived mesenchymal stem cell lines demonstrated impaired ability to respond to noradrenaline compared to primary adMSCs. These data draw attention to the usage of immortalized cells for MSCs-based regenerative medicine, especially in the field of pharmacology.Primary adipose tissue-derived multipotent stem/stromal cells (adMSCs) demonstrate unusual signaling regulatory mechanisms, i.e., increased of sensitivity to catecholamines in response to noradrenaline. This phenomenon is called “heterologous sensitization”, and was previously found only in embryonic cells. Since further elucidation of the molecular mechanisms that are responsible for such sensitization in primary adMSCs was difficult due to the high heterogeneity in adrenergic receptor expression, we employed immortalized adipose-derived mesenchymal stem cell lines (hTERT-MSCs). Using flow cytometry and immunofluorescence microscopy, we demonstrated that the proportion of cells expressing adrenergic receptor isoforms does not differ significantly in hTERT-MSCs cells compared to the primary adMSCs culture. However, using analysis of Ca Adipose tissue renewal, as well as its growth during high-fat diet, strongly depends on the activity of progenitor cells, residing in adipose tissue stroma, known under various names, including mesenchymal stem/stromal cells (MSCs) or adipose derived stromal cells, etc. ,2. HormoPreviously, we demonstrated that the ratio of adipose derived MSCs (adMSCs) subpopulations expressing α1A, α1B, α2A, α2B, β1, β2, and β3 adrenergic receptors vary substantially from donor to donor. Besides, not every cell which expresses a particular receptor was able to respond to the appropriate hormone . adMSCs Further investigation of signaling mechanisms of adrenergic regulation of adMSCs required the population with more homogenous expression of adrenoceptors. In this study, we used a commercially-available cell line of immortalized adipose tissue-derived MSCs (hTERT-MSCs) with defined characteristics . AlthougTo identify adrenergic receptor isoforms expressed by hTERT-MSCs, we performed immunofluorescent staining using specific antibodies followed by confocal microscopy analysis. hTERT-MSCs expressed α1A, α1B, α2A, α2B, β1, β2, and β3 adrenergic receptors at their surface and intracellular, with the pattern similar to primary adMSCs . Flow cy2+ release. As shown in 2+ signaling in single cells, we demonstrated that hTERT-MSCs respond to noradrenaline in a non-uniform manner; only 1.9 ± 0.3% of these cells responded to noradrenaline by calcium release . For the experiments, adMSCs cultured up to 3rd- 4th passages were used.ASC52telo, hTERT immortalized adipose derived mesenchymal stem cells (ATCC® SCRC-4000™) were obtained from ATCC  and maintained in Advance Mesenchymal Cell Medium  in a humidified COiversity . All donThe ratio of cells expressing particular adrenergic receptor was examined using flow cytometry as previously described. Briefly, adrenergic receptors were detected using specific antibodies α1A , α1B , α2A , α2B , β1 , β2 , β3, following by secondary antibodies AlexaFluor 594  on formalin fixed non permeabilized cells. Normal rabbit IgG  and normal mouse IgG1  were used as a negative control. Stained cells were analyzed using flow cytometry scanner BDLSR Fortessa Special Order Research Product . Finally, 20,000–40,000 events were acquired and analyzed for antigen expression. Adrenergic receptors were stained using specific antibodies α1A , α1B , α2A , α2B , β1 , β2 , β3 , following by secondary antibodies AlexaFluor 594  on formalin fixed non permeabilized cells. Normal rabbit IgG  and normal mouse IgG1  were used as a negative control. The presence of adrenergic receptors was analyzed using immunofluorescent staining, and analyzed using a Leica DMI6000D  microscope equipped with a camera Leica DFC 7000T.2+ imaging at the single cell level. Cells grown in HyClone Advance Stem medium with a Supplement in 24 well plates were loaded with Fluo-8 , 4 µM in Hanks Balanced Salt Solution with 20 mM Hepes, for 1 h. Cells were grown at low densities to prevent cell-to-cell communications during the calcium imaging. To analyze the functional activity, cells were treated with either noradrenaline , or α1-agonist phenylephrine  or α2-adrenergic receptor agonist clonidine . To analyze the amount of