AIM To investigate the pharmacokinetic behaviors of total flavonoids from Epimedii Folium in osteoporotic rats in vivo.METHODS Twelve rats were divided into model group and sham group,after which the osteoporotic rat model was established by removing bilateral ovaries.After intragastric administration with total flavonoids (500 mg/kg),HPLC-DAD was applied to detecting the plasma concentrations of total flavonoids' prototype glycosides (epimedin A,epimedin B,epimedin C,icariin) and their metabolites (sagittatoside A,sagittatoside B,2-O-rhamnosylicariside Ⅱ,baohuoside Ⅰ,icaritin) at thirteen time points (0.083,0.25,0.5,0.75,1,2,3,4,6,8,12,24 and 48 h),then the plasma concentration-time curves were drawn,followed by the calculation of pharmacokinetic parameters.RESULTS Doubled peaks were observed in the plasma concentration-time curves of total flavonoids' prototype glycosides and their metabolites in both groups.Compared with the sham group,the integrated AUC and Cmax in the model group were significantly decreased (P ＜ 0.01),as well as the prolonged t1/2 (P ＜ 0.01).CONCLUSION The in vivo absorption and metabolism of total flavonoids from Epimedii Folium are much slower in the state of osteoporosis,thus affects the efficacy.

This study was performed to prepare immobilized β-glucosidase and snailase, then optimize and compare the process conditions for conversion of icariin. Immobilized β-glucosidase and snailase were prepared using crosslink-embedding method. The best conditions of the preparation process were optimized by single factor analysis and the properties of immobilized β-glucosidase and snailase were investigated. The reaction conditions including temperature, pH, substrate ratio, substrate concentration, reaction time and reusing times of the conversion of icariin using immobilized β-glucosidase or snailase were optimized. Immobilized β-glucosidase and snailase exhibited better heat stabilities and could remain about 60% activity after storage at 4 degrees C for 4 weeks. The optimized conditions for the conversion of icariin were as follows, the temperature of 50 degrees C, pH of 5.0, enzyme and substrate ratio of 1 : 1, substrate concentration of 0.1 mg x mL(-1), reaction time of 6 h for β-glucosidase and 2 h for snailase, respectively. In 5 experiments, the average conversion ratio of immobilized β-glucosidase and snailase was 70.76% and 74.97%. The results suggest an effect of promoted stabilities, prolonged lifetimes in both β-glucosidase and snailase after immobilization. The immobilized β-glucosidase and snailase exhibited a higher conversion rate and reusability compared to the free β-glucosidase and snailase. Moreover, the conversion rate of immobilized snailase was higher than that of immobilized β-glucosidase. The process of icariin conversion using immobilized β-glucosidase and snailase was moderate and feasible, which suggests that immobilized enzymes may hold a promise for industrial usage.

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.
Adenosine Triphosphate ; pharmacology ; Amino Acid Substitution ; Biological Transport ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; physiology ; HeLa Cells ; Humans ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; physiology ; Membrane Potentials ; drug effects ; physiology ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Plasmids ; Transfection

Liver cancer is one of the common malignant tumors in clinic, and the incidence rate has been increasing year by year. Most patients are diagnosed at the advanced stage, and the prognosis is poor. The occurrence and development of hepatocellular carcinoma is closely associated with immune elimination, balance and escape. In recent years, checkpoint inhibitors have been used in clinic and immune checkpoint inhibitors have made extensive progress and application in clinical research, which has brought a significant opportunity to improve the survival rate of patients with advanced liver cancer. However, HCC patients develop immunotherapy resistance afterwords. The poor drug response to tumor cells is a serious problem affecting the clinical prognosis. This review briefly summarizes the potential mechanism of innate resistance and acquired resistance in immunotherapy of liver cancer.

