| Kallistatin?KAL?is one of the serine protease inhibitors.In recent years,it has been found that serum KAL concentration is correlated with high-density lipoprotein cholesterol?HDL-C?level,and reduces the risk of cardiovascular disease,It is closely related to atherosclerosis.ATP-binding cassette transporter A1?ABCA1?is a membrane transporter protein,which plays a key role in the generation of high-density lipoprotein?HDL?.The effect of KAL on the expression of ABCA1 and atherosclerosis and its mechanism were investigated in cell and animal experiments.It was found that Kallistatin promoted the expression of ABCA1 against atherosclerosis through the pathway of mir-29c-3p/ lymphoid transcription repressor BTB and CNC homology 2?BACH2?,providing a new strategy for the prevention and treatment of atherosclerosis.Part ? The changes of KAL levels in the serum of patients with CHDObjective: To observe the changes of KAL content in the serum of normal coronary artery and CHD population,and to detect the HDL-C level of the population at the same time,to confirm the correlation between KAL and atherosclerosis,as well as KAL and HDL-C.Methods: The subjects were 401 patients who were examined by coronary angiography?CAG?and intravascular ultrasound?IVUS?in the cardiovascular department of the First Affiliated Hospital of South China University.The degree of stenosis in each coronary artery was quantitatively evaluated by Gensini integral system.The higher the Gensini score,the more severe the coronary stenosis,and the more severe the atherosclerotic lesions.The subjects were divided into four groups: 1.Control group: Gensini score 0,no stenosis of coronary artery;2.Low Gensini score?LGS?group: Gensini score 1-20,and coronary artery stenosis was mild;3.Middle Gensini score?MGS?group: Gensini score 20-40,and coronary artery stenosis was moderate;4.High Gensini score Score?HGS?group: Gensini score of 40-160 points,and coronary artery stenosis was severe.Blood samples of each group were collected,centrifuged to obtain serum,and the serum KAL concentration was measured by ELISA.At the same time,HDL-C level was detected.It was verified the relationship between KAL and atherosclerotic lesions and the relationship between KAL and HDL-C.Results: The results showed that there were no significant differences in age,gender,smoking history,hypertension,BMI,WBC,ALT,BUN,Cr,etc.The results showed that the KAL concentration in the control group was?45.69 ± 9.69??g/ml,while the serum KAL concentration in the three groups of CHD patients decreased significantly,LGS group was?34.91 ± 7.72??g/ml,MGS group was?25.36 ± 7.06??g/ml,HGS group was?17.90 ± 4.35??g/ml,which showed that the serum KAL concentration in the three groups of CHD patients was significantly different from that in the control group,and decreased with the increase of Gensini score Trends.It suggests that KAL may have an inhibitory effect on As.After further study on the correlation between KAl concentration and HDL-C,it was found that there was a statistically positive correlation between KAL concentration and HDL-C?r=-0.4981,P<0.01?.It is suggested that HDL may be involved in the inhibition of As lesions by KAL.Summaries:?1?The content of KAL in CHD patients is significantly decreases;?2?KAL concentration is positively correlates with the level of HDL-C.Part ? KAL regulates ABCA1 expression and lipid accumulation inmacrophages through mi R-29c-3p/BACH2 pathwayObjective: To observe the effect of KAL on ABCA1 expression and lipid accumulation in macrophages,and to elucidate the role of mi R-29c-3p/BACH2 pathway in the regulation of ABCA1 expression by KAL.Methods: THP-1 monocytes were cultured and then treated with 50 n M PMA for 48 hour to induce their differentiation into THP-1 macrophages.Incubated with ox-LDL?50g/ml?,and foam cells were formed after loading the lipid.Different concentrations?0,0.1,0.2,0.4,0.8,1.6 ?mol/L?of KAL were used to treat THP-1 macrophage derived foam cells 24 hour,or THP-1 KAL macrophage derived foam cells were treated with 0.8umol/L KAL at different time?0,6,12,24,24 hour?.The cells were collected,the expression of ABCA1 was detected by q RT-PCR and Western blot,the lipid accumulation was detected by oil red O staining,the content of cholesterol in cells was detected by HPLC,and the efflux of cholesterol was detected by liquid scintillation