functionally active cells after pretreatment with noradrenaline, cells were stimulated with noradrenaline for 1 h, washed three times, and then incubated in full growth medium for an additional 5 h. To measure the percent of responding cells, we recorded the baseline for 5 min, and then once added noradrenaline. Ca2+ transients were measured in individual cells using an inverted fluorescent microscope Nikon Eclipse Ti equipped with an objective CFI Plan Fluor DLL 10X/0.3  and with digital EMCCD camera Andor iXon 897 . We used the simultaneous measuring of 6×6 fields of view in Large Image mode to increase the number of analyzed cells. Movies were analyzed using NIS-Elements (Nikon) and ImageJ software . Alterations of cytosolic Ca2+ from the resting level were quantified by a relative change in the intensity of Fluo-8 fluorescence (ΔF/F0) recorded from an individual cell. The percent of responding cells was measured as a ratio of the number of responding cells to the number of all analyzed cells.Adrenergic receptor activation was assessed using noradrenaline and CaTo measure cAMP-dependent response of the cells, we used cAMP Direct Fluorometric Immunoassays Kit . Cells were grown in a 96-well plate to a near confluent monolayer a day before the experiment. Cells were treated with noradrenaline for 15 min and lysed with Cell Lysis Buffer for 10 min. Samples were prepared, and a cAMP Assay procedure was performed according to the manufacturer’s protocol. cAMP concentrations in the samples were calculated using a calibration curve preparing in every experiment independently. t-test for normally-distributed data, and Mann–Whitney U-criteria (M-U test) for not normally distributed data. Variation of calcium responses were measured for normally-distributed samples as relative standard deviations (RSDs), calculated as standard deviations divided by the mean value. Statistical significance was defined as p-value < 0.05. Statistical analysis was performed using SigmaPlot 11.0 software . Data was assessed for normality of distribution using the Kolmogorov-Smirnov test. Values are expressed as mean ± standard error of mean (SEM). Comparison of two independent groups was performed by Student


We found that OVX mice decreased levels of calcium, phosphorus, and estradiol-17β in the blood, decreased uterine weight and endometrial thickness, and decreased expressions of ERα, ERβ, PR, and SOD2 in the uterine tissue but increased blood ROS levels compared with those of WT mice. In addition, OVX mice with the herbal formula B401 therapy can increase levels of calcium, phosphorus, and estradiol-17β in the blood, increase uterine weight and endometrial thickness, and increase expressions of ERα, ERβ, PR, VEGF, and SOD2 in the uterine tissue but decrease blood ROS levels. Our results may provide reasonable explanation for the reproductive regulation of the herbal formula B401 therapy.In Taiwan, the herbal formula B401 is considered as a health supplement for middle-aged women that can alleviate sweating, anxiety, and sleep disorders. However, the relevant mechanisms are still unclear. In this study, we evaluated the beneficial effects of the herbal formula B401 therapy in the reproductive regulation of ovariectomised mice. Female ICR mice were randomised into four groups: wild-type (WT) mice with sham treatment, wild-type mice treated with the herbal formula B401, bilateral ovariectomised (OVX) mice with sham treatment, and bilateral ovariectomised mice treated with the herbal formula B401. Mice were orally given the herbal formula B401 at a dose of 30 mg/kg bw/day for 2 weeks. At the end of oral treatment with sham or the herbal formula B401, levels of reactive oxygen species (ROS), calcium, phosphorus, and estradiol-17 Our cell viability assay experiment was approved by the Committee on Biological Research of National Taiwan Normal University and was implemented under the guidelines of the committee.SH-SY5Y cells were treated with and without (control) the herbal formula B401 at indicated doses for 24 h to determine the IC ad libitum access to water and food. Mice received oral herbal formula B401 treatment at a dose of 30 mg/kg bw/day for 2 weeks.All ICR mice were purchased from BioLASCO Taiwan Yi-Lan Breeding Center, which has been awarded the Full Accreditation of AAALAC International. Our