Erratum: [Corrigendum] Macleaya cordata extracts exert antiviral effects in newborn mice with rotavirus­induced diarrhea via inhibiting the JAK2/STAT3 signaling pathway.

[This corrects the article DOI: 10.3892/etm.2020.8766.].

Freeze-thawing impairs the motility, plasma membrane integrity and mitochondria function of boar spermatozoa through generating excessive ROS.

BACKGROUND: Cryopreservation is an efficient way to store spermatozoa and is closely associated with the quality of sperm after the freeze-thaw process. During freeze-thaw cycling, excessive reactive oxygen species (ROS) are produced, and the effects of ROS on boar sperm during cryopreservation have not been identified. RESULTS: In this study, we evaluated the quality of boar spermatozoa in different steps of cryopreservation (extension, cooling, and thawing for 30 min and 240 min) with or without boar-sperm antioxidant (N-acetylcysteine (NAC)). The ROS levels, sperm motility, plasma membrane integrity, mitochondrial activity, sperm chromatin structure, ATP content, and sperm apoptosis were assayed. After thawing, the ROS level and sperm apoptosis were significantly increased, and the sperm motility, plasma membrane integrity, mitochondrial activity, sperm chromatin structure, and ATP content were significantly impaired compared with those at the extension period and cooling period. Moreover, the addition of N-acetyl L-cysteine (NAC) reversed these changes. CONCLUSION: The freeze-thawing of boar spermatozoa impaired their motility, plasma membrane, mitochondrial activity, sperm chromatin structure and apoptosis by producing excessive ROS. Thus, the downregulation of ROS level by antioxidants, especially the NAC, is important for manufacturing frozen pig sperm to increase reproductive cells and livestock propagation, as well as to improve the application of frozen semen in pigs worldwide.

Valeriana jatamansi Jones Inhibits Rotavirus-Induced Diarrhea via Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway.

Rotavirus (RV), as the main cause of diarrhea in children under 5 years, contributes to various childhood diseases. Valeriana jatamansi Jones is a traditional Chinese herb and possesses antiviral effects. In this study we investigated the potential mechanisms of V. jatamansi Jones in RV-induced diarrhea. MTT assay was performed to evaluate cell proliferation and the diarrhea mice model was constructed using SA11 infection. Mice were administered V. jatamansi Jones and ribavirin. Diarrhea score was used to evaluate the treatment effect. The enzyme-linked immunosorbent assay was performed to detect the level of cytokines. Western blot and quantitative reverse transcription-PCR were used to determine protein and mRNA levels, respectively. Hematoxylin-eosin staining was applied to detect the pathological change of the small intestine. TdT-mediated dUTP nick-end labeling was conducted to determine the apoptosis rate. The results showed V. jatamansi Jones promoted MA104 proliferation. V. jatamansi Jones downregulated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in protein level, which was consistent with the immunohistochemistry results. Moreover, V. jatamansi Jones combined with ribavirin regulated interleukin-1ß (IL-1ß), interferon γ, IL-6, tumor necrosis factor α, and IL-10, and suppressed secretory immunoglobulin A secretion to remove viruses and inhibit dehydration. V. jatamansi Jones + ribavirin facilitated the apoptosis of small intestine cells. In conclusion, V. jatamansi Jones may inhibit RV-induced diarrhea through PI3K/AKT signaling pathway, and could therefore be a potential therapy for diarrhea.

Investigations on residual elimination of altrenogest oral solution in pigs and the withdrawal time.

The residual elimination of altrenogest in swine was investigated, preparing for the determination of withdrawal time. The residues of altrenogest in sebum, muscle, liver and kidney were extracted by optimized methods and further analyzed by UPLC-MS/MS. Under experimental conditions, the LOD and LOQ of altrenogest in sebum, muscle, liver and kidney were 0.5 and 1.0 µg/kg, respectively. The recoveries were in the range of 65 and 95% and the inter- and intra-RSD were less than 15%. The established method for the extraction, purification and detection of altrenogest is suitable for the determination of the residue of altrenogest in edible tissues of pigs. It was showed that altrenogest had the highest residual concentration level in liver, followed by kidney, sebum and muscle. Then withdrawal time was set at 9 days. The study provides an effective basis for elimination of altrenogest in swine.

