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ObjectiveTo explore the biological mechanism of drought improving the quality of Rhizoma Atractylodis Chinensis and establish a new method for the production of high-quality medicinal materials. MethodThe fresh roots of Atractylodes chinensis were soaked in 0 （control）， 5%， 10%， and 20% PEG-6000 solutions. The changes in reactive oxygen species （ROS） level， antioxidant enzyme activity， activities of key enzymes in primary metabolism and secondary metabolism， and content of secondary metabolites were compared. ResultCompared with the control group， the treatment with 20% PEG for 2 days elevated the levels of superoxide anion radicals （O2-·）， hydrogen peroxide （H2O2）， and malondialdehyde （MDA） by 172.5%， 56.9%， and 14.7%， respectively. The treatment did not change the activity of superoxide dismutase （SOD）， reduced the peroxidase （POD） activity， and increased the catalase （CAT） activity by 10.8%. It increased the activities of 3-hydroxy-3-methylglutaryl-CoA reductase （HMGR）， phosphoenolpyruvate carboxylase （PEPC）， and acetyl-CoA carboxylase （ACC） by 49.9%， 12.1%， and 19.0%， respectively. Furthermore， the content of atractylodin， β-eudesmol， atractylone， and atractylenolide Ⅱ was increased by 51.0%， 36.9%， 47.1%， and 91.5%， respectively. The simulated drought stress can cause the burst of ROS in the fresh roots of A. chinensis， induce the physiological state of plants under drought， change the antioxidant system， and promote the massive synthesis of secondary metabolites in a short time. ConclusionPEG-6000-simulated drought stress can greatly improve the quality of A. chinensis in cultivation.
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Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia seen in clinical settings, which has been associated with substantial rates of mortality and morbidity. However, clinically available drugs have limited efficacy and adverse effects. We aimed to investigate the mechanisms of action of andrographolide (Andr) with respect to AF. We used network pharmacology approaches to investigate the possible therapeutic effect of Andr. To define the role of Andr in AF, HL-1 cells were pro-treated with Andr for 1 h before rapid electronic stimulation (RES) and rabbits were pro-treated for 1 d before rapid atrial pacing (RAP). Apoptosis, myofibril degradation, oxidative stress, and inflammation were determined. RNA sequencing (RNA-seq) was performed to investigate the relevant mechanism. Andr treatment attenuated RAP-induced atrial electrophysiological changes, inflammation, oxidative damage, and apoptosis both in vivo and in vitro. RNA-seq indicated that oxidative phosphorylation played an important role. Transmission electron microscopy and adenosine triphosphate (ATP) content assay respectively validated the morphological and functional changes in mitochondria. The translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the molecular docking suggested that Andr might exert a therapeutic effect by influencing the Keap1-Nrf2 complex. In conclusions, this study revealed that Andr is a potential preventive therapeutic drug toward AF via activating the translocation of Nrf2 to the nucleus and the upregulation of heme oxygenase-1 (HO-1) to promote mitochondrial bioenergetics.
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Animals , Rabbits , Atrial Fibrillation/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , Signal Transduction , NF-E2-Related Factor 2/pharmacology , Molecular Docking Simulation , Oxidative Stress , Energy Metabolism , Mitochondria/metabolism , Inflammation/metabolism , Heme Oxygenase-1ABSTRACT
Objective:To compare the differences in the safety, efficacy and protective effects of rabies vaccine using the current pre-exposure prophylaxis schedule in China (0-7-21 or 28) and the newly recommended immunization program of WHO (0-7), aiming to provide data support for modifying the related content of Technical Guideline for Human Rabies Prevention and Control. Methods:The mice were randomly divided into five groups, namely 0-7-21 group (3-injection regimen), 0-7 group (2-injection regimen), 0-14 group, 0-21 group and control group, according to the current 3-injection regimen (0-7-21) in China and the 2-injection regimen (0-7) recommended by WHO. The survival status of the mice was observed. The mice were weighed every five days to compare the safety of different immunization procedures. Rabies virus neutralizing antibodies (RVNA) were detected 7, 14, 21, 28 and 35 d after the initial immunization. On day 35, the mice in each group were challenged with lethal dose of CVS-11 rabies virus to evaluate the protective effects of different pre-exposure immunization procedures.Results:There was no significant difference in weight gain of mice after vaccination. The positive rate of RVNA was 100% in all immunized groups from day 14, which could provide complete protection to mice. There was a significant difference in RVNA levels between 0-7-21 and 0-7 groups at 35 d( P<0.05), but there was no statistical difference at other time points ( P>0.05). RVNA level had a significant difference between 0-7 and 0-21 groups at 21 d and 35 d ( P<0.05). There was no statistical difference in RVNA level between other groups at each time point ( P>0.05). In the protective test, the survival rates of mice in all immune groups were 100%. Conclusions:The current 3-injection pre-exposure immunization procedure for rabies vaccine (0-7-21) and the newly recommended 2-injection immunization procedure (0-7) had similar efficacy and protective effects in animal tests. In view of the cost saving and better compliance of the 2-injection immunization procedure, it was recommended that relevant departments should conduct clinical trials as soon as possible to promote the implementation of this program.
