Objective To study the chemical constituents of Dysosma pleiantha.Methods Acidic resins were precipitated by 1% HCl solution, then the constituents were isolated and purified on repeated silica gel column.They were identified and structurally elucidated by physicochemical constants and spectral analysis.Results Ten compounds were obtained and identified: podophyllotoxone (Ⅰ), dehydropodophyllotoxin (Ⅱ), podophyllotoxin (Ⅲ), 4′-demethylpodophyllotoxin (Ⅳ), 4′-demethyldehydropodophyllotoxin (Ⅴ), kaempferol (Ⅵ), quercetin (Ⅶ), n-hexadecanoic acid (Ⅷ), ?-sitosterol (Ⅸ), and daucosterol (Ⅹ).Conclusion Compound Ⅴ is a new compound, compounds Ⅷ-Ⅹ are first separated from the plants of Dysosma Woodson and other compounds are first separated from D.pleiantha.

Objective: To investigate the inhibitory effect of Rh2 on HepG2 cells and explore the underlying mechanism.Methods: We used lentivirus carrying β-catenin to infect HepG2 cell, and detected expression of β-catenin using fluorescence microscopy.The effect of Rh2 on proliferation of HepG2-β-catenin and HepG2 cells was measured by CKK-8 assay,and flow cytometry was used to detect cell cycle and apoptosis.The activity of Gsk-3βwas checked by ELISA kit.The expression of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 genes were measured by qRT-PCR.In order to checked the relationship between β-catenin and TCF4,CHIP assay kit was used,the expression of Bax,Bcl2,CyclinD1,MMP3 genes were measured by PCR.The expressions of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 proteins were examined by Western blot.Results:HepG2 cells were successfully infected by pLOV-EF1a-MCS-3FLAG-β-catenin lentivirus,named HepG2-β-catenin.CCK-8 showed that ginsenoside Rh2 could effectively inhibit the proliferation of HepG2 and HepG2-β-catenin cells in vitro,which exhibits a dose-dependent manner at range of 10-160 μmol/L Rh2.The IC50 of Rh2 exposure on HepG2 cell for 48,72 h were 100 μmol/L and 58.12 μmol/L,but the IC50 of Rh2 exposure on HepG2-β-catenin for 48,72 h were 129.2 μmol/L,83.33 μmol/L,respectively.The IC50 of Rh2 exposure on HepG2-β-catenin cell was higher than HepG2 cell, compared with HepG2 group the differences was statistically significant ( P<0.01 ).Flow cytometry indicated that Rh2 could arrest HepG2 and HepG2-β-catenin cells in G0/G1 phase;the cell population in G0/G1 phase of HepG2+Rh2 group was(64.57±0.65)%,HepG2-β-catenin+Rh2 group was(58.61±2.01)%.Flow cytometry indicated that Rh2 could induced early apoptosis in HepG2 and HepG2-β-catenin cells.The apoptosis rate of HepG2 +Rh2 group was (17.27 ±2.77)%,HepG2-β-catenin +Rh2 group(9.02 ±1.76)%.The ELISA results indicated that HepG2 cells was induced by Rh2 for 12,24,48,72 h,the activity of Gsk-3βgradually increased,peak in 48 h,then decreased.Compared with control group,Rh2 induced HepG2 and HepG2-β-catenin cells for 48 hours, Gsk-3βactivity were increased, and their activity reduced after adding Bio, there were no significant differences between HepG2+Rh2 and HepG2-β-catenin+Rh2 groups.The PCR,CHIP and WB results showed that the expression of Gsk-3β,Bax gene and proteins increased,while theβ-catenin,CyclinD1,Bcl2,MMP3 gene and proteins down-regulation in HepG2 and HepG2-β-catenin cell induced by Rh2.Compared with HepG2-β-catenin +Rh2 group, the expression of other gene and proteins changed significantly,however,Gsk-3βwas no significant difference.Conclusion:Over-expression of β-catenin may weaken the phar-macological effects of ginsenoside Rh2 on HepG2 cells.The activity of Gsk-3βwas increased by ginsenoside Rh2 to degradeβ-catenin, affecting the expression of downstream genes,promoting apoptosis of liver cancer cells and inhibiting metastasis.