Macleaya cordata extracts exert antiviral effects in newborn mice with rotavirus-induced diarrhea via inhibiting the JAK2/STAT3 signaling pathway.

Accumulating evidence demonstrates that Macleaya cordata extract exerts antiviral and anti-inflammatory effects in various diseases. The present study aimed to investigate the potential effects of M. cordata on rotavirus SA11-induced diarrhea in mice. Diarrhea severity, levels of inflammatory cytokines, histological changes in the small intestine and the underlying mechanisms were evaluated in rotavirus-stimulated mice treated with 1, 2 and 4 mg/kg/day M. cordata or 4 mg/kg/day ribavirin (positive control). M. cordata treatment effectively ameliorated rotavirus-induced diarrhea in a dose-dependent manner by decreasing viral RNA levels. In addition, M. cordata reduced the release of pro-inflammatory cytokines including migration inhibitory factor, interleukin (IL)-8, IL-ß, interferon-γ and tumor necrosis factor-α, and elevated the secretion of the anti-inflammatory cytokine IL-10 following rotavirus infection. M. cordata inhibited intestinal epithelial cell apoptosis and improved intestinal inflammation after rotavirus infection. The study also revealed that M. cordata exerted antiviral and anti-inflammatory effects on rotavirus-induced diarrhea by suppressing the Janus kinase 2 (JAK2)/STAT3 pathway, as reflected by decreased protein expression of phosphorylated (p)-JAK2 and p-STAT3. Overall, M. cordata effectively inhibited the inflammation caused by rotavirus, which was closely associated with the suppression of JAK2/STAT3 phosphorylation. These data suggested that M. cordata may be applied as a treatment for rotavirus-induced diarrhea.

Purification, characterization and in vitro and in vivo immune enhancement of polysaccharides from mulberry leaves.

Mulberry leaf polysaccharide (MLP) was extracted and purified by DEAE-52 cellulose and Sephadex G-100 column chromatography to afford two major purified polysaccharides (MLP-1 and MLP-2). The purified polysaccharides were characterized, and their immune-enhancing properties were investigated. MLP-1 had a molecular weight of 9.31×104 Da and was composed of mannose, rhamnose, glucose, galactose, xylose, and arabinose in a molar ratio of 0.71:1.00:2.76:1.13:3.70:2.81. The molecular weight of MLP-2 was 2.22×106 Da, and its monosaccharide constituents were mannose, rhamnose, glucose, galactose, and arabinose in a molar ratio of 1.31:8.45:6.94:1.00:11.96. Infrared spectroscopy showed that each MLP had a typical absorption peak characteristic of sugars, and ultraviolet (UV) spectroscopy showed that neither MLP contained nucleic acid or protein components. Then, the abilities of these polysaccharides to stimulate spleen lymphocyte proliferation in mice in vitro were compared by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. MLP-2 was more effective than MLP-1; therefore, MLP-2 was chosen for the study of its immune-enhancing effects in vivo. For the in vivo experiments, 14-day-old chickens immunized with Newcastle disease (ND) vaccine were orally administered MLP-2, and Astragalus polysaccharide (APS) was used as the control. Each chicken was orally administered 4 mg or 8 mg of MLP-2 for seven consecutive days starting three days before ND vaccine immunization. MLP-2 significantly improved the ND serum antibody titer and interleukin-2 (IL-2), interferon-γ (IFN-γ) and immunoglobulin A (sIgA) concentrations in tracheal and jejunal wash fluids, and increasing numbers of immune globulin A-positive (IgA+) cells in cecal tonsils and increased body weight. These results indicated that MLP-2 could significantly enhance immune activity and could therefore be utilized as an immunopotentiator drug candidate.

Optimization of selenylation modification for garlic polysaccharide based on immune-enhancing activity.

Garlic polysaccharide (GPS) was modified in selenylation respectively by nitric acid-sodium selenite (NA-SS), glacial acetic acid-selenous acid (GA-SA), glacial acetic acid-sodium selenite (GA-SS) and selenium oxychloride (SOC) methods each under nine modification conditions of L9(3(4)) orthogonal design and each to obtain nine selenizing GPSs (sGPSs). Their structures were identified, yields and selenium contents were determined, selenium yields were calculated, and the immune-enhancing activities of four sGPSs with higher selenium yields were compared taking unmodified GPS as control. The results showed that among four methods the selenylation efficiency of NA-SS method were the highest, the activity of sGPS5 was the strongest and significantly stronger than that of unmodified GPS. This indicates that selenylation modification can significantly enhance the immune-enhancing activity of GPS, NA-SS method is the best method and the optimal conditions are 0.8:1 weight ratio of sodium selenite to GPS, reaction temperature of 70 °C and reaction time of 10h.

