Andrographis paniculata (Burm.f.) Nees has been found to have anti-inflammatory and immunostimulatory effects. This study was to investigate antihyperuricemic and anti-inflammatory effects of A. paniculata leaf extracts. Andrographolide, 14-deoxy-11,12-didehydroandrographolide, and neoandrographolide were quantified in 80% ethanol (EtOH80) and water extracts using High Performance Liquid Chromatography (HPLC) analysis. Antihyperuricemic activity was evaluated using a spectrophotometric in vitro inhibitory xanthine oxidase (XO) assay. The most active extract and andrographolide were further investigated in a hyperuricemic rat model induced by potassium oxonate to determine serum uric acid levels, liver XO activity, followed by Western blot analysis for renal urate transporter URAT1, GLUT9, and OAT1 to investigate the excretion of uric acid via kidney. Anti-inflammatory activity was assessed by in vitro interleukin assay for interleukin , and tumor necrosis factor (TNF-α) in monosodium urate (MSU) crystal-induced human fibroblast-like synoviocyte (HFLS) cells using ELISA-kits, followed by Western blot analysis for the expression of MyD88, NLRP3, NF-κB p65, and caspase-1 proteins to investigate the inflammation pathway. In vivo assay of the most active extract and andrographolide were performed based on the swelling rate and inhibition of pro-inflammatory mediator release from synovial fluid of a rat knee joint induced by MSU crystals. The results showed that the EtOH80 extract had a greater amount of andrographolide (11.34% w/w) than the water extract (1.38% w/w). In the XO inhibitory activity, none of the samples exhibited greater than 50% inhibition. However, in a rat model, EtOH80 extract (200 mg/kg/day) and andrographolide (30 mg/kg/day) decreased serum uric acid levels and reduced liver XO activity, reduced the protein expression levels of URAT1 and GLUT9, and restored the decrease in OAT1 levels. In the in vitro anti-inflammatory study, EtOH80 extract and andrographolide significantly decreased production of IL-1α, IL-1β, IL-6, and TNF-α, as well as inhibited the synthesis of MyD88, NLRP3, NF-κB p65, and caspase-1 in a concentration-dependent manner, almost comparable to dexamethasone. The EtOH80 extract (200 mg/kg/day) and andrographolide (30 mg/kg) significantly decreased swelling rate and IL-1α, IL-1β, IL-6, and TNF-α in the synovial fluid of rat models in a time-dependent manner, comparable to indomethacin (3 mg/kg/day). In conclusion, the findings show that EtOH80 extract has a substantial anti-gout effect by lowering uric acid levels and suppressing pro-inflammatory mediator production due to the andrographolide content, that might be beneficial in the treatment of gouty-inflammation. Andrographis paniculata (Burm.f.) Nees  and Lalapan (fresh vegetable) (f.) Nees  (Acanthaf.) Nees . It is af.) Nees . Many megetable) .A. paniculata , promoting expression of genes involved in cell survival, proliferation, and angiogenesis  is an eicosanoid that is, biosynthesised from arachidonic acid precursor by the action of cyclooxygenase-2 (COX-2) enzyme and PGE2 synthase in endothelial cells.Interleukin (IL)-1β has been reported to have an important role in MSU-induced inflammation in gout. Thus, the inhibition of IL-1β is able to downregulate the inflammatory responses related to gout . MSU cryA. paniculata extract maybe useful as an anti-inflammatory and anti-hyperuricemic agent in the treatment of gouty arthritis. Thus, the aim of this study was to determine the anti-hyperuricemic effect of A. paniculata leaf extracts by exploring the xanthine oxidase (XO) inhibitory and uricosuric effects, asl well as the anti-inflammatory effect on the release of pro-inflammatory markers via the MyD88, NLRP3, NF-κB p65 and caspase-1 pathways in MSU-induced HFLS.Based on this information, it is hypothesized that Salmonella abortusequii, uric acid, DMSO, and tryphan blue® were purchased from Sigma-Aldrich . Synoviocyte growth medium was procured from Cell Applications . 100× Halt Protease and Phosphatase Inhibitor Cocktail were purchased from Thermo Fisher Scientific . Xanthine oxidase from bovine milk (20 U/mL) was purchased from Roche Diagnostic GmbH . Lymphoprep was purchased from Axis-Shield PoC AS . Phosphate buffer saline (PBS) was purchased from MP Biomedicals (USA). Uric acid and xanthine oxidase kits were purchased from BioVision . The concentration of cytokine was tested using appropriate ELISA kits for human. IL-8 ELISA kits were purchased from Abnova, Germany, while all other kits were purchased from Cayman, USA. Limit of detection of these kits were: 2.0 ρg/mL for IL-8, 7.8 ρg/mL for IL-6, and 3.9 ρg mL for IL-1α, IL-1β, and TNF-α. IL-1α, IL-1β, IL-6, and TNF-α in rats were measured using a multi-cytokine bead array detection system .All analytical grade solvents and HPLC-grade acetonitrile were purchased from Merck  while pure water for HPLC was obtained from ultrapure water system machine . Andrographolide , 14-deoxy-11,12-didehydroandrographolide  and neoandrographolide  were HPLC grade and acquired from Chromadex . Dexamethasone was purchased from CCM Duopharma Biotech Bhd. Xanthine, Tween 20, potassium oxonate, 20 mM HEPES, FBS, pen-strep, RPMI-1640 medium containing L-glutamine, MTT, LPS from Andrographis paniculata was cultivated at Field 2 Universiti Putra Malaysia (UPM) in Serdang, Selangor. A voucher specimen (No. SK965/04) was deposited at the Herbarium of the Laboratory of Natural Products, Institute of Bioscience, UPM. The leaves were dried in an oven dryer at 40°C for 3 days before being ground in an electric grinder. Dried powder was extracted with ethanol (80%) at a 1:10 ratio by rigorous maceration for 3 days at room temperature and repeated five times. To eliminate any remaining organic solvent, the organic filtrates were collected and concentrated under reduced vacuum pressure. The solvent-free extract was mixed with water and then freeze-dried (Labconco’s FreeZone 4.5 L Freeze Dry Systems) to get the crude ethanol 80% (EtOH80) extract. Meanwhile, the water extract was obtained from the UPM laboratory of Prof. Dr. Johnson Stanslas. All crude extracts were kept at 4°C until further usage.Male Sprague-Dawley rats  were procured from Universiti Kebangsaan Malaysia’s Laboratory Animal Resource Unit (LARU-UKM). The animals were kept in plastic cages on a 12 h/12 h light/dark cycle. The temperature and relative humidity were maintained at 25 ± 2°C and 50%, respectively. They were fed a commercial laboratory diet and were given access to food and drink ad libitum during the study. They were given 1 week to acclimate to their surroundings before the experiment. All procedures followed the A CIOMS Ethical Code for Animal Experimentation  and wereA. paniculata leaves was carried out using RP-HPLC based on a slightly modified method described by Phytochemical analysis of ethanol 80% (EtOH80) and water extracts of XO inhibitory activity of the extracts (400 μg/ml), chemical markers (100 μg/ml) and allopurinol (100 μg/ml) as a positive control was measured in 96-well plates using a spectrophotometric technique previously published by in vivo anti-hyperuricemic assay extract was carried out for 14 days in accordance with a previous study by Only the most active EtOH80 extract  and AP  were investigated to assess the anti-hyperuricemic effect compared to the positive control allopurinol . The Kidney samples from each treatment group were homogenized in RIPA lysis buffer with protease and phosphatase inhibitors, and chilled for 30 min. The lysate was then centrifuged at 13,000 rpm for 10 min at 4°C to extract total proteins, which were then quantified using the Bradford assay. Total proteins were incubated in boiling water for 7 min with loading buffers. On a 10% SDS-PAGE, an equal quantity of total proteins was isolated and transferred onto a PVDF membrane. Membranes were blocked in TBST containing 5% skimmed milk powder for 1 h. They were then incubated with the primary antibodies againts rabbit anti-SLC22A12 (URAT1) antibody , rabbit anti-GLUT9 antibody , rabbit anti-OAT1 antibody , and rabbit anti- β-actin (13E5) antibody  in TBST containing 5% skimmed milk powder overnight at 4°C. After three TBST washes of the membranes, immunoreactive bands were identified using HRP conjugated goat anti-rabbit IgG  as a secondary antibody that was diluted in a ratio of 1:5,000 in TBST for 1 h at room temperature. ECL detection reagent (Bio-Rad) was used to view the proteins, while ChemiDoc XRS + was used to evaluate the density of bands, which was then normalized to β-actin.2 at 37°C. Every other day, the medium was changed. When HFLS cells achieved 80% confluence, they were sub-cultured.Human fibroblast-like synoviocyte cells (HFLS) were purchased from Cell Applications . HFLS were grown in synoviocyte growth medium with 10% synoviocyte growth supplement in 5% CO6 per mL in 96-well plates with the same volume of extracts , chemical markers  and dexamethasone as a positive control at concentrations of 5 and 10 μg/ml and 0.5% DMSO as a negative control (5 μg/ml), and pre-incubated for 27 h at 37°C with 5% CO2. After adding 20 µL of MTT reagent (5 mg/ml), the plates were incubated for an additional 4 h at 37°C with 5% CO2. The supernatant was discarded carefully, and the cells’ formazan blue crystals were dissolved in 100 ml DMSO. Finally, the absorbance was determined using Tecan’s Infinite 200PRO NanoQuant microplate reader at a wavelength of 570 nm.The MTT assay was used to determine HFLS cell viability following a method described by 6 per mL) were pre-treated with extracts, chemical markers and dexamethasone at a concentration of 10 μg/ml, as well as complete medium with 0.5% DMSO for 3 h at 37°C in 5% CO2, and then stimulated for 24 h with MSU crystal suspension (200 mg/ml). Cells were centrifuged for 10 min at 300 × g and 4°C following incubation. The supernatant was carefully transferred to a sterile tube, and the concentration of cytokines was determined using suitable human ELISA kits. The amounts of cytokine secretion were compared to a negative control, which was assumed to have 100% cytokine secretion. The IC50 values for the active extracts were obtained from five different concentrations (0.625–10 μg/ml).The concentrations of IL-1α, IL-1β, IL-6, and TNF-α were measured in the HFLS culture supernatant using the ELISA technique described before . The HFL6 cells/well) for 24 h before being pre-treated for 3 h with 1.25, 2.5, and 5 μg/ml concentrations of extracts and chemical markers. MSU crystals were then used to stimulate cells, which were then incubated for 24 h. The cells were rinsed with cold PBS and disrupted in a lysis buffer containing protease and phosphatase inhibitors for the radioimmunoprecipitation assay (RIPA). On ice, harvested cells were lysed. The lysate was then centrifuged at 13,000 rpm for 10 min at 4°C to extract total proteins, which were then quantified using the Bradford assay. Total proteins were incubated in boiling water for 5 min with loading buffers. Equal quantity of total proteins was then separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in TBST  with 5% skimmed milk powder for 1 h. They were then incubated overnight at 4°C in 3% BSA with primary antibodies from Cell Signalling, USA against MyD88 (D80F5) , NLRP3 (D2P5E) , NF-κB p-p65 (D14E12) , caspase 1 (D7F10) , and β-actin (13E5) . After washing the membrane three times with TBST, immunoreactive bands were detected using a secondary antibody, anti-rabbit IgG HRP-linked antibody  that was diluted in a ratio of 1:3,000 in BSA and incubated for 1 h at room temperature. ECL detection reagent (Bio-Rad) was used to visualize the proteins and ChemiDoc XRS+ was used to analyze the density of bands, which was normalized to β-actin.Cells were seeded in 6-well plates (1 × 10in vivo assay was carried out for 14 days in accordance with the previously published protocol (n = 6 per group): normal control, hyperuricemia-induced control, indomethacin treatment (3 mg/kg/day) as a positive control, andrographolide treatment (30 mg/kg/day) and extract treatment of three different doses . Feeding was stopped 2 h before oral administration of the allopurinol, andrographolide, or extract. Except for the normal control group, all rats were anaesthetized with isoflurane on the 11th day of the experiment, and inflammation was induced by intra-articular injection of 50 µL of MSU crystals suspension (100 mg/ml) into a rat’s right knee joint 1 h after oral treatment. A caliper was used to determine the swelling rate of a rat’s knee joint at 2, 4, 8, 12, 24, 48, and 72 h after induction. Rats were anaesthetized with isoflurane and killed by cervical dislocation at the end of experiment. After shaving the skin around the knee, 100 µL sterile saline was injected into the synovial cavity using a 27 G needle, followed by the insertion of a second needle to collect synovial fluid. Inflammation was determined by measuring the IL-1α, IL-1β, IL-6, and TNF-α levels in synovial fluid according to the manufacturer’s instructions using a multi-cytokine bead array detection system . The PGE2 level was quantified using a single ELISA assay as directed by the manufacturer. The liver, kidneys, spleen, heart, and lung were promptly removed, rinsed in cold saline (0.9%), dried with a paper towel, and weighed.Only the most active EtOH80 extract and AP were investigated to assess the anti-gouty inflammatory properties. An experimental model of gouty inflammation was used with MSU crystals as the inducer. The protocol . Briefly50 values. Every experiment was performed three times (n = 3) in this study, and the results were presented as mean ± SEM. The test samples and controls were compared using one-way analysis of variance (ANOVA) and post hoc Tukey. If the p value was less than 0.05 (p < 0.05), the variance was considered significant.GraphPad Prism 5 was used to analyse all of the data and to calculate the ICA. paniculata leaf extracts were detected using HPLC in this work, namely andrographolide (AP), 14-deoxy-11,12-didehydroandrographolide (DAP), and neoandrographolide (NAP)  in EtOH80 extract, which was greater than the concentrations of the other identifiable phytochemicals.Three diterpene lactones previously discovered in de (NAP) . Chemica50 values were not determined.p < 0.05) decreased to 2.59, 2.54, and 2.48 mg/dl, respectively, compared to the hyperuricemic control group. However, none of the extract treatments could lower serum uric acid levels to the baseline value. When compared to the hyperuricemic control group, allopurinol (5 mg/kg) resulted in a significant (p < 0.05) reduction in serum uric acid levels. This decrease was seen even after 1 day of allopurinol treatment, and normal serum uric acid levels were maintained throughout the 14-days experiment with values of 1.70, 1.69, and 1.57 mg/dl on days 1, 7, and 14, respectively.As indicated in p < 0.05). Treatment with EtOH80 extract at a dose of 200 mg/kg and AP reduced XO activity in the liver by only 17.01 and 19.79%, respectively, which were not comparable to the 48.61% inhibition observed with allopurinol.The inhibitory action of liver XO in rats was investigated to determine the antihyperuricemic efficacy of the EtOH80 extract. As demonstrated in A. paniculata extracts, chemical markers, and dexamethasone, showing that such concentrations had no influence on HFLS viability after 27 h of exposure . Meanwhile, EtOH80 extract possessed a comparable inhibitory activity to dexamethasone for IL-1β, IL-6 and TNF- α (p ≥ 0.05).A. paniculata and its main constituents reduced MSU-induced inflammation in HFLS, western blot analysis was used to determine the MyD88, NLRP3, NF-κB, and caspase-1 protein levels. The results (p < 0.05) increased protein expression levels for all protein targets when compared to untreated cells. The treatment of EtOH80 extract, AP, DAP, and NAP significantly (p < 0.05) reduced the expression of all target proteins following MSU induction in a concentration dependent manner. However, treatment with water extract had no significant effect on the expression of MyD88, NF-κB, or caspase-1 in MSU-induced HFLS. In fact, it reduced NLRP3 expression considerably only at concentrations of 5 and 2.5 μg/ml.To elucidate the mechanisms of action by which  results  demonstrp < 0.05) decreased levels of IL-1α, IL-1β, IL-6, TNF-α, and PGE2 in a dose-dependent manner, equivalent to indomethacin and normal control groups (p > 0.05)  and AP (30 mg/kg) reduced swelling to normal levels, equivalent to the indomethacin (3 mg/kg) group . In addi > 0.05) .p > 0.05). Physical observations revealed no changes in the rats’ skin, hair, eyes, mucous membranes, behavioral pattern, tremor, salivation, or diarrhea. There was no death or substantial weight loss in the rats at the tested dosages (p > 0.05).During the 14-days experiment, changes in body weight  and orgaA. paniculata is a widely used traditional medicine. Despite its unpleasant bitter taste, the plant is often consumed as a herbal drink and fresh salad among other vegetables. A. paniculata has been extensively used empirically to treat a variety of illnesses, including inflammation, allergic reactions, and as an immunomodulator. Numerous publications have reported on the anti-inflammatory effects of A. paniculata and its bioactive components. Although A. paniculata has many anti-inflammatory chemicals, andrographolide is the most prevalent  a signal from TLR activates NF-κB, which activates pro-IL-1β production via the MyD88/TRIF pathway. MSU phagocytosis results in cathepsin B leaking into the cytoplasm, which activates the inflammasome. Finally, non-hematopoetic cells or IL-1R produce mature IL-1β as an inflammatory mediator  from arachidonic acid precursor. PGE2 plays a crucial role as a pro-inflammatory mediator in inflammation. As a result, a PGE2 production inhibitor might be useful as a treatment for inflammation  secretion. As a result, inhibiting PGE2 production is crucial in the therapy of gouty inflammation.When MSU crystals interact with cells, particularly monocytes, they upregulate IL-6 and TNF-α secretion . In addiammation . One of ormation . VariousThe kidneys excrete uric acid with the aid of uric acid transporters. Recent studies have demonstrated that hyperuricemia is related with alterations in the expression and function of urate transporters. Uric acid transporters are classified into two types: those that promote urate reabsorption, such as urate anion transporter 1 (URAT1), organic anion transporter 4 (OAT4), and glucose transporter 9 (GLUT9), and those that promote urate excretion, such as OAT1, OAT3, urate transporter (UAT), multidrug resistance protein 4 (MRP4/ABCC4), ABCG-2, and sodium-dependent phosphate transport protein.A. paniculata extracts and its bioactive compounds, such as andrographolide, 14-deoxy-11,12-didehydroandrographolide, and neoandrographolide, on anti-gout activity in terms of lowering uric acid and reducing crystal-induced inflammation were investigated in this study. The anti-inflammatory effect of this study was focused on the MyD88 pathway, which regulates the synthesis of NLRP3, NF-κB, caspase-1, and IL-1β. Meanwhile, the anti-hyperuricemic effect was attributed to its ability to modulate uric acid transporters, including URAT1, GLUT9, and OAT1.The effects of A. paniculata and one of its primary constituents, andrographolide, inhibited expression of MyD88, NLRP3, NF-κB, and caspase-1 proteins in MSU-induced HFLS. Additionally, the extract and andrographolide reduced MSU-induced cytokine production of IL-1α, IL-1β, IL-6, and TNF-α in HFLS and hyperuricemic rats, as well as PGE2 in rats. Furthermore, the extract and andrographolide decreased the protein expression levels of the renal urate transporters URAT1 and GLUT9 in hyperuricemic rats induced by potassium oxonate, but the reduction was not comparable to that of allopurinol. Meanwhile, renal OAT1 protein levels were considerably reduced in potassium oxonate-induced hyperuricemic rats compared to the normal control rats, which were significantly recovered by the extract, andrographolide and allopurinol.Our findings indicated that 80% ethanol extract of A. paniculata leaves and its main diterpene lactone compound, andrographolide, may have antihyperuricemic and anti-inflammatory properties and therefore be used to treat gout. In vivo, the antihyperuricemic action was mediated by xanthine oxidase inhibition or maybe in synergy with other mechanisms such as uricosuric effect. Meanwhile, anti-inflammatory action was demonstrated by inhibitory activity of monosodium urate crystals-induced cytokines and prostaglandin E2 secretion. In vitro assays with HFLS may indicate that EtOH extract of A. paniculata leaves and andrographolide inhibited gouty inflammation by inhibiting the production of cytokines and protein expression of MyD88, NLRP3, NF-κB, and caspase-1. This research establishes a foundation for the development of a novel anti-inflammatory phytotherapy. It is believed that well characterized standardised A. paniculata herbal product gives an excellent opportunity to further investigate its potential treatment of gouty-inflammatory conditions.Overall, the results indicated that an ethanol (80%) extract of


