Studies On The Gene Expression Of Hypercholesterolemia Mice Treated With Electroacupuncture And Simvastatin

The efficiency of electroacupuncture(EA) has been concerned in treating clinical hypercholesterolemia. In order to provide with molecular evidence to understand the mechanism of acupuncture and the side function of simvastain in treating hypercholesterolemia, identification of differentially expressed genes responding to the hypercholesterolemia have been carried out in hypercholesterolcmic mice liver after acupuncture and simvastatin treatment.In previous study, the mice model of hypercholesterolemia group(HC), normal group(NM), EA treatment group(EA), simvastatin treatment group(ST), EA preventive group(EP), EA non-acupoint group(EN) had been established, and the results of blood biochemical indicators manifested that the treatment of electroacupuncture on fenglong acupoint or simvastatin could markedly reduce the levels of total cholesterol, high density cholesterol and low density cholesterol of plasma. In this study, four groups of NM, HC, EA, and ST mice was chosen for constructing Suppression Subtractive Hybridization(SSH) libraries. Mirror orientation selection(MOS) method was applied to subtracted cDNA populations to reduce the level of false positives before the ligation between vector and cDNA. A total of five independent SSH libraries were constructed and named as ssh-EH and ssh-HE, ssh-SH and ssh-HS, ssh-SN from reciprocal substractive hybridization between EA and HC, EA and ST, NM and ST.Targeted Display(TD), a novel procedure of differentially displaying method was used to select for and amplify truc differentially expressed cDNAs. Total 142 differential displayed fragments were cloned and sequenced. The corresponding genes of 116 were identified by BLAST with the sequenced fragments against GenBank and the mice genome sequence. Forty and thirty-seven genes were up-and down-regulated, respectively, in response to electroacupuncture. However, functions of 52 genes are known and 25 unknown among them. After simvastatin treatment, 16 genes were up-regulated and 23 down-regulated, while among them the functions of 23 genes are known and the others unknown. The differentially expressioned genes were found to be involved in a lot of physiological processes in EA treated mice. The genes related to metabolism(major of lipid metabolism), gene expressed regulation and immunity were differentially expressed after EA stimulation. Simvastatin treatment was mainly relateted to those genes involved in metabolism, signal transduction, immune response, and so on.The number of down-regulated genes after simvastatin treatment was exceeded that of up-regulated. It was suggested that this might be caused by the down-expressed nuclear factor I. At the same time, the number of differentially expressed genes in up-and down-regulation was almost equal after EA treatment, and the genes encoding key enzyme of acyl-Coenzyme A oxidase(Acox) and stearoyl-Coenzyme A desaturase 1(Scd 1) in lipid metabolism were down-regulated, implying that the modulated action to balance body metabolism was contributed to electroacupuncture treatment.The expression of genes encoding adiponectin recptor 2(Adipo R2), lysyl-tRNA synthetase(Krs), tumor necrosis factorα(TNF-α), Utrophin(Utr) was analyzed by real-time RT-PCR in the livers of groups NM, HC, EA, ST, EP and EN. Adipo R2 and Krs genes were lowest expressed in EA group. The genes of TNF-αand Utr were lowest expressed in the groups of EN and ST, respectively. This result was coincidence with that of SSH. It means that decreased expression of Adipo R2, Krs, TNF-αmight be relateted to the action of lipid metabolism by EA treatment, and the down-regulation of Utr gene expression was might be involved in cell damage after simvastatin treatment.To confirm the effect of simvastatin on the changes of gene expression and the state of cell growth, C2C12 was treated in 0(control group), 1, 3, 10, 30 and 100μmol/L of simvastatin. The depressant effect was found dose and time dependent as increase in concentration increased the inhibition. The RNA level of Utr and other three genes compared with control cells were all increased after 24h treatment with different doses of drug. It was suggested that simvastatin coule be able to result in different changes of genes expression between liver and C2C12 cells.This study was the first time using SSH accompanyed by TD to study gene expression of liver in hypercholesterolemic mice. The results demonstrated that EA could modulate gene expression of key enzymes in lipid oxidation, and simvastatin reduced the expression of transductional factor gene to accommodate the gene expression in tissues and C2C12 striated muscle cells.The consequence obtained from EA stimulation and simvastatin treatment might be able to arouse our interest to further study the mechanism of acupuncture on gene level. At the same time, it also help us understand the pleiotropic action of simvastatin.