Hypolipidemic Activity And Mechanisms Of The Ligustrum Robustum(Roxb.) Blume

With the rapid development of economy and people's lifestyle changing,cardiovascular and cerebrovascular disease has become the number one of health killers.Therefore,it is very important to explore natural products to reduced lipids.Ligustrum robustum(Roxb.)Blume is one of the main origin plants of Kudingcha in the southwest of China.It is widely used as the tea for hypertension,hyperlipoidemia and obese people,with a long history of drinking in China,whereas its mechanism of effect is not clear.Therefore,it is vital to carry out a systematical pharmacodynamics evaluation on the hypolipidemic effect and a study on the relevant mechanism.In this study,first of all,we evaluated the hypolipidemic effect of the total phenylpropanoid glycosides of Ligustrum robustum(Roxb.)Blume(CNTG)by in vivo experiment and preliminaryly studied the relevant mechanism.The Syrian hamsters were randomly divided into the control group,the model group,the positive control group(fenofibrate),and CNTG high,middle and low dose groups.To administrate drug for 4 weeks,and detect serum lipid levels at 2 weeks,3 weeks and 4 weeks.After the experiment,take the liver to determine lipid content and take pathological examination.The expression relevant gene mRNA in the liver was detected by real-time quantitative PCR;the relevant protein expression level were detected by Western blot.The results showed that the levels of serum TC,TG and LDL-C of CNTG high dose group were significantly lower than those in model group.Real-time quantitative PCR results showed that CNTG significantly increased the relative expression of AMPK(P<0.01),but the relative expression of CD36,ACC and CPT1 did not change significantly in contrast with the model group.Western blot results showed that the relative expression of LKB1,phospho-LKB1 and phospho-AMPK in the liver of the high-dose group was significantly higher than that in the model group(P<0.05)and the difference was significant.In addition,the insulin resistance model and the lipid accumulation model were established to further evaluate the activity of improving insulin resistance and lipid accumulation of different fractions of Ligustrum robustum(Roxb.)Blume's ethanol extract and its active ingredients.The insulin resistance HepG2 cell model was induced by Hyperglycemia,and glucose consumption was detected on the insulin resistant HepG2 cells.Liposomal accumulation of HepG2 cells was induced by oleic acid and the effect of samples on lipid accumulation of HepG2 cells was detected by Oil Red O Method for Lipofuchsin.The results showed that there was no significant effect on the cell proliferation in the range of effective concentration,and had no significant effect on the glucose consumption of HepG2 cells.Compared with the model group,the total extract,ethyl acetate,n-butanol and water fractions could increase the glucose consumption and improve the lipid accumulation in HepG2 cells with varying concentration except the petoleum ether fraction.Liguopurpuroside A,C,D and actoside can significantly improve lipid accumulation of HepG2 cells.Therefore,the Ligustrum robustum(Roxb.)Blume can improve HepG2 cell insulin resistance and lipid accumulation.To further explore the bioactivity and the target of those phenylpropanoid glycosides,the virtual screening and RNA-seq experiments were designed in this paper.In the virtual screening experiment,9 compounds were found to catch 15 targets.These selected targets were related with hypercholesterolemia,Alzheimer's disease and tumor and so on.To further explore the effect of phenylethano glycosides on the metabolism of glycolipids and the preliminary mechanism,the RNA-seq were performed.The results showed that there were a large number of differentially expressed genes between Control vs Model and AKG vs Model group.Further analysis of these differential genes using KEGG and GO showed that in the AKG group a large number of pathways related to lipid metabolism:metabolic pathway,glycerol phospholipid metabolism,fatty acid degradation,etc.In addition,there are also other differential expression gene in AKG group compared to Model.9 genes were selected to be verified by qPCR,including SCARB1,SCARB2,ACAT2,DHCR7,FDFT1,LSS,HMGCR,SREBF1 and CPT1A.The results of real-time PCR were consistent with the results of RNA-seq sequencing,indicating that RNA-seq sequencing results were credible.AKG may affect the expression of SCARB1,SCARB2,ACAT2,DHCR7,FDFT1,LSS,SREBF1 and HMGCR,but has no effect on CPT1A.These research provides reliable data for the studies of Hypolipidemic mechanism of phenylethanosine compounds.