Solomonseal polysaccharide and sulfated Codonopsis pilosula polysaccharide synergistically resist Newcastle disease virus.

Five combinations of three ratios (PS9-sPS1, PS7-sPS3 and PS6-sPS4) were prepared with polysaccharide (PS) and sulfated polysaccharide (sPS). The antiviral activities of these compounds were subsequently compared in vitro using the MTT assay, observation of the virus structure and immunofluorescence. The results demonstrated that SP9-sCP1, CP7-sCA3, EP7-sAP3, CA9-sEP1 and EP7-sCA3 presented higher activities, and SP9-sCP1 displayed the highest virus inhibition rate and clearly killed the virus and inhibited viral antigen expression. In an in vivo test, 28-day-old chickens were challenged with Newcastle disease virus (NDV) and were administered the five drug combinations. On day 14 after the challenge, the morbidity, mortality and cure rate in each group were calculated. The results indicated that SP9-sCP1 presented the lowest morbidity and mortality and the highest cure rate. These results indicate that Solomonseal polysaccharide and sulfated Codonopsis pilosula polysaccharide synergistically resist NDV. Moreover, SP9-sCP1 had the highest efficacy and may be used as a new antiviral drug.

The comparison of antioxidative and hepatoprotective activities of Codonopsis pilosula polysaccharide (CP) and sulfated CP.

Codonopsis pilosula polysaccharide (CP) was extracted, purified and modified by chlorosulfonic acid-pyridine method to obtain a sulfated CP (sCP). Their antioxidative activities in vitro were compared through the free radical-scavenging test. The results demonstrated that the scavenging capabilities of sCP were significantly stronger than those of CP. In vivo test, the mice hepatic injury model was prepared by BCG/LPS method, then administrated respectively with sCP and CP at three dosages, the biochemical indexes in serum, antioxidative indexes in liver homogenate and histopathological change in liver of the mice were compared. The results showed that in high (200mg/kg) and middle (150mg/kg) dosages of sCP groups, the contents of ALT, AST and TNF-α in serum and MDA in liver homogenate were significantly lower than those in the model group and numerically lower than those in the CP groups, the activities of SOD and GSH-Px in liver homogenate were significantly higher than those in the model group and numerically higher than those in the CP groups. In the model group there were obvious pathological changes in the liver, while in the sCP groups were near normal. These results indicate that sCP and CP possess antioxidative activity in vitro and in vivo, the activity of sCP is stronger than that of CP and sulfation modification can enhance the antioxidative and hepatoprotective activities of Codonopsis pilosula polysaccharide.

Optimization of selenylation conditions for lycium barbarum polysaccharide based on antioxidant activity.

Lycium barbarum polysaccharide (LBP) was modified by HNO3-Na2SeO3 method according to L9(3(4)) orthogonal design to obtain nine selenizing LBPs (sLBPs), sLBP1-sLBP9. Their antioxidant activities in vitro were compared by free radical-scavenging test. sLBP6, sLBP8 and sLBP9 presented stronger activity. In vivo test, 14-day-old chickens were injected respectively with sLBP6, sLBP8 and sLBP9 taking LBP as control, and serum GSH-Px and SOD activities and MDA content were determined. The results showed that three sLBPs could significantly enhance GSH-Px and SOD activities and decrease MDA content. The actions of sLBPs were significantly stronger than that of unmodified LBP. These results indicated that selenylation modification could significantly enhance the antioxidant activities of LBP, sLBP6 possessed the best efficacy and could be exploited into an antioxidant. The optimal modification conditions were 400mg of sodium selenite for 500 mg of LBP, reaction temperature of 70 °C and reaction time of 6h.

Effects of selenylation modification on immune-enhancing activity of garlic polysaccharide.

