| BackgroundStatins are considered as the most common treatment to reduce low density lipoprotein cholesterol(LDL-C).Statins inhibit HMG-Co A reductase(HMGCR)to reduce cholesterol biosynthesis and activate sterol regulatory element binding protein 2(SREBP2)to increase the expression of LDL receptor(LDLR)in cell membrane,and ultimately achieve the purpose of lowering LDL-C.However,activation of SREBP2 also up-regulates LDLR,it also up-regulates proprotein convertase subtilisin/kexin type 9(PCSK9).PCSK9 can bind to LDL receptor(LDLR)and the complex of PCSK9 and LDL receptor(LDLR)is transported to lysosome for degradation.This process eventually leads to the decrease of the number of LDLR in the liver,which limits the efficacy of statins.Aerobic exercise has been shown to significantly benefit patients with cardiovascular disease.Researches indicate that aerobic exercise down-regulates the expression of PCSK9,but the specific mechanism is still unclear.On this basis,our previous studies have shown that aerobic exercise significantly up-regulates the level of epoxyeicosatrienoic acids(EETs).EETs are a class of lipid epoxides with strong cardiovascular protection and are closely related to lipid metabolism.Therefore,we speculate that statin combined with aerobic exercise may negatively regulate PCSK9 through EETs.There is still a lack of research on the regulation of PCSK9 by EETs.Hepatocyte nuclear factor-1α(HNF-1α)is a transcription factor mainly expressed in the liver.Research shows that HNF-1α is closely related to cholesterol metabolism.It is one of the main transcription factors of PCSK9 and can positively regulate the expression of PCSK9.Moreover,there are many studies have confirmed that aerobic exercise is closely related to HNFs.Therefore,we speculate that statin combined with aerobic exercise up-regulates EETs,and EETs affect the expression of PCSK9 through HNF-1α.Sirtuins(Sirt)are proteases that depend on nicotinamide adenine dinucleotide(NAD+)histone deacetylase and ADP ribosyltransferase.Silent mating type information regulation 2 homolog 6(Sirt6)is an important member of them.Studies have shown that Sirt6 can be recruited by the PCSK9 transcription factor Fox O3 a and bind to the distal promoter region of the PCSK9 gene,inhibiting PCSK9 gene expression by deacetylating specific sites.On the other hand,studies have confirmed that aerobic exercise can significantly up-regulate the expression of Sirt6.Based on the above background and our previous research results,we put forward the scientific hypothesis: statin combined with aerobic exercise regulates the Sirt6-HNF-1α axis through EETs,thereby down-regulating PCSK9,and ultimately achieving the goal of further lowering cholesterol.PurposeThis study intends to clarify the effects of statin combined with aerobic exercise on blood lipids,PCSK9,HNF-1α and Sirt6 in hypercholesterolemic mice through animal experiments,and to explore in vivo and in vitro experiments whether statin combined with aerobic exercise could regulate Sirt6-HNF-1α-PCSK9 axis through EETs,and ultimately further enhanced the lipid-lowering effect of statin.Methods1.Determine whether statin combined with aerobic exercise can down-regulate the expression of PCSK9 in hypercholesterolemic mice through EETs,and evaluate the lipid-lowering effect of statin combined with aerobic exercise:3-week ICR male mice were fed a normal diet(NCD)and a high cholesterol diet(HFD)for 4 weeks to establish a hypercholesterolemia mouse model.The results of HE staining and Oil Red O staining of the liver were observed and the blood lipids were detected.The hypercholesterolemic mice were randomly divided into groups and then treated with statin,aerobic exercise and statin combined with aerobic exercise for 8 weeks.The blood lipids,PCSK9,LDLR,HMGCR and SREBP2 expression of the mice were detected after 8 weeks.Different concentrations of soluble epoxide hydrolase inhibitor TPPU(0mg/L,0.1mg/L,1mg/L,10mg/L)and EETs antagonist14,15-EEZE were to treat HFD+statin+Exercise mice.After 8 weeks,the