Pain is a significant problem worldwide that affects the quality of life of patients. Dezocine is a non-addictive analgesic drug with kappa-opioid antagonist activity and has been successfully used to alleviate of postoperative pain. In addition, dezocine has an analgesic effect similar to that of morphine, alleviating moderate to severe pain. Rap guanine nucleotide exchange factor 3 (RAPGEF3) is a guanine nucleotide exchange factor for GTPases Rap1 and Rap2, which could enhance the activity of Rap1 to promote cell adhesion and axon regeneration, as well as promote neurite extension by interacting with nerve growth factors. Here, we first observed that overexpression of RAPGEF3 increased cell viability, as shown by a CCK-8 assay, and recovered brain function in rats. The expression of inflammation-related factors at the mRNA level was detected using qPCR, and the concentration of these factors in a cultured cell medium and rat serum samples were decreased as shown by ELISA after RAPGEF3 overexpression. Through western blotting, we further found that pro-inflammatory proteins were decreased, and these effects might be mediated by inhibition of the Ras/p-38 MAPK signaling pathway. Taken together, we speculated that RAPGEF3overexpression enhances the therapeutic effect of dezocine on neuropathic pain by inhibiting the inflammatory response through inhibition of the Ras/p-38 MAPK signaling pathway. Neuropathic pain (NP) is normally caused by lesions in the nervous system and leads to long-term debilitating conditions, which reduce the quality of life. Pathophysiological changes in patients with NP result in anxiety, depression, and elevated blood pressure . NP is aH-DMEM (11965092) and FBS (10093) were purchased from Thermo Fisher Scientific . BsmBI (R0739S), KpnI (R3142S), and BamHI (R3136S) were purchased from Mew England Biolabs . Dezocine was acquired from the Dezocine Yangtze River Pharmaceutical Group . TNF-α (H8916), IL-1β (SRP6169), CCL2 (MSST0036), and CXCL1 (SRP3216) were purchased from Sigma-Aldrich . Anti-SOCS3 (ab16030), Epac1 (ab21236), GRK2 (ab137666), PDE4B (ab14611), PACAP (ab181205), PKCα (ab32376), p38 (ab31828), p38 (phospho T180 + Y182) (ab4822), Ras (ab180772), Raf (ab137435), RAP1(ab113480) antibody and IL-1β (ab197742), IL-6 (ab100713), TNF-α (ab208348) and COX-2 (ab210574) ELISA kits were purchased from Abcam . 2. After 2 days of culture, the medium was changed to remove unattached cells.Primary glial cells were acquired previously described . The cerRAPGEF3 gene was acquired using PCR with the following primers: Forward: 5′-CCGAAGCTGCTCCTACCA-3′, Reverse: 5′-ACTCCTCGCTGTTGGTGAGT-3′ (reaction steps: 50 ºC for 30 min and 85 ºC for 5 min. Amplified size: 594bp). Thereafter, the PCR products and pcDNA3.1 blank vector were digested with KpnI and BamHI, and linked with DNA ligase. Next, the pcDNA3.1-RAPGEF3 overexpression vector was transfected into primary glial cells using Lipofectamine 2000 Transfection Reagent for 48 h, and the stably expressed cells were screened using G418. The RAPGEF3 knockdown vector was constructed according to a previous study . The CCK-8 assay was performed according to the manufacturer’s instructions . Briefly, cells were seeded into each well of a 96-well plate at a concentration of 1×10RAPGEF3 overexpression group (TO), and dezocine treatment combined with RAPGEF3 inhibition group (TI). In the dezocine treated group, rats were injected with dezocine via intraperitoneal injection at a concentration of 3 mg/kg 30 min before the procedure.After the confluence of glial cells reached 70-80%, cells were treated with TNF-α (10 ng/mL), IL-1β (10 ng/mL), CCL2 (100 ng/mL), and CXCL1 (312 ng/mL) to mimic the inflammatory conditions of neuropathic pain before performing subsquent experiments. The cells were then divided into four groups: normal group (NC), dezocine treatment group (DT), dezocine treatment combined with RAPGEF3 overexpression group (DO), and dezocine treatment combined with RAPGEF3 inhibition group (DI). In the dezocine treated group, the cells were treated with 9 ng/mL dezocine for 10 consecutive days. Ten SD rats, five RAPGEF3 overexpression and five RAPGEF3 knockdown rats were purchased from Cyagen . Rats were kept in a 22-24 ℃, 50-60% humidity atmosphere with a 12-h light/dark cycle, with freely available water and food. The neuropathic pain after sciatic nerve injury model was constructed according to a previous study . BrieflyMechanical allodynia in rats was detected using von Frey filaments. Rats in each group were divided and treated as previously described, and then the rats were placed in transparent Plexiglas compartments on a metal mesh. The surface of the left paw was subjected to von Frey hairs with logarithmically increasing stiffness (0.4-26 g). Rapid withdrawal or flinching of the paw was considered a positive response. Thereafter, the 50% paw withdrawal threshold (PWT) was detected using Dixon’s up-down method . ThermalThe experiments were performed in accordance with the guidelines of the ethics committee of the Affiliated Hospital of Qingdao University , and all animals were treated under the guidelines of the Public Health Service Policy on the Humane Care and Use of Laboratory Animals of the National Institutes of Health.Rats and glial cells were grouped and treated as previously described, and the RNA extraction process was performed according to the protocol of the Total RNA Extraction Kit . Briefly, the cells and brain tissues of rats were lysed with lysis buffer, followed by incubation at room temperature for 5 min. After incubation with chloroform, the samples were centrifuged at 12,000 rpm for 5 min (4 ℃). The water phase of the samples was then removed into the absorption tube and centrifuged at 12,000 rpm for 2 min, washed with washing buffer, and then eluted using elution buffer and stored at -80 ℃ until subsequent experiments. The quality and integrity of RNA samples were determined using agarose gel electrophoresis. If the 28S and 18S bands were clearly observed in the sample and the intensity of the 28S band was more than 2-fold that of the 18S band, we considered the quality and integrity of RNA samples as acceptable for further experiments. IL-1b: Forward: 5’-GACTTCACCATGGAACCCGT-3’, reverse: 5’-GGAGACTGCCCATTCTCGAC-3’; IL-6: Forward: 5’-ACCCCAACTTCCAATGCTCT-3’, reverse: 5’-GGTTTGCCGAGTAGACCTC-3’; TNFA: Forward: 5’-AACACACGAGACGCTGAAGT-3’, reverse: 5’-TCCAGTGAGTTCCGAAAGCC-3’; PTGS2: Forward: 5’-CACGGACTTGCTCACTTTGTT-3’, reverse: 5’-AAGCGTTTGCGGTACTCATT-3’. The reaction was performed using the following steps: reverse transcription at 45 ℃ for 10 min, pre-degeneration at 95 ℃ for 5 min. Subsequently, the following steps were repeated for 36 cycles: denaturation at 95 ℃ for 15 s, annealing at 58 ℃ for 30 s and extension at 72 ℃ for 30 s. Each experiment was replicated thrice independently, and GAPDH was used as an internal control. The expression of each gene was determined using the 2-ΔΔCq method . Briefly, the reaction mixture was prepared as recommended, and the primers used were as follows: q method . The cells and brain tissues were cultured and grouped as previously described. After treatment, cells and brain tissues were lysed with lysis buffer . After centrifugation, the samples were collected into a new tube, and the concentration of proteins was determined using the BCA assay. Protein samples (60 μg) were loaded and separated by 10% SDS-PAGE, and proteins were transferred onto nitrocellulose membranes. Membranes were then incubated with 5% skim milk for 1 h at room temperature, followed by incubation with primary antibodies at 4 ℃ overnight. After incubation with a secondary antibody for 1 h at room temperature, the gray value of proteins was determined using a chemiluminescent immunoassay, and the expression of each target protein was measured using IPP 6.0. ELISA was performed according to the ELISA kit protocol. Briefly, serum samples and cultured cell media were added into each well of a 96-well plate, and the samples were incubated with an antibody at room temperature for 1 h. Afterward, samples were washed with washing buffer thrice, incubated with TMB substrate for 10 min, and the OD value at 450 nm was measured using a SpectraMax iD3 microplate reader (Molecular Devices).P < 0.05. Data of these experiments are presented as mean ± SEM. Each experiment was replicated thrice independently. Differences between groups were analyzed using one-way ANOVA in SPSS . Statistical significance was set at As shown in As shown in As shown in IL-1b, IL-6, TNFA and PTGS2 was determined using qPCR. The fold change of IL-1b was 1.00 ± 0.08, 0.70 ± 0.06, 0.40 ± 0.03, and 0.76 ± 0.06 in the NC, DT, DO, and DI groups, respectively, and was significantly decreased in all treatment groups relative to that in the NC group (P<0.05), and in the DO group compared with that in the DT group (P<0.05) in the cell model. The fold change of IL-1b was 1.00 ± 0.07, 0.87 ± 0.07, 0.48 ± 0.02, and 1.00 ± 0.07 was significantly decreased in the TO group compared with the SH and TD groups (P<0.05) in the rat model. The fold change of IL-6 was 1.00 ± 0.07, 0.85 ± 0.06, 0.51 ± 0.04, and 0.97 ± 0.07, and was significantly decreased in the DO group compared with that in the NC and DT groups (P<0.05). The fold change of IL-6 was 1.00 ± 0.08, 0.77 ± 0.06, 0.53 ± 0.04, and 0.88 ± 0.07 and was significantly decreased in the TD and TO groups compared with that in the SH group (P<0.05) in cell model, as well as in the TO group compared with that in the TD group (P<0.05) in the rat model. The fold change of TNF-A was 1.00 ± 0.08, 0.80 ± 0.06, 0.55 ± 0.04 and 0.89 ± 0.07, respectively, and was significantly decreased in the DT and DO groups than that in the NC group (P<0.05), as well as in the DO group compared with that in the DT group (P<0.05) in the cell model. The fold change of TNF-A was 1.00 ± 0.07, 0.87 ± 0.07, 0.59 ± 0.05, and 0.95 ± 0.07, respectively, and was significantly decreased in the TO group relative to that in the SH and TD groups (P<0.05) in the rat model. The fold change of PTGS2 was 1.00 ± 0.08, 0.79 ± 0.06, 0.53 ± 0.04, and 0.95 ± 0.08, and was significantly decreased in the DT and DO groups compared with that in the NC group (P<0.05), significantly decreased in the DO group and significantly increased in the DI group than that in the DT group (P<0.05) in the cell model. The fold change of PTGS2 was 1.00 ± 0.07, 0.83 ± 0.07, 0.62 ± 0.05, and 0.96 ± 0.08, and was significantly decreased in the DT and DO groups compared with that in the SH groups (P<0.05), as well as in the DO group compared with that in the DT group (P<0.05). As shown in As shown in As shown in As shown in Injury of the peripheral nervous system induced by traumatic, metabolic, and toxic factors, is the leading cause of neuropathic pain development . DezocinDuring chronic pain, inflammatory mediators are continuously produced, and pro-inflammatory factors, including TNF-α and IL-1β, induce pain through activation and sensitization of nociceptors . The cycet al., 2011). A previous study found that RAPGEF3 could inhibit the inflammation process induced by IL-6 through upregulation of SOCS3  are known regulators of the Janus kinase (JAK)/ signal transducer and activators of the transcription (STAT) pathway through a negative feedback mechanism . After dof SOCS3 . Anotherof SOCS3 . Using Rof SOCS3 . Furtherof SOCS3 . G proteof SOCS3 . Under tof SOCS3 . Phosphoof SOCS3 . A previof SOCS3 . After iof SOCS3 , promotiof SOCS3 . Proteinof SOCS3 . Furtherof SOCS3 . Here, w+ channels to promote cellular proliferation (RAPGEF3 is a sensor of the cellular second messenger cAMP and acts as the exchange factor of the Rap and Ras families; increased cAMP expression promotes the translocation of RAPGEF3 to the cellular membrane, further inducing Rap1 activation . Rap1 isferation . In addiferation . Here, wferation . Accumulferation . A recenferation , and alsferation , which iferation . Here, we demonstrated that dezocine treatment combined with overexpression of RAPGEF3 significantly increased the viability of cells, decreased the expression of inflammation-related cytokines at the transcriptional level and the concentration of inflammatory cytokines in cultured medium and serum samples of cells and rats. In contrast, the expression of anti-inflammatory and neuroprotective proteins increased. We further revealed that these effects might be mediated by inhibition of the Ras/Raf/MAPK signaling pathway. Thus, we hypothesized that dezocine treatment combined with RAPGEF3 overexpression might have a therapeutic effect on NP by increasing the proliferation ability of neuronal cells. However, the present results were limited by the small sample size of the rats and the limitation of the research methods. Thus, further experimentation with human samples is required.


