The Cholesterol-lowering Effect And Mechanisms Of Columbamine

Hyperlipidemia is one of the major factors that contribute to cardiovascular diseases with heavy harmfulness.Hyperlipidemia is one of of systemic metabolic disorders,which is associated with higher levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-c)and lower levels of high-density lipoprotein cholesterol(HDL-c)in serum.Therefore,controlling the metabolism level of TC is the key to prevent and interfere with the progression of hyperlipidemia and cardiovascular events.Rhizoma Coptidisis a traditional Chinese medicine and has been used among the peoplefor a quite long history.Modern research shows that Rhizoma Coptidis has anti-inflammatory,antibacterial,antihyperglycemic and hypolipidemic and other effects.Columbamine(COL)is one of the main bioactive compounds of Rhizoma Coptidiswith a isoquinoline structure.The aim of this study was to investigate the antihypercholesterolemic activity and potential molecular mechanism of COL in Syrian golden hamsters and HepG2 cells.The following is the main contents and results of the study:(1)We investigated the safety of the COL.According to the accessment on the acute toxicity of Kunming mice and sub-chronic toxicity of SD rats,COL showed a high LD50 value which was about 1524.6 mg/kg and there were no significant differences in organ weight changes,hematological analysis and other clinical indicators in COL-treated animals compared to the control group during the 90-day period.The HepG2 cells with a good status in the exponential phase were used to investigate the cell toxicity ofCOL in different concentrations on HepG2 cells by MTT assay.The results showed that there was no obvious influence on the viability in Hep G2 cellsafter treated with COL within 5 μg/ml,and more than 80% of cells were survived.Therefore,COL is a relatively safe compoundwith a very low toxicity.(2)Preliminarily study on the antihypercholesterolemic activity of COL.Firstly,we used Syrian golden hamsters as experimental model to investigate the effect of COL on lipid profiles.It was found that serum lipid levels including TC,TG,LDL-c were significantly decreased and HDL-c level was distinctly increased in hamsters after administration of COL in hypercholesterolemic hamsters induced by HFHC diet.Moreover,COL at medium and high dosage could enhanced the fecal contents of TBA by 21.8% and 25.1% respectively,compared with HFHC group hamsters.Subsequently,we used Hep G2 cells by Oil Red O staining to research the effect of COL on lipid accumulation in cells.The results showed that COL can reduce the lipid and cholesterol accumulation in HepG2 cells effectively.The result both in vivo and in vitro proved that COL has a good cholesterol-lowering activity.(3)Effects of COL on the key genes involved in the cholesterol metabolism in hamsters and HepG2 cells.The liver homogenate of hamstersand HepG2 cells lysatewere excised for qRT-PCR,western blot and immunofluorescence analysis to test expression of the pivotal genes involved in cholesterol biosynthesis,transport,uptake and efflux pathway,such as HMGCR,LDLR,ABST,APOA4,CYP8B1,CYP7A1 and CYP27A1.The study demonstrated that COL regulated the mRNA and protein expression of those key genes to different degrees.Remarkably,among those key genes,the COL exhibited the notably coincident effect on regulating the expression of CYP7A1 both in hamsters and HepG2 cell,compared with the HFHC group.(4)To further explore how COL upregulatedthe expression of CYP7A1,we detected its transcriptional factors.The half-time of CYP7A1 mRNA was very short,its expression was mainly mediated by transcriptional level,therefore,the transcription factors HNF-4α,FTF,FXR,Prox1,SHP and SREBP2 related to CYP7A1 were investigated by qRT-PCR,western blot and immunofluorescence analysis.Results indicated that COL obviously activated the expression of the transcription factors FTF,HNF-4α,and inhibited those of SHP.The luciferase assay for promoter activity indicated that,after treated with COL,the promoter activity was enhanced to a degree.Especially,in the presence of HNF-4? and FTF,COL significantly increased luciferase activity.Furthermore,the result of LibDock analysis for the interaction between receptor and ligand was match with the previous experiments,suggested that COL promoted CYP7A1 transcription with the action of transcriptional factor HNF-4? and FTF.In conclusion,our results suggested that COL showed high lipid-lowering activities through transactivating CYP7A1 by upregulating FTF and HNF-4?,and activating CYP7A1 catalytic activity by strongly interacting with receptor and ligand,therefore promoting cholesterol catabolism and accelerating the excretion of bile acids.