胃祺饮乙醇提取物可诱导AGS细胞G2/M 期阻滞及凋亡

Objective:To evaluate the effects of the ethanol extract isolated from Weiqi Decoction(胃祺饮,WQD-EE)on AGS cell proliferation and apoptosis.Methods:By using high-performance liquid chromatography with ultraviolet detectors(HPLC-UV)assay and MTT method,the main compounds in WQD-EE and cell viability were detected.And cell cycle distributions were determined by flow cytometry with propidium iodine(PI)staining while apoptosis was detected by flow cytometry with annexin V/Pl double staining.Finally,caspase-3 activities were measured by calorimetric method and protein expression was determined by Western blotting.Results:HPLC analysis showed that naringin(35.92μg/mg),nobiletin(21.98μg/mg),neohesperidin(17.98μg/mg)and tangeretin(0.756μg/mg)may be the main compounds in WQD-EE.WQD-EE not only inhibited AGS and MCF7 cell proliferation in a dose-dependent manner,but also blocked cell cycle progression at G_2/M stage as well as inducing cell apoptosis at concentrations triggering significant inhibition of proliferation and cell cycle arrest in AGS cells.While at 0.5 mg/mL,WQD-EE significantly increased caspase-3 activity by 2.75 and 7.47 times at 24 h and 48 h,respectively.Moreover,WQD-EE in one hand reduced protein expressions of p53 and cyclin B1,and in other hand enhanced protein expressions of cytochrome c and Bax.Protein levels of Bcl-2,Fas L and Fas were not significantly affected by WQD-EE.Conclusions:WQD-EE inhibits AGS cell proliferation through G_2/M arrest due to down-regulation of cyclin Bi protein expression,and promotes apoptosis by caspase-3 and mitochondria-dependent pathways,but not by p53-dependent pathway.     

Time-effect Relationship of Extracts from Ginseng, Notoginseng and Chuanxiong on Vascular Endothelial Cells Senescence

Objective:To observe the time-effect relation of extracts from ginseng,notoginseng and chuanxiong on angiotensin II(AngⅡ)-induced senescence of vascular endothelial cells and explore the feature of Chinese medicine against vascular diseases.Methods:Human umbilical vein endothelial cells(HUVECs)cultured in vitro were stimulated with 10~(-6) mol/L Ang Ⅱ to induce cell senescence,which were divided into 4groups,the blank control group,the Ang Ⅱ model group,the extracts group and the telmisartan group.The(J-gal was used to identify senescence of cells,the cell counting kit-8 method was applied to assess the cell viability,the cell function was examined with the level of endothelial nitric oxide synthase(eNOS) and the flow cytometry was used for analyzing the cell cycle changes.Results:Compared with the control cells,the cells positive for β-gal staining was significantly increased in the Ang Ⅱ model group,and showed cell cycle arrest at G_0/G_1 phase with decreased S and G_2/M phase ceil percentage,eNOS expression and cell viability(P0.05).The extracts and telmisartan treatment of Ang II-induced cells resulted in decreased β-gal positive cells with a reduction in G_0/G_1phase cells and an increasing in S,G_2/M phase ceils and eNOS expression(P0.05).At 24 h,the extracts were more effective than telmisartan(P0.05);while telmisartan was more effective at 48 h(P0.05).Conclusion:Extracts from ginseng,notoginseng and chuanxiong can delay Ang II-induced aging of HUVECs and may play an important role in early senescence.     

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats

Objective: To observe the effect of norcantharidin(NCTD) on collagen-induced arthritis(CIA) rats. Methods: Sixty Sprague-Dawley(SD) rats were randomly divided into 6 groups(n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg·d)], NCTD middle-dose group [2.7 mg/(kg·d)], NCTD high-dose group [5.4 mg/(kg·d)] and methotrexate(MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin(HE) staining. The serum levels of interleukin(IL) 1β, IL-6, tumor necrosis factor(TNF)-α, vascular endothelial growth factor(VEGF), IL-17 and transform growth factor(TGF) β were detected by enzyme linked immunosorbent assay(ELISA). The mRNA expression of retinoid-related orphan nuclear receptor γ t(ROR γ t) and forkhead box P3(Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. Results: MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group(P0.05 or P0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats(P0.05). Only middle-and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats(P0.05). However, NCTD has no effect on vascular endothelial growth factor(VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group(P0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group(P0.05). Conclusion: NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.     