animal experiments were approved by the Institutional Animal Care and Use Committee of National Taiwan Normal University . In accordance with the Institutional Guidelines of the Animal Care and Use Committee of NTNU, ICR mice were maintained in the animal facility at NTNU under specific pathogen-free conditions. A total of 12 female ICR mice were randomised into four groups: wild-type mice with sham treatment (WT + sham), wild-type mice with B401 treatment (WT + B401), bilateral ovariectomised mice with sham treatment (OVX + sham), and bilateral ovariectomised mice with B401 treatment (OVX + B401). All mice were housed in a constant temperature environment at 22°C ± 2°C with a 12-h light/dark cycle, and mice hadβ was assayed using an Assay Design EIA kit . Measurements of blood calcium and phosphorus levels were manipulated in line with the manufacturer's protocol. In addition, levels of ROS in the blood of the four groups of ICR mice were determined using lucigenin- and luminol-amplified chemiluminescence (CL) methods to measure O2•− and H2O2 activity. Blood levels of ROS were measured using a CL analyzer  after 1.0 mL of 0.1 mM lucigenin in phosphate-buffered saline  was added to the blood samples of mice. The total CL counts of the four groups were calculated by integrating the area under the curve within 600 s.Levels of calcium and phosphorus were assayed using colorimetric assay kits , and the concentration of estradiol-17μm-thick sections. Some sections were mounted on slides and then assessed through hematoxylin and eosin (H&E) staining with a kit-based approach (Sigma-Aldrich Corporation). For immunohistochemistry (IHC) staining, following the heat-induced epitope retrieval method, uterine tissue sections were separately stained at room temperature for 1 h with antibodies for estrogen receptor α (ERα), estrogen receptor β (ERβ), progesterone receptor (PR), and vascular endothelial growth factor (VEGF) . Then, immunostaining was detected through incubation with biotinylated secondary antibodies  for 30 min and the avidin–biotin–horseradish peroxidase (HRP) complex  for additional 30 min. Immunostaining was visualised using DAB Chromogen , and the slides were counterstained with hematoxylin .After herbal formula B401 or sham treatment, the four groups of ICR mice were anaesthetised with urethane (1.5 mg/kg); subsequently, cardiac perfusion with PBS containing 4% formaldehyde (Sigma-Aldrich Corporation) was conducted. Uterine tissue specimens of mice were obtained and fixed with 4% formaldehyde (EM grade) (Sigma-Aldrich Corporation). The uterine tissue specimens were embedded in paraffin and then cut into 5-β-actin (Thermo Fisher Scientific Inc.) and superoxide dismutase 2 (SOD2) , which were detected using suitable HRP-conjugated secondary antibody (Santa Cruz Biotechnology Inc.). Immunostaining was visualised using the enhanced chemiluminescence (ECL) substrate  and was quantified using ImageJ analysis software .After herbal formula B401 or sham treatment, the four groups of ICR mice were anaesthetised with urethane (1.5 mg/kg); subsequently, transcardial perfusion with physiological saline was conducted. Uterine tissue specimens were homogenised in a buffer solution. Then, proteins in the separated solution were quantified using a BCA protein assay kit  and separated on 12.5% or 15% SDS polyacrylamide gels . The separated proteins were transferred to polyvinylidene difluoride membranes . The antibodies used in this study were P value of less than 0.05 was considered significant.All data are presented as mean ± SEM. The data were analysed using one-way or two-way ANOVA followed by Student–Newman–Keuls multiple comparison post-test. A  Ang. sinensis; astragaloside, isoastragaloside, astragaloside, and astragaloside IV from Ast. membranaceus; calycosin-7-O-β-D-glucoside, ononin, and wedelolactone from E. prostrata; oleanolic acid from L. fructus; ginsenoside Rc and ginsenoside Rb2 from P. ginseng; and calycosin, formononetin, forsythiaside, and acteoside from R. glutinosa.50 value of the herbal formula B401 was 305.3 mg/mL for SH-SY5Y cells.P < 0.01), whereas quantified blood levels of calcium and phosphorus in OVX + B401 mice significantly increased compared with those in OVX + sham mice (P < 0.01). β in WT + sham, WT + B401, OVX + sham, and OVX + B401 