Objective To explore the effect and preliminary mechanism of the plant-derived immunostimulatory molecule,ophiopogonin,on enhancing the immune response of a subunit vaccine with the receptor-binding domain(RBD)of coronavirus spike protein as the antigen.Methods CCK-8 assay was used to determine the cytotoxicity of ophiopogonin D'(OPD')on bone marrow-derived dendritic cells(BMDCs).Female Balb/c mice were randomly divided into RBD,RBD/OPD',RBD/Alum,and control groups.The immunization dose was 5 μg of antigen per mouse and 100 μg of adjuvant per mouse,and immunization was carried out according to the intramuscular injection immunization procedure on days 0,21,and 42.The titers of specific IgG and its subtype antibodies were detected by ELISA.The cytokine levels in the supernatant of splenocytes were detected using ELISA.The number of splenocytes secreting IFN-γ was detected by ELISpot.Laser confocal microscopy was employed to observe the uptake of antigen by BMDCs.The phagocytic ability of BMDCs for antigen was quantitatively analyzed by flow cytometry.The mechanism of its enhanced immune effect was preliminarily explored using transcriptomics technology combined with bioinformatics research.Results When the concentration of OPD'was less than 5 μg/mL,the survival rate of BMDCs was 100%.After a single intramuscular injection in mice,except for a slight decrease in body weight,the other biochemical indicators were within corresponding normal ranges.After intramuscular injection immunization of the vaccine,the titers of serum-specific IgG,IgG1,and IgG2a in the RBD/OPD'group were significantly higher than those in the RBD group(P<0.05).Compared with the RBD group,the RBD/OPD'group induced a high-level Th1 cell immune response of IL-1β,TNF-α,and IFN-γ(P<0.01)and had more lymphocytes secreting IFN-γ(P<0.001).Laser confocal microscopy displayed that BMDCs took up more antigens after OPD'treatment,which was further confirmed with flow cytometry in quantitative analysis on antigen uptake rate(P<0.01).Transcriptomics results indicated that there was more significant enrichment of the PPAR signaling pathway in the RBD/OPD'group than the RBD group,suggesting that OPD'may activate the PPAR signaling pathway to exert its adjuvant effect.Conclusion OPD'effectively enhances the immune response of the RBD subunit vaccine,and its action mechanism may be related to the activation of the PPAR signaling pathway.

Objective To establish a mouse model of infection with the minimum lethal dose of Vibrio vulnificus(V.vulnificus)and to evaluate the protective efficacy of inactivated whole-cell(IWC)vaccine using this model.Methods A mouse model of lethal-dose infection was established by intraperitoneal injection of different doses of V.vulnificus.Bacterial colonization in the organs was detected with tissue homogenate plating,and pathological changes in the organs were observed after tissue section staining.Flow cytometry was used to detect immune cell responses after liver tissues were digested into single-cell suspension.IWC vaccine of V.vulnificus was prepared,and the mice were immunized through different routes to observe the protective efficacy of the vaccine.Results A mouse model of infection with the minimum lethal dose at 1×106 CFU of V.vulnificus was successfully established.After infection,the bacteria were mainly colonized in the liver of mice and caused severe pathological damages.Compared with the uninfected mice,the proportion of neutrophils in the liver was significantly increased in the infected mice,whereas the proportions of B cells and T cells were correspondingly decreased(P<0.05).A single intramuscular or intraperitoneal injection of the IWC vaccine could protect the mice effectively against lethal infection of V.vulnificus in 7 d later(P<0.01),although the level of serum IgG having no significant increase.Conclusion A mouse model of lethal-dose infection with V.vulnificus is successfully established,with histopathological characteristics.The IWC vaccine of V.vulnificus rapidly mediates immune protection in this model probably independent of IgG.