Effect of snake venom of agkstrodon halys pallas in

Objective

To study the effect and mechanism of snake venom ofAgkistrodon Halys Pallas in Zhejiang in inducing human leukemic Jurkat cell apoptosis.

Methods

IC₅₀ and growth curve of Jurkat cells were detected by MTT; percentage of apoptotic cells was analyzed by flow cytometry (FCM) using Annexin V FITC/PI staining method; cell cycle was also detected by FCM and protein expression of Bcl-2 gene was detected by FCM and Western-blot.

Results

Zhejiang viper venom could inhibit the growth of Jurkat cells dose-dependently, induce the cell apoptosis, with the bcl-2 protein expression down-regulated. The strongest effect was shown at 48 hrs, it weakened gradually, and the Jurkat cells restored slowly in low concentration of snake venom.

Conclusion

Zhejiang viper venom could induce apoptosis of human leukemic Jurkat cell in dose-dependent manner, the effect is related to the down-regulation of Bcl-2 protein expression.     

Effect of prevention and treatment of murine acute viral myocarditis and protection of lymphoid organ atrophy with xinkang oral liquid

Objective

The effect of prevention and treatment of Xinkang oral liquid, ( , XKOL) on experimental coxsackievirus B₃ (CVB₃) myocarditis mice model were investigated.

Methods

The mice were inoculated intraperitoneally with 0.3 ml of 10⁵ TCID₅₀ of CVB₃ to induce acute viral myocarditis model. These mice were divided into model control group (Group A), prevention high dosage group (Group B) and prevention low dosage group (Group C), treatment high dosage group (Group D) and treatment low dosage group (Group E), respectively. In addition, XKOL control group (Group F) and normal control group (Group G) were not infected with CVB₃ intraperitoneally. The administration of XKOL in Group B and C began 2 days before virus infection. All animals were sacrificed on day 20 for evaluation.

Results

Histological examination showed extensive myocardial necrosis and cell infiltration in most of Group A mice, but necrosis and cell infiltration were less severe in Group B,C,D and E mice. Thymus weight in Group B,C,D and E mice were heavier and less cell depletion occurred than those in Group A.

Conclussion

The XKOL could effectively inhibit myocardial CVB₃ replication, reduce the myocardial inflammatory response, lower incidence rate of myocarditis and prevent the disease associated lymphoid organ atrophy in this animal models.     

顺铂联合虫草素对OC3人口腔癌细胞的诱导凋亡作用

Objective:To evaluate apoptotic effects of cisplatin and cordycepin as single agent or in combination with cytotoxicity in oral cancer cells.Methods:The influences of cisplatin(2.5μg/mL)and/or cordycepin treatment(10 or 100μmol/L)to human OC3 oral cancer cell line were investigated by morphological observation for cell death appearance,methylthiazoletetrazolium(MTT)assay for cell viability,flow cytometry assay for cell apoptosis,and Western blotting for apoptotic protein expressions.Results:Data demonstrated that co-administration of cisplatin(2.5μg/mL)and cordycepin(10 or 100μmol/L)resulted in the enhancement of OC3 cell apoptosis compared to cisplatin or cordycepin alone treatment(24 h),respectively(P0.05).In flow cytometry assay,percentage of cells arrested at subG1 phase with co-treatment of cordycepin and cisplatin(30%)was significantly higher than cisplatin(5%)or cordycepin(12%)alone group(P0.05),confirming a synergistically apoptotic effect of cordycepin and cisplatin.In cellular mechanism study,co-treatment of cordycepin and cisplatin induced more stress-activated protein kinase/Jun terminal kinase(JNK),the expressions of caspase-7,and the cleavage of poly ADP-ribose polymerase(PARP)as compared to cisplatin or cordycepin alone treatment(P0.05).Conclusion:Cisplatin and cordycepin possess synergistically apoptotic effect through the activation of JNK/caspase-7/PARP pathway in human OC3 oral cancer cell line.     

