Anti-hepatocarcinoma HepG2 Cell Mechanism of Jaranol： Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo study the in vitro anti-hepatocarcinoma HepG2 cell mechanism of Jaranol. MethodThe methyl thiazolyl tetrazolium （MTT） assay was employed to examine the inhibition of Jaranol （0， 5， 10， 25， 50， 100， 150， 300 μmol·L-1） on HepG2 cell proliferation at different time （24 ， 48 ， 72 h）， annexin V-fluorescein isothiocyante/propidium iodide （Annexin V-FITC/PI） kit to detect the effect of Jaranol （0， 3， 15， 75 μmol·L-1） on HepG2 cell apoptosis， and Western blot to determine the influence of Jaranol on the expression of B-cell lymphoma 2 （Bcl-2） and Bcl-2-associated X protein （Bax） in HepG2 cells. Transcriptome sequencing was performed to analyze the differential expression of genes and changes of related signaling pathways after the treatment of HepG2 cells with Jaranol （15 μmol·L-1）. Real-time PCR was carried out to verify the relative mRNA content of differential genes ［TEK， platelet-derived growth factor receptor α （PDGFRA）， spleen tyrosine kinase （SYK）， phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit gamma （PIK3CG）， Janus kinase 3 （JAK3）， membrane-associated guanylate kinase inverted 2 （MAGI2）］. ResultCompared with the blank group， Jaranol decreased HepG2 proliferation （P<0.05， P<0.01）， increased apoptosis rate of HepG2 cells （P<0.05， P<0.01）， raised Bax expression （P<0.05， P<0.01）， and reduced Bcl-2 expression （P<0.05， P<0.01）. Transcriptome sequencing yielded 59 000 regulated genes， 125 of which showed significantly different expression， with 47 up-regulated and 74 down-regulated. Kyoto Encyclopedia of Genes and Genomes （KEGG） analysis showed that the differential genes related to apoptosis in the phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway changed significantly after drug addition. The mRNA expression of TEK， PDGFRA， SYK， PIK3CG， JAK3， and MAGI2 decreased in Jaranol （15 μmol·L-1） group compared with that in the control group （P<0.05）. ConclusionIn vitro cytological experiment verified that Jaranol inhibited the proliferation of HepG2 cells and promoted the apoptosis， possibly by influencing the expression of some differential genes in the PI3K/Akt signaling pathway. The result lays an experimental basis for the follow-up study of the anti-tumor effect of Jaranol， and the further development and utilization of flavonoids.

Research on autophagy induced by two xanthone compounds in HepG2 cells / 中国中药杂志

To investigate the inhibitory effects of two xanthone compounds, 1-hydroxy-2,3,4,8-4 methoxy xanthone(here in after referred to as Fr15) and 1-hydroxy-2,3,4,6-4 methoxy xanthone(here in after referred to as Fr17), on the proliferation of hepatocellular carcinoma cells HepG2, and to further investigate their mechanism in combination with transcriptomics. Cell counting was used to detect the effects of two kinds of xanthone compounds Fr15 and Fr17(0, 0.03, 0.15, 0.3 mmoL·L~(-1)) on the proliferation of HepG2 cells; the effects of the two compounds Fr15 and Fr17 on HepG2 cell cycle were detected by flow cytometry; the changes of autophagosomes count in cells were observed under fluorescence microscope; the expression of autophagy marker proteins autophagy marker proteins SQSTM 1(p62) and microtubule associated protein 1 light chain 3 Ⅰ/Ⅱ(LC3 Ⅰ/Ⅱ) in the cells was detected by Western blot; the differentially expressed genes between the control group and the experimental group were analyzed by RNA-seq transcriptome sequencing; qRT-PCR was used to verify the differentially expressed genes in sequencing. The results showed that compounds Fr15 and Fr17 inhibited the proliferation of HepG2 cells with the increase of drug concentration and time. Flow cytometry showed that compounds Fr15 and Fr17 had little effect on HepG2 cell cycle. Fluorescence microscopy results showed that the number of autophagosomes in cells increased with the increase of drug concentration. Western blot showed that the expression of p62 protein was decreased and the expression of LC3-Ⅱ protein was significantly increased after drug addition. The results of RNA sequencing showed that 26 102 and 52 351 differentially expressed genes were obtained in Fr15 and Fr17 respectively. Analysis of KEGG showed that drug treatment had a great effect on autophagy pathway. qRT-PCR verified that 6 up-regulated genes were related to autophagy, and their trend was consis-tent with sequencing results, where all 6 genes showed an up-regulated trend. Two xanthone compounds Fr15 and Fr17 may inhibit proliferation of HepG2 cells by inducing autophagy.

Triptolide, A Potential Autophagy Modulator / 中国结合医学杂志

As a major active component extracted from traditional Chinese herb Tripterygium wilfordii Hook F, triptolide exhibits multiple pharmacological effects. Autophagy is an evolutionary conserved intracellular catabolic process involved in cytoplasmic materials degradation. Autophagic dysfunction contributes to the pathologies of many human diseases, which makes it a promising therapeutic target. Recent studies have shown that triptolide exerts neuroprotection, anti-tumor activities, organ toxicity, and podocyte protection by modulating autophagy. This article highlights the current information on triptolide-modulated autophagy, analyzes the possible pathways involved, and describes the crosstalk between autophagy and apoptosis modulated by triptolide, in hope of providing implications for the roles of autophagy in pharmacological effects of triptolide and expanding its novel usage as an autophagy modulator.