The corresponding plasmids were transfected, and the cell growth was detected, including inflammation and oxidative stress, etc. The targeting of miR-30 c-5p with HIPK1 was examined. The results clarified that reduced miR-30 c-5p and elevated HIPK1 were manifested in CIRI. SEV could improve CIRI and modulate the miR-30 c-5p-HIPK1 axis in vitro and in vivo, and miR-30 c-5p could target HIPK1. Depressed miR-30 c-5p could eliminate the protection of SEV in vitro and in vivo. Repression of HIPK1 reversed the effect of reduced miR-30 c-5p on CIRI. Therefore, it is concluded that SEV is available to depress CIRI via targeting HIPK1 through upregulated miR-30 c-5p.Sevoflurane (SEV) has been reported to be an effective neuroprotective agent for cerebral ischemia/reperfusion injury (CIRI). However, the precise molecular mechanisms of Sev preconditioning in CIRI remain largely unknown. Therefore, CIRI model was established via middle cerebral artery occlusion method. SEV was applied before modeling. after successful modeling, lentivirus was injected into the lateral ventricle of the brain. Neurological impairment score was performed in each group, and histopathologic condition, infarct volume, apoptosis, inflammation, oxidative stress, microRNA (miR)-30 c-5p and homeodomain-interacting protein kinase 1 (HIPK1) were detected. Mouse hippocampal neuronal cell line HT22 cells were pretreated with SEV, and the  SevofluAccumulated studies have assured the crucial micrornas (mirnas) and their target genes in controlling i/r damage in animal or cell models ,12. MeanHomeodomain-interacting protein kinase 1 (hipk1) is a modulator of homologous domain transcription factors modulating various cell biological processes associated with inflammation and anti-stress responses . A studyThe study was to explore the beneficial function of sev on ciri in rats. The molecular mechanism of sev in ciri was further studied, with emphasizing on the regulation of mir-30 c-5p/hipk1 axis. It was hypothesized that sev protected ciri via modulating hipk1 expression through mir-30 c-5p. Taken together, in our results novel insights will be provided on the mitigation of sev on ciri.2.2.1.in vivo with 2.5% sev  inhalational administration system in 33% oxygen and air mixture 30 min a day for 4 consecutive days [9 infected units/ml)  [A total of 60 male c57bl/6 rats , weighing 250–280 g, was raised in a cage with relative humidity of 50%-60%, at room temperature: 20°C-23°C, with 12-h light cycle, and free to gain food and water. the rats were randomly assigned into 5 groups. Middle cerebral artery occlusion (mcao) was applied for establishment of ciri model as set forth. the rats were anesthetized with 10% chloral hydrate (350 mg/kg), then the midline skin was cut open to expose the external carotid artery (eca) and the internal carotid artery (ica), which were then ligated. the heparinized endovascular wire  was inserted to ica via eca to block the origin of middle cerebral artery. After sealing for 2 h, slowly pulling out the suture was conducted. Then reperfusion was performed, and the rats were placed in an intensive care incubator for 24-h restoration. The sham rats were conducted with same operation without inserting sutures. Before mcao, the rats were pretreated , china)  were imp, china) : all mic2.2.Obeying former literature , neurolo2.3.The fresh brain sections with 1 mm thickness were soaked in 2% ttc-phosphate buffered saline (PBS) solution for 20 min in darkness at room temperature. After a brief wash with PBS, the sections were fixed in 4% paraformaldehyde  for 30 min at room temperature. These sections were shot via a digital camera. ImageJ 1.51 was used to measure each brain section and the infarct area (unstained area). The infarct volume was calculated as 100% .2.4.The rat brain tissue was immersed in 4% paraformaldehyde for preparation of paraffin sections, cut into 4 μm, roasted, de-waxed and hydrated regularly. then the paraffin sections were stained with he solution, after which, the damaged neurons manifested nuclear constricting, cell edema, vacuolization and nucleus .2.5.Tunel staining was conducted with the Apoptosis Kit  with the manufacturer’s instructions. Briefly, after dewaxing, the sections were incubated with protease K, membrane breakdown solution, tunel reagent, 3% hydrogen peroxide, and the conversion agent-pod, and developed via diaminobenzidine. tunel positive neurons around the damaged area  were observed and counted under fluorescence microscope .2.6.2/5% CO2 for 4 h. Then the cells were transferred to a complete DMEM containing 10% FBS, and cultured under 37°C with normoxia  for 24 h. Then, the cells were introduced with miR-30 c-5p inhibitor, si-HIPK1 and its Nc (RiboBio) with 30 μL Lipofectamine 3000 reagent . The collection of cells was conducted for later experiments. After incubation for 24 h, the cells were subjected to subsequent experiments.Mouse Hippocampal Neuronal Cell Line HT22 cells , were cultured in dulbecco modified eagle medium (dmem) incubator consisting of 10% fetal bovine serum (FBS) and 100 u/ml penicillin and streptomycin. As set forth , in the 2.7.2/95% air, and immersed for 30 min, with 3 times’ repetition, and rinsed for 15 min after each exposure. Twenty-four h after SEV pretreatment, the cells were received with Ogd/R [The cells were placed in a sealed anesthetic chamber containing 2.5% SEV and 5% COth Ogd/R .2.8.4 HT22 cell suspension was gained and supplied with ROS probe dichlorofluorescin diacetate (DCFDA)  as set forth. After incubation, the cells were centrifuged at 300 g and re-suspended. Relative ROS levels were determined by Fluorescence-activated cell sorter (FACS)  [The 1 × 102.9.Measurement of interleukin (IL)-1β, tumor necrosis factor α (TNF-α) and IL-6 in OGD/R-treated HT22 cells and rat brain tissue was with elisa kits obeying manufacturer’s protocol. The content of malondialdehyde (MDA), glutathione peroxidase (Gsh-Px) and superoxide dismutase (SOD) were detected with the kit with the manufacturer’s instructions.2.10.CCK-8  reagents were mixed with cell medium with manufacturer’s instructions. the cells were then incubated. Absorbance was recorded at 450 nm applying a microplate reader .2.11.3 cells were placed in 6-well plates and the colonies were stained with the staining solution composed of methanol (20%) and crystal violet (0.1%). The cells were counted and analyzed [HT22 cells were treated with trypsin. The 1 × 10analyzed .2.12.5 cells/well) were seeded into the upper chamber of transwell and cultured in serum-free medium . for the assay of cell invasion, transwell’s upper chamber was covered with 50 µL Matrigel (BD Biosciences) at 37°C for 2 h. The complete medium supplemented with 10% FBS  was added to transwell’s lower chamber. after culture for 24 h, the cells were fixed with 4% paraformaldehyde  at room temperature for 20 min, and stained at room temperature with 0.1% crystal violet  for 10 min. The number of migrating and invading cells was determined under a light microscope (zeiss AG) under five random fields  at 10 min [Transwell inserts  with 8 µm aperture was inserted into a 6-well plate. HT22 cells  [Apoptotic cells were quantified via flow cytometry applying the Annexin V- fluoresceinisothiocyanat (FITC) Apoptosis Assay Kit  as per instructions. Simply put, the cells were harvested by trypsin detachment, seeded into 6-well plates at a density of 2.0 × 10ositive) .2.14.Extraction of total RNA was from tissues or cells via TRIzol reagents . Detection of the concentration and purity of RNA was via nano-droplet spectrophotometer. With the instructions, reverse transcription of 1 μg total RNA was via the PrimeScript RT kit  and synthesis of its complementary DNA (cDNA) was conducted. performance of RT-qPCR was via SYBR Premix Ex TaqTM kit . The loading control of HIPK1 was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that of miR-30 c-5p was U6. The primers were synthesized by RiboBio  .GenesFo2.15.Total protein was extracted from tissues or cells via radioimmunoprecipitation lysis buffer . Measurement of protein concentration was via the bicinchoninic acid Protein Determination Kit (Beyotime Biotechnology). separation of the protein samples of 30 μg was via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted on polyvinylidene membrane (Millipore). After block with 5% skim milk, incubation of the membrane was with anti-HiPK1  and GAPDH , and a horseradish peroxidase conjugated goat anti-rabbit Immunoglobulin G secondary antibody. Then enhanced chemiluminescence reagent (Thermo Fisher Scienti) was applied .2.16.4 cells/well, and transfection of the cells was with HIPK1-WT and -MUT, and miR-30 c-5p mimic or its NC (RiboBio). Detection of the luciferase activity was via dual luciferase reporter detection system (Promega) [HIPK1 3ʹuntranslated region (UTR) consisting of wild-type (WT) or mutant (MUT) binding sites of miR-30 c-5p was cloned into the pmirGLO vector (Promega), named HIPK1-WT and -MUT. HT22 cells were seeded into 24 well plates at a concentration of 2 × 10Promega) .2.17.P was a bilateral test, and P < 0.05 was considered statistically significant.SPSS 21.0  statistical software was applied for analysis of data. After examination of the kolmogorov smirnov test, the data were normally distributed, expressed as mean ± standard deviation (SD). Application of t test was in the comparison between the two groups, and one-way analysis of variance (ANOVA) in comparison among multiple groups, and Fisher’s least significant difference t test (LSD-t) for pair comparison after ANOVA analysis. 3.in vitro and in vivo experiments, it was found that SEV protected CIRI via modulating the expression of HIPK1 through miR-30 c-5p. Therefore, in the data, the function and mechanism of miR-30 c-5p and HIPK1 in CIRI were first studied, providing new insights into the pathogenesis of CIRI.The study was to explore the beneficial effects of SEV on CIRI in rats. The molecular mechanism of SEV in CIRI was further investigated. Meanwhile, through a series of 3.1.in vivo was explored from the above two angles. The data manifested that CIRI rats had inflammation and oxidative stress, and after SEV intervention, TNF-α, IL-1β, IL-6 and MDA were apparently reduced, whereas GSH-Px and SOD were distinctly elevated (in vivo.SEV, a crucial volatile anesthetic, has been affirmed to ameliorate myocardial I/R injury and reduce infarct size . For undelevated . The patelevated , affirmielevated . Moreoveelevated . These e3.2.in vitro, establishment of an in vitro OGD/R model was also presented. It was manifested the similar inflammatory and oxidative stress responses in the in vitro OGD/R mode. In the cell supernatant in the SEV intervention group, apparent decline in inflammatory factors and MDA, and elevation in GSH-Px and SOD were revealed (in vivo and in vitro, thereby repressing apoptosis and protecting Ogd/R-induced HT22 cell damage.For exploration of the protective mechanism of SEV revealed . Former revealed . Moreoverevealed . In shor3.3.in vitro and vivo, the influence of SEV on mir-30 c-5p and HIPK1 was discussed. It was affirmed clear repression of miR-30 c-5p and elevation of HIPK1 in CIRI in vitro and vivo models, whereas distinct amelioration of miR-30 c-5p and HIPK1 with different degrees were manifested after SEV intervention. In brief, it was suggested that the protective mechanism of SEV against CIRI may be implicated with the regulation of miR-30 c-5p and HIPK1 (For exploration of the molecular mechanism of the protection of SEV on CIRI nd HIPK1 .Figure 33.4.in vivo, distinct repression of miR-30 c-5p was affirmed (in vivo.Further discussion of the function of miR-30 c-5p was in the protective mechanism of SEV affirmed . It was affirmed . In the affirmed . As maniaffirmed . Tunel saffirmed . Briefly3.5.in vitro, and acquisition of depressive miR-30 c-5p was manifested (Next, the role of miR-30 c-5p was also discussed in the protective mechanism of SEV nifested . Elisa rnifested , and augnifested . Meanwhinifested . Moreovenifested . In sum,3.6.Through bioinformatics analysis was forecasted the binding site of miR-30 c-5p with HIPK1 , which w3.7.in vitro.Further research of the regulatory role of HIPK1, the downstream target gene of miR-30 c-5p, was on CIRI, then transfection of in-miR-30 c-5p + si-HIPK1 was implemented in HT22 cells , clarify4.In this study, SEV was available to ameliorate CIRI by regulating oxidative stress, inflammatory response, cell proliferation, apoptosis, etc. Other experiments have manifested that SEV can repress the decline of miR-30 c-5p stimulated via I/R, thereby restraining HIPK1, namely, SEV can mitigate CIRI through the miR-30 c-5p/HIPK1 axis.in vitro model of OGD/R cells were constructed, and the therapeutic effect of SEV on CIRI was originally verified, manifesting the amelioration of oxidative stress, inflammatory response and histopathologic condition, and reduction of apoptosis, and facilitation of cell growth in vitro.CIRI is a crucial pathological factor exacerbating brain dysfunction, leading to unpleasing treatment and prognosis of ischemic stroke . CIRI, ain vitro and vivo models of CIRI, and restrains the down-regulation of miR-30 c-5p caused via I/R, thereby repressing HIPK1.Studies have assured that the therapy of diseases via drugs is often achieved through downstream molecular mechanisms. For instance, notoginsenoside R1 protects the injury induced by hypoxia and re-oxygen deprivation via upregulation of miR-132 in H9c2 cells , and demin vivo and vitro models pretreated by SEV, oxidative stress, inflammatory response and histopathologic condition were aggravated, and apoptotic cells in tissues were strengthened. For the cell model, the degree of cell damage was augmented, with restrained cell growth, manifesting that repressive miR-30 c-5p is available to reverse the influence of SEV pretreatment on neuronal cells.miR-30 c-5p, a member of the miRNA family, has been reported as a key mediator of several cardiac abnormalities, and is commonly applied as a therapeutic target for ameliorating myocardial ischemia-reperfusion injury . In addiin vitro and in vivo modulation of CIRI. Of course, there are still some shortcomings in this study. For example, only male rats were applied in animal selection. Cell culture was conducted using higher oxygen concentrations relative to brain tissue; in the meantime, the latent clinical efficacy of this axis will be further complemented later as conditions permit.The miR-30 c-5p-HIPK1 axis was a novel molecular regulatory network of CIRI pathogenesis, which was originally discovered in the study. HIPK1 is a serine/threonine kinase affiliated to the CMGC superfamily. a study has testified that HIPK1, as a target gene of miR-495-3p, takes part in the modulation of the development of acute kidney injury . In this5.All in all, the study demonstrates that SEV is available to ameliorate CIRI, and this effect may be achieved through miR-30 c-5p/HIPK1 axis. These new findings and mechanisms may provide a potential therapeutic approach for CIRI in the future.