Pretreatment with Baicalin Attenuates Hypoxia and Glucose Deprivation-Induced Injury in SH-SY5Y Cells

Objective:To explore the neuroprotective effects of baicalin against hypoxia and glucose deprivationreperfusion(OGD/RO)-induced injury in SH-SY5 Y cells.Methods:SH-SY5 Y cells were divided into a control group,a OGD/RO group,which was subject to OGD/RO induction;and 3 baicalin groups subject to baicalin(1,5,25 μ mol/L) for 2 h before induction of OGD/RO(low-,medium-,and high-dose baicalin groups).Cell viability was detected by thiazolyl blue tetrazolium bromide(MTT) assay and flow cytometric analysis was used to detect cell apoptosis.Real-time polymerase chain reaction was performed to determine the mRNA expression of caspase-3gene.Western blot analysis was conducted to determine the expression of nuclear factor(NF)- k B and N-methyl-daspartic acid receptor-1(NMDAR1).Results:Baicalin could significantly attenuate OGD/RO mediated apoptotic cell death in SH-SY5 Y cells;the apoptosis rates in the low-,medium- and high-dose groups were 12.1%,7.9%,and 5.4%,respectively.Western blot and real-time PCR analysis revealed that significant decrease in caspase-3 expression in the baicalin group compared with the OGD/RO group(P0.01).Additionally,down-regulation of NF-κB and NMDAR1 was observed in the baicalin group compared with those obtained from the OGD/RO group.Compared with the low-dose baicalin group,remarkable decrease was noted in the medium- and high-dose groups(P0.01).Conclusion:Baicalin pre-treatment attenuates brain ischemia reperfusion injury by suppressing cellular apoptosis.     

Anticancer Effects of Crude Extract from Melia toosendan Sieb. et Zucc on Hepatocellular Carcinoma In Vitro and In Vivo

Objective:To investigate the anti-cancer effects of crude extract from Melia toosendan Sieb.et Zucc and its possible molecular mechanisms in vitro and in vivo.Methods:Transonic alcohol-chloroform extraction method was used to extract toosendanin from the bark of Melia toosendan Sieb.et Zucc,and the content of toosendanin in the crude extract was measured by high performance liquid chromatography(HPLC).Anti-cancer effects of crude extract from Melia toosendan Sieb.et Zucc were investigated in in vivo and in vitro studies.In the in vitro experiment,human hepatocellular carcinoma cell lines SMMC-7721 and Hep3 B were co-incubated with toosendanin crude extract of different concentrations,respectively.In the in vivo experiment,BALB/c mice were subcutaneously inoculated with mouse hepatocellular carcinoma H22 cells and treated with crude extract.Results:HPLC revealed the content of toosendanin was about 15%.Crude extract from Melia toosendan Sieb.et Zucc inhibited cancer cells growth in a dose- and time-dependent manner.The 50%inhibitory concentration(IC_(50),72 h) was 0.6 mg/L for SMMC-7721 cells and 0.8 mg/L for Hep3 B cells.Both high-dose[0.69 mg/(kg·d)]and low-dose[0.138 mg/(kg·d)]crude extract could markedly suppress cancer growth,and the inhibition rate was greater than 50%.Hematoxylin and eosin staining showed necrotic area in cancers and transmission electron microscopy displayed necrotic and apoptotic cancer cells with apoptotic bodies.Immunohistochemistry showed that the expression of Bax and Fas increased and the expression of Bcl-2 reduced.Conclusions:Toosendanin extract has potent anti-cancer effects via suppressing proliferation and inducing apoptosis of cancer cells in vivo and in vitro.The mechanism of apoptosis involves in mitochondrial pathway and death receptor pathway.     