Objective To observe the effects of tetrandrine on the proliferation,migration,and invasion of melanoma cell B16,and to explore its effects on epithelial mesenchymal transition(EMT)and potential regulatory mechanisms.Methods(1)The proliferation of B16 cells was detected by CCK-8 assay after 0,2,4,6,8 and 10 μmol·L-1 of tetrandrine intervention for 24 and 48 hours.The colony formation ability of B16 cells was detected by plate clone formation assay after 1,2 and 4 μmol·L-1 of tetrandrine intervention;the migration and invasion ability of B16 cells was detected by cell scratch assay and Transwell invasion assay;the expressions of N-cadherin,Vimentin and E-cadherin related to EMT in B16 cells were detected by Western Blot assay.The mouse melanoma lung metastasis model was replicated by tail vein injection of B16 cells to observe the effects of tetrandrine(50 and 100 mg·kg-1)administered by gavage on the number of metastatic tumor nodules in the lungs of mice.(2)The CTD,SwissTargetPrediction and Similarity Ensemble Approach databases were used to predict the targets of tetrandrine;the GeneCards database was used to search for targets related to melanoma disease;the intersection of these two databases was taken as the potential target of tetrandrine for melanoma treatment.The intersected targets were imported into STRING database to construct protein-protein interaction(PPI)network and screen the core targets;the intersected targets were imported into DAVID database for GO function and KEGG pathway enrichment analysis;and molecular docking between tetrandrine and the core targets was verified by Autodock software.(3)In vivo experimental validation:after intervention of 1,2 and 4 μmol·L-1 tetrandrine,Western Blot method was used to detect the expression of the key pathway AKT/NF-κB/CREB pathway-related proteins;and AKT agonist SC79 was used to validate the replication experiments.Results(1)The IC50 of B16 cells intervened by tetrandrine was 4.273 and 4.085 μmol·L-1 at 24 and 48 hours.Compared with the control group,the colony forming ability,scratch healing rate and invasion rate of cells in the 1,2 and 4 μmol·L-1 tetrandrine group were all significantly reduced(P＜0.05,P＜0.01,P＜0.001);the expressions of cellular Vimentin and N-cadherin protein expressions were significantly down-regulated(P＜0.01,P＜0.001),and E-cadherin protein expression was significantly up-regulated(P＜0.01,P＜0.001).Compared with the model control group,the number of melanoma lung metastatic nodules was significantly reduced in the mice in the high-dose group of tetrandrine(P＜0.05).(2)A total of 60 potential targets were obtained for the treatment of melanoma with tetrandrine;core targets such as AKT1,TNF,CCND1,RELA,CASP9,CHUK,and CREBBP were further screened,among which AKT1 was the most strongly interacting target;the signaling pathways such as apoptosis,FoxO,TNF,PI3K-AKT,and NF-κB were mainly involved.The molecular docking showed that tetrandrine had strong binding activity with AKT1,TNF,RELA and other core targets.Compared with the control group,protein expressions of p-AKT/AKT,p-NF-κB p65/NF-κ B p65,and p-CREB/CREB were significantly down-regulated in the cells of the tetrandrine 1,2,and 4 μmol·L-1 groups(P＜0.05,P＜0.01);protein expressions of p-AKT and p-NF-κB p65 were significantly up-regulated in the cells of the SC79 group(P＜0.001).Compared with the SC79 group,protein expressions of p-AKT,p-NF-κB p65,and p-CREB were significantly down-regulated in the cells of the 2 μmol·L-1 tetrandrine+ SC79 group(P＜0.001).Conclusion Tetrandrine may inhibit the proliferation,migration,invasion and EMT of mouse melanoma by regulating the AKT/NF-κB/CREB pathway,and thus inhibit the lung metastasis of mouse melanoma.

Objective:To establish an analytical method for the simultaneous determination of five fat-soluble vitamins in serum using isotope dilution ultra high performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MSMS).Methods:Fat-soluble vitamins were obtained from serum samples which collected from Shanghai Tenth People′s Hospital between April 2019 and August 2019 by the extraction method, and were detected by ID-UPLC-MSMS. The performance of the method was verified by referring to the relevant documents of the Clinical and Laboratory Standards Institute (CLSI).Results:The ID-UPLC-MSMS method for the rapid detection of various fat-soluble vitamins in serum was proposed and successfully verified. The linear range of the method: vitamin A: 25-2 500 μg/L, 25(OH)D 2: 2-200 μg/L, 25(OH)D 3: 2-200 μg/L, vitamin E: 0.25-50 mg/L, vitamin K1: 0.1-20 μg/L. The intra- and inter-assay precision standard deviations of the five analytes were within ± 15%, and the accuracy of the test results of the 25(OH)D 2 and 25(OH)D 3 standards was 96.44%-102.37%. Conclusion:The performance of ID-UPLC-MSMS method for the simultaneous determination of five fat-soluble vitamins is satisying, and the result is accurate and reliable, which suggested it can be used for the clinical sample.

This study was aimed to prepare tripterine-coix seed component microemulsions (TCC-MEs) with coix seed oil and coix seed polysaccharide as bimodal excipients and evaluate its activity on anti-lung cancer in vivo.Coix seed oil was extracted by supercritical extraction methods and coix seed polysaccharide was obtained through hot water extraction and alcohol precipitation from coix seed.Then,coix seed oil was used as the oil phase,and coix seed polysaccharide aqueous solution was used as the aqueous phase.RH40 was used as the surfactant.PEG400 was used as cosurfactant to prepare microemulsion.TCC-MEs were made by water titration method and characterized by size,polymer dispersity index (PDI),zeta potential,encapsulation efficiency and drug loading capacity.And then,anti-tumor activity of TCC-MEs was evaluated on the xenograft Lewis tumor mouse models.The liver and kidney toxicity was evaluated by HE staining and biochemical indicators.The results showed that the optimized prescription for TCC-MEs was coix seed oil 400 mg,RH40 300 mg,PEG 400 100 mg,coix seed polysaccharide 50 mg,tripterine 10 mg;and the tripterine encapsulation efficiency was (90.72 ± 0.28)％;the drug loading capacity was (1.08 ± 0.17)％;the mean diameter of microemulsion was (43.86 ± 0.22) nm;PDI was 0.10 ± 0.01;the zeta potential was (-13.14 ± 1.35) mV.The activity of anti-lung cancer in TCC-MEs was significantly better than that of the control group,with no significant liver and kidney toxicity after treatment with TCC-MEs.It was concluded that the prepared TCC-MEs had advantages of small amount of conventional auxiliary materials,small particle size and high stability.This study showed that the combination of triptolide and coix seed to microemulsion system has synergistic and attenuated effect.

Ambystoma mexicanum/immunology* ; Amputation ; Animals ; Biomarkers/metabolism* ; Blastomeres/immunology* ; Cell Lineage/immunology* ; Connective Tissue Cells/immunology* ; Epithelial Cells/immunology* ; Forelimb ; Gene Expression ; High-Throughput Nucleotide Sequencing ; Humans ; Immunity ; Peroxiredoxins/immunology* ; Regeneration/immunology* ; Regenerative Medicine/methods* ; Single-Cell Analysis/methods*

Lipid Droplets/metabolism* ; Bacteria ; Lipid Metabolism