Objective To evaluate the stability,safety and immune enhancement and anti-tumor effects of Wilms'tumor gene 1(WT1)peptide combined with AddaVaxTM emulsion vaccine for acute myeloid leukemia.Methods The stability of WT1 peptide in the adjuvant vaccine was evaluated using MALDI-TOF-MS time-of-flight mass spectrometry.Female C57BL/6 mice were randomly divided into PBS group,WT1 peptide group,and WT1 peptide+AddaVaxTMemulsion adjuvant vaccine group.The immunization was performed at a dose of 50 μg/mouse for antigen and 50 μg/mouse for adjuvant,with intramuscular injection on days 0,14,and 28.HE staining was used to assess the toxicity of intramuscular vaccination on mouse organ tissues.Cytokine levels were detected by ELISA,and the number of IFN-γ-secreting splenocytes was measured by ELISpot.Flow cytometry was employed to detect the maturation of bone marrow-derived dendritic cells(BMDCs)promoted by the vaccine in vitro and the promotion for lymphocyte activation,and H-2Db WT1 tetramer was utilized to detect the proportion of specific CD8+T cells.After establishing a mouse leukemia tumor model using the C1498-mWT1 stable cell line,the anti-tumor effects of the vaccine for prevention and treatment were evaluated.Results The WT1 peptide stably existed in the vaccine without causing significant organ tissue changes in mice after intramuscular injection.Compared to the mice immunized with WT1 aqueous solution,the mice after intramuscular injection of the WT1 peptide emulsion adjuvant vaccine showed stronger immune responses of Th1 cells,including IFN-γ and TNF-α,as well as Th17 cells of IL-17A(P＜0.05),and the mice had not only promoted number of IFN-γ secreting splenocytes(P＜0.01)but also enhanced maturation of BMDCs,as indicated by an increase in the proportions of CD40+/CD11c+and CD86+CD80+/CD11c+ cells(P＜0.05).Additionally,there were increases in both the proportion of CD4+/CD3+T and CD69+/CD8+T cells(P＜0.05)and the proportion of specific CD8+T cells(P＜0.05).In the anti-tumor effect study using the C1498-mWT1 mouse model,the median survival time of the WT1+AddaVaxTM group was extended by 6 d compared to the WT1 aqueous solution group.At day 50,the survival rate of mice in the WT1+AddaVaxTM group was still 28.5％,while all mice in the other groups had died(P＜0.05).Conclusion The vaccine with the WT1 peptide and AddaVaxTM emulsion adjuvant exhibits good immunological and anti-tumor effects.

Objective To investigate the antibacterial effect and its preliminary mechanism of lavender essential oil on multi-drug resistant Acinetobacter baumannii.Methods Micro-dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)of lavender essential oil against multi-drug resistant Acinetobacter baumannii,and bactericidal kinetic study was employed to determine the onset and maintenance time of lavender essential oil.Meanwhile,the promoting and therapeutic effects of lavender essential oil on wound healing were observed in a mouse model of infection.Subsequently,crystal violet staining was used to determine the inhibition and clearance of multi-drug resistant Acinetobacter baumannii biofilm by lavender essential oil,and laser confocal microscopy was utilized to observe the survival of bacteria in biofilms.NanoDrop instrument was utilized to quantify the leakage of bacterial DNA nucleic acid and protein after intervention with 3 and 6 mg/mL lavender essential oil,and the leakage of bacterial potassium ion was measured by potassium ion test kit.Proteomics technology combined with bio-informatics were applied to explore the action mechanism of lavender essential oil against multi-drug resistant Acinetobacter baumannii.Results The MIC and MBC of lavender essential oil were both 6 mg/mL,which could kill almost all multi-drug resistant Acinetobacter baumannii at the time point of 120 min,and showed an obvious dose-and time-dependent manner.The overall animal model evaluation showed that both 3 and 6 mg/mL lavender essential oil could promote wound healing,and the curative effect was obvious.Further studies confirmed that 3 mg/mL lavender essential oil had a certain biofilm inhibitory effect on multi-drug resistant Acinetobacter baumannii,and 6 mg/mL also had a certain biofilm clearance effect under the same conditions.Meanwhile,when incubated at 37℃ for 1 h,the dose of 3 mg/mL could increase the leakage of DNA nucleic acid and protein,and significantly promote the efflux of potassium ions.Proteomic analysis suggested that the antibacterial effect of lavender essential oil may be related to affecting the oxidorereductase activity and cell metabolic process of multi-drug resistant Acinetobacter baumannii,and interfering with the biosynthesis of cell wall/membrane/envelope and other structures.Conclusion Lavender essential oil at 3 mg/mL can play an antibacterial effect against multi-drug resistant Acinetobacter baumannii,and its mechanism may be related to the destruction of bacterial biofilm and interference with bacterial metabolism.

