| Background：ApoE is an important component of plasma lipoproteins which regulates theplasma lipid level in the body by binding to LDL receptor. Clinical studies havereported that the ApoE gene polymorphism had a significant effect on the efficacy ofstatins in lipid regulating, and other studies suggested that differences in lipidregulating effect of atorvastatin may not related with the ApoE gene polymorphism.Further evidence is needed to judge which is true. It should be noted that the bindingof ApoE to LDL receptor is involved in lipid metabolism, and regulation of bloodlipids by HMG-CoA reductase which is the rate-limiting enzyme of cholesterolsynthesis can be inhibited by statins. The association between the effects of these twocompletely different pathways attracts more concerns. Therefore, we try to explorethe exact role of ApoE gene polymorphism in lipid-lowering effect of statins and itsmolecular mechanism taking ApoE gene polymorphism as a starting point and usingcellular and molecular biological methods, which will provide new ideas for the studyof lipids regulating effect of statins.Objective:In this study, action site of ApoE lipid-lowering effect was analyzed through thecomparison of competitive binding to LDL between different ApoE genetic subtypes.The expression of HMG-CoA reductase mRNA was tested by RT-PCR to compare theeffect of different genetic subtypes of ApoE on the inhibition of HMG-CoA reductaseby fluvastatin and Danshensu or both. Moreover, the exact action site of ApoE genepolymorphism affecting HMG-CoA reductase and its molecular mechanism wasexplored.Method:1. ApoE competitive binding assay: the optimum conditions such as the bindingtime of LDL with LDL receptor, LDL concentration were explored systematically byestablishing a HSF cell model. ApoE-DMPC complexes of different genetic subtypeswere prepared. ELISA method was used to determine the replacement amount of LDL in the competitive binding of ApoE-DMPC complexes of different genetic subtypeswith LDL.2. HMG-CoA reductase mRNA expression determination: the optimumconditions such as cell culture serum, drug action time and ApoE concentration wereexplored systematically by establishing an L-02cell model. HMG-CoA reductasemRNA expression in L-02cells was determined by RT-PCR, expression quantity wasindicated by gray value.3. In ApoE competitive receptor binding assay, the difference in the competitivebinding activity of ApoE-DMPC complexes of3ApoE isoforms (ApoE2(Arg at112→Cys),ApoE3andApoE4(158Cys→Arg)) with LDL was observed.4. HMG-CoA reductase mRNA expression was determined by RT-PCR toinvestigate the effect of3isoforms of ApoE2ApoE3, ApoE4on the inhibition ofHMG-CoA reductase expression by fluvastatin, Tanshinol and both. The action siteand molecular mechanisms of ApoE gene polymorphism on the inhibition ofHMG-CoA reductase by fluvastatin was comprehensively analyzed.5. Data processing and statistical analysis. IC50was calculated by doublereciprocal plot. Inhibition rate=(gray value ratio of blank group-gray value ratio ofexperimental group)/gray value ratio of blank group*100%. All the data are indicatedas mean±standard deviation (mean±SD). The statistical analysis was performedusing SPSS software for data processing. The difference between groups wasanalyzed using independent samples t-test, and for statistical results, P>0.05indicatedthe difference was not statistically significant, P <0.05indicated the difference wasstatistically significant, and P<0.01indicated the difference was extremelystatistically significant.Result:1. The ELISA method to determine LDL established in this study has highsensitivity, precision and accuracy, which are all in line with the experimentalrequirements. The linear range was0.2-12.8μg/ml. The best binding time forcompetitive receptor binding assay was120min, and the binding amount of LDL withthe receptor was1.59±0.07μg/mg protein.2. In L-02cells cultured in the presence of serum, the gray value ratio of HMG-CoA reductase mRNA expression determined by RT-PCR was0.38±0.02,which increased to0.86±0.01after induced in the absence of serum for24hours.However, the gray value ratio of HMG-CoA reductase mRNA expression in L-02cells treated with fluvastatin for24hours was0.49±0.01. The optimum experimentalcondition was culture with serum for24hours. The concentration of ApoE3, ApoE2,ApoE4were0.36,0.5,0.63μg/ml.3. The maximum replacement of LDL in the competitive binding of3kinds ofgene subtypes wild-type E3, mutant E2and mutant E4with LDL was6.84±0.23,3.92±0.40,6.30±0.32μg/mg protein, respectively; IC50was0.05±0.01,0.35±0.02,0.24±0.01μg/ml, respectively. Homogeneity test of variance test showed that the threegroups were not identical (P=). The difference between the pairwise comparison ofmutant E2, E4with wild-type E3was statistically significant (P <0.01, P <0.01). Thereceptor binding activity of mutation E2and E4was lower than that of wild-type E3,and the receptor binding activity of mutant E2was lower than that of mutant E4(P<0.01).4.IC50of fluvastatin and Tanshinol inhibiting the expression of HMG-CoAreductase mRNA was1.62±0.11and3.00±0.14, the corresponding inhibition rate was24.13±0.20%and15.11±0.31%, and the maximum inhibition rate was48.26±0.40%and30.21±0.62%, respectively. These results