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OBJECTIVE：To study the protective effect of timosaponin BⅡ（TB-Ⅱ）on blood vessels and explore its possible mechanism. METHODS ：Using aquaculture water as blank control ，the effects of 100，200 and 400 μg/mL TB-Ⅱ treatment for 48 h on the situation of subintestinal veins （SIVs）in normal zebrafish embryos 24 h after fertilization （24 hpf）were investigated. PTK787（0.06 μg/mL），a tyrosine kinase inhibitor ，was used to induce the model of zebrafish intestinal vascular injury ；using combing with 0.1％ dimethyl sulfoxide but no PTK 787 as blank control ，combing with PTK 787 but no drug as model control ，the effects treatment of 100，200 and 400 μg/mL TB-Ⅱ for 48 h on the SIVs of zebrafish model with vascular injury were investigated. Relative expressions of fam-like tyrosine kinase 1（Flt-1），kinase insert domain containing receptor （Kdr），kinase insert domain containing receptor l （Kdr-l），vascular endothelial growth factor A （VEGF-A），tumor necrosis factor α（TNF-α）and interleukin 6 （IL-6）mRNA were detected by RT-PCR. RESULTS ：100 μg/mL TB-Ⅱ could significantly increase the sprouting vessel of normal zebrafish SIVs sprouting vessel number （P＜0.05），and 200 μg/mL TB-Ⅱ could significantly increase SIVs number of normal zebrafish （P＜0.05）. Compared with blank control ， SIVs treatment （P＜0.01），and the relative expressions of Flt-l ， Kdr，Kdr-l，VEGF-A，TNF-α and IL-6 mRNA were alse decreased significantly （P＜0.05 or P＜0.01）. After treated 化。E-mail：pn333@163.com with different concentrations of TB- Ⅱ ，SIVs number of vascular injury model zebrafish increase d to different extents ；relative expressions of Flt-l ，Kdr，Kdr-l，VEGF-A，TNF-α and IL-6 mRNA were increased to different extents. There was no significant difference in SIVs number and the expression of Flt-l ，TNF-α mRNA in zebrafish treated with 100 μg/mL TB-Ⅱ and the expression of TNF-α mRNA in zebrafish treated with 400 μg/mL TB-Ⅱ， but there was statistical significance in other indexes （P＜0.05 or P＜0.01）. CONCLUSIONS ：TB-Ⅱ has a certain function of promoting angiogenesis and repairing damaged blood vessels ，and its mechanism is related to the up-regulation of vascular endothelial growth factor receptor and pro-inflammatory cytokine expression.
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Objective To explore the inhibitory mechanism of timosaponin B-Ⅱ against the proliferation and migration of human gastric cancer cell lines BGC-823 and MGC-803. Methods BGC-823 and MGC-803 cells were treated with timosaponin B-Ⅱ (50 ng/mL) for 48 h, and the mRNA and protein expressions of scavenger receptor A5 (SCARA5) were measured by qPCR and Western blotting, respectively. The binding site of hsa-miRNA-766-3p in SCARA5 gene 3′UTR was predicted by bioinformatics, and was validated by luciferase report assay. After transfecting with hsa-miRNA-766-3p mimic or siRNA-SCARA5 for 24 h, the BGC-823 and MGC-803 cells were treated with timosaponin B-Ⅱ(50 ng/mL) for 48 h. The relative levels of hsamiRNA-766-3p and SCARA5 protein expression were detected by qPCR and Western blotting, respectively. The proliferation and migration abilities of cells were determined by MTT. Results The expressions of SCARA5 protein in BGC-823 and MGC-803 cells treated with timosaponin B-Ⅱ (50 ng/mL) for 48 h were significantly increased versus the control group (P0.01), while no significant difference was found in relative mRNA level of SCARA5 between the timosaponin B-Ⅱ treated cell group and the control group (P0.05). Compared with transfection of reporter gene expression vector alone group, the luciferase activity was significantly inhibited or enhanced in the cells co-transfected with hsa-miRNA-766-3p mimic or hsa-miRNA-766-3p inhibitor and wild-type luciferase reporter gene (P0.05, P0.01). No change was observed between the cells co-transfected with hsa-miRNA-766-3p mimic or hsa-miRNA-766-3p inhibitor and mutant-type luciferase reporter gene expression vector and the cells transfected with reporter gene expression vector alone (P0.05). Hsa-miRNA-766-3p levels were significantly decreased and SCARA5 protein expressions were significantly increased in BGC-823 and MGC-803 cells treated with 50 ng/mL timosaponin B-Ⅱ (P0.01). Compared with the timosaponin B-Ⅱ treatment group, hsa-miRNA-766-3p levels were significantly increased and SCARA5 protein expressions were significantly decreased in the BGC-823 and MGC-803 cells of the hsa-miRNA-766-3p mimic transfection＋ timosaponin B-Ⅱ treatment group (P0.01). There were no differences in the hsa-miRNA-766-3p levels between the hsa-miRNA-766-3p mimic transfection＋timosaponin B-Ⅱtreatment group and the timosaponin B-Ⅱ treatment group (P0.05), but SCARA5 protein expressions were significantly decreased (P0.01). Compared with cell control group and vehicle control group, the proliferation and migration abilities of BGC-823 and MGC-803 cells were significantly inhibited by timosaponin B-Ⅱ (P0.01). Compared with the timosaponin B-Ⅱ treatment group, the proliferation and migration abilities of BGC-823 and MGC-803 cells were significantly increased in the siRNA-SCARA5 transfection＋timosaponin B-Ⅱ treatment group (P0.01). Conclusion Timosaponin B-Ⅱ can inhibit the proliferation and migration of BGC-823 and MGC-803 cells via suppressing hsamiRNA-766-3p and upregulating the target gene SCARA5.
