| PurposeTo investigate the effect of the method of activating spleen-energy and dissolving turbidity on the expression of microRNAs and target genes in hyperlipidemia rats.Method1.84 male Wistar rats were randomly divided into for the normal group (n= 10) and high fat group (n= 74), and normal control group rats were fed with normal diet, high-fat group were given high fat diet. After 4 weeks,serum TC, TG, HDL-C and LDL-C were measured to evaluate the establishment of hyperlipidemia rats model. Tnen, using Huazhuo Tiaozhi Decoction(HZTZD) to intervene for 8 weeks, as well as using pravastatin sodium tablets for western medicine and Xuezhikang Capsule for Chinese medicine control.2. Extracting miRNAs in liver tissues of rats, differentially expressed miRNAs between two samples were filtered through Fold change(Fold-change≥2.0 when up-regulated, Fold-change≤0.5 when down-regulated). Then, using bioinformatics database to detected differentially expressed miRNAs, and selected miRNAs and target genes which were closely related to the lipid metabolism.3. Real time fluorescent quantitative PCR method to detect the expression of miRNAs.Result1.miRNA expression profileScanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction, screening a total of 700 miRNA information.2.differentially expressed miRNAs① in HFM:dyslipidemia can change the expression of miRNAs in rat liver, compared with the normal group,10 miRNAs were up regulated,42 miRNAs were down regulated. The most obviously increased miRNA was rno-miR-342-5p, the most obviously down-regulated was rno-miR-10a-3p.② in WMCM:Pravastatin Sodium Tablets can change the expression of miRNAs in rat liver, compared with the HFM,71 miRNAs were up regulated,35 miRNAs were down regulated. The most obviously increased miRNA was rno-miR-10a-3p, rno-miR-214-5p was up regulated too, and the most obviously down-regulated was rno-miR-9a-5p, rno-miR-155-5p、rno-miR-148b-3p were down regulated either.③ in TCMCM:Xuezhikang Capsule can change the expression of miRNAs in rat liver, compared with the HFM,93 miRNAs were up regulated,76 miRNAs were down regulated. The most obviously increased miRNA was rno-miR-10a-3p, rno-miR-214-5p was up regulated too, and the most obviously down-regulated was rno-miR-184, rno-miR-155-5p、 rno-miR-122-5p were down regulated either.④ in TCMGM1:HZTZD NO.1 can change the expression of miRNAs in rat liver, compared with the HFM,53 miRNAs were up regulated,24 miRNAs were down regulated. The most obviously increased miRNA was rno-miR-10a-3p, rno-miR-214-5p、 rno-miR-30b-5p were up regulated too, and the most obviously down-regulated was rno-miR-7a-1-3p, rno-miR-122-5p wad down regulated either. Compared with the WMCM, 42 miRNAs were up regulated, and 69 miRNAs were down regulated. The most obviously increased miRNA rno-miR-9a-5p, no-miR-27b-3p、rno-miR-148b-3p、rno-miR-30b-5p were up regulated too, and the most obviously down-regulated was rno-miR-471-3p, rno-miR-122-5p wad down regulated either. Compared with the TCMCM,82 miRNAs were up regulated, and 159 miRNAs were down regulated. The most obviously increased miRNA rno-miR-880-3p, rno-miR-148b-3p、rno-miR-155-5p、rno-miR-30b-5p were up regulated too, and the most obviously down-regulated was rno-miR-193a-5p,rno-miR-33-3p was down regulated either.⑤ in TCMGM2:HZTZD NO.2 can change the expression of miRNAs in rat liver, compared with the HFM,99 miRNAs were up regulated,63 miRNAs were down regulated. The most obviously increased miRNA was rno-miR-10a-3p, rno-miR-214-5p、 rno-miR-30b-5p were up regulated too, and the most obviously down-regulated was rno-miR-184, miR-155-5p was down regulated either. Compared with the WMCM,50 miRNAs were up regulated, and 29 miRNAs were down regulated. The most obviously increased miRNA rno-miR-122-3p, rno-miR-30b-5p was up regulated too, and the most obviously down-regulated was rno-miR-lb, rno-miR-155-5p was down regulated either. Compared with the TCMCM,54 miRNAs were up regulated, and 45 miRNAs were down regulated. The most obviously increased miRNA rno-miR-144-3p, rno-miR-30b-5p was up regulated too, and the most obviously down-regulated was rno-miR-193a-5p, rno-miR-155-5p was down regulated either.3.MiRNA target gene prediction① HZTZD NO.1:put rno-miR-411-3p、rno-miR-329-3p、rno-miR-877、rno-miR-345-5p、 rno-miR-30b-5p、rno-miR-20b-5p、rno-miR-122-5p、rno-miR-214-5p into database, and a total of 777 target genes related to miRNA were detected.② ZTZD NO.2:put rno-miR-10a-3p、rno-miR-147、rno-miR-142-3p。rno-miR-300-5p、 rno-miR-125b-5p、rno-miR-324-3p、rno-miR-199a-3p、rno-miR-340-5p、rno-miR-17-5p、 rno-miR-339-5p、rno-miR-148b-3p、rno-miR-184 etc. into database, and a total of 1109 target genes related to miRNA were detected.③ Comprehensive prediction results, then made GO/KEGG pathway analysis, and PPARD, ABCG1 and LDLR were selected as 3 target genes which were closely related with lipid metabolism.4.PCR results① rno-miR-155-5p:compared with HFM, the expression of each group was down regulated, and the difference was not statistically significant (P>0.05);② rno-miR-10a-3p:compared with HFM, the expression of each group was down regulated, and the difference was not statistically significant (P>0.05);③ rno-miR-214-5p:compared with the HFM, the expression of each group was down regulated, P=0.013, the difference was statistically significant. Among them, compared with the HFM, the expression of the HZTZD NO.1 and NO.2 were markedly down regulated, the difference was statistically significant (P=0.008,0.014);④ no-miR-148b-3p:compared with the HFM, the expression of each group was down regulated, the difference was not statistically significant (P>0.05);⑤ rno-miR-33-3p:compared with the HFM, the expression of each group was down regulated, the difference of no statistical significance (P> 0.05); but compared with the HFM, the expression of HZTZD No.2 was decreased significantly, P=0.028, the difference was statistically significant;⑥ rno-miR-33-5p:compared with the HFM, the expression of each group was down regulated, the difference of no statistical significance (P>0.05);⑦ rno-miR-122-5p:compared with HFM, the expression of each group was down regulated, P= 0.048, difference has statistical significance; the expression of HZTZD No.1 and No.2 was decreased significantly, the difference is statistically significant (P= 0.039, 0.006);⑧ rno-miR-30b-5p:compared with HFM, the expression of each group was up-regulated, the difference of no statistical significance (P> 0.05).Conclusion1. Dyslipidemia can affect the expression of microRNAs in liver tissue of rats;2. The method of activating spleen-energy and dissolving turbidity can affect microRNAs expression in liver tissue of hyperlipidemia rats;3. The possible mechanism of regulating the metabolism in the method of activating spleen-energy and dissolving turbidity was inhibiting the expression of rno-miR-122-5p and rno-miR-33-3p. |
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