counter.It was confirmed that KAL can up regulate the expression of ABCA1 and promote cholesterol efflux.The binding sites of ABCA1 promoter and BACH2 were predicted by JASPAR online website.The promoter of ABCA1 gene was amplified from THP-1 genomic DNA by PCR and inserted into p GL3-ABCA1-promoter,the luciferase reporter gene p GL3-ABCA1-promoter and the transcription factor BACH2 expression plasmid were cotransfected into 293 T cells.The luciferase activity was determined by luciferase detection kit to confirm whether there was BACH2 binding site in ABCA1 promoter region.BACH2 was transfected into THP-1 macrophage derived foam cells,and chromatin immunoprecipitation?CHIP?was used to further analyze the interaction between BACH2 and ABCA1 promoter.Subsequently,BACH2 or BACH2 si RNA were transfected into THP-1 macrophage derived foam cells,the expression of ABCA1 was detected by fluorescence quantitative PCR and Western Blot,lipid accumulation was detected by oil red O staining,cholesterol content in cells was detected by high performance liquid chromatography,and cholesterol flow was detected by liquid scintillation counter.It was confirmed whether BACH2 can directly up regulate ABCA1 expression and promote cholesterol outflow.THP-1 macrophage derived foam cells were incubated with KAL.The expression of BACH2 was detected by fluorescence quantitative PCR and Western Blot,and the effect of KAL on BACH2 was confirmed.After the expression of BACH2 was silenced,THP-1 macrophage derived foam cells were incubated with KAL,the expression of ABCA1 was detected by fluorescence quantitative PCR and Western Blot.Lipid accumulation was detected by oil red O staining,cholesterol content in cells was detected by high performance liquid chromatography,and cholesterol efflux was detected by liquid scintillation counter.It was determined whether KAL affected ABCA1 expression and cholesterol efflux through BACH2.The binding sites and free energy of mi R-29c-3p and BACH2 3'UTR were predicted by RNAhybrid and other bioinformatics online websites.PCR method was used to amplify the 3'UTR sequence of BACH2 gene from macrophage genomic DNA,insert p GL3-BACH2-3'UTR into luciferase reporter vector,and construct the p GL3-BACH2-3'UTR mutant?p GL3-BACH2-3'UTR-m?vector.Luciferase reporter gene p GL3-BACH2-3'UTR or p GL3-BACH2-3'UTR-m and mi R-29c-3p mimics were cotransfected into 293 T cells.Luciferase activity was measured by luciferase detection kit.It was confirmed whether mi R-29c-3p can directly affect BACH2.THP-1 macrophage derived foam cells were treated with mi R-29c-3p mimics or anti-mi R-29c-3p respectively.The expressions of BACH2 and ABCA1 were detected by fluorescence quantitative PCR and Western Blot,Lipid accumulation was detected by oil red staining,and the cholesterol content in cells was detected by high performance liquid chromatography.It was understood the effect of overexpression or silence of mir-29c-3p on the expression of BACH2,ABCA1 and cholesterol efflux.Finally,THP-1 macrophagy-derived foam cells were incubated with KAL,and the expression of mi R-29c-3p was detected by fluorescence quantitative PCR to determine the effect of KAL on the level of mi R-29c-3p.Mi R-29c-3p mimics and KAL were co-incubated with THP-1 macrophagy-derived foam cells,and fluorescence quantitative PCR Western was used.The expressions of BACH2 and ABCA1 were detected by Blot,lipid accumulation was detected by oil red O staining,intracellular cholesterol content was detected by HPLC,and cholesterol efflux was detected by liquid scintillation counter.It was determined whether KAL could silence BACH2 through mi R-29c-3p to promote the expression of BACH2 and affect the expression of ABCA1 and the effusion of cholesterol.Results: KAL increased the expression of ABCA1 in THP-1 macrophage derived foam cells in a concentration-and time-dependent manner.KAL increased ABCA1 mediated cholesterol efflux,decreased the content of TC,CE,FC and inhibited lipid accumulation.Bioinformatics predicted that there was binding site of BACH2 in the ABCA1 promoter region.Furthermore,luciferase reporter gene and chip experiments confirmed that BACH2 had binding site with ABCA1 promoter,while overexpression of BACH2 promoted ABCA1 expression and increased cholesterol outflow of macrophages,suggesting that BACH2 could bind with ABCA1 and promote its transcription..After KAL treatment of THP-1 macrophage derived foam cells,the m RNA and protein levels of BACH2 and ABCA1 increased significantly,suggesting that KAL could promote the expression and binding capacity of BACH2 and ABCA1.After si BACH2 pretreatment,the effect of KAL on the expression of ABCA1 was reversed,as was the effect of cholesterol efflux.The results showed that KAL increased the expression of ABCA1 and the outflow of cholesterol by increasing BACH2.Bioinformatics online websites predicted that there was a binding site between mi R-29c-3p and BACH2 3'UTR,and the binding free energy was low?