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Objective To optimize the method for integration of field processing and crude drug preparation of Anemarrhenae Rhizoma (AR) and to provide scientific evidence for the procedure of AR. Methods Four main factors including drying temperature of the fresh herbal medicine, the water content of the medicine, thickness of the products, and the drying temperature of decoction pieces were studied by using single factor experiment and orthogonal test. The content of timosaponin B-Ⅱ and mangiferin and the appearance of the products were selected as evaluation indexes to optimize the integral processing of AR by using comprehensive evaluating method. The changing of the body temperature of yeast-induced rat model was used to compare the antipyretic activity between the two processing technologies (the integration technology and traditional technology). The content of the blood glucose, insulin, and glycosylated serum proteins (GSP) in the blood of the STZ-induced diabetic rat model were selected as the evaluation indexes to compare the hypoglycemic activity between the two processing technologies. Results AR was dried at 50 ℃ for 11 h (water content of medicinal materials was 45%—50%), and then the flosses were knocked off in the drum for 30 min. The results showed that 40—50 ℃ was the best drying temperature, 45%—50% was the best water content, and then unhairing process were conducted for AR for 30 min. The thickness of the slice was 4 mm and the best drying temperature was 50 ℃. There were no significant differences in chemical composition and hypoglycemic activity between the integration technology and traditional technology, while the antipyretic effect of the integrated processing was better than that of the traditional technology. Conclusion The technology of integration of field processing and crude drug processing is feasible and it can be used in industrial production.
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OBJECTIVE:To study the inhibitory mechanism of timosaponin B-Ⅱ(TB-Ⅱ) on the proliferation and migration of human lung cancer A549 cells. METHODS:A549 cells were treated with TB-Ⅱ [0(blank control),1,10 and 100 μg/ml] for 48 h,and total RNA and total protein were extracted respectively. Real time fluorescence quantitative-PCR and Western blot were used to detect mRNA and protein levels of IL-18. IL-18 in A549 cells was silenced by transfection;the expression of IL-18 mRNA and protein were compared among untransfection group,negative control group and transfection group;and then human lung can-cer A549 cells with silenced gene were treated with 10 μg/ml TB-Ⅱ for 24,48 and 72 h. The activity of cell proliferation was de-tected with CCK-8,and the change of cell migration ability was observed by streak method. RESULTS:Compared with blank con-trol,the expression of IL-18 mRNA and protein in A549 cells all increased after treated with TB-Ⅱ(P<0.05 or P<0.01),and were positively correlated with concentration. Compared with untransfection group,the expression of IL-18 mRNA and protein de-creased in transfection group(P<0.01). Compared with untransfected cell treated with TB-Ⅱ,the viability and migration ability of A549 cells with transfection gene increased after treated with TB-Ⅱ for 72 h(P<0.01). CONCLUSIONS:TB-Ⅱ can inhibit the proliferation and migration of A549 cells by up-regulating IL-18 gene expression.
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This study was aimed to establish a rapid detection method for timosaponin BⅡ in Anemarrhenae Rhizoma in order to determine its concentration quickly,conveniently and efficiently.The concentration of timosaponin BⅡ in A.Rhizomadetected by HPLC in the Chinese Pharmacopeia was used as the actual measured value.The near-infrared spectroscopy (NIRS) was used to collect the spectrogram of A.Rhizomasamples.The partial least squares (PLS) of TQ Analyst 8.0 were used in the data analysis.Through the pretreatment,wavelength range and principal component number selection,the actual measured value and NIRS information were associated for the establishment of the optimal quantitative analysis model of timosaponin BⅡ.The results showed that the correlation coefficients (R2),root-mean-square error of calibration (RMSEC),root-mean-square error of prediction (RMSEP),root-mean-square error of cross-validation (RMSECV) and the performance index (PI) of the established model were 0.975 15,0.094 2,0.080 0,0.369 20,and 91.0,respectively.It was concluded that the established quantitative analysis model by NIRS with HPLC was able to determine the concentration of timosaponin BⅡ in A.Rhizomaquickly and accurately.