We aimed to determine the prognostic values of the National Early Warning Score 2 (NEWS2) and laboratory parameters during the first week of COVID-19.All adult patients who were hospitalized for confirmed COVID-19 between 11 March and 11 May 2020 were retrospectively included.Overall, 611 patients were included. Our results showed that NEWS2, procalcitonin, neutrophil/lymphocyte ratio and albumin at D0, D3, D5 and D7 were the best predictors for clinical deterioration defined as a composite of ICU admission during hospitalization or in-hospital death. Procalcitonin had the highest odds ratio for clinical deterioration on all days.This study provides a list of several laboratory parameters correlated with NEWS2 and potential predictors for clinical deterioration in patients with COVID-19. The COVID-19 pandemic is a grueling problem worldwide. There is a lack of knowledge about the predictive value of National Early Warning Score 2 (NEWS2) for severe COVID-19 illness. We analyzed the prognostic value of NEWS2 and laboratory parameters during the clinical course of COVID-19. This study provides a list of several laboratory parameters correlated with NEWS2 and potential predictors for intensive care unit admission during hospitalization or in-hospital death. The COVID-19 pandemic due to the SARS-CoV-2 virus causes high rates of mortality, morbidity, longer duration of hospitalization and increased need for intensive care unit (ICU) admission . NEWS2 iTo date limited data exists on the NEWS2 and laboratory parameters in patients with COVID-19. In this study, we aimed to determine prognostic value of NEWS2 and laboratory parameters during the clinical course of COVID-19. Additionally, the correlation between NEWS2 and laboratory parameters at admission (D0), day 3 (D3), day 5 (D5) and day 7 (D7) were evaluated. To our knowledge, this is the first study evaluating both NEWS2 and laboratory parameters on the clinical course of COVID-19 in Turkey.In this retrospective and single-center study, all adult patients (≥18 years old) who were hospitalized for a laboratory confirmed COVID-19, between 11 March and 11 May 2020 were included. SARS-CoV-2 testing was performed using real-time reverse transcription-PCR of samples collected by nasopharyngeal and/or oropharyngeal swabs.Patients with COVID-19 requiring hospitalization were included in the study. Outpatients and asymptomatic patients were excluded. Also we excluded patients if oropharyngeal or nasopharyngeal swab samples were repeatedly negative for SARS-CoV-2 by reverse transcription-PCR. Our primary outcome was the occurrence of clinical deterioration defined as a composite of ICU admission during hospitalization or in-hospital death.The criteria for ICU transfer in our hospital were the following parameters (at least one or more); dyspnea and respiratory distress; respiratory rate ≥30/min; oxygen saturation <90% or partial oxygen pressure <60 mmHg despite oxygen support (≥5 l/min); septic shock and/or multiple organ dysfunction.Epidemiological and demographic characteristics, clinical, laboratory, radiological findings and outcomes were collected from medical records. Vital signs including respiratory rate, peripheral capillary oxygen saturation, heart rate, blood pressure, body temperature and consciousness  were recorded. Laboratory parameters including albumin, C-reactive protein (CRP), procalcitonin, hemoglobin, hematocrit, neutrophil count, lymphocyte count, platelet count, neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), urea, ferritin, albumin, fibrinogen, D-dimer, aspartate aminotransferase (AST) and alanin aminotransferase at D0, D3, D5 and D7 were included. The NEWS2 score was calculated at D0, D3, D5 and D7. These parameters were obtained at the same time.post hoc analysis. To evaluate the factors in prognosis which were admission ICU and in-hospital death, univariate logistic regression analysis was performed. Afterward, these dependent groups were handled one by one, receiver operating characteristic (ROC) curves were drawn and cut-off values, sensitivity and specificity and area under the curve (AUC) were demonstrated. To predict clinical deterioration, the prognostic accuracy of NEWS2 and laboratory parameters at D0, D3, D5 and D7 was evaluated by ROC analyses.NEWS2 and laboratory parameters at D0;NEWS2 and laboratory parameters at D3 after excluding from the analysis patients with clinical deterioration within the first 3 days of hospitalization;NEWS2 and laboratory parameters at D5 after excluding from the analysis patients with clinical deterioration within the first 5 days of hospitalization;NEWS2 and laboratory parameters at D7 after excluding from the analysis patients with clinical deterioration within the first 7 days of hospitalization.Quantitative variables are expressed as mean and standard deviation when they contain continuous and normal distributed data. When the data were not distributed normally, median and interquartile range (IQR) were used. When they contained categorical data, they were expressed as a percentage (%) and frequency (n). Comparison of qualitative variables was performed by Pearson’s Chi-square test. The normal distribution questioning the necessity of using the parametric test was examined by Kolmogrov–Smirnov, Shapiro–Wilk, Kurtosis–Skewness tests and box plot distribution. When normally distributed data could not be determined, nonparametric tests and spearman correlation were used. Kruskal–Wallis test was used for the analysis of continuous and more than two independent nonparametric groups (Bonferroni correction was used when necessary) and Mann–Whitney test was used for Additionally, the association of the parameters at admission with the clinical deterioration was evaluated to predict the 3-day, 5-day and 7-day end points by ROC analyses.The results were evaluated in 95% CI and statistical significance level was defined as p < 0.05. The analyzes were performed using IBM SPSS-21 .Supplementary Table 1.Overall, 611 patients were included. Of whom, 329 (53.8%) were male, the mean age was 52.53 ± 15.07 years. Seventy-three patients (11.9%) were admitted to the ICU. In-hospital death occurred in 46 (7.5%) patients. Among 73 patients (11.9%) admitted to the ICU, 40 patients (54.8%) died during hospitalization. Clinical deterioration was observed in 79 patients (12.9%) during hospitalization, 36 (5.9%), during the first 3 days, 54 (8.8%) during the first 5 days and 62 (10.1%) during the first week of hospitalization. NEWS2 was calculated at D0, D3, D5 and D7 of hospitalization. Patients were stratified into three risk groups: low risk from zero to four; medium risk from five to six and high risk above seven. Of 611 patients, 505 (82.7%) at D0, 411 (91.9%) at D3, 375 (92.2%) at D5, 284 (93.8%) at D7 had a NEWS2 score <7. The median length of hospital stay was 8.9 days and 332 patients (54.3%) who did not have fever and did not need oxygen in the last 48–72 h and meet the criteria for home monitoring were discharged within the first 7 days. Demographic characteristics of hospitalized patients with COVID-19 are available in The parameters associated with admission ICU and in-hospital death at D0, D3, D5 and D7 were NEWS2, lymphocyte count, neutrophil count, platelet count, NLR, PLR, CRP, procalcitonin, D-dimer, troponin, AST, urea, lactate dehydrogenase (LDH) and albumin. The median and IQR values of the laboratory parameters and NEWS2 are represented in In univariate analysis, among parameters associated with ICU admission or in-hospital death at D0, D3, D5 and D7, best predictors were NEWS2, procalcitonin, NLR and albumin. Additionally, D-dimer  and hemoglobin (at D3 and D5) were valuable predictors in univariate analysis .Laboratory parameters correlated with NEWS2 at D0, D3, D5 and D7 were lymphocyte count, neutrophil count, NLR, PLR, CRP, procalcitonin, ferritin and urea  & 2.ROC curves of NEWS2 at D0, D3, D5 and D7 to predict clinical deterioration are shown in ROC curves of procalcitonin at D0, D3, D5 and D7 to predict clinical deterioration are shown in ROC curves of albumin at D0, D3, D5 and D7 to predict clinical deterioration are shown in ROC curves of NLR ratio at D0, D3, D5 and D7 to predict clinical deterioration are shown in The combined parameter of procalcitonin, NLR, albumin and NEWS2 at D0, D3, D5 and D7 to predict clinical deterioration yielded an increased AUC of 0.838 , 0.947 , 0.989  and 0.868 , respectively. The sensitivities and specificities of combined use were 70.4 and 88.1 at D0, 95.2 and 82.5 at D3, 83.3 and 98.9 at D5, 66.7 and 97.4% at D7, respectively.Additionally, we analyzed the association of NEWS2, procalcitonin, albumin and NLR at admission with the end point events at D3, D5 and D7. Albumin was the best predictor for the 3-day end-point. AUC was 0.849 (95% CI: 0.755–0.944), sensitivity of 84.1% and specificity of 72.2% (p < 0.001). Procalcitonin was the best predictor for the 5-day and 7-day end points. AUC was 0.835 (95% CI: 0.748–0.821), with a sensitivity of 79.2 and specificity of 77.1% (p < 0.001) for the 5-day end-point. AUC was 0.849 (95% CI: 0.773–0.924), sensitivity of 79.3 and specificity of 78.2% (p < 0.001).In this study, we presented a detailed analysis of the NEWS2 score and laboratory parameters in hospitalized patients with COVID-19. Our results showed that NEWS2, procalcitonin, NLR and albumin at D0, D3, D5, and D7 were the best predictors for clinical deterioration . The combined effect of NEWS2, procalcitonin, NLR and albumin were more valuable to predict clinical deterioration. Procalcitonin had the highest odds ratio (OR) for clinical deterioration at D0, D3, D5 and D7 in univariate analysis. ROC analyses showed that NEWS2 at D7, procalcitonin at D5, albumin at D7 and NLR at D5 had highest AUC values. Additionally, we detected a strong correlation between NEWS2 and laboratory parameters including lymphocyte