大黄蛰虫丸血清活性成分对血小板源生长因子介导血管平滑肌增殖的作用及其机制

Objective: To investigate and compare the effects and mechanisms of three functional parts of Dahuang Zhechong Pill (DHZCP), including drugs with the function of removing blood stasis and promoting blood circulation (FP-Ⅰ), drugs with the function of expelling heat and moistening dryness (FP-Ⅱ), and drugs with the function of nourishing yin and replenishing blood (FP-Ⅲ) of DHZCP, on platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs) proliferation with the method of serum pharmacology. Methods: VSMCs proliferation of rat was assayed by measuring the cell viability with the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) method. DNA synthesis in VSMCs was examined by detecting 5''-bromo-2''-deoxyuridine incorporation with the immunocytochemical method. Cycle of VSMCs was evaluated with flow cytometry. Expression of cyclin D1, p27, PKCα, and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) was quantified by the Western blotting method. Results: The FP-Ⅰ and FP-Ⅲ containing serum was capable of inhibiting PDGF-stimulated proliferation and DNA synthesis of VSMCs, arrested VSMCs in G1 phase, downregulated cyclin D1, and upregulated p27 expression (P<0.01 or P<0.05). The FP-Ⅰ and FP-Ⅲ containing serum also inhibited the PDGF-induced phosphorylation of tyrosine of ERK1/2 and PKCα expression (P<0.01 or P<0.05). Conclusions: FP-Ⅰ and FP-Ⅲ of DHZCP are able to inhibit VSMCs proliferation via interrupting PKCα-ERK1/2 signaling, modulating the expression of cell cycle proteins to result in arresting the cells in G1 phase. The inhibitory effect is mainly related to the function of removing blood stasis and promoting blood circulation, slightly to the function of nourishing yin and replenishing blood, but not to the function of expelling heat and moistening dryness.     

Tonglian Decoction(通莲汤) Arrests the Cell Cycle in S-phase by Targeting the Nuclear Factor-Kappa B Signal Pathway in Esophageal Carcinoma Eca109 Cells

Objective:To investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction(通莲汤,TLD) on esophageal carcinoma Eca109 cells.Methods:Eca109 cells were treated with TLD and its separated formulae,including the clearing-heat and detoxification formula(Q),activating-blood and promoting-qi formula(H) and nourishing-yin and blood formula(Z).Cell proliferation was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay,cell morphology was observed using a microscope,the cell cycle was measured using flow cytometry and the activity of the nuclear factor-kappa B(NF-κB) signal pathway was detected by Western blot.Results:The half maximal inhibitory concentrations of TLD,Q and H were 386,771 and 729 mg/L,respectively.TLD,Q and H significantly inhibited cell proliferation,with 69.43%,60.84%and 61.90%of treated cells in the G1 phase of the cell cycle.The percentage of cells in S phase increased significantly after treatment with TLD,Q,and H compared with the control group(P0.05),and TLD showed the strongest effect.Z had no influence on the cell cycle compared with the control group(P0.05).Western blot detection indicated slight differences in the inhibition of the NF- k B pathway by the different formulae.TLD formula strongly inhibited IKKβ,NF-κB,interleukin-6 and tumor necrosis factor-α expression compared with the control group.Conclusions:TLD inhibited Eca109 cell proliferation by arresting cells in S phase.The possible mechanism might be related to inhibiting the NF- κB transduction cascade.The combination of the herbs found in the three separate formulae,H,Q and Z,work synergistically in TLD to produce the inhibitory effects of TLD treatment on Eca109 proliferation.     

Experimental study of the inhibitory effect of gantai capsule on HBsAg and HBeAg expression

Objective

To study the inhibitory effect of Gantai capsules (GTC) on replication and expression of HBsAg and HBeAg.

Methods

The 2.2.15 cell line was chosen as a in vitro cell culture system, and by means of radioimmunoassay, the hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in cell cultured medium were examined.

Results

HBsAg, HBeAg values of 2.2.15 cells treated by GTC (200 μg/ml- 1200 μg/ml) were lower than those of the control. And this inhibitory effect was in a dose-dependent manner under experimental condition, GTC had no cytotoxicity.

Conclusion

GTC could inhibit HBV replication and expression.     

下瘀血汤减少CCl4诱导小鼠肝纤维化肝星状细胞激活及肝窦内皮细胞死亡的相关研究

Objective： To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction （下瘀血汤, XYXD） on activation of hepatic stellate cells （HSCs） and defenestration of sinusoidal endothelial cells （SECs） in CCI4-induced fibrotic liver of mice. Methods： High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCI4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 （CD31）. Whole liver lysates were detected of α-smooth muscle actin （α-SMA） and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） assay and cellomics arrayscan, respectively. Results： Amygdalin and emodin in XYXD were identified. XYXD （993 mg/kg） inhibited Sirius red positive area up to 70.1% （P〈0.01）, as well as protein levels of α-SMA and type- I collagen by 42.0% and 18.5% （P〈0.05） respectively. In vitro, XYXD （12.5 μg/mL, 50 μg/mL） suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% （P〈0.05）. Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% （P〈0.05） and 49.5% （P〈0.01）, evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 （P〈0.05）. Conclusions： XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.     