The garlic polysaccharide was modified by HNO3-Na2SeO3 method according to orthogonal design L9(3(4)) to obtain nine selenizing garlic polysaccharides, sGPS1-sGPS9. Their effects on chicken peripheral lymphocytes proliferation in vitro were compared by MTT assay. The results showed that sGPSs could significantly promote lymphocytes proliferation, sGPS3, sGPS5 and sGPS6 presented stronger efficacy. In vivo experiment, 14-day-old chickens were injected respectively with sGPS3, sGPS5 and sGPS6 when they were vaccinated with ND vaccine taking unmodified GPS as control. The results showed that three sGPSs could significantly promote lymphocyte proliferation, enhance serum antibody titer, IFN-γ and IL-2 contents. These results indicated that selenylation modification could significantly enhance the immune-enhancing activity of GPS, sGPS6 possessed the best efficacy and could be as a candidate drug of immunoenhancer. Its optimal modification conditions were 400 mg of sodium selenite for 500 mg of GPS, reaction temperature of 70°C and reaction time of 6 h.

Effects of epimedium polysaccharide-propolis flavone oral liquid on mucosal immunity in chickens.

A previous study found that epimedium polysaccharide (EP)-propolis flavonoid (PF) injection (EPI) produced reliable immunoenhancement. In this study, we investigate the effects of EP-PF oral liquid (EFO) on mucosal immunity in the chicken small intestine while using EPI, EP and PF as controls. Groups of fourteen-day-old chickens were given EFO orally at one of the three doses when they were vaccinated with ND vaccine. On days 7, 21 and 35 after the first vaccination, six chickens were selected randomly from each group for measurements of the sIgA and IL-17 contents of the washing liquors of the duodenum and jejunum, counts of the lymphocytes in the duodenal endothelium and counts of the IgA(+) cells in the jejunal endothelium and cecum tonsil. The results indicated that EFO significantly promoted the secretion of sIgA and IL-17 and increased the numbers of lymphocyte and IgA(+) cells. Furthermore, EFO was more efficient than EPI at the high and medium doses. These findings indicate that EPO may enhance intestinal mucosal immunity and may be exploited as an oral immunopotentiator.

Adjuvanticity of compound astragalus polysaccharide and sulfated epimedium polysaccharide per os.

On the basis of previous researches, compound astragalus polysaccharide (APS) and sulfated epimedium polysaccharide (sEPS) oral liquid (AEO) was prepared. Three hundred and twenty 14-day-old chickens were randomly assigned into eight groups and vaccinated with ND vaccine except for blank control (BC) group, repeated vaccination at 28 days old. At the same time of each vaccination, the chickens in three experimental groups were taken orally with AEO, respectively, at three doses, in two component control groups with APS and sEPS, once a day for three successive days; in injection control group were injected with AEI once, and in vaccination control (VC) and BC groups were not administrated. On days 7, 14, 21, 28 and 35 after the first vaccination, peripheral lymphocyte proliferation, the serum antibody titer, IFN-γ and IL-2 concentrations and on day 35 immune organ index were measured. The results showed that AEO at high and medium doses could significantly promote lymphocyte proliferation and development of immune organ, enhance antibody titer and IFN-γ and IL-2 concentration, which was stronger than actions of AEI and two components. The results confirmed that AEO possessed reliable immunoenhancement and could be exploited into an oral immunopotentiator.

Selenylation modification can enhance immune-enhancing activity of Chinese angelica polysaccharide.

On the basis of previous test that selenizing Chinese angelica polysaccharides (sCAPs) with stronger immune-enhancing activity in vitro were picked out, the immune-enhancing activity in vivo of three sCAPs, sCAP2, sCAP6 and sCAP8, at high and low dosage were compared taking the unmodified Chinese angelica polysaccharide (CAP) as control by determination of peripheral lymphocyte proliferation, serum antibody titer, IFN-γ and IL-6 contents in chicken vaccinated with Newcastle Disease vaccine. The results showed that three sCAPs at suitable dosage could significantly promote lymphocyte proliferation, enhance serum antibody titer, IFN-γ and IL-6 contents as compared with unmodified CAP, sCAP2 at low dosage possessed the strongest action. These results indicated that selenylation modification could significantly enhance the immune-enhancing activity of CAP, sCAP2 possessed the best efficacy and would be as a component drug of new-type immunoenhancer.