count, neutrophil count, NLR, PLR, CRP, procalcitonin, ferritin and urea at D0, D3, D5 and D7.et al., they suggested that NEWS2 score was not a valuable tool to predict clinical deterioration in elderly patients with COVID-19 [et al. showed that NEWS2 scores on D0 significantly differed in noncritical and critical patients  [et al. the AUC value of NEWS score to predict ICU admission or death was 0.74, in consistent with our result. However, the AUC values of NEWS2 at D3, D5 and D7 were 0.798, 0.833 and 0.842 whereas NEWS2 at D0 was not accurate (AUC <0.750) [et al. showed that AUC values of NEWS2 was 0.74 at D0, with a best cutoff of six and was 0.98 at D7, with a best cut off of seven. They reported high sensitivity and specificity at D7  [Early and accurate discrimination of need for ICU improves the clinical course of COVID-19 and reduce unnecessary use of ICU beds. There are several published studies on the use of NEWS2 in COVID-19 patients ,7–13. HoCOVID-19 . Howeveret al. found an association between the severity of COVID-19 and the following laboratory parameters NLR, LDH, D-dimers, CRP, fibrinogen and ferritin [et al. procalcitonin, CRP and NLR were valuable predictors for COVID-19 mortality. They showed that the AUC from highest to lowest was combined effect >CRP >procalcitonin >NLR, respectively [et al. reported that NLR, thrombocytopenia, prothrombin time and D-dimer were associated with death. They showed that increased NLR (≥9·13) was associated with fivefold increased mortality risk [et al. demonstrated that higher levels of leukocyte (OR: 5.21), neutrophil (OR: 6.25), D-dimer (OR: 4.19) and prolonged PT (OR: 2.18) was associated with ICU admission. IL-6 (OR: 13.87), CRP (OR: 7.09), D-dimer (OR: 6.36), and neutrophils (OR: 6.25) had the highest ORs for mortality [et al. showed that increased procalcitonin values are associated with a nearly fivefold higher risk for need for ICU or use of mechanical ventilation  [et al. found that procalcitonin , CRP  and NLR  had higher HRs of 12.82, 12.30 and 8.6 for mortality, respectively. Additionally, procalcitonin (≥0.10 ng/ml) and CRP (≥52.14 mg/l), but not NLR exhibited independent increasing risks of mortality, with HRs of 52.68 (95% CI: 1.77–1571.66) and 5.47 (95% CI: 1.04–28.72), respectively [Due to the physiopathological changes, deterioration in different laboratory parameters occurs while the disease progresses. Therefore, laboratory parameters are commonly used for assessing disease severity. Lagadinou ferritin . In the ectively . Liao etity risk . SimilarPT OR: 2. was assoectively .et al. Spearman’s rank correlation analysis revealed that leukocyte, neutrophil, CRP, procalcitonin and LDH were positively correlated and albumin was negatively correlated with mortality in patients with receiving maintenance hemodialysis. Additionally, they showed that CRP had the highest AUC value (0.895) and the values of AUC of neutrophil count, LDH, leukocyte, albumin and procalcitonin were 0.813, 0.758, 0.757, 0.743 and 0.728, respectively [In the study of Shang ectively . In contet al., they suggested that a limited increase in procalcitonin levels  could be associated with increased IFN-γ [Procalcitonin is not well studied for COVID-19 cases. However, some studies suggested that increased procalcitonin levels were found to be associated with the disease severity in patients with COVID-19. A meta-analysis showed that severe patients with COVID-19 had increased procalcitonin levels ,19. Simied IFN-γ .et al. reported that albumin was associated with clinical deterioration and significantly higher in patients with the improvement/stabilization than in those with disease progression  [et al., mean albumin at D0 was 3.50 g/dl (CI: 3.26–3.74 g/dl) in the severe group and 4.05 g/dl (CI: 3.82–4.27 g/dl) in the nonsevere group (p < 0.001). They reported that hypoalbuminemia was associated with 12.6-fold increased risk of mortality [Low-serum albumin levels in studies with COVID-19 patients are suggested to be associated with an increased risk of mortality . In cons= 0.006) . In the ortality .et al. showed that NLR was significantly correlated with all-cause in-hospital mortality  [An increase in neutrophils and a decrease in lymphocytes have been found in various studies. Some studies have shown that NLR may be an important indicator for the severity of COVID-19 patients. Yan 425.088) . The NLR425.088)  and incr425.088) ,26.This study has several limitations. First, it was retrospectively conducted in a single center. Second, this study had a small sample size and a control group was not included. The generalizability of our results may be limited. Thus, we need new large scale studies providing important information to better understand COVID-19 pandemic. Our study has also several strengths. First, we were able to admit all critically ill patients requiring intensive care to the ICU during the first months of pandemic. This prevents a selection bias. Second, longitudinal evaluation of the association between clinical deterioration and the dynamic changes of laboratory parameters was performed, since we regularly monitored laboratory parameters during the clinical course.This study provides a list of several laboratory parameters correlated with NEWS2 and potential predictors for ICU admission or in-hospital death during the clinical course of COVID-19. NEWS2, procalcitonin, NLR and albumin have a high accuracy to predict clinical outcomes/disease progression in hospitalized patients and should be considered in the clinical decision of ICU admission. In conclusion, dynamic monitoring of NEWS2 and laboratory parameters is vital for improving clinical outcomes.The parameters associated with admission intensive care unit (ICU) and in-hospital death at (D0), day-3 (D3), day-5 (D5) and day-7 (D7) were National Early Warning Score 2 (NEWS2), lymphocyte count, neutrophil count, platelet count, neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), C-reactive protein (CRP), procalcitonin, D-dimer, troponin, aspartate aminotransferase, urea, lactate dehydrogenase and albumin.Laboratory parameters correlated with NEWS2 at D0, D3, D5 and D7 were lymphocyte count, neutrophil count, NLR, PLR, CRP, procalcitonin, ferritin and urea.Procalcitonin had the highest odds ratio for clinical deterioration on all days.Receiver operating characteristic analyses showed that NEWS2 at D7, procalcitonin at D5, albumin at D7 and NLR at D5 had highest area under the curve values.In univariate analysis, among parameters associated with ICU admission or in-hospital death at D0, D3, D5 and D7, best predictors were NEWS2, procalcitonin, NLR and albumin.This study provides a list of several laboratory parameters correlated with NEWS2 and potential predictors for ICU admission or in-hospital death during the clinical course of COVID-19.


The 3-year overall survival (OS) rate of patients with previously treated or untreated stage III or IV melanoma has by now reached 63% using ipilimumab and nivolumab therapy. However, immune-related adverse events (irAEs) of grade 3 or 4 occurred in 59% of patients leading to discontinuation of therapy in 24.5% of patients and one death. Therapy with checkpoint inhibitors could be safer and more effective in combination with hyperthermia and fever inducing therapies. We conducted a retrospective analysis to test the safety and efficacy of a new combination immune therapy in 131 unselected stage IV solid cancer patients with 23 different histological types of cancer who exhausted all conventional treatments. Treatment consisted of locoregional- and whole-body hyperthermia, individually dose adapted interleukin 2 (IL-2) combined with low-dose ipilimumab (0.3 mg/kg) plus nivolumab (0.5 mg/kg). The objective response rate (ORR) was 31.3%, progression-free survival (PFS) was 10 months, survival probabilities at 6 months was 86.7% , at 9 months was 73.5% , at 12 months was 66.5% , while at 24 months survival was 36.6% . irAEs of World Health Organization (WHO) Toxicity Scale grade 1, 2, 3, and 4 were observed in 23.66%, 16.03%, 6.11%, and 2.29% of patients, respectively. Our results suggest that the irAEs profile of the combined treatment is safer than that of the established protocols without compromising efficacy.The online version of this article (10.1007/s00262-020-02751-0) contains supplementary material, which is available to authorized users. Concurrent ipilimumab and nivolumab treatment has by now achieved a 3-year overall survival (OS) rate of 63% for patients with advanced melanoma . Howeverallogeneic bone marrow transplantation (BMT)  P: 0.661).Stage IV cancer patients . Twenty-four percent of our patients had to receive antibiotics. Antibiotic treatments administered within 30 days from commencement of ICI therapy has been known to be associated with significantly worse overall survival in patients with nonsmall cell lung cancer (2.5 vs. 26 months), melanoma (3.9 vs. 14 months), and other tumor types (1.1 vs. 11) who received antibiotic treatment vs. those who did not. In addition, patients had a higher risk of disease refractory to treatment .Table 2ODuring a follow-up period of up to 5 years, the ORR and OR were 31.3% and 49.62%, respectively, while the OS at 12 months was 65.9%  and low microsatellite instability (MSIlow) was determined only in a small subgroup of patients. However, TMBlow and MSIlow can be expected in the majority based on published evidence [high, and MSIhigh [Antibiotic use in 24%;Low PD-1/PD-L1 expression;Only stage IV patients, with 35.1% liver metastasis, a specifically bad prognosis;Only 35% ECOG 0;Heavily pretreated patients.Importantly, the overwhelming majority (> 98%) of the evaluated patients in this study had low PD-L1 expression as confirmed by immunohistochemistry (determined as ≤ 1%). Low tumor mutational burden Below is the link to the electronic supplementary material.