mice. Quantified serum concentrations of estradiol-17β in OVX + sham mice significantly decreased compared with those in WT + sham mice (P < 0.01), whereas quantified serum concentrations of estradiol-17β in OVX + B401 mice significantly increased compared with those in OVX + sham mice (P < 0.01).Figures P < 0.01), whereas the quantified uterine weight of OVX + B401 mice significantly increased compared with that of OVX + sham mice (P < 0.01).As shown in α, ERβ, and PR in the uterine tissue in the four groups , whereas quantified ROS counts in the blood of OVX + B401 mice significantly decreased compared with the counts in the blood of OVX + sham mice (P < 0.01). SOD2 is an antioxidant enzyme for oxidative stress. P < 0.01), whereas quantified uterine SOD2 expression was significantly increased in OVX + B401 mice than in OVX + sham mice (P < 0.01).To study the effects of the herbal formula B401 on mitochondrial oxidative stress in ovariectomised mice, we examined and then compared the blood ROS count and uterine SOD2 expression among the four groups Figures  and 7.  50 value of the herbal formula B401 was 305.3 mg/mL for SH-SY5Y cells (50 dose of SH-SY5Y cells converted to the IC50 dose of ovariectomised mice should be 305 g/Kg bw/day. If safety factor calculated from 1/10 as the starting dose of human, the safety dose of the herbal formula B401 for an adult woman weighing 60 kilograms should be 1,830 g per day. In this study, those ovariectomised ICR mice received oral herbal formula B401 treatment at a dose of 30 mg/kg bw/day for 2 weeks. The converted edible dose of the herbal formula B401 for an adult woman weighing 60 kilograms is 1.8 g per day that should be much smaller than the dose of IC50 value of the herbal formula B401. Thus, the herbal formula B401 should be quite safe dosage for menopausal women.In our previous studies, the herbal formula B401 treatment should be viable alternative treatment for the management of neurodegenerative diseases such as Alzheimer's, Huntington's, and Parkinson's diseases –11, 13. 5Y cells . The IC5β in the blood, while showing significant increased levels of calcium, phosphorus, and estradiol-17βin the blood after the herbal formula B401 therapy (Ovariectomised female rats have a higher risk of osteoporosis . In a pr therapy .β therapy can increase endometrial blood flow and reduced endometrial vascular resistance [It has been reported that the reduction of uterine weight in ovariectomised rats might be due to reduced uterine epithelial height, expanded uterine stroma, reduced uterine myometrial thickness, and atrophic vaginal epithelium , 18. In sistance . The incsistance .α and ERβ [α, ERβ, and PR in the uterine tissue. In this study, ovariectomised mice showed significantly decreased levels of estradiol-17β in the blood. We further found that ovariectomised mice significantly decrease expressions of uterine ERα, ERβ, and PR, whereas the herbal formula B401 therapy could enhance expressions of uterine ERα, ERβ, and PR (β levels and increase the expression of ERα, ERβ, and PR in the uterine tissue. It is reasonable that the herbal formula B401 can be an alternative treatment for reproductive regulation.It has been reported that estrogen may play multiple biological functions by modulating ER and ERβ , and PR  and ERβ . Thus, w, and PR . These r2− dismutation to H2O2 [2− and inhibit other antioxidant enzymes [It has been reported that menopause is associated with metabolic disorders and oxidative stress . A previ to H2O2 . Reduced enzymes . The enhβ in the blood, increase uterine weight and endometrial thickness, and improve expressions of ERα, ERβ, PR, and SOD2 in the uterine tissue but decrease blood ROS levels in ovariectomised mice. Thus we suggested that the herbal formula B401 can be a health supplement for alleviating discomfort symptoms in middle-aged women.The herbal formula B401 therapy can enhance levels of calcium, phosphorus, and estradiol-17


