Studies On The Mechanism By Which Surf4 Regulates Pcsk9 Secretion

ObjectiveAs a clinical first-line drug,statins play an important role in reducing the incidence and mortality of atherosclerotic cardiovascular and cerebrovascular diseases.But statins also improve the level of PCSK9?proprotein convrtase subtilisin/kexin type 9?in plasma while reducing blood lipids.However,the downregulation of LDLR?low density lipoprot-ein receptor?on the cell membrane by PCSK9,to a certain extent,weakens the effect of statins on lowering blood lipid.In our previous study,we found that the Cargo receptor Surf4 could promote the secretion of PCSK9.It is suggested that statins may mediate the secretion of PCSK9 throught Surf4,but no literature has been confirmed so far.This resea-rch takes the apolipoprotein E gene knockout mice(ApoE^-/-)as animal model,with the help of RNA interference,Surf4 interference respectively from the overall and cell level,and to observe the effect of lovastatin on cholesterol and PCSK9 levels in plasma;detect the effect of lovastatin on the expression level of liver Surf4,PCSK9,LDL receptor and ERS?Endoplasmic Reticulum Stress?related protein.In addition,the possible molecular mechanism of lovastatin to promote the secretion of PCSK9 was observed and analyzed by using HepG2 cell model and interfering with Surf4.Method1.Construct a adenovirus vector of Surf4 shRNAFirstly,the AAV-H1-SV40 adenovirus vector was digested and inserted into the desi-gned gene fragment that interfered with Surf4.Then,the recombinant adenoviral vector plasmid was mixed with Phelper plasmid and DJ/8 plasmid and then transfected into HEK293 cells.After the adenovirus was packed,the transfected HEK293 cells were collected,the cells were lysed,the virus was purified and the titer was measured.2.Animal experimentsThirty-two ApoE^-/-mice were randomly divided into 4 groups:control group?Con?,lovastatin group?Lov?,negative interference+lovastatin group?si-NC+Lov?,Surf4 interf-erence+lovastatin group?si-Surf4+Lov?,they were fed with high-fat diet,si-Surf4+Lov and si-NC+Lov groups were injected Surf4 or negative interference virus(1.0×10¹⁰GC/?l)from the inner canthus,Con and Lov groups were injected with equal volume of saline;The drug group was given the lovastatin suspension?2.6mg/kg/d?and the control group was given the same amount of saline.Blood samples were taken before AAV injection and at 10d,20d,30d,40d after injection,the animals were taken blood,killed and collected after 60d.The levels of cholesterol and PCSK9 in mouse plasma were detected using kits;the expression of PCSK9 and Surf4 mRNA in liver tissue of mice was detected by real-ti-me PCR;and the expression levels of Surf4,PCSK9,LDLR and ERS-related protein in liver tissue of mice was detected by Western blot.3.cell experimentsFirst,lovastatin with different concentrations?0?mol/L,0.625?mol/L,1.25?mol/L,2.5?mol/L,5?mol/L,10?mol/L?was used to treat HepG2 cells for 24 hours,to observe the effect of lovastatin on the expression of PCSK9 protein.The effect of si-Surf4 on the intracellular calcium fluorescence intensity was observed by a calcium ion fluorescent probe method.To observe the effect of PKA inhibitor on the level of phosphorylation of ERK1/2 and the expression of PCSK9 protein in HepG2 cells induced by lovastatin.HepG2cells were divided into 4 groups:control group?Con?,lovastatin group?Lov?,negative interference+lovastatin group?si-NC+Lov?,Surf4 interference+lovastatin group?si-Surf4+Lov?,the expression of Surf4,PCSK9,LDLR and ERS related protein in cell were detected by Western blot.Result1.Transfection efficiency and titer results of interfering with Surf4 adenovirusAfter transfection of adenovirus to 48h,the green fluorescence in human embryonic kidney cells was observed using inverted fluorescence microscope.The results showed that the green fluorescence of both the negative adenovirus transfection group?si-NC?and the interference Surf4 adenovirus transfection group?si-Surf4?could reach 70%⁸0%;The experiment successfully constructed the interfering Surf4 adenovirus,where the titer of the negative adenovirus?si-NC?was 1.149×10⁸GC/?l,and the titer of the interference Surf4adenovirus?si-Surf4?was 1.097×10⁹GC/?l.2.Interfering with Surf4 weakened the effects of lovastatin on the levels of cholesterol and PCSK9 in the plasma