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Objective:To analyze the in vitro inhibitory effects of resveratrol on rabies virus. Methods:The challenge virus standard (CVS)-11 strain of rabies virus and BHK-21 cells were used to establish the infection model. In vitro inhibitory effects of resveratrol on rabies virus were analyzed at different stages of infection by direct immunofluorescence and cell fluorescence focus unit assay. Results:Without affecting cell growth, resveratrol could block the adsorption of virus, interfere with the entry of virus into cells and inhibit virus proliferation in a concentration-dependent manner. The inhibition rate could reach up to about 95%. The results of co-incubation experiment showed that 40 μmol/L resveratrol could directly kill the virus.Conclusions:This study indicated that resveratrol inhibited the activity of rabies virus in a concentration-dependent manner.
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Objectives@#. The simulation of microlaryngeal skills is rarely seen in surgical training, but it is particularly important in phonomicrosurgery. This study described and validated the laryngeal surgical simulator through surgical training. @*Methods@#. A simple and low-cost simulator was developed for the fixation of the suspension laryngoscope and porcine larynges. Twenty participants with work skills and experience did preparation before training, and performed suture and carbon dioxide (CO2) laser cordectomy for simulator evaluation. The results were proposed by the aspects of time taken for each procedure, the global rating scale, a procedure-specific assessment, and a post-simulation questionnaire. @*Results@#. All participants completed the preparation within 9 minutes and reached the conclusion that the microlaryngeal surgical simulator was helpful in improving their surgical skills. The performance of experts was superior to that of novices in both suture and CO2 laser cordectomy. @*Conclusion@#. This simulator could be easily assembled and was successfully validated by microlaryngeal surgical training both subjectively and objectively. It may be helpful to clinicians in microlaryngeal skills.
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Objective:To establish a rabies virus CVS-11 challenge model in BALB/c mice through intramuscular or intracerebral injection.Methods:The CVS-11 strain propagated in BSR cells with a titer of 2.7×10 7 FFU/ml was serially diluted 10 -1-10 -7 times to infect 4-week-old female mice through intramuscular or intracerebral injection. The morbidity and mortality of mice were observed after virus challenge. Moreover, brain tissues of all challenged mice were subjected to direct immunofluorescence assay (DFA) and reverse transcription polymerase chain reaction (RT-PCR) to analyze the cause of death. The median lethal doses (LD 50) in mice under different challenge methods were determined. Mouse challenge models were established to evaluate the immunoprotective effects of four domestically available rabies vaccines on mice after CVS-11 exposure. Results:BALB/c mice developed typical neurological symptoms and died 6-12 d after intracerebral challenge and the LD 50 was 18.3/0.1 ml. The mice intramuscularly challenged with CVS-11 showed clinical symptoms on 8-15 d and the LD 50 was 2.7×10 5/0.1 ml. DFA results showed that specific yellow-green fluorescence appeared in the brain tissue prints of all dead mice. RT-PCR results showed that all amplified products showed bright bands at about 250 bp. These results suggest that rabies virus infection was the cause of death in mice. The protective effect test results of four different rabies vaccines on the market without immunoglobulin application showed that the survival rate of mice after exposure to one of the vaccines was 50%, and the survival rates of mice immunized with the other three vaccines were all 30%. The above results indicated that the four rabies vaccines provided partial protection for mice exposed to CVS-11 without the use of rabies passive immunization preparations. Conclusions:This study established rabies virus CVS-11 challenge models in BALB/c mice under different challenge methods and provided a technical platform for related research on rabies and rabies vaccines.