Lactobacillus. Lactobacillus plantarum strain PS128TM isolated from spontaneously fermented mustard greens in Taiwan was recently reported to exhibit probiotic properties. In this study, we aimed to assess the safety of strain PS128TM for use in humans via examining genotoxic and oral toxic effects using in vitro and in vivo testing. Five strains of Salmonella typhimurium were evaluated by the Ames test; no signs of increased reverse mutation were observed following exposure to PS128TM. Additional testing of Chinese hamster ovary (CHO) cells exposed to PS128TM revealed that the incidence of chromosomal aberrations in CHO cells had not increased. PS128TM treatment also did not affect the proportion of immature to total erythrocytes or the number of micronuclei in the immature erythrocytes of ICR mice. Moreover, following a 28 day study involving repeated oral dose toxicity tests  utilizing an ICR mouse model, no observable adverse level (NOAEL) was found at any of the doses. PS128TM was sensitive to antibiotics; however, genes related to the production of biogenic amines were absent. While further research is required, these toxicological assessments suggest that PS128TM could be safe for human consumption.Probiotics are extensively available to consumers; however, the use of probiotics may not always be safe, and there are few reports on their side effects, including those of  Probiotics are defined as live microorganisms that, when administered in adequate amounts, exert beneficial health effects on the host . NumerouLactobacillus plantarum PS128TM was isolated from spontaneously fermented mustard greens in Taiwan [TM treatment. Prefrontal cortex analysis showed increased levels of dopamine and serotonin [TM exerts psychotropic effects that could reduce anxiety-like behavior and may be helpful in alleviating neuropsychiatric disorders by decreasing stress-related symptoms. Recently, evidence also showed that there are potentials for PS128TM to improve cerebral dopamine-related symptoms in mice such as Tourette syndrome [Lactobacillus [L plantarum strain PS128TM.n Taiwan . Daily in Taiwan . Serum an Taiwan . In naïverotonin , suggestbacillus . Thus, tTM powder was provided by Bened Biomedical Co., Ltd. . This bacterium was isolated from fermented mustard greens, which is a traditional Hakka ethnic food product. This novel strain was identified by phylogenetic classification of the 16S rDNA sequence. The isolated PS128TM was inoculated into Man, Rogosa, and Sharpe broth (MRS) from BD Difco , cultured at 37 °C for 18 h and then harvested by centrifugation at 6000× g for 10 min. The pellet was re-suspended in MRS plus 12.5% glycerol to a final concentration of 5 × 109 colony-forming units (CFU) per milliliter. The re-suspended solution was then aliquoted in freezer tubes and stored at −20 °C until use. Before use, the aliquot was pre-warmed to 37 °C for 1 h and resuspended in saline. PS128TM contains 100 billion CFU L. plantarum PS128TM in 1 g powder (1 × 1011 CFU/g).PS128Salmonella typhimurium—TA-102, TA-97, TA-98, TA-1535, and TA-100--were obtained from MolTox Inc. . The plate incorporation test performed both in the presence (0.5 mL S9 mix) and in the absence  of metabolic activation was previously described [9 cells/mL), 2 mL soft agar , 0.5 mL S9 mixture (if necessary), and five concentrations of PS128TM ranging from 0.3125-5 mg/plate were combined in a test tube separately. After mixing, the sample was then poured into minimal glucose agar plates (Vogel-Bonner E medium containing 2% glucose with 1.5% agar). After incubating for 48 h at 37 °C in the dark, the number of his+ revertants for Salmonella typhimurium strains were counted macroscopically and compared to the negative control. A sample was considered positive when (1) a p-value less than 0.05 was found, (2) the number of revertants was at least double the spontaneous yield, and (3) a reproducible positive dose-response was present.Five strains of histidine-deficient escribed . BrieflyTM as described previously [Chinese Hamster Ovary (CHO) K1 cell line purchased from the American Type Culture Collection (ATCC) was seeded into plastic 96-well plates and treated with different concentration of PS128eviously . After tTM, chromatid breaks and polyploidy in metaphases were analyzed in CHO epithelial cells. Thus, 2 × 105 cells/mL of cells were seeded in 6-cm dishes. PS128TM powder was dissolved in distilled water before treatment and used at concentrations of 0.3125, 0.625, or 1.25 mg/mL. Mitomycin C (1 μg/mL) and benzo(a)pyrene (5 μg/mL) were added as positive control without or with S9 metabolic activation, respectively. After treatments, cells were exposed to Colcemid  for 2 h of