of ApoE^-/-miceThe results showed that plasma levels of cholesterol were significantly lower in the Lov group than in the Con group on the 10th,20th,30th,40th,and 60th days,while the PCSK9 level was significantly higher than that in the Con group?P<0.05?;There was no significant change in plasma levels of cholesterol and PCSK9 in the Lov group compared with the si-NC+Lov group?P>0.05?;Compared with Lov group,the plasma levels of chol-esterol and PCSK9 in si-Surf4+Lov group were significantly decreased?P<0.05?.3.The effect of interfering with Surf4 on the expression of Surf4,PCSK9 and LDLR in liver of ApoE^-/-mice induced by lovastatinCompared with the Con group,the expression of Surf4 and PCSK9 mRNA and protein in the Lov group was significantly increased?P<0.05?,but there is no change in the expression of LDLR protein?P>0.05?;The mRNA and protein expression of Surf4 and PCSK9 in liver tissue of the si-Surf4+Lov group was significantly lower than that in the Lov group?P<0.05?,and the expression level of LDLR protein was significantly increased?P<0.05?.4.The effect of interfering with Surf4 on the expression of ERS-related proteins?ATF-6,p-PERK,GRP78,p-EIF2??in the liver of ApoE^-/-mice induced by lovastatinCompared with Con,the expression of ATF-6 and GRP78 protein in Lov group was significantly increased,and the protein expression of p-PERK and p-EIF2?was also increased?P<0.05?;Compared with Lov,interfering Surf4 could decrease the expression of endoplasmic reticulum stress protein induced by lovastatin,and the difference was signific-ant?P<0.05?.5.The effect of iovastatin on the expression of PCSK9 protein in HepG2 cellsThe expression of PCSK9 was up-regulated in different concentrations of lovastatin?0.625?mol/L,1.25?mol/L,2.5?mol/L,5?mol/L,and 10?mol/L?compared with 0?mol/L,among of them,the effect of 2.5?mol/L lovastatin up-regulated PCSK9 significantly?P<0.001?.6.The effect of interfering with Surf4 on the expression of Surf4,PCSK9 and LDLR protein in HepG2 cells induced by lovastatinCompared with Con group,the expression levels of Surf4 and PCSK9 protein in Lov group were increased?P<0.05?,and the expression of LDLR was also increased,but the difference was not statistically significant?P>0.05?;compared with Lov group,the expres-sion levels of Surf4 and PCSK9 protein in si-Surf4+Lov group was significantly decreased?P<0.05?,and the expression levels of LDLR was significantly increased?P<0.05?.7.The effect of interfering with Surf4 on the expression of ERS-related proteins?ATF-6,p-PERK,GRP78,p-EIF2??in HepG2 cells induced by lovastatinThe expression of endoplasmic reticulum stress protein in lov group was significantly higher than that in control group?P<0.05?;Compared with the Lov group,interfering Surf4 can weaken the expression level of the endoplasmic reticulum stress protein induced by lovastatin?P<0.05?,and has significant statistical significance.8.The effect of interfering with Surf4 on the expression levels of ERK1/2 phosp-horylation and PKA and SREBP2 protein in HepG2 cells induced by lovastatinThe phosphorylation level of ERK1/2 and the protein expression level of PKA and SREBP2 in the Lov group were increased than that in Con group?P<0.05?;Compared with the Lov group,interfering Surf4 could significantly inhibit the phosphorylation of ERK1/2and PKA and SREBP2 protein expression level?P<0.05?.9.PKA may be involved in the process of ERK1/2 phosphorylation and the expression of PCSK9 protein in HepG2 cells induced by lovastatinCompared with the Con group,the expression level of ERK1/2 and PCSK9 in the Lov group were significantly increased?P<0.05?;Compared with the Lov group,the phosphor-ylation of ERK1/2 and PCSK9 protein expression levels were significantly reduced after the PKA inhibition?P<0.05?.10.Interfering with Surf4 enhanced fluorescence intensity of intracellular calciu-m in HepG2 cellsThe results showed that the green fluorescence intensity of intracellular Ca²⁺in si-Surf4 group was significantly higher than that in Con group.Conclusion1.Interfering with Surf4 can inhibit the protein expression of PCSK9 in the liver of ApoE^-/-mice induced by lovastatin.2.Lovastatin promotes the synthesis of PCSK9,which may be induced by the expression of Surf4,causing calcium depletion of hepatocytes and activating ERS-PKA-ERK1/2-SREBP2 signaling pathway.