Tumor has become the major reasons cause of death,and its vaccine has become the effective tracts of treatment and prevention by enhancing the immune response of patients.However,most vaccines which are recombination subunit protein antigens are poorly immunogenic and difficult to induce a robust immune response in patients with compromised immune systems,resulting in poor marketing approval.The core component of the vaccine adjuvant can greatly enhance the strength,speed and duration of the immune response,thus becoming the key to the development of an ideal tumor vaccine.Most tumor vaccines are combined with tradition adjuvant such as aluminum,MF59 and AS adjuvant,but their products and patents are monopolized by large foreign companies.We found that natural adjuvants have many unique advantages,such as good biocompatibility and biodegradability,promoting the maturation of dendritic cell and the secretion of immune cytokines,significantly enhancing the tumor vaccine immune response,etc.In this paper,the application and future development of natural polysaccharides,saponins,flavonoid and plant virus-like particles in cancer vaccines were reviewed,which may lay a solid foundation for the development of the original and innovative adjuvants with domestic independent intellectual property rights.

In this research,a new phospholipid based monolith was fabricated by in situ co-polymerization of 1-dodecanoyl-2-(11-methacrylamidoundecanoyl)-sn-glycero-3-phosphoethanolamine and ethylene dimethacrylate to mimick bio-membrane environment.Excellent physicochemical properties of this novel monolith that were achieved included column efficiency,stability,and permeability.Moreover,the biomimetic monolith showed outstanding separation capability for a series of intact proteins and small molecules.In particular,it exhibited good potential as an alternative to the commercial immobilized artificial membrane(IAM)column(IAM.PC.DD2)for studying drug-membrane interactions.This study not only enriched the types of IAM stationary phases,but also provided a simple model for the prediction of phosphatidylethanolamine related properties of drug candidates.

OBJECTIVETo observe the clinical efficacy of acupuncture combined with western medicine in the treatment of rheumatoid arthritis (RA) and its effect on blood stasis, and to explore ways to improve the clinical curative effect.

METHODSA total of 56 patients of RA were randomly divided into an observation group and a control group, 28 cases in each one. ① ibuprofen sustained-release tablets, 2 times a day, each time 0.3 g; ② methotrexate tablets (MTX), once a week, each time 10 mg ③ folic acid tablets, once a week, each time 5 mg were given in the control group, 30 days as one course, a total of 3 courses were required. In the observation group, acupuncture was adopted on the basis of the treatment as the control group. The main acupoints were Ganshu (BL 18), Pishu (BL 20), Shenshu (BL 23), Hegu (LI 4), Quchi (LI 11), Zusanli (ST 36) combined with local points. The treatment was given once every day for continuous 6 days a week, the treatment for 30 days as one course, a total of 3 courses were required. The serological indexs were evaluated before and after treatment, including the rheumatoid factor (RF), hypersensitive C-reactive protein (hs-CRP), erythrocyte sedirnentation rate (ESR), platelet (PLT), fibrinogen (FBG) and D-dimer (D-D), the changes of disease activity score (DAS-28), symptom grade quantitative score, blood stasis syndrome symptom (the joint tingling, lip color, tongue, pulse, subcutaneous ecchymosis, squamous and dry skin) score were observed.

RESULTS① The scores of RF, hs-CRP, ESR, PLT, D-D, FBG, DAS-28 and symptom grade quantitative were significantly improved in the two groups compared with those before treatment (all <0.05), and the scores of hs-CRP, ESR, DAS-28 and symptom grading in the observation group were more better than those in the control group (all <0.05). ② The total score of joint tingling, lip color, tongue, pulse, subcutaneous ecchymosis, squamous and dry skin and blood stasis syndrome in both groups were decreased after treatment (all <0.05), the joint tingling, tongue, lip color and subcutaneous ecchymosis were improved obviously in the observation group than those in the control group (all <0.05). ③ The total effective rate in the observation group was 85.7% (24/28), which was better than 75.0% (21/28) in the control group (<0.05).

CONCLUSIONAcupuncture combined with western medicine can not only improve the clinical efficacy of RA patients but also improve the blood stasis.