Objective:To study the mechanism of ginsenoside Rh2 inhibiting HepG2 cells migration.Methods:HepG2 cells in logarithmic growth phase were cultured in 96-well plates,which were induced by different concentration Rh2,respectively for 24,48,72 hours.The cell inhibition was detected by Cell Counting Kit.Transwell chambers was used to checked HepG2 cell migration ability;luciferase was tested by Luciferase Reporter Assay system reagent;The expressions of P-ERK,ERK,P-P38,P-38,P-JUK,JUK,MMP3 proteins were detect by Western blot;the expression of AP1,MMP3 gene were detected by Quantitative PCR;The expression of AP1, MMP3 fluorescence protein were observed by fluorescence microscopy.Results:Administrated with different concentration of Rh2 after 24 ,48 ,72 h,the proliferation of HepG2 cells were inhibited ( P<0.05) ,and in dose-and time-dependent manner.Transwell assay showed Rh2 could significantly inhibited migration of HepG2 cells.The expressions of P-ERK , MMP3 proteins were significantly decreased,the expressions of P-JUK, P-P38 proteins were significantly increased, expression levels of ERK, P-38, JUK were no significant difference.Expression of AP1,MMP3 gene were significantly decreased,the expressions of AP1,MMP3 fluorescence proteins were significantly decreased.Conclusion:Ginsenoside Rh2 can activate MAPK pathway to inhibit the migration of HepG2 cells.

OBJECTIVETo investigate the inhibitory effect of trichostatin A (TSA) on the proliferation of HepG2 cells and explore the underlying mechanism.

METHODSHepG2 cells exposed to different concentrations of TSA for 24, 48, or 72 h were examined for cell growth inhibition using a cell counting kit, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under inverted microscope. The expressions of beta-catenin, HDAC1, HDAC3, H3K9, cyclinD1 and Bax proteins in the exposed cells were detected by Western blotting, and the expressions of HDAC1 and HDAC3 mRNAs by quantitative fluorescent PCR.

RESULTSExposure to TSA caused significant dose- and time-dependent inhibition of HepG2 cell proliferation (P<0.05) and resulted in increased cell percentage in G0/G1 and G2/M phases and decreased cell percentage in S phase. The apoptotic index in the control group was (6.22 ± 0.25)%, which increased to (7.17 ± 0.20)% and (18.14 ± 0.42)% after exposure to 250 and 500 nmol/L TSA, respectively. Exposure to 250 and 500 nmol/L TSA also caused cell morphology changes with numerous floating cells. The expressions of beta-catenin, H3K9 and Bax proteins were significantly increased and CyclinD1, HDAC1, and HDAC3 protein expressions decreased in TSA-treated cells, but the expressions of HDAC1 and HDAC3 mRNAs showed no significant changes.

CONCLUSIONSTSA can inhibit the proliferation of HepG2 cells and induce cell cycle arrest and apoptosis by inhibiting HDAC activity, promoting histone acetylation, and activating Wnt/beta-catenin signaling pathway.

Acetylation ; Apoptosis ; Cell Cycle Checkpoints ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Hep G2 Cells ; drug effects ; Histone Deacetylase 1 ; metabolism ; Histone Deacetylases ; metabolism ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Wnt Signaling Pathway ; bcl-2-Associated X Protein ; metabolism ; beta Catenin ; metabolism

Objective: To investigate the differences of clinicopathological features, diagnosis, treatment and prognosis between patients with extra-gastrointestinal stromal tumors (EGIST) and duodenal gastrointestinal stromal tumors (DGIST). Methods: A retrospective case - control study was performed. Case inclusion criteria: (1) tumor confirmed by histology and pathology; (2) primary tumor locating in the extra - gastrointestinal tract or duodenum; (3) without other synchronous tumors; (4) complete clinical and pathological data. Clinical data of 20 EGIST patients and 32 DGIST patients from March 2011 to September 2016 at Department of Gastrointestinal Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine were retrospectively collected and analyzed. The observational parameters included clinicopathological characteristics, treatment and prognosis conditions. Continuous data of abnormal distribution were expressed as median (range) and compared using the Mann-Whitney U-test. Survival curves were drawn by the Kaplan-Meier method and compared with the Log-rank test. Results: Of the 20 EGIST patients, 8 were males and 12 were females with age of 61.0 (30.0 to 86.0) years and of the 32 DGIST patients, 12 were males and 20 were females with age of 55.5 (27.0 to 70.0) years. Compared with DGIST patients, EGIST patients were older (U=188.000, P=0.012], had larger tumor size [10.0 (3.0 to 29.0) cm vs. 4.0 (1.5 to 10.0) cm, U=98.500, P<0.001] and higher ratio of high risk classification [85.0% (17/20) vs. 12.5% (4/32), χ²=26.870, P<0.001]. Among the 20 EGIST patients, 5 were diagnosed with distal metastasis and received imatinib (400 mg/d), and the other 15 patients underwent radical resection who were included in survival analysis. All the 32 DGIST patients underwent radical resection. The median follow-up of whole group was 43 (14 to 76) months. The 3-year recurrence/metastasis-free survival rate of 15 cases undergoing radical resection in the EGIST group was 85.6%, which was lower than that of the DGIST group (88.6%), and the difference was not statistically significant (P=0.745). There was no significant difference in the 3-year overall survival rate between the EGIST group (92.9%) and the DGIST group (100%) (P=0.271). Conclusions As compared to DGIST, EGIST mostly occurs in those with older age, larger tumor size and higher risk grade. The prognosis of EGIST patients after radical resection is similar to that of DGIST patients.