ABSTRACT
Using the concepts and methods of epigenetics and metabolomics, to investigate the overall action molecular mechanism of Chrysanthemi indici C (CIC), the anti-hepatitis B virus (HBV) active extracts from Flos chrysanthemi indici. The inhibitory effects of CIC on proliferation and hepatitis B surface antigen (HBsAg), hepatitis B envelope antigen (HBeAg) and HBV-DNA of HepG2.2.15 cells were detected by CCK-8 and antigen kit. The DNA methyltransferases (DNMTs)/ten-eleven-translocation-2 (TET2) equilibrium was detected by ELISA. Illumina 850K methylation chip, pyrosequencing and qPCR were used to determine the action pathway and target of CIC by GO and KEGG analysis. Cell metabolites were extracted with 80% methanol, and the changes of differential metabolites, differential metabolic pathways and cell microenvironment were detected by LC-MS and other metabolomics methods. The results showed that CIC could inhibit the proliferation, HBsAg, HBeAg and HBV-DNA of HepG2.2.15 cells obviously, down-regulate DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a) and DNA methyltransferase 3b (DNMT3b), up-regulate TET2, and restore the balance of DNMTs/TET2. The action targets of CIC were phospholipase C gamma 2 (PLCG2), phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), 5-hydroxytryptamine receptor 2B (HTR2B), nerve growth factor (NGF), mainly involved in lipid metabolism, inflammation mediated regulation of transient receptor potential (TRP), phospholipase D signaling and advanced glycation end product-receptor for AGE (AGE-RAGE) signaling in diabetic complications pathways. CIC could significantly affect fatty acid metabolism and had great influence on phenolic acid, alkaloid and lipid metabolites in cell microenvironment. These results suggest that the action mechanism of CIC may be the synergistic action of multiple pathways and multiple targets, including related inflammatory pathways, immune pathways and lipid metabolism, through regulating epigenetic expression balance and restoring the balance of cell microenvironment.
ABSTRACT
Objective To investigate the effects of extractum trametes robiniphila murr combinated with hydroxyurea on proliferation and apoptosis of K562 cells and whether the 2 drugs have synergistic effects,and to detect the antiproliferation and apoptosis ratio of cells in diffe-rent groups were detected.Methods K562 cells were cultured in vitro,and logarithmic phase cells were used for study.MTT-inhibitory test and cell morphological analysis were employed to examine the effects of extractum trametes robiniphila murr alone or in combination with hydroxyurea on proliferation of K562 cells.The effects of extractum trametes robiniphila murr alone or in combinated with hydroxyurea on apoptosis of K562 cells were examined by flow cytometry.The expressions of bcr-abl,bax and bcl-xl mRNA levels were detected by way of reverse transcription polymerase chain reaction(RT-PCR).Results Extractum trametes robiniphila murr could inhibit proliferation and induce apoptosis of K562 cells,and extractum trametes robiniphila murr combinated with hydroxyurea had a synergistic effects on cell prolife-ration and apoptosis.The RT-PCR displayed that either extractum trametes robiniphila murr alone or in combinated with hydroxyurea could down-regulate the expression of bcl-xl mRNA and up-regulate the expression of bax mRNA and the effect of the 2 drugs had a synergistic effect on bcl-xl and bax mRNA expressions.Extractum trametes robiniphila murr alone could also down-regulate the expression of bcr-abl mRNA,but hydroxyurea alone did not show any effect on the expression of bcr-abl mRNA and the 2 drugs had no synergistic effect.Conclusions The effect of extractum trametes robiniphila murr combinated with hydroxyurea on the proliferation and apoptosis of K562 cells has a synergistic effect.The mechanism of antiproliferation and inducing apoptosis of extractum trametes robiniphila murr is probably related to down-regulating the expression of bcl-xl and bcr-abl mRNA and up-regulating the expression of bax mRNA.The synergistic effect of antiproliferation and inducing apoptosis of extractum trametes robiniphila murr combinated with hydroxyurea on K562 cells is probably related to down-regulating the expression of bcl-xl mRNA and up-regulating the expression of bax mRNA.