suggest that fluvastatin and Tanshinolsignificantly inhibit HMG-CoA reductase expression, and fluvastatin has strongerinhibitory effect with statistical difference (P <0.01).The inhibition rate of1.62μg/ml fluvastatin in combination with3.00μg/mlTanshinol on HMG-CoA reductase mRNA expression was34.61±0.45%, which wassignificantly higher than that of fluvastatin (24.13±0.20%) alone and Tanshinol(15.11±0.31%) alone, with significant difference in t test (P <0.01), clearly showingsynergistic effect in the inhibition of HMG-CoA reductase activity.5. When wild-type E3, mutant E2and E4were added, the inhibition rate offluvastatin (IC501.62μg/ml) on HMG-CoA reductase was18.59±1.09%,23.49±0.37%and12.13±0.91%, all lower than that of non-ApoE group. Significantdifference (P <0.01) was observed in the inhibition rate of HMG-CoA reductasebetween wild-type E3group and non-ApoE group, while the difference of mutant E2 group and mutant E4group was not the same (P>0.05, P <0.01). The comparisonbetween mutant E4group and wild-type E3group showed that the difference in theinhibition rate of fluvastatin on HMG-CoA reductase was statistically significant(P<0.01). Therefore, the mutation of Arg to Cys at ApoE2site112is unlikely relatedto the change of inhibition rate of fluvastatin on HMG-CoA reductase.When wild-type E3, mutant E2and mutant E4mutant were added, theinhibition rate of Tanshinol (IC503.00μg/ml) on HMG-CoA reductase was9.94±1.05%,6.54±0.47%and22.46±1.21%. The inhibition rate of wild-type E3andmutant E2group were lower than that of non-ApoE group, both with significantdifference (P<0.01). However, the result of mutant E4group was opposite whichshowed significantly higher inhibition rate on HMG-CoA reductase, and significantdifference (P <0.01) was observed when compared with wild-type E3group.Therefore, the mutation of Arg to Cys at ApoE2site112increases the inhibition rateof Tanshinol on HMG-CoA reductase.6. When wild-type E3, mutant E2and mutant E4were added, the inhibition rateof1.62μg/ml fluvastatin in combination with3.00μg/ml Tanshinol on HMG-CoAreductase mRNA expression was24.68±2.63%,11.08±1.38%and33.69±1.65%. Theinhibition rates on HMG-CoA reductase in wild-type E3group and mutant E2groupwere lower than non-ApoE group, with significant difference (P <0.01). However, nosignificant difference was observed between mutant E4group and non-ApoE group(P>0.05). This is likely related to the increased inhibitory effect of Tanshinol onHMG-CoA reductase by the mutation of Cys to Arg at ApoE4site158.Conclusion:From the cellular and molecular level to systematically study how ApoE genepolymorphism affecting the inhibition fluvastatin on HMG-CoA reductase is theinnovation of this research.1. The results showed that the mutation of Arg (arginine) to Cys (cysteine) atApoE2site112and the mutation of Cys to Arg at ApoE4site158may both result indecreased receptor binding activity than that of wild-type E3, however, the differencebetween mutant ApoE2and wild-type E3was more significant than that betweenmutant E4and wild-type E3, and significant difference was observed between mutant E2and mutant E4. These results suggest that site112may be the primary mutationsite of ApoE.2. The results showed that both fluvastatin and Tanshinol inhibited HMG-CoAreductase expression and fluvastatin had a stronger inhibitory effect than Tanshinol.Fluvastatin had a synergistic effect with Tanshinol in the inhibition of HMG-CoAreductase.3. The results suggest that the inhibitory effect of fluvastatin on HMG-CoAreductase mRNA is not related to the mutation of Arg to Cys at ApoE2site112,however, the inhibitory effect of Tanshinol on HMG-CoA reductase mRNA is likelyrelated to the mutation of Cys to Arg at ApoE4site158. Compared with ApoE, in thepresence of mutant E2and E4, most of the inhibitory effect of fluvastatin andTanshinol on HMG-CoA reductase decreased. It can be inferred that the mutations atApoE site112and158which leads to decreased LDL replacement may reduce theinhibitory effect of fluvastatin and Tanshinol on HMG-CoA reductase expression.However, it is still unclear that mutant E2almost had no effect on the inhibitoryeffect of fluvastatin on HMG-CoA reductase, while in the presence of mutant E4theinhibitory effect of Tanshinol significantly increased than that of non-ApoE group,which was probably because mutant E2and fluvastatin, mutant E4and Tanshinolformed a complex which may affected the whole process. The exact relationshipbetween them requires further in-depth studies.4. Experiment results further illustrated that when fluvastatin was combined withTanshinol, the inhibition rates on HMG-CoA reductase of wild-type E3group andmutant E2group were lower than that of non-ApoE group, while no significantdifference was observed between mutant E4group and non-ApoE group. This may berelated to the enhanced effect of Tanshinol on HMG-CoA reductase by the mutationof Cys to Arg at ApoE4site158.5. The mutations both at ApoE site112and158may lead to the change ofinhibitory effect of fluvastatin and Tanshinol on HMG-CoA reductase. Therefore, the mutations at different ApoE sites in the patient should be closely observed during theclinical administration of fluvastatin and Tanshinol. |
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