This new information about the β-glucan content of Thai rice cultivars could be used for the development of cereal-based functional food.β-glucan is a dietary fiber that is beneficial to human health, and its content varies according to its different parts, type of cereal grain, and growing environment. In this study, the β-glucan of milled rice, rice husk, and rice bran fractions, as well as the amylose content of milled rice fraction, from 38 selected rice-paddy grains from six regions of Thailand were quantitatively determined. The milled rice of the Sakon Nakhon (SN) cultivar grown in the northeast contained the highest β-glucan content (0.88 ± 0.03%), followed by the milled rice of the Jow Khao Chiangmai (JKC) cultivar (0.71 ± 0.03%) and rice bran of the Sew Mae Jan (SMJ) cultivar (0.67 ± 0.03%) grown in the north. The results reveal that the rice cultivars from each region showing variation in the β-glucan level in each fraction, which is mainly found in milled rice and rice bran, are similar to those found in other cereal grains, although low amounts are found in the husk. The amylose and β-glucan contents in the milled rice fraction showed a strong negative correlation (r = −0.805;  Thailand is an agricultural country that produces rice as its main staple food, which is grown on approximately 10 million ha of land  ,4,5,6. MThailand has 138 varieties of rice that are classified based on the ecosystem of the riceland on which they were cultivated and their photoperiod sensitivity and cooked texture . There aBased on the flowering response to light, rice cultivars are categorized into photosensitive and non-photosensitive; accordingly, the cultivars’ planting times are different . PhotopeAmylose is a major component of rice and strongly influences its cooked texture. Glutinous rice and non-glutinous rice are classified based on their amylose/amylopectin ratio: glutinous rice has low (<5%) amylose in its starch but is high in amylopectin . Finallyd-glucan (hereafter referred to as “β-glucan”) and are found as a chain of linear homopolysaccharide glucopyranosyl residues linked by several (most often two or three) consecutive (1–4)-β-d-linkages separated by a single (1–3)-β-d-linkage [β-glucan is a significant component of the cell walls of higher plants that belong to the grass family (Poaceae), such as rice, wheat, barley, and oat; therefore, it is a common component of many human diets and feed formulations . Structu-linkage ,20. The -linkage ,22,23. C-linkage ,25,26. In general, β-glucan in cereal is found in the sub-aleurone layer and endosperm cell walls . DiffereThailand not only has several rice cultivars distributed over entire regions but is also rich in rice milling fractions from these rice cultivars. Two major varieties are the Khao Dawk Mali 105 (KDM105) and RD6, which together cover approximately 64% of the cultivation area and have the greatest yield of paddy rice , so they have the most rice milling fractions compared to others . These fThe percentage yields of rice paddy fractions of milled rice, rice husk, and rice bran, and the yield loss of all 38 cultivars are given in β-glucan is abundant in the cell walls of cereal grains, such as oat, barley and wheat. In rice, β-glucan is distributed throughout the grain, including in the starchy endosperm and sub-aleurone layer of milled rice, aleurone layer and maternal pericarp of the bran, as well as the husk ,41. In TThe β-glucan distribution of each paddy-rice fraction is ranked in the following descending order: milled rice (0.11–0.88%) > rice bran (0.11–0.67%) > rice husk (0.02–0.11%). The measured contents in milled rice and rice bran are relatively similar to the findings of Demirbas  and JungKDM105 and RD6 had β-glucan contents in milled rice of 0.41 ± 0.02 and 0.46 ± 0.02%, respectively; rice bran of 0.20 ± 0.02 and 0.31 ± 0.01%, respectively; and rice husk of 0.06 ± 0.00 and 0.07 ± 0.00%. The total β-glucan contents in milled rice, rice husk, and rice bran of KDM105 were 842.02 ± 18.98, 46.11 ± 0.97, and 38.36 ± 0.70 mg, respectively. The total β-glucan contents in milled rice, rice husk, and rice bran of RD6 were 854.67 ± 26.14, 56.45 ± 3.97, and 79.80 ± 1.67 mg, respectively. Although these two cultivars have moderate β-glucan contents in each fraction, they are two major cultivars of Thailand. Therefore, they are another source of β-glucan content as they have a rich annual yield of rice paddy.For each of the six regions, the highest amounts of β-glucan for selected rice cultivar fractions, in descending order, for milled rice, were found as follows: northeastern (0.88 ± 0.03% for SN) > northern (0.71 ± 0.03% for JKC) > southern (0.45 ± 0.02% for NDC4) > central plain (0.42 ± 0.02% for PT1) > eastern (0.29 ± 0.02% for At1) > western (0.21 ± 0.03% for CN1). For rice bran, the highest amounts were found in the following order: northern (0.67 ± 0.03% for SMJ) > southern (0.57 ± 0.02% for Dpy) > northeastern (0.52 ± 0.02% for SN) > central plain (0.40 ± 0.01% for JC1) = eastern region (0.40 ± 0.01% for PB2) > western region (0.39 ± 0.02% for CN1). For the most part, the rice husk showed a very low β-glucan content. In descending order, the highest levels were northeast (0.11 ± 0.01% for SN) > northern (0.09 ± 0.01% for JKC) > southern (0.08 ± 0.00% for DPy) > central plain  = eastern (0.07 ± 0.00% for PB1) > western (0.06 ± 0.01% for CN1).The β-glucan content of the cultivars, as classified by the riceland ecosystem, photoperiod sensitivity, and cooked texture, was also investigated, as shown in Based on the flowering response to light, rice cultivars are grouped into photosensitive and non-photosensitive. Photoperiod-sensitive rice has a longer growing period, resulting in only one crop per year. In contrast, photoperiod-insensitive rice can be grown at any time, even during the dry season when lowland rice areas are being irrigated . In ThaiBased on the texture of cooked rice, rice cultivars are grouped into glutinous and non-glutinous rice. The non-glutinous cultivars were grown in all regions of the country, while the glutinous cultivars were mainly grown in the northeast . HoweverThe SN cultivar showed the highest β-glucan of all rice fractions in the photoperiod-insensitive and glutinous rice groups, but its rice bran fraction had a lower β-glucan content than SMJ, which is in the same glutinous rice group, but is photoperiod sensitive. In the photoperiod-sensitive and non-glutinous rice groups, JKC also has the highest β-glucan content for all fractions. These results suggest that the milled rice fractions of SN and JKC, as well as the rice bran fraction of SMJ, are better sources of β-glucan than the other cultivars. According to photoperiod sensitivity , SN can The Kolmogorov–Smirnov test for normality and skewness and kurtosis statistics were applied to the analysis of the β-glucan content of each rice fraction sample. The significant value of the test is 0.05, and an acceptable range for skewness and kurtosis was from +1.96 to −1.96 . The datThe box plot in To evaluate the relationship between amylose and β-glucan content, amylose in milled rice was determined using an amylopectin/amylose assay kit, and the results are shown in Amylose level distribution in milled rice was ranked in the following ascending order: cluster 1 (2.45–5.77%) < cluster 2 (10.07–13.85%) < cluster 3 (15.19–19.97%) < cluster 4 (20.03–27.39%). For the glutinous rice group (cluster 1), RD6 had the lowest amylose content of 2.45 ± 0.08%, close to that of SN with 2.59 ± 0.02%. Glutinous rice RD14 had the highest amylose content of 5.77 ± 0.06%. Among the non-glutinous cultivars, RD41 had the highest amylose content of 27.39 ± 0.25%, close to that for LPt123 with 27.10 ± 0.28%, while JKC had the lowest: 10.07 ± 0.03%. Cluster 1 contained only glutinous rice cultivars, which mostly had less than 6% amylose, whereas clusters 2–4 contained non-glutinous cultivars with amylose in the range of 10–30%. This was consistent with the findings of Chung et al. [In contrast to the amylose level, the distribution of β-glucan content was ranked in the following descending order: cluster 1 0.34–0.88%) > cluster 2 (0.24–0.71%) > cluster 3 (0.21–0.34%) > cluster 4 (0.11–0.18%). This inverse relationship between amylose and β-glucan content was consistent with the findings of Islamovic et al.  and lzyd–0.88% > p < 0.0001). In the case of the four clusters, classified by differences in amylose level, the relationship between amylose and β-glucan was separately evaluated, as shown in p < 0.001). Cluster 2 showed a strong negative correlation , followed by cluster 3, which showed a good negative correlation . Clusters 1 and 4, however, showed only a fairly negative correlation . These results exhibited a relationship similar to that observed by Hang et al. [p < 0.01, respectively. The glutinous rice cultivars (cluster 1) revealed a lower correlation of amylose and β-glucan content than for non-glutinous rice cultivars (clusters 2–4). However, the β-glucan level in cluster 1 was higher than for the other clusters. As shown in To date, much research has found an inverse relationship between amylose and β-glucan content, especially for barley cultivars, but data for rice are lacking. Hence, the correlation as well as linear regression analysis were studied in the milled rice fraction to confirm the inverse relationship. In the milled rice of all the cultivars, amylose content showed a strong negative correlation with β-glucan . Ethanol, glacial acetic acid, and dimethyl sulfoxide were purchased from Merck . Sodium dihydrogen orthophosphate dihydrate, sodium hydroxide, anhydrous sodium acetate, and sodium chloride were purchased from Qrec (Qrec (Asia) SDN BHD, Selangor, Malaysia) and Ajax Finechem Pty Ltd. . Calcium chloride dihydrate, magnesium chloride hexahydrate, and manganese chloride tetrahydrate were obtained from Sigma-Aldrich Co. . All chemicals and reagents used in the study were of analytical grade.The 38 paddy-rice samples in this study were obtained from 12 rice research centers in each province of Thailand. A list of the selected rice cultivars is shown in Based on information from the Rice Department at the Ministry of Agriculture and Cooperatives , the ricRF is the weight of each paddy-rice fraction  of the cultivars (g) after rice milling, and WR is the weight of the paddy-rice sample (g).The 38 paddy-rice samples (300 g) were de-hulled and polished using a rice milling machine  to obtain the fractions of milled rice, rice husk, and rice bran, the latter of which includes the rice germ  aqueous ethanol and incubated at 100 °C for 5 min. After mixing the sample on a vortexer , 5.0 mL of 50% (v/v) aqueous ethanol was then added, followed by mixing. The sample was then centrifuged at 1000g × for 10 min . The pellet was resuspended in 10.0 mL of 50% (v/v) aqueous ethanol and the centrifugation repeated. The pellet was further mixed and incubated with 5.0 mL of 20 mM sodium phosphate buffer (pH 6.5)  at 100 °C for 2 min, then vigorously stirred and incubated again for 3 min. Then, 0.2 mL of lichenase  was added to the sample and incubated at 40 °C for 1 hr. Distilled water was added to the sample in a tube to adjust the final volume to 30.0 mL, followed by centrifugation at 1000g × for 10 min. Furthermore, 3 of 4 supernatant tubes (0.1 mL) were added to 0.1 mL of β-glucosidase  plus 50 mM sodium acetate buffer (pH 4.0) , while another was added to 0.1 mL of 50 mM sodium acetate buffer (pH 4.0). Then, these samples were incubated at 40 °C for 15 min. Finally, 3.0 mL of GOPOD reagent, containing 36 U of glucose oxidase (EC. 1.1.3.4) plus 1.95 U of peroxidase (EC 1.11.1.7) and 0.24 g of 4-aminoantipyrine was added and incubated at 40 °C for 20 min. An amount of 100 µg of D-glucose standard was added to GOPOD reagent and incubated at 40 °C for 20 min. The β-glucan content was measured at 510 nm using a spectrophotometer  and reported on a moisture-free basis. The moisture content was assayed following approved method 44-15.02 [A mixed-linkage β-glucan assay for cereal grains kit  was used for the analysis of β-glucan content in each fraction of the 38 rice cultivars. This method uses a highly specific enzyme for cereal mixed-linkage β-glucan hydrolysis without affecting other polysaccharides. To carry out the reaction, 1.0 g of rice fraction sample, after passing through a 0.5 mm sieve screen, was mixed with 5.0 mL of 50% (44-15.02 .d-glucosidase treatment—the absorbance of a blank (non-treatment by β-d-glucosidase); mg is the weight of the sample (mg); and F is a factor for the conversion of absorbance values to µg glucose, which is calculated from 100 (µg of d-glucose)/ absorbance of 100 µg of d-glucose.w/w), and Y is the fraction yield of paddy rice (% w/w).β-glucan content was calculated using the glucose quantity determined with the described assay in Equation (2) and expressed as total β-glucan (mg) in 300 g of paddy-rice using Equation (3).v/v) aqueous ethanol for 15 min while lipids remaining in the supernatant were removed by centrifugation at 2000g × for 10 min. The starch pellet was mixed with 2.0 mL of dimethyl sulfoxide and incubated at 100 °C for 15 min. A measure of 4.0 mL of Con A solvent was immediately added to this mixture. The Con A solvent was freshy prepared by diluting 30 mL of concentrated Con A solvent in 100 mL of distilled water .The amylose content in the milled rice fraction of each rice cultivar was determined using an amylose/amylopectin assay kit . Starch pretreatment was first performed, whereby a 20 mg milled rice sample was completely dispersed in 1.0 mL of dimethyl sulfoxide by heating at 100 °C for 1 min. Then, the mixture was vigorously stirred and incubated at 100 °C for 15 min. After incubating the mixture at room temperature for 5 min, the starch was precipitated out by adding 4.0 mL of 95%   to 1.0 mL of the supernatant to decrease the pH to 5.0, the remaining Con A was denatured by heating at 100 °C for 5 min. An amount of 0.1 mL of a mixture of amyloglucosidase  and α-amylase  enzymes was added to the supernatant, and then incubated at 40 °C for 30 min. After centrifugation at 2000g × for 5 min, 1.0 mL of supernatant was mixed with 4.0 mL of GOPOD and incubated at 40 °C for 20 min. The absorbance of Con A supernatant (EA) was measured at 510 nm.Amylopectin specifically forms a complex with lectin concanavalin A (Con A). To the starch suspension, 0.50 mL of Con A solution (4 mg/mL) was added, gently mixed, and allowed to stand at room temperature for 1 h. Then, the amylose supernatant was collected following centrifugation at 4400S) was measured at 510 nm.Total starch was further determined for the calculation of amylose content. The starch suspension (0.5 mL) was mixed with 4.0 mL of 100 mM sodium acetate buffer (pH 4.5) plus 0.1 mL of amyloglucosidase/α-amylase solution and incubated at 40 °C for 10 min. Then, 1.0 mL of the mixture was added to 4.0 mL of GOPOD reagent and incubated at 40 °C for 20 min. The absorbance of total starch  and ES is the absorbance of total starch.The amylose content was estimated using the glucose quantity found in the Equation (4)p < 0.05 level using the Duncan multiple-range test (DMRT). Pearson coefficient (r) studies were carried out to determine the relationship between amylose and β-glucan contents using bivariate significance at the p < 0.01 level.Statistical analyses were conducted using SPSS . The results were calculated as an average of three replicate samples and the results expressed as mean ± standard deviation. A significant difference among each paddy-rice fraction yield, β-glucan, and amylose contents of 38 rice cultivars was determined using the one-way analysis of variance (ANOVA) at the In this study, 38 paddy-rice samples grown in six regions of Thailand  were selected to determine and evaluate the difference in β-glucan contents in three of their fractions . All the cultivars grown in those regions contained variations in β-glucan levels in each fraction, which were milled rice, rice bran, and rice husk in ranges of 0.11–0.88%, 0.11–0.67%, and 0.02–0.11%, respectively. A slight difference was found in milled rice and rice bran fractions, but both clearly differed from rice husk, which had a low level of β-glucan. Therefore, the β-glucan mainly found in milled rice and rice bran is similar to that found in other cereal grains, generally in the starchy endosperm, sub-aleurone, and aleurone cell walls. The milled rice of the SN cultivar, lowland rice grown in the northeastern region, contained the highest β-glucan content, followed by the milled rice of the JKC cultivar and rice bran of the SMJ cultivar, which are upland rice grown in the northern region. The environmental classification, based on riceland ecosystem and photoperiod sensitivity, showed some patterns with the level of β-glucan content for each paddy-rice fraction, while the cooked texture of glutinous and non-glutinous rice, which is strongly influenced by amylose, was obviously related to the β-glucan content in the milled rice fraction. The significant correlation between β-glucan and amylose content was strongly negative. This research suggests that Thai rice cultivars could be a potential source of beneficial β-glucan, especially in their milled rice and rice bran fractions, for further use as functional ingredients for cereal-based products, supplement products and cosmetics. The application of milled rice of SN and JKC cultivars, as well as rice bran of SMJ, as functional food ingredients could be studied in the future.