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Objective@#To make etiological diagnosis and evaluate the protective effects of post-exposure prophylaxis(PEP) in an event of one dog injured seven persons.@*Methods@#Direct immunofluorescence assay (DFA) and nested polymerase chain reaction (PCR) were employed to detect nucleoprotein and nucleoprotein(N) gene of rabies virus in the brain tissues of the dog, the positive samples were sequenced for the full length of N gene of rabies virus, then the homology of the N gene of rabies virus was analyzed after the phylogenetic tree was constructed. Rapid fluorescent focus inhibition test (RFFIT) was applied to detect the rabies virus neutralizing antibodies(RVNA) on day 0, 14 and 40 after PEP.@*Results@#The cerebral, cerebellar and hippocampal tissues were positive by DFA and nested PCR. The phylogenetic tree indicated the rabies virus belonged to the rabies virus genotype I. The homology of the nucleotide and amino acid of the rabies virus N gene were over 86% with the vaccine strains. The titer of the RVNA increased significantly from the day 0 to day 14 after PEP, the lowest was 5.78 IU/ml and the highest was 26.15 IU/ml. On the day 40, the highest RVNA titer was 51.96 IU/ml. No rabies cases occurred in a one year follow-up visit.@*Conclusions@#Normative PEP can effectively prevent the occurrence of rabies cases.
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Objective@#To investigate the inactivating effect of soap solution on rabies virus and to explore the concentration of soap solutions which could be effective in post-exposure prophylaxis (PEP) of rabies virus infection.@*Methods@#The BSR and N2a cells were respectively infected by the mixture of different concentrations of soap solution and rabies virus CVS-11. Based on the direct immunofluorescent method (DFA) and reverse transcription PCR (RT-PCR), the inactivating effects of soap solutions on rabies viruse in BSR and N2a cells were detected, respectively.@*Results@#This experiment established the BSR or N2a cells model in 24 well cell culture plates, and we found that the upper limit of soap solution concentration which BSR or N2a cells could tolerate was 1%. The inhibitory effect test of different soap solution on rabies virus showed that the 0.5% concentration of soap solution could completely inhibit the survival of CVS-11 strain in both the BSR and N2a cells, but the 0.1% concentration of soap solution could not inhibit the rabies viruses completely.@*Conclusions@#The 0.5%-1% concentration of soap solutions could inhibit the survival of CVS-11 strain in three minutes in vivo experiment.
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Objective To establish a CVS-11 pseudovirus particles ( pp)-based assay for detec-tion of neutralizing antibody against rabies virus. Methods An improved rapid fluorescence focus inhibition test ( RFFIT) for detection of neutralizing antibody against rabies virus ( RVNA) was established based on the CVS-11 pseudovirus expressing a luciferase reporter gene. Forty-six human serum samples were analyzed with the improved RFFIT and the results were compared with those by using standard RFFIT. Moreover, the improved RFFIT was used to detect the titers of RVNA in 91 serum samples collected from pet dogs and pet-breeders in Beijing. Results The coincidence rate of the improved RFFIT and the standard RFFIT was 100% regarding to the analysis of 46 human serum samples and 5 negative reference serum samples. Moreo-ver, the RVNA titers of all serum samples obtained with CVS-11 pseudovirus-based assay showed a signifi-cant high correlation with those obtained with standard RFFIT (n=46, r=0. 94, P<0. 000 1). All of the 91 serum samples collected from pet dogs and pet-breeders in Beijing were positive for RVNA as indicated by the improved RFFIT with a mean titer of 33. 01 IU/ml. Conclusion We established an improved RFFIT based on the CVS-11 pp expressing luciferase reporter gene, which might be used as a reliable alternative RFFIT for measuring RVNA titer. Analysis of the 91 serum samples collected in Beijing with the improved RFFIT showed that all samples were positive for RVNA.