the incubation period. The metaphase cells were then harvested by trypsinization and fixed with Carnoy’s solution . The incidences of chromosomal aberrations from 100 metaphases per treatment were collected and scored. Data were shown as the average number of chromosomal aberrations per treatment ± standard deviation (SD) from 300 cells scored in three independent experiments.To investigate the clastogenic effects of PS128TM at doses of 0.5, 1, and 2 g/kg body weight were given by oral gavage in 5 male ICR mice once. Cyclophosphamide at 200 mg/kg body weight was intraperitoneal-injected as the positive control. At 24 h, 48 h, and 72 h, 10 μL blood samples from the tail vein were carefully collected onto a slide pre-coated with 40 μg/mL acridine orange. The incidence of micronucleated polychromatic erythrocytes  and polychromatic erythrocytes  to total erythrocytes were measured by calculating a total of 1000 erythrocytes or PCEs per animal by fluorescence microscopy, respectively.Twenty-five male ICR mice  were acclimatized for 7 days and then randomly assigned to five cages for in vivo micronucleus assay. PS128TM by oral gavage at doses of 0 (control), 40, 400, and 2400 mg/kg body weight. Body weight was measured twice a week. Water and food consumption were measured at 2 and 4 weeks. On day 29, all mice were sacrificed. Cage-side observations were conducted daily and included eyes, respiration, fur, motion, and the activity of all animals.Forty male and forty female ICR mice  were randomly allocated to cages (4 mice/cage) and underwent the 28 day subacute toxicity study treated with PS128Animals were fasted overnight (16–18 h) and placed in an induction chamber with 2% isoflurane in 100% oxygen at a constant flow of 1.0 L/min using a Matrx VIP 3000 vaporizer  for general anesthesia at the end of the 28 day toxicity test. Blood samples from the abdominal aorta were collected for serum chemistry and hematology using an automatic analyzer. Clinical biochemistry analysis was performed using Fuji Dri-chem 4000i , and sysmex XT-1800iv  was utilized for complete blood count and differential count (CBC/DC) analysis from whole blood.Gross necropsy was performed for analyzing external and internal features macroscopically ,17. AfteThe biopsy sections of heart, lung, liver, spleen, kidney, adrenal gland, and gonads were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) at pH 7.4. The fixed tissue segments were embedded in paraffin and stained with H&E  for histological assessment under a light microscope .All the animals were kept under a 12:12 h light/dark cycle  with ad libitum access to regular chow and water. Room temperature was maintained at 22.0 ± 2.0 °C with a relative humidity between 39 and 43%. The mice were maintained under a 12 h light/dark cycle at 22 ± 2 °C and 39–43% relative humidity. All animal handling procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University . All surgeries were executed under isoflurane anesthesia to minimize suffering. Animal testing criteria mandated that a loss over 20% of body weight related to the original weight was considered a humane endpoint in this study. None of the tested mice reached this humane endpoint during our study.TM was evaluated according to the standard method procedure ISO 10932 . The minimum inhibitory concentration (MIC) was determined after 48 h growth at 28 °C in an anaerobic chamber  for the following nine antimicrobial agents as recommended by European Food Safety Authority (EFSA) . For each agent, the concentration range was expressed in μg/mL (given in brackets): gentamicin (1–256); kanamycin (2–256); streptomycin (4–256); tetracycline (0.5–128); erythromycin (0.0625–16); clindamycin (0.0625–16); chloramphenicol (0.5–32); ampicillin (0.25–32); and vancomycin (0.5–16). L. plantarum ATCC 14917T was used as the reference strain for quality control purposes .The antibiotic resistance profile for PS128histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc), were detected by Polymerase chain reaction (PCR). Briefly, a 25 μL amplification reaction mixture containing 10 ng of template DNA, 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 200 μM of each deoxynucleoside triphosphate, 1 μM of each primer, and 1 U of AmpliTaq Gold DNA polymerase was performed in a PCR cycler  using the following