Promotion of HepG2 cell apoptosis by Sedum emarginatum Migo and the mechanism of action.

BACKGROUND: Sedum emarginatum Migo(S. emarginatum) has anti-tumor and anti-oxidant effects. This study aimed to screen the extractions of S. emarginatum against liver cancer in vitro and explore its anti-liver cancer mechanism. METHODS: The CCK-8(Cell Counting Kit-8) method was used to detect the inhibitory effect of different extracts of S. emarginatum on the proliferation of liver cancer HepG2 cells. The morphological changes of the cells after administration were observed with microscopy, cell apoptosis was detected by flow cytometry, and the expression of Bax, Bcl-2 and Caspase-3 mRNA in the cells were detected by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) to explore the mechanism of action. RESULTS: CCK-8 method test results showed that among the different extracts of S. emarginatum, the ethyl acetate extract(1000 µg/ml, 2000 µg/ml, 2500 µg/ml, 3000 µg/ml) and n-butanol extract(1000 µg/ml, 2000 µg/ml, 2500 µg/ml, 3000 µg/ml) have the strongest inhibitory effect on the proliferation of HepG2 cells. In these 4 concentrations, the inhibitory effect increased as the concentration increased. The IC50 of the ethyl acetate extract on HepG2 cells was less than that of the n-butanol extract, so the ethyl acetate extract has a better proliferation inhibitory effect on HepG2 cells than the n-butanol extract, followed by the 70% ethanol extract(3000 µg/ml) and the water extract(3000 µg/ml), petroleum ether extract was the weakest. The results of microscopy showed that ethyl acetate extract caused hepatocarcinoma HepG2 cell morphology changed, cell density decreased, and suspension cells increased. Moreover, the results of flow cytometry showed that the ethyl acetate extract of S. emarginatum could induce HepG2 cell apoptosis at the concentrations of 2500µg/ml and 3000µg/ml. RT-PCR results showed that the expression of Bax mRNA was up-regulate by the middle(2500 µg/ml) and high(3000 µg/ml) dose groups of ethyl acetate extract. The expression of Caspase-3 mRNA was up-regulated by the low(2000 µg/ml), medium(2500 µg/ml) and high(3000 µg/ml) dose groups of ethyl acetate extract. The expression of Bcl-2 mRNA was down-regulated by the high(3000 µg/ml) dose group of ethyl acetate extract. CONCLUSION: The ethyl acetate extract of S. emarginatum has the best effect on human liver cancer HepG2 cells. Its anti-hepatocellular mechanism may be related to affect the expression of apoptosis genes (Bax, Bcl-2 and Caspase-3mRNA) and promote the apoptosis of liver cancer cells. It provided a reference for the research and development of drugs for the treatment of liver cancer.

The effect of 8-OH-DPAT and dapoxetine on gene expression in the brain of male rats during ejaculation.

The 5-HT1A receptor agonist 8-hydroxy-2-[di-n-propylamino] tetralin (8-OH-DPAT) promotes ejaculation of male rats, whereas dapoxetine delays this process. However, the gene expression profile of the brain at ejaculation following administrationof these two compounds has not been fully elucidated. In the present study, a transcriptomic BodyMap was generated by conducting mRNA-Seq on brain samples of male Sprague-Dawley rats. The study included four groups: pre-copulatory control (CK) group, ejaculation (EJ) group, 0.5 mg/kg 8-OH-DPAT-ejaculation group (DPAT), and 60 mg/kg dapoxetine-ejaculation (DAP) group. The resulting analysis generated an average of approximately 47 million sequence reads. Significant differences in the gene expression profiles of the aforementioned groups were observed in the EJ (257 genes), DPAT (349 genes) and the DAP (207 genes) compared with the control rats. The results indicate that the expression of Drd1 and Slc6a3 was significantly different after treatment with 8-OH-DPAT, whereas the expression of Drd4 was significantly different after treatment with dapoxetine. Other genes, such as Wnt9b, Cdkn1a and Fosb, exhibited significant differences in expression after the two treatments and are related to bladder cancer, renal cell carcinoma and sexual addiction. The present study reveals the basic pattern of gene expression that was activated at ejaculation in the presence of 8-OH-DPAT or dapoxetine, providing preliminary gene expression information during rat ejaculation.

Profiles of brain central nervous system gene expression associated with ejaculation behavior in male rats.

The male rat has been used extensively as a model for evaluating the neurophysiology of sexual behavior. However, gene expression in the brain throughout the process of sexual intercourse has yet to be elucidated. In the present study, we created a transcriptomic BodyMap by performing mRNA-Seq on brain samples from pre-copulatory control (CK), fourth intromission (CR4), ejaculation (EJ) and post-ejaculatory interval 1-min (PEI1) Sprague Dawley rats (n=40, all male, each 10). The resulting analysis generated an average of approximately 47 million sequence reads, indicating changes in roughly 21,255 genes for each sample. Among of them, significant differences in gene expression relative to control rats were observed in the CR4 (139 genes), EJ (257 genes), and PEI1 (130 genes) groups. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis identified 22 pathway-related genes. We further identified eight important genes related to neural pathways using RT-qPCR and Western blot, ruling out the possibility of false positives. The results of the present study not only revealed the basic pattern of gene expression during male rate sexual activity but also provide preliminary data and methodology for further research regarding animal sexual activity.

Liquid chromatography with tandem mass spectrometry method for the simultaneous determination of multiple sweet mogrosides in the fruits of Siraitia grosvenorii and its marketed sweeteners.

A high-performance liquid chromatography with electrospray ionization tandem mass spectrometry method has been developed and validated for the simultaneous quantification of eight major sweet mogrosides in different batches of the fruits of Siraitia grosvenorii and its marketed sweeteners. The sample preparation procedure and chromatographic and mass spectrographic conditions were optimized. Chromatographic separation was achieved on a reversed-phase Poroshell 120 SB C18 column in 10.0 min with gradient elution with acetonitrile and water, both of which contained 0.1% formic acid. The multiple reaction monitoring scanning mode was employed for quantification in the negative ion mode. The developed method was validated with acceptable linearity (r2 = 0.9984-0.9998) over a wide concentration range, precision (relative standard deviations = 1.09-3.91%), stability (relative standard deviations = 1.21-3.01%), and recovery in the range of 91.22-106.58% (relative standard deviations ≤ 3.79%) under optimum conditions. The proposed method was demonstrated to be simple, rapid, specific, and reliable and was successfully applied for the quality control of the fruits of S. grosvenorii and its marketed sweeteners.