expressions of blood lipids,PCSK9,LDLR,HMGCR and SREBP2 in the mice were checked.2.To clarify whether EETs affects the expression of PCSK9 in the liver of hypercholesterolemic mice through HNF-1α,and to further observe its mechanism in a cell model co-intervention of statin and oxidized LDL(ox-LDL):In vivo experiments,the 3-week ICR male mice were randomly divided into four groups and were given NCD,HFD,HFD+statin and HFD+statin+Exercise intervention for 8 weeks.The overall level of HNF-1α in mouse liver and the levels of nuclear and cytoplasmic HNF-1α proteins were detected.On this basis,under the premise of HFD+statin+Exercise intervention,the EETs antagonist 14,15-EEZE and s EHi preparation TPPU were further used to intervene in hypercholesterolemic mice.After8 weeks,Real-time PCR and Western blot(WB)were used to detect the overall level of HNF-1α,and WB was used to check nuclear and cytoplasmic HNF-1α protein levels.In vitro experiment,Hep G2 cells were co-intervened with ox-LDL(TC)and statin to establish a cell model.After 24 hours,Oil Red O staining was used to test whether the model was successfully constructed.The model cells were intervened with different concentrations of TPPU(0μM,0.5μM,5μM,50μM)and 50μM TPPU combined with14,15-EEZE,and the expressions of PCSK9,LDLR,global HNF-1α,nuclear and cytoplasmic HNF-1α were detected 24 hours later.Finally,TC,statin,TC+statin,TC+statin+TPPU,TC+statin+TPPU+14,15-EEZE were used to treat the cells respectively.After 24 hours,the HNF-1α/PCSK9 complex was extracted by Ch-IP method,and the level of the complex in each group was detected by Real-time PCR.3.To verify whether statin combined with aerobic exercise affects the Sirt6-HNF-1α axis through EETs,and finally reduces the expression of PCSK9:In vivo experiment,3-week ICR male mice were randomly divided into four groups,which were given NCD,HFD,HFD + statin and HFD +statin + Exercise intervention for 8 weeks.The expression level of Sirt6 in mouse liver was detected.On this basis,under the premise of HFD+statin+Exercise intervention,the EETs antagonist 14,15-EEZE and s EHi preparation TPPU were further used to intervene in in hypercholesterolemic mice.After 8 weeks,Real-time PCR and Western blot were used to detect Sirt6 in mouse liver.In vitro experiment,the cells were divided into four groups: normal culture medium(NC),TC,statin,and TC+statin.Sirt6 in each group was detected after 24 hours.Model cells were intervened with different concentrations of TPPU(0μM,0.5μM,5μM,50μM)and 50μM TPPU combined with 14,15-EEZE,and Sirt6 expression was detected 24 hours later.Subsequently,cells were transfected with Negative Control si RNA and the three constructed si Sirt6 sequences with a final concentration of100 n M.The effective silencing sequences were screened by detecting the expression of Sirt6,and the Sirt6 silencing cell model was constructed.Next,the effect of TPPU on PCSK9,LDLR,and HNF-1α in a Sirt6-silenced cell model were examined.Finally,the Co-IP method was used to detect whether there is a protein-protein interaction between HNF-1α and Sirt6 in the four groups which were treated with NC,TC+statin,TPPU and TC+statin+TPPU,respectively.Results1.Part1(1)Compared with the NCD mice,the lipid droplets in hepatocytes of the HFD mice were significantly increased,and TC,HDL-C and LDL-C were significantly increased(all P<0.05),indicating that the hypercholesterolemia mouse model was successfully established.(2)Compared with the HFD group,the TC,HDL-C and LDL-C of the HFD+statin group were significantly decreased,while PCSK9,LDLR,HMGCR,and SREBP2 were increased(all P<0.05).Compared with the HFD+statin group,the TC and LDL-C of the HFD+statin+Exercise group were further decreased,PCSK9 was decreased and LDLR was increased(all P<0.05),and the expressions of HMGCR and SREBP2 had no significant changes(all P>0.05).