Studies On The Chinese Electro-acupuncture's Molecular Mechanism Of Treating Mice Hypercholesterolemia By Using Gene Microarray

The molecular mechanism of the effect of electro-acupuncture (EA) induced hypocholesterolemia on the hypercholesterolemia mice induced by hypercholesterolemic diet was studied by using cDNA microarray.Male C57BL/6J mice were first randomly divided into three groups: normal group (NG), hypercholesterolemia group and EA preventive group (EPG). NG mice were fed normal chow, hypercholesterolemia group were fed a hypercholesterolemic diet (HD), EPG mice were fed the same HD and received EA treatment on Fenglong acupoint (ST40). After 2 weeks , the plasma cholesterol levels in hypercholesterolemia mice markedly raised to 6.87 ± 0.36 mmol/L, which was more than two-fold high in the NG mice. It indicated that the hypercholesterolemia model was successfully constructed. The mice of hypercholesterolemia group were then randomly divided into model group (MG), EA treatment group (ETG), EA non-acupoint group (ENG) and drug control group (DG). Mice of ETG received EA treatment, ENG received EA on non-acupoint, DG received simvastatin treatment, EPG received continuous EA treatment. During the treatments, the mice were continuously fed HD. NG mice were still fed normal chow, and received no treatment. After 16 days, all mice were killed, plasma was obtained for biochemicalanalysis, liver were excised for the analysis of histology, lipid content, cDNA microarray and real-time quantitative RT-PCR.The result from 12 items of plasma biochemical analysis indicated that plasma total triglyceride (TG) level had no statistically significance, and there were 8 items with no significant difference among 6 groups. However, the levels of total cholesterol (TC), high density cholesterol , and low density cholesterol of plasma, TC and TG of liver in MG mice were significantly elevated compared with those of NG, and those of EPG, ETG and DG were decreased when compared with MG mice. The hepatic steatosis of MG mice was severe, and that of EPG, ETG and DG was ameliorated. It indicated that EA at the Fenglong acupoint had the same hypocholesterolemic effect as that of simvastatin. All above-mentioned levels of ENG mice were not significantly different from those of MG. The results indicated that acupoint is important and necessary in the hypocholesterolemic treatment.Although EA or simvastatin had significant hypocholesterolemia effect, the above-mentioned levels of EPG, ETG and DG were not restored to the normal level. The microarray analysis of the expression of 16,463 murine genes was studied in the 6 groups' mice. The most number of expressed gene detected was 5,766(35%), the least number was 3,104 (19%) . The numbers of significantly expressed genes were different in various combinations of hybridizations as follows: NG/MG, 98 genes; EPG/MG, 101 genes; ENG/MG, 63 genes; ETG/MG, 29 genes; DG/MG, 27 genes. The function of the significantly expressed genes were associated with metabolism(including cholesterol metabolism), response to stress, immunity, signal transduction, transcription regulation, transport, cell apoptosis, cell cycle, development, cell organization, cell differentiation, cell adhesion.The result of clusters analysis of microarray data indicated that the various combinations of hybridization were clustered into different branch in the denbrogram: ENG/MG, a single branch; EPG/MG and DG/MG, one branch; EPG/MG and NG/MG, one branch.The result of the function analysis of differentially expressed genes in the EPG and ETG indicated that EA not only affected the gene expression of cholesterol metabolism, but also that of signal transduction, cell apoptosis and response to stress.4 differentially expressed genes in the cDNA microarray and 10 other genes associated with the liver cholesterol metabolism were analyzed by using real-time quantitative RT-PCR in their expression level. The result not only confirmed the reliability of microarray data, but also indicated that the molecular mechanism of hypocholesterolemia effect of EA was different from that of simvastatin. EA effect was associated with higher expression levels for LXRcc and genes encoding bile acid synthetic enzymes. DG treatment had the same lower plasma cholesterol levels as EPG, but it was associated with the lower FXR mRNA level and higher expression levels for genes encoding membrane-associated bile acid transporters.This work is the first time to study the molecular mechanism of the effect of EA-induced hypocholesterolemia. The results in the present study provide more clues on not only how hypocholesterolemia is induced by EA, but also the exploration of target gene of treating hypercholesterolemia.