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BACKGROUND:Fisher-Lewis rat kidney transplant models are the international common chronic renal alograft rejection models, but their application is greatly limited because of difficulty in model preparation and high costs. OBJECTIVE:To explore a new method of establishing SD-Wistar rat models of chronic renal alograft rejection. METHODS: Fifty-six pairs of SD-Wistar rats were subjected to left kidney orthotopic transplantation. The right kidneys of the recipients were intact and used as internal controls. 23 rat recipients were randomly divided into model group (n=15) and control group (n=8). The rats in the model group were injected with cyclosporine microemulsion for 10 days (2 mg/kg/day,i.p.) after kidney transplantation. The rats in the control group were not treated with immunosuppressive therapy. RESULTS AND CONCLUSION:The irreversible acute rejection occurred in al the transplanted kidneys of rats in the control group within 4 weeks, leading to the necrosis of transplanted kidney. Moderate inflammatory cel infiltration appeared in the transplanted kidneys of rats in the model group at 4, 8 and 12 weeks after transplantation. Typical histopathological changes of chronic rejection were observed within 12 weeks after transplantation. The Banff total scores were increased with time after transplantation. Al these histopathological changes were not observed in the intact right kidneys of rat recipients in both groups. The valey value of
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<p><b>OBJECTIVE</b>To elucidate the characteristics of genetic variability and its relationship with prevalence, through sequencing and analysis of N gene among street rabies virus isolated from different hosts (homo sapiens, ferret badger, dog) in Zhejiang province.</p><p><b>METHODS</b>Samples were screened and confirmed by direct fluorescence assay and reverse transcript PCR. Sequences were analyzed using bio-information software.</p><p><b>RESULTS</b>Eighteen street rabies virus strains were identified, including 2 from homo sapiens, 5 from ferret badger, and 11 from dog. Similarities of N gene and N protein were calculated to be 89.7%-100.0% and 98.4%-100.0% respectively. Mutations occurred in N gene were almost non-sense mutations. In addition,Data from phylogenetic analysis showed that all these strains could be classified into traditional genotype 1.</p><p><b>CONCLUSION</b>The prevalence of rabies viruses among different hosts in Zhejiang province had certain regional properties. Rabies viruses isolated from the same kind of host or from the same/adjacent county/counties had the closest relationship. However, the characteristics of rabies virus prevalent in homo sapiens were somewhat complicated. In summary, the transmission of street rabies virus in Zhejiang province was from dogs to ferret badgers and homo sapiens, and the virus could circulate and cross-regional transmit among dogs and ferret badgers.</p>
Subject(s)
Animals , Dogs , Humans , China , Epidemiology , DNA Mutational Analysis , Virology , Mustelidae , Virology , RNA, Viral , Genetics , Rabies , Epidemiology , Rabies virus , Genetics , Viral Envelope Proteins , GeneticsABSTRACT
To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection,anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice.Spleen cells and SP2/0 myeloma cells were fused according to conventional methods:the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally,systematic identification of subclass,specificity and sensitivity was carried out.Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12,with ascitic fluid titers of 1∶8000 and 1∶10000,respectively.Both belonged to the IgG2a subclass.These strains secrete potent,stable and specific anti-rabies virus monoclonal antibodies,which makes them well suited for the development of rabies diagnosis reagents.
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The World Health Organization (WHO) standard assay for determining antibody level is the rapid fluorescent focus inhibition test (RFFIT) and is used to determine the degree of immunity after vaccination against rabies.To compare the difference in RFFIT results between the laboratories of The National Institute of Infectious Disease in Japan (NIID) and the Chinese Centre for Disease Control (CCDC) as well the influence of the choice of standard serum (STD) for the detection,the two laboratories detection methods were simultaneously manipulated by RFFIT.The reference serums used in NIID and the WHO standard serum used in CCDC were compared in the same RFFIT detection to determine the titer of four sera samples C1,S1,S2 and S4 in parallel,and the titers of the detected sera samples were calculated using the standard formula for neutralizing antibody titer.No significant difference was found in RFFIT methods from the two laboratories and the RFFIT testing procedures of the two laboratories have good consistency.However,different titers were obtained with the tentative internal standard serum (TI-STD) produced by adjusting to 2.0 IU of WHO standard serum in NIID and the WHO STD.The titer determined with the TI-STD was higher than that determined with WHO STD,This difference appears to be significant and requires further investigation.