(3)Compared with the HFD+statin+Exercise group,TPPU further reduced TC,LDL-C,PCSK9 and increased LDLR in a drug concentration-dependent manner,and 10 mg/L TPPU significantly decreased TC,LDL-C,PCSK9 and increased LDLR(all P<0.05).Compared with the HFD+statin+Exercise group,further intervention with14,15-EEZE increased TC,LDL-C and PCSK9,and decreased LDLR(all P<0.05).Neither TPPU nor 14,15-EEZE significantly changed HMGCR and SREBP2(all P>0.05).2.Part2Animal experiments(1)Compared with the HFD group,the overall expression of HNF-1α and nuclear HNF-1α in the HFD+statin group increased(all P<0.05),while the cytoplasmic HNF-1α had no significant change(P>0.05).Compared with the HFD+statin+Exercise group,the overall expression of HNF-1α had no significant change(P>0.05),the expression of nuclear HNF-1α decreased and the cytoplasmic HNF-1α increased(all P<0.05).(2)Compared with the HFD+statin+Exercise group,there was no significant change in the overall expression of HNF-1α in mice after further administration of 14,15-EEZE or TPPU(all P>0.05)but14,15-EEZE increased nuclear HNF-1α and decreased cytoplasmic HNF-1α,while TPPU decreased nuclear HNF-1α and increased cytoplasmic HNF-1α(all P<0.05).Cell experiments(3)Compared with the NC group,a large number of lipid droplets were observed in the cells of the TC group.Compared with the TC group,the lipid droplets in the cells of the TC+statin group decreased(all P<0.05),suggesting that the cell model of ox-LDL combined with statin intervention is successfully established.(4)Compared with TC+statin group,further administration of TPPU decreased PCSK9,nuclear HNF-1α and increased LDLR in a drug concentration-dependent manner.When the concentration of TPPU reached 50 μM,PCSK9 and nuclear HNF-1α were significantly decreased,LDLR and cytoplasmic HNF-1α were significantly increased(all P<0.05),and the overall expression of HNF-1α had no significant change(P>0.05).Compared with TC+statin+TPPU group,after further administration of14,15-EEZE,PCSK9 and nuclear HNF-1α increased,LDLR and cytoplasmic HNF-1α decreased(all P<0.05),and the overall expression of HNF-1α also changed not significantly(P>0.05).(5)Ch-IP results showed that statin intervention increased the level of HNF-1α/PCSK9 complex;further administration of TPPU reduced HNF-1α/PCSK9 complex,and the effect of TPPU could be counteracted by 14,15-EEZE(all P<0.05).3.Part3Animal experiments(1)HFD and statin intervention both down-regulated the expression of Sirt6 in mice,and aerobic exercise intervention can increase the expression of Sirt6(all P<0.05).(2)On the basis of HFD+statin+Exercise,further administration of14,15-EEZE reduced Sirt6,and further administration of TPPU increased Sirt6(all P<0.05).Cell experiments(3)Compared with cells in NC group,both TC and statin intervention reduced the expression of Sirt6 in cells,and the two had a synergistic effect(all P<0.05).(4)Compared with TC+statin group,Sirt6 in TC+statin+0.5μM TPPU group showed an upward trend(P>0.05).The expression of Sirt6 in TC+statin+5μM TPPU group and TC+statin+50μM TPPU group was significantly increased(all P<0.05).Compared with TC+statin+TPPU group,the expression of Sirt6 decreased after adding 14,15-EEZE(P<0.05).(5)Compared with the Negative Control si RNA,the Sirt6 m RNA and protein in cells transfected with si Sirt6 effective sequence were significantly decreased(P<0.05),and the Sirt6 silencing cell model was successfully constructed.Compared with TC+statin+TPPU group,after silencing Sirt6,PCSK9 increased and LDLR decreased(all P<0.05),and the levels of nuclear,cytoplasmic and overall HNF-1α had no significant changes(all P>0.05).(6)Co-IP test showed that in the NC group and the TC+statin group,there was no interaction between the Sirt6 protein and HNF-1α protein,and in the TPPU group and the TC+statin+TPPU group,there was a protein-protein interaction between Sirt6 and HNF-1α.Conclusions:1.Statin combined with aerobic exercise negatively regulates PCSK9 through EETs,and finally further reduces LDL-C.2.Aerobic exercise regulates the Sirt6-HNF-1α axis via EETs,reduces the formation of HNF-1α/PCSK9 complex,and significantly down-regulates the expression of PCSK9. |
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