Purification and biochemical function of Astragalus membtanaceus pathogenesis-related protein 10 / 中国中药杂志

Astragalus membranaceus pathogenesis-related protein 10 (AmPR-10) is largely expressed in case of environmental pressure and pathogen invasion. This study aims to explore the biochemical functions of AmPR-10. The dried root of Astragalus membranaceus was mechanically homogenized and extracted by Tris-HCl buffer to obtain its crude extract, which was then purified by anion exchange chromatography and gel filtration chromatography to obtain electrophoretically pure AmPR-10. The nuclease activity of AmPR-10 was tested with different RNAs by detecting the absorption value at 260 nm. The results demonstrated potent nuclease activity toward yeast tRNA, yeast RNA, Poly (A) and Poly (C). The optimum reaction temperature was 50 °C and pH was 7-8. EDTA showed no effect on its activity, while Mg²⁺ exhibited potent activation effect on the activity, and Co²⁺, Ca²⁺ and Zn²⁺ manifested moderately inhibition of the activity. Since AmPR-10 had no sequence homology with other known nucleases, AmPR-10 was probably a novel nuclease. The inhibition kinetic data against papain was analyzed by Lineweaver-Burk plots, and the results showed that the inhibition of papain followed noncompetitive-type kinetics. AmPR-10 played an important role in Astragalus membranaceus defense mechanism against environmental pressure and pathogen invasion, which may be achieved by inhibiting cycteine enzymes activity.

Research on purification and activity characterization of AmPR-10 ribonuclease from Astragalus membranaceus / 中草药

Objective To establish a stable and rapid separation and purification method for Astragalus membranaceus (Am) pathogenesis-related protein-10 (AmPR-10) using an automatic intelligent protein purification system AKTA Avant 25, and analyze its physiochemical and biological activity. Methods Am was extracted by Tris-HCl buffer. The crude extract was captured by anion exchange chromatography, and finely separated by hydrophobic chromatography and gel filtration chromatography. The relative molecular weight of AmPR-10 was measured by MALDI-TOF/TOF mass spectrometry, the protein identification was determined by mass spectrometry and MS/MS Ion Search, the glycoprotein identification was estimated by periodic acid-Schiff method, and the ribonuclease activity and effect factors were analyzed by agarose gel electrophoresis. Results The electrophoretically pure AmPR-10 was obtained by three-step purification of Q Sepharose Fast Flow, Butyl Sepharose High Performance and SuperdexTM 75 10/300 GL from the crude extraction. The relative molecular weight of AmPR-10 was 16 801. AmPR-10 was highly homologous to PR-10 and has no carbohydrate chains. Incubated at 56 ℃ for 30 min, AmPR-10 exhibited significant ribonuclease activity to total RNA of mammalian cells. The activity was insensitive to NaCl, pH value and mental ions, and weekly inhibited by 0.5 mol/L NaCl, pH 9.0, Mg2+ and Co2+. The activity was the same at EDTA as high as 20 mmol/L. Conclusion The three-step method of exchange chromatography-hydrophobic chromatography-gel filtration chromatography, a stable and rapid separation and purification method of AmPR-10, can be applied for other Chinese herbs. AmPR-10 might play an important role in resistance against virus.

Resistance reversal effect of a novel taxane compound NPB304 and its collaboration with verapamil / 药学学报

The tumor multidrug resistance reversal effect of NPB304, a novel taxane, was studied. MTT assay was used to determine the IC50 of chemotherapy drugs. Western blotting assay was applied to analyze the expression of P-glycoprotein (P-gp). The effect of compounds on the P-gp function and P-gp ATPase activity was determined by rhodamine 123 (Rh123) accumulation assay and analysis kit, respectively. Molecular docking was employed to predict the binding force between compounds and P-gp. Transmembrane transport of NPB304 was analyzed using MDCK II and MDR1-MDCK II cell model. NPB304 displayed multidrug resistance reversal effect on KBV cells and MCF-7/paclitaxel cells, NPB304 collaborative with P-glycoprotein (P-gp) inhibitors verapamil enhanced the reversal activity, specifically, 10 μmol x L(-1) verapamil in combination with paclitaxel reversed resistance by 56.5-fold, while combined with NPB304 increased the reversal fold; NPB304 synergistically increased Rh123 accumulation in the resistant cells when combined with verapamil, and NPB304 at 0-1 μmol x L(-1) enhanced the ATPase activity activated by verapamil was observed. NPB304 existed the hydrophobic interactions with the TM regions of P-gp, and the binding force between NPB304 and the A chain of the TM region was stronger. P-gp ATPase activity assay demonstrated NPB304 at lower concentrations (0-1.5 μmol x L(-1)) could activate the P-gp ATPase, playing a role on inhibition of P-gp function. However, NPB304 did not have an obvious feature of P-gp substrate. NPB304 exerted itself and synergy with verapamil activity on reversing tumor resistance via inhibiting the P-gp function.