Tang, Feng, Lin Feng, Runle Li, Wei Wang, Huihui Liu, Quanyu Yang, and Ri-Li Ge. Inhibition of suicidal erythrocyte death by chronic hypoxia. High Alt Med Biol. 00:000-000, 2018. Background/Aims: High-altitude polycythemia is defined by the increase of hematocrit and hemoglobin at high altitudes caused by production of excessive erythrocytes. Eryptosis is a process by which mature erythrocytes undergo self-destruction sharing several features with apoptosis. However, the eryptosis in high-altitude hypobaric hypoxia is unknown. Thus, the main purpose of this study was to investigate whether chronic hypoxia affected eryptosis and, if so, by what mechanisms. Methods: Biotin labeling technology was utilized to study the survival of red blood cells in chronic hypoxia. Flow cytometry was used to determine the volume of mature erythrocytes from forward scatter, phosphatidylserine scrambling from annexin-V-binding, intracellular Ca2+ from Fluo-3-AM, reactive oxygen species (ROS) abundance from ROS-probe, and ceramide and CD47 abundance utilizing specific antibodies. Results: The volume of mature erythrocytes was significantly changed, and the percentage of annexin-V-binding cells was significantly decreased under chronic hypobaric hypoxia. Erythrocyte survival was improved under chronic hypoxia, and chronic hypoxia resulted in a decrease in intracellular Ca(2+)in vivo and influenced eryptosis which was induced by the Ca2+-ionophore ionomycin (1M, 60 minutes) in vitro. Chronic hypoxia also resulted in an increase in CD47 and ceramide abundance, but it had no effect on ROS formation. Conclusions: Chronic hypobaric hypoxia can inhibit eryptosis by decreasing intracellular Ca2+ and increasing integrin-associated protein CD47.

Ultrasound-assisted extraction is evaluated as a simpler and more effective alternative to conventional extraction methods for the extraction of bioactive compounds from natural product. This study investigated the use of ultrasound-assisted extraction to extract three dibenzylbutyrolactone lignans, including tracheloside, hemislienoside, and arctiin from Hemistepta lyrata. Factors such as extraction solvent, solvent concentration, solvent to material ratio, and extraction time were examined. High-performance liquid chromatography with photodiode array detection was used for simultaneous determination of the target compounds in the corresponding extracts. Results showed that the optimal parameters to extract the target compounds from H. lyrata were as follows: extraction solvent: 70% aqueous ethanol; solvent to material ratio: 20:1 (v/w, ml/g); extraction time: 20 min under the conditions: ultrasonic frequency: 40 Hz; extraction temperature:30 °C. With all these merits, ultrasound-assisted extraction should be considered for wider application in the extraction of tracheloside, hemislienoside, and arctiin from other medicinal plants.

BACKGROUND: Harmine has antitumor and antinociceptive effects, and inhibits human DNA topoisomerase. However, no detailed data are available on the mechanisms of action of harmine in hepatocellular carcinoma. This study aimed to investigate the effects of harmine on proliferation and apoptosis, and the underlying mechanisms in the human hepatocellular carcinoma cell line HepG2.METHODS: The proliferation of HepG2 cells was determined by the cell counting kit-8 (CCK-8) assay and the clone formation test. The morphology of HepG2 cells was examined using fluorescence microscopy after Hoechst 33258 staining. Annexin V/propidium iodide (PI) was used to analyze apoptosis and PI to analyze the cell cycle. Western blotting was used to assess expression of the apoptosis-regulated genes Bcl-2, Bax, Bcl-xl, Mcl-1, caspase-3, and caspase-9. Mitochondrial transmembrane potential (Psi(m)) was determined using JC-1.RESULTS: Harmine inhibited the proliferation of HepG2 cells in a dose-dependent manner. Hoechst 33258 staining revealed nuclear fragmentation and chromosomal condensation, cell shrinkage, and attachment loss in HepG2 cells treated with harmine. The percentage of the sub/G1 fraction was increased in a concentration-dependent manner, indicating apoptotic cell death. PI staining showed that harmine changed the cell cycle distribution, by decreasing the proportion of cells in G0/G1 and increasing the proportion in S and G2/M. Harmine induced apoptosis in a concentration-dependent manner, with rates of 20.0%, 32.7% and 64.9%, respectively. JC-1 revealed a decrease in Psi(m). Apoptosis of HepG2 cells was associated with caspase-3 and caspase-9 activation, down-regulation of Bcl-2, Mcl-1, and Bcl-xl, and no change in Bax.CONCLUSIONS: Harmine had an anti-proliferative effect in HepG2 cells by inducing apoptosis. Mitochondrial signal pathways were involved in the apoptosis. The cancer-specific selectivity shown in this study suggested that harmine is a promising novel drug for human hepatocellular carcinoma.