Acupuncture Points ; Acupuncture Therapy ; Arthritis, Rheumatoid ; therapy ; Combined Modality Therapy ; Folic Acid ; therapeutic use ; Humans ; Ibuprofen ; therapeutic use ; Methotrexate ; therapeutic use ; Treatment Outcome

Objective To explore clinical application value of Traditional Chinese Medicine nursing technology to decrease postoperative complication after breast cancer surgery.Methods Randomized controlled trial,large sample size,multicenter study design were adopted,and 200 patients who met inclusion criteria were randomly divided into the control group and the treatment group (100 cases in each group).The control group received routine nursing methods after breast cancer surgery.The treatment group received routine nursing methods as well as Traditional Chinese Medicine nursing intervention,such as auricular application pressure,acupoint sticking,meridian moxibustion.Data of arm circumference,Athens insomnia scale(AIS),symptom in affected arm were collected a week before and after surgery,and range of motion of shoulder joint was evaluated 3 months after surgery to compare postoperative subcutaneous effusion,skin flap necrosis,limb edema,sleep disorder and occurrence of shoulder joint dysfunction in two groups.Results The cases developed postoperative subcutaneous effusion,skin flap necrosis,limb edema were different in two groups,but the differences were not statistically significant(P＞0.05).There were significant differences between two groups in affected arm pain,swelling,skin tension,sleep,and shoulder joint dysfunction after 3 months(P＜0.05).Conclusion Traditional Chinese Medicine nursing technology can effectively decrease postoperative complication after breast cancer surgery.

Objective To explore the correlation between ATF5 protein expression level and drug sensitivity of gastric cancer AGS cells.Methods We established the high,middle and low ATF5 expression groups by transfecting the AGS cells with ATF5 pcDNA3.1 plasmid,ATF5 siRNA and empty plasmid using the liposome transfection technique.Then we detected the expression of the ATF5 protein of the 3 groups using the Western Blot.The drug sensitivity of AGS cells in 3 groups to paclitaxel and cisplatin was detected using the MTT experiments and plate clone formation experiments then the correlation between ATF5 protein expression level and drug sensitivity of gastric cancer AGS cells was analyzed.Results With the effect of paclitaxel or cisplatin on AGS cells in 3 groups,the surviving fraction and the IC50 of high ATF5 expression group were higher than those of control group,which indicated statistical significance (all P ＜ 0.05);while the surviving fraction and the IC50 of low ATF5 expression group were lower than those of control group (all P ＜ 0.05,P cisplatin =0.00,P paclitaxel =0.002).We found that cell clone formation of AGS cells to paclitaxel and cisplatin was significantly increased after the transfection with ATF5 protein plasmid and decreased after transfection with ATF5 siRNA plasmid (all P ＜ 0.05).Conclusion The expression of ATF5 protein is related to the drug sensitivity of gastric cancer AGS cells that upregulation of ATF5 protein expression can decrease the drug sensitivity of AGS cells to paclitaxel and cisplatin in gastric cancer.

OBJECTIVETo explore the clinical efficacy of moxibustion on rheumatoid arthritis (RA) in the patients and the impacts on the inflammatory indices.

METHODSForty RA patients were randomized into an observation group and a control group, 20 cases in each one. In the control group, diclofenac sodium sustained release tablets were used, 0.3 g each time, twice a day; methotrexate tablets were used, 10 mg each time, once a week; folic acid was used, 5 mg each time, once a week. In the observation group, on the basis of the treatment as the control group, moxibustion was added specially on the swelling and painful joints, once a day, 15-20 min each time till: the local skin turned slightly red. The treatment was given once a day, at the interval of 1 day after every 6 treatments. In the two groups, the efficacy was observed after 30-day treatment. Before and after treatment, the rheumatoid factor (RF), high sensitivity C-reactive protein (hs-CRP), erythrocyte sedimentation rate (ESR), disease activity score-28 (DAS-28) and the score of joint symptom and physical signs were observed.

RESULTSCompared with those before treatment, the levels of RF, hs-CRP, ESR, DAS-28 and the symptom scale score were all reduced in the RA patients of the two groups after treatment (all P < 0.05). Compared with the control group, the levels of RF, hs-CRP, ESR, DAS-28 and the symptom scale score in the observation group were improved much apparently (all P < 0.05). The total effective rate was 85.0% (17/20) in the observation group and was 80.0% (16/20) in the control group. The total effective rate in the observation group was apparently better than that in the control group (P < 0.05).

CONCLUSIONMoxibustion as supplement relieves the joint symptoms, reduces the inflammatory reactive indices and improves the clinical efficacy in the RA patients.

Adult ; Aged ; Arthritis, Rheumatoid ; blood ; therapy ; C-Reactive Protein ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Moxibustion ; Rheumatoid Factor ; blood ; Treatment Outcome ; Young Adult