cycling parameters: 10 min for enzyme activation at 95 °C followed by 35 cycles of 60 s at 95 °C, 90 s at 53 °C, and 75 s at 72 °C, and a final extension step of 10 min at 72 °C. PCR products were resolved in 1% agarose gels and stained with ethidium bromide.Genes related to the production of biogenic amines, including p < 0.05 (*).Data are expressed as means ± SD. The differences between means were tested for statistical significance using a one-way ANOVA followed by a Dunnett’s multiple comparison test. Differences between the control group and other groups were considered statistically significant when TM at doses of 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate both in the presence (0.5 mL S9 mix) and in the absence  of metabolic activation were tested for gene mutation using Salmonella typhimurium strains TA-97, TA-98, TA-102, TA-100, and TA1535. Results showed that the number of revertants was not elevated, while a significantly increased number of revertants was observed in positive controls  for 24 h, and the cell viability was 100 ± 0.0, 74.1 ± 15.6, 58.0 ± 5.4, 52.1 ± 8.8, 35.6 ± 10.1, and 26.9 ± 3.6, respectively, indicating the insoluble precipitation of PS128TM may result in growth inhibition of CHO-K1 cells.PS128controls . In the TM at a dose of 1.25 mg/mL demonstrated cell viability of 52.1 ± 8.8 following treatment for 24 h and was used as the highest concentration for the chromosome aberration test. Results showed that treatment of PS128TM did not increase the number of abnormal chromosomes  by in vivo micronucleus assay . Although the body weight of each group increased, it was not significant . In female mice, the aspartate aminotransferase (AST) values for low-, medium-, and high-dose groups were found to be lower than the control group (p < 0.05). The lactate dehydrogenase (LDH) value in low- and high-dose groups was lower than the control group (p < 0.05). The triglycerides value in the high-dose group was higher than the control group (p < 0.05). The Ca value in the high-dose group was higher than that of the control group (p < 0.05). These changes were not dose-dependent, and the values were in the normal range [Following 28 days of repeated oral administration of nificant . Food anal range ; therefop < 0.05). Histological assessment of heart, lung, liver, spleen, kidney, adrenal gland, and gonads  was performed to estimate the effect of 28 day repeated oral administration of L. plantarum PS128TM. Low-, medium-, and high-dose groups showed no significant differences in either male or female mice when compared with the control group. Histopathological photographs of the control and the high-dose (2400 mg/kg) group were presented in After the mice were euthanized, heart, lung, liver, spleen, kidney, adrenal gland, and gonads  were carefully collected, and the organ weights were measured. The results are shown in TM was challenged with nine antimicrobial agents and grown for 48 h growth at 28 °C in an anaerobic chamber, and then the MIC was determined for each agent. MIC values for antibiotic susceptibility of PS128TM against the tested antibiotics are depicted in TM exhibited MIC values lower than the MIC breakpoint values recommended for heterofermentative Lactobacillus strains by EFSA for all the tested antibiotics . Since PS128TM was not found to be resistant to the tested antibiotics, no further studies on their antibiotic resistance are required according to EFSA.PS128Lactobacilli are capable of producing biogenic amines [L. plantarum PS128TM harbors genes related to the production of biogenic amines , PCR-based detections were performed, and the results showed the absence of these genes in the genome of L. plantarum PS128TM (Some c amines , which ac amines . Thus, t PS128TM .Salmonella/microsome mutagenicity assay, the chromosome aberration test, and the mammalian in vivo micronucleus assay showed that the probiotic PS128TM isolated from fermented mustard greens did not exert a genotoxic effect. The NOAEL of PS128TM was set to be greater than 2.4 g/kg body weight for both male and female ICR mice. PS128TM was sensitive to antibiotics, and no genes related to the production of biogenic amines were detected. These results were summarized and indicated the safety of PS128TM for human consumption (The results of the Ames sumption .