熊胆粉通过线粒体依赖性途径诱导人肝癌细胞凋亡

Objective:To evaluate the effect of Bear Bile Powder(熊胆粉,BBP) on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells,and investigate the possible molecular mechanisms mediating its anti-cancer activity.Methods:HepG2 cells were treated with 0.4-1.0 mg/mL of BBP for 24,48 and 72 h.The viability of HePG2 cells was determined by MTT assay.Cellular morphology was observed via phase-contrast microscopy.Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide(JC-1) staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential,respectively.Activation of caspase-9 and-3 was evaluated by a colorimetric assay.Results:The treatment with 0.4-1 mg/mL of BBP for 24,48,or 72 h respectively reduced cell viability significantly by 7%-60%,20%-90%or 25%-98%,compared with the untreated control cells(P0.01).In addition,BBP treatment induced morphological changes in HepG2 cells.Furthermore,after treated with 0,0.4,0.6,0.8 and 1.0 mg/mL of BBP,apoptosis cells(including early and late apoptotic cells) were 18.0%±1.3%,34.9%±2.2%,33.9%±2.8%,37.4%±2.8%and 46.0%±2.5%,respectively(P0.05);and the percentage of cells with reduced JC-1 red fluorescence were 6.6%±0.8%,8.5%±0.8%,13.5%±1.6%,17.6%±2.3%and46.7%±3.6%,respectively(P0.01).Finally,BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells(P0.05).Conclusions:BBP could inhibit the growth of HepG2hepatocellular cancer cells through mitochondrion-mediated apoptosis,which may,in part,explain its anti-cancer activity.BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.     

Ethanol Extract of Ilex Hainanensis Merr. Exhibits Anti-Melanoma Activity by Induction of G1/S Cell-Cycle Arrest and Apoptosis

Objective: To evaluate anti-melanoma effect of ethanol extract of Ilex hainanensis Merr. (IME) and elucidate its underlying mechanism. Methods: Thirty-six tumor-bearing mice were randomized into 6 groups (n=6) as follows: model group, IME 25, 50, 100, and 200 mg/kg groups and dacarbazine (DTIC) 70 mg/kg group. The mice in the IME treatment groups were intragastrically administered with IME 25, 50, 100 or 200 mg/kg per day, respectively. The mice in the DTIC group were intraperitoneally injected with DTIC 70 mg/kg every 2 days. The drug administration was lasting for 14 days. The cell viability was evaluated by 3-(4,5-dime-thylthylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect cell cycle and apoptosis. The gene and protein expressions of nuclear factor κB-p65 (NF-κB-p65), Bcl-2, B-cell lymphoma-extra large (Bcl-xL) and Bax were detected by quantitative real-time polymerase chain reaction and Western blot analyses. Caspases-3, -8, and -9 activities were detected using the colorimetric method. In addition, a B16-F10 melanoma xenograft mouse model was used to evaluate the anti-cancer activity of IME in vivo. Furthermore, a survival experiment of tumor-bearing mice was also performed to evaluate the possible toxicity of IME. Results: IME significantly inhibited the proliferation of B16-F10 cells (P<0.01). Flow cytometric analysis showed that IME induced G1/S cell cycle arrest and apoptosis (both P<0.01). IME inhibited activation of NF-κB, decreased the gene and protein expressions of Bcl-2, Bcl-xL, and increased the gene and protein expressions of Bax (all P<0.01). In addition, IME induced the activation of Caspases-3, -8, and -9 in B16-F10 cells. The study in vivo showed that IME significantly reduced tumor volume (P<0.01), and the inhibitory rate came up to 68.62%. IME also induced large areas of necrosis and intra-tumoral apoptosis that correlated with a reduction in tumor volume. Survival experiment showed that treatment with IME for 14 days significantly prolonged survival time and 20% of mice in the IME 200 mg/kg group were still alive until the 50th day. Notably, IME showed no apparent side-effects during the treatment period. Conclusion: IME exhibited significant anti-melanoma activity in vitro and in vivo, suggesting that IME might be a promising effective candidate with lower toxic for malignant melanoma therapy.