P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus–oocyte complexes (COCs) to 20 µM ( Hydroxyurea (HU), a United States Food and Drug Administration-approved drug , is an iAll chemicals and reagents, except those specifically noted, were purchased from Sigma–Aldrich .in vitro maturation (IVM) medium supplemented with 1, 10, 20, or 50 µM HU for 46–48 h.Previous studies exposed embryos to 2 mM HU for 2 h  or 0.237IVM media supplemented with different concentrations of HU  were prepared prior to incubation with oocytes to examine maturation. We analyzed numbers of metaphase II (MII) oocytes, as well as glutathione (GSH) and ROS levels, Δφm, and apoptosis.in vitro culture (IVC) medium without HU] to test the effect of HU on development of MII oocytes to blastocysts. We analyzed rates of blastocyst formation, total blastocyst cell numbers, and apoptosis of blastocyst cells.Next, we cultured parthenogenetically activated MII oocytes . After 4 h, activated MII oocytes were transferred into 450 μl IVC medium and covered with 500 μl mineral oil, and culture dishes were placed in an embryo incubator at 38.5°C with 5% CO2 and 95% humidity. Two-celled, four- to eight-celled, blastocyst, and hatching rates were analyzed at 46, 46–72, 168, and 192 h.The parthenogenetic activation system employed followed our previously described method ,20. IsolTotal blastocyst cell numbers were determined for 21 control group blastocysts  and 21 HU-exposed blastocysts . Blastocysts were fixed with 4% (w/v) paraformaldehyde, washed three times with phosphate-buffered saline mixed with 1g/l PVA (PBS-PVA), and incubated in 10 μg/ml Hoechst 33342 for 5 min at 37°C. Finally, blastocysts were placed on a glass slide under a glass coverslip, and images were acquired with a digital camera and fluorescence microscope .MII oocytes were washed three times with PBS-PVA, fixed with 4% (w/v) paraformaldehyde solution, washed, and incubated with 0.2% (v/v) Triton X-100 for 15–20 min. Fixed oocytes were washed and incubated in 1% (w/v) BSA for 1 h at room temperature to block nonspecific binding. Oocytes were incubated with anti-P53  and anti-Caspase-3  antibodies at 4°C overnight. The following day, oocytes were washed, incubated with a secondary antibody  at 37°C for 1–2 h, washed three times, and placed in Hoechst 33342 for 5 min at 37°C. We used 24  and 24  MII oocytes to examine P53 expression, and 21  and 21  MII oocytes to examine Caspase-3 expression. Immunostained oocytes were placed on glass slides and covered with a glass coverslip. Images were acquired with a digital camera with a fluorescence microscope.4 digested cumulus cells (from 20 MII oocytes) using a microRNA extraction kit . mRNA was reverse transcribed into cDNA using a reverse transcription kit . SYBR green fluorescent dye (Tiangen Biotech), cDNA, ddH2O, and primers (Supplementary Table S1) were added to the samples for PCR using a reverse transcription polymerase chain reaction (RT-PCR) instrument . RT-PCR cycles included pre-denaturation at 95°C for 15 min followed by 45 cycles of 95°C for 10 s (denaturation), 60°C for 20 s , and 72°C for 30 s (extension), followed by melting curve analysis. The β-Actin gene was used for standardization. Three independent experiments were performed, and the 2Ct−ΔΔ [ΔΔCt = ΔCt (case) − ΔCt (control)] method was used to calculate relative mRNA expression.Total mRNA was extracted from 5 × 102+-dependent phospholipid binding protein; Solarbio Life Sciences, Beijing, China) was added to samples, which were mixed at 20°C for 10 min. Next, 5 µl of propidium iodide (Solarbio Life Sciences) was added to samples for incubation at 20°C for 5 min. Finally, samples were gently mixed with 500 µl of PBS  and analyzed within 1 h by flow cytometry.Digested cumulus cells were collected using a low-speed centrifuge and washed in a 1.5-ml tube. The PBS supernatant was gently decanted after centrifugation. PBS-PVA cleaning solution was added and the cell slurry was gently mixed by pipetting. Following three washes, a solution containing 5 µl of Annexin V-FITC  and 18 (6 per group) blastocysts from parthenogenetically activated MII oocytes  were washed three times with PBS-PVA, fixed with 4% (w/v) paraformaldehyde solution, washed, and incubated with 0.2% (v/v) Triton X-100 for 15–20 min. After washing three times with PBS-PVA, fixed blastocysts were incubated with TdT and fluorescein-conjugated dUTPs  in the dark for 30 min at 37°C. Next, blastocysts were washed three times, placed in Hoechst 33342 for 5 min at 37°C, and washed again three times with PBS-PVA for 10 min each. Immunostained blastocysts were placed on glass slides and covered with glass coverslips. Images were acquired with a digital camera and fluorescence microscope.http://imagej.nih.gov).To measure GSH levels, 48 (15–17 per group) and 50 (16–18 per group) MII oocytes from control and 20 μM HU groups, respectively, were incubated with IVM medium containing 10 µM of 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin  cell tracer blue dye for 20 min, washed three times, and analyzed by spectroscopy . To examine ROS levels, 48 (15–17 per group) MII oocytes from control and 20 μM HU groups were washed three times with PBS-PVA and incubated with IVM medium containing 10 µM of 2,7-dichlorodihydrofluorescein diacetate  for 15 min in non-treated four-well plates. Following incubation, plates were washed three times and analyzed using a fluorescence microscope . All images were stored as TIFF files and analyzed by ImageJ software . Images were stored as TIFF files and analyzed by ImageJ software. Approximately 1–5 × 104 cumulus cells incubated with H2DCFDA were placed on ice in the dark for analysis by flow cytometry . Expression of 2′,7′-dichlorofluorescein is presented as the peak of the median FITC-A signal.Cumulus cells were washed three times for 5 min at 600×Δφm was calculated as the ratio of red fluorescence (corresponding to activated mitochondria) to green fluorescence . Forty-five (15 per group) MII oocytes from control and 20 μM HU groups were washed three times with PBS-PVA, then incubated with IVM medium containing JC-1 fluorescent probe (Solarbio Life Sciences) at 37°C for 20 min. After washing three times with JC-1 staining buffer, 15 oocytes per well were placed in 15 μl of JC-1 staining buffer and covered with 50 μl of mineral oil in six-well plates.g in 1.5-ml microcentrifuge tubes. Next, cells were incubated with IVM medium containing JC-1 fluorescent probe at 37°C for 20 min, and then washed three times with JC-1 staining buffer. Cells were placed in 1 ml of JC-1 staining buffer per tube and transferred in 200-μl aliquots to wells of a six-well plate for analysis. Cells were analyzed using a fluorescence microscope with 490-nm (green fluorescence) and 530-nm (red fluorescence) excitation. Images stored as TIFF files were analyzed by ImageJ software.For measurement of Δφm, cumulus cells were washed three times for 5 min at 600×t test to analyze comparisons of two groups and ANOVA test to analyze comparisons of more than two groups. P<0.05 was considered statistically significant.Each experiment was repeated at least three times and data were analyzed by SPSS 20.0 software . We used Student’s Porcine COCs were cultured in IVM media supplemented with 1, 10, 20, or 50 µM HU to determine the effect of HU on MII oocyte development. Exposure to 20 or 50 µM HU significantly decreased the percentage of MII oocytes compared with the control group . These results indicate that 20 µM HU affected post-meiotic development of MII oocytes.MII oocytes cultured in IVM media with (20 µM) or without HU were subsequently cultured in IVC media to examine post-meiotic development to the blastocyst stage. Percentages of blastocysts were significantly decreased in the 20-µM HU group compared with the control group , 35.55 ± 1.33% (P<0.01), and 27.33 ± 5.90% (P<0.001) vs. 46.96 ± 1.53%, respectively, P<0.001), 16.04 ± 1.02% (P<0.001), 13.46 ± 0.11% (P<0.001), and 9.77 ± 0.93% (P<0.001) vs. 22.79 ± 2.45%, respectively, Porcine COCs were cultured in IVM media without HU, and the resulting MII oocytes were cultured in IVC media supplemented with 0, 1, 10, 20, or 50 µM HU to examine the effect of HU on two-celled to hatching stage embryonic development. Percentages of two- and four- to eight-celled embryos were significantly decreased in the 50-µM HU group compared with the control group  through regulation of production and binding to fetal hemoglobin. Increased NO may result in oxidative stress and damage to the fetus. During oxidative stress, ROS levels increase and GSH levels decrease ,37. Sevein vitro is modulated through increased apoptosis of oocytes and cumulus cells, and increased oxidative stress. These effects hinder polar body extrusion in oocytes, which negatively influences their maturation and embryo development. The current study helps with risk assessment associated with HU exposure during oocyte maturation.The current study shows that the toxic effect of HU on porcine oocyte maturation Click here for additional data file.