In this study, the effects of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation and apoptosis of human Lovo cells in vitro were investigated. Apollon ASODN was incubated with human colorectal Lovo cells for 48 h, the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay, respectively. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry. The morphology of apoptotic cells was examined by fluorescence microscope. Lovo cells incubated with apollon ASODN combined with 5-fluorouracil (5-FU), cisplatin (DDP) or epirubicin (EPI) of different concentrations, cell proliferation inhibition rates were detected with WST method and IC50 was calculated. It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly (P < 0.05). After Lovo cells treated with apollon ASODN for 48 hours, the expression of the apollon mRNA level was suppressed significantly. And a marked concentration-dependent decline of cell proliferation and clone forming, increasing of cell apoptosis levels were observed. The percentage of G0/G1 phage cells was abated and that of S phage cells was increased and the Lovo cells arrested at S phage of the cell cycle detected with flow cytometry. Many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation. Cell proliferation inhibition was detected and their chemo-therapeutic effects of 5-FU, DDP and EPI on Lovo cells combined with apollon ASODN (0.08 micromol x L(-1)) were enhanced independently compared with single 5-FU, DDP and EPI groups, and the sensitivity enhanced about 2.58, 4.47, and 5.33 times respectively. It can be concluded that ASODN targeting apollon can suppress the expression of apollon mRNA, and inhibit the proliferation, induce apoptosis, arrest cell cycle at S phase of colorectal cancer Lovo cells in vitro and enhance the chemo-sensitivity to 5-FU, DDP and EPI.

In order to explore the function of PCNA gene on the proliferation and apoptosis of hepatocelluar carcinoma HepG2 cells, to provide the experimental evidence and a new tool to further explore the function of PCNA gene and the feasibility of its gene therapy, the eukaryotic expression vector targeting PCNA was constructed. According to the sequence screened in previous work, PCNA siRNA was converted into cDNA coding expression of shRNA (small hairpin RNA). The cDNA was synthesized and inserted into plasmid pSilencer2.0-U6 to construction of eukaryotic expression vector of siRNA specific for targeting PCNA gene, the negative plasmid pShPNA was also constructed. The plasmids were identified by sequence analysis. The plasmid was transiently transfected into hepatocelluar carcinoma HepG2 cells for 48 It, proliferation and the clone formation of HepG2 cells were detected by CCK-8 and clone formation assay respectively. The percentage of hypodiplold cells and early apoptotic cells was detected by flow cytometry. The morphology was examined by fluorescence microscope after Hoechst 33258 staining. Compared with control group and negative control group, PCNA mRNA level was reduced by pShPCNA detected by RT-PCR. The proliferation and clone formation of HepG2 cells treated with pShPCNA for 48 It were significantly inhibited (P < 0.01). The migration was also attenuated when PCNA was knocked down by shRNA, and flow cytometry analysis showed ail increase of the percentage of G0/G1 phase cells, along with a decrease of cell population in the S phase. Percentages of hypodiploid cells and early apoptosis rates were significantly higher in treatment groups than those in control and negative control group (P < 0.01). Mitochondrial membrane potential was reduced in pShPCNA group detected by JC-1 fluorescent staining. Apoptotic morphology such as cell shrinkage, nuclear condensation, nuclear fragmentation, chromatin condensation and apoptotic bodies were also observed by staining with Hoechst33258 under fluorescence microscope. The study indicated the eukaryotic expression vector, PCNA shRNA has been successfully constructed, and effectively inhibits proliferation and induces apoptosis and arrested HepG2 cells in G0/G1 phage.

OBJECTIVE To establish the optimum preparative procedure for ultrasonic extracting bergenin from Rodgersia aesculifolia Batal. METHODS The optimum conditions of extraction process for bergenin from Rodgersia aesculifolia Batal were selected with the extractive yield of bergenin as marks by orthogonal experimental design, investigating the ratio of extracting temperature, solid to liquid, extracting power, extracting time and extracting times. RESULTS The result showed that the most important factors were extracting times, extracting time and solid to liquid. The optimum preparative procedure was 70 ℃ and 60 min for four times. CONCLUSION The process is feasible and useful for extracting bergenin from Rodgersia