ABSTRACT
ObjectiveTo study the effect of C3G gene on apoptosis and proliferation of H9C2 cardiomyocytes and its mechanism. MethodsThe RNA lentivirus was constructed, and pCXN2-Flag plasmid (empty plasmid) and pCXN2-Flag-hC3G plasmid (over-expressed human C3G mRNA) were purchased. H9C2 cardiomyocytes were respectively infected and transfected with blank reagent, negative lentivirus, C3G siRNA lentivirus, C3G siRNA lentivirus+pCXN2-Flag plasmid and C3G siRNA lentivirus+pCXN2-Flag-hC3G plasmid with stochastic method. Thus the experiments were randomly divided into five groups, namely blank group, negative control group, C3G silence group, C3G silence+empty plasmid group, and C3G silence+C3G overexpression group. Seventy two hours after plasmid transfection, the expression of C3G mRNA was detected by PT-RCR, cell apoptosis was determined by flow cytometry, cell proliferation rate was determined by MTT, and the protein levels of C3G, p-ERK1/2, Bax and Flag were determined by Western blotting. Results As screened by puromycin, it was found that more than 85% of the cells in negative control group and C3G silence group were labeled with green fluorescent protein, showing that more than 85% of the cells were infected with lentivirus. Compared with blank group and negative control group, the expressed Bax protein and cell apoptosis rate were increased significantly (P<0.01, P<0.05), and the expression levels of C3G mRNA, C3G protein and p-ERK1/2 protein and the cell proliferation rate were remarkably decreased in C3G silence group and C3G silence+empty plasmid group (P<0.01, P<0.05); Compared with C3G silence group and C3G silence+empty plasmid group, the expression level of Bax protein and the cell apoptosis rate were decreased obviously (P<0.01, P<0.05), while the expression levels of C3G mRNA, C3G protein and p-ERK1/2 protein and the cell proliferation rate were remarkably increased in C3G silence+C3G overexpression group (P<0.01, P<0.05). Conclusion C3G gene silence can induce apoptosis and inhibit proliferation in H9C2 cardiomyocytes, and overexpression of C3G gene can reverse the effects of C3G gene silence affecting in H9C2 cardiomyocytes, characterized by a reduction of apoptosis rate and promotion of proliferation, and they may be related to p-ERK1/2 protein and pro-apoptotic molecule Bax.
ABSTRACT
Objective: To investigate the effects of the guanine nucleotide exchange factor C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) overexpression on the survival of cardiomyocytes and the underlying mechanisms. Methods: H9C2 cardiomyocytes were transiently transfected with pCXN2-Flag or PCXN2-Flag-hC3G (overexposing human C3G mRNA) plasmids, and then were exposed to hypoxia/reoxygenation (H/R) treatment. The cardiomyocytes were divided into blank group, empty plasmid group, C3G overexpression group, blank + H/R group, empty plasmid + H/R group, and C3G overexpression+H/R group. The expression of C3G mRNA was detected by RT-PCR, and the expression of C3G and p-ERK1/2 protein was detected by Western blotting analysis. The apoptosis rates of cardiomyocytes were analyzed by flow cytometry in each group, and the cell proliferation rates were analyzed by MTT. Results: C3G overexpression increased cell proliferation rate and expression of C3G mRNA and protein, p-ERKl protein(all P<0. 01), and decreased apoptosis rate (P< 0. 05,P<0. 01) compared to the blank and empty plasmid groups; the same was true for C3G overexpression+H/R group when compared to the blank+H/R and empty plasmid+H/R groups. Expression of p-ERK2 protein in C3G overexpression+ H/R group was increased compared to blank+H/R and empty plasmid+H/R groups (P< 0. 01). Conclusion: C3G overexpression can promote cell survival in the cardiomyocytes, which might be mediated by the increase of p-ERKl/2 protein expression and apoptosis inhibition.
ABSTRACT
We analyzed the pharmacokinetics of C3G on data from twelve subjects, after 2-week multiple dosing of black bean (Phaseolus vulgaris, Cheongjakong-3-ho) seed coat extract, using the mixed effect analysis method (NONMEM, Ver. 6.2), as well as the conventional non-compartmental method. We also examined the safety and tolerability. The PK analysis used plasma concentrations of the C3G on day 1 and 14. There was no observed accumulation of C3G after 2-week multiple dosing of black bean seed coat extract. The typical point estimates of PK were CL (clearance)=3,420 l/h, V (volume)=7,280 L, Ka (absorption constant)=9.94 h(-1), ALAG (lag time)=0.217 h. The black bean seed coat extract was well tolerated and there were no serious adverse events. In this study, we confirmed that a significant amount of C3G was absorbed in human after given the black bean seed coat extract.