-23.6kcal/mol?.Luciferase reporter gene and other experiments further found that mi R-29c-3p could directly affect BACH2,inhibit its expression,and significantly reduce ABCA1.Inhibition of mi R-29c-3p could increase the m RNA and protein levels of BACH2 and ABCA1,and promote the increase of cholesterol efflux in THP-1 macrophage derived foam cells.It is suggested that mi R-29c-3p can inhibit the expression of BACH2 and ABCA1,and reduce the outflow of cholesterol.Incubation of THP-1 macrophage derived foam cells with KAL showed that KAL significantly decreased the expression of mi R-29c-3p.After co-incubation with KAL and mi R-29c-3p mimic,compared with mi R-29c-3p mimic group,the decreased m RNA and protein levels of BACH2 and ABCA1 were restored to a certain extent,and the decreased cholesterol outflow was also restored to a certain extent.These results showed that KAL increased the expression of BACH2,ABCA1 and cholesterol efflux by inhibiting mi R-29c-3p.Summaries:?1?KAL up-regulates the expression of ABCA1,promotes the outflow of cholesterol,and inhibits the accumulation of lipid in macrophages.?2?KAL increases the expression of macrophage ABCA1 through mi R-29c-3p /BACH2 pathway.Part ? The effect of KAL on ABCA1 and atherosclerotic plaquearea in apoE-/-miceObjective: To observe the effects of KAL on the expression of mi R-29c-3p BACH2 and ABCA1,lipid level,RCT efficiency and atherosclerotic plaque area of apoE-/-mice,and to reveal the anti-atherosclerotic effect and molecular mechanism of KAL.Methods: 60 male Apo E-/-mice were randomly divided into three groups after 8 weeks of high-fat diet feeding: 1.The control group?n = 20?was injected with 200?l normal saline twice a week;2.The low-dose KAL group was injected with 0.5mg/kg recombinant KAL protein twice a week by intraperitoneal injection;3.The high-dose KAL group was injected with 2mg/kg KAL protein twice a week,and the rest was the same as the low-dose group.The treatment lasted for 4 weeks.B-ultrasound was used to examine the plaques of aorta and carotid artery in mice.The contents of TG,TC,LDL-C and HDL-C in plasma and the function of liver and kidney were measured by enzyme oxidation.Following intraperitoneal injection with [3H] cholesterol-labelled J774 cells,radioactivity in plasma,liver and feces was detected to calculate RCT.After sacrifice,aortic arch was isolated,and stereoscopic microscope was used to observe plaque formation.Masson staining,oil red O and HE was used to detect plaque area,lipid accumulation,and collagen content in aortic sinus.Lipid deposition in aortic wall was observed using oil red O staining.Quantitative PCR was used to detect mi R-29c-3p in aorta,BACH2 and ABCA1 m RNA expression in aorta and peritoneal macrophages,Western blot and immunohistochemistry were used to detect BACH2 and ABCA1 protein expression in aorta and peritoneal macrophages.Results: Both low-dose and high-dose KAL decreased the area of plaques in the aortic arch and sinus of Apo E-/-mice,inhibited the lipid deposition in the aortic wall and plaques,and increased the collagen content of plaques.It is confirmed that KAL can inhibit the progression of As lesions in vivo.KAL decreased the level of mi R-29c-3p in aortic tissues of apoE-/-mice,increased the levels of BACH2 and ABCA1 in aortic tissues and peritoneal macrophages,increased plasma HDL-C levels,and promoted RCT.It is suggested that KAL also inhibits the expression of mi R-29c-3p in vivo,thereby reducing the inhibition of BACH2 by mi R-29c-3p,promoting the expression of ABCA1,increasing cholesterol efflux to HDL,promoting RCT,and finally inhibiting the progression of As lesions in apoE-/-mice.KAL had no significant effect on body weight,liver and kidney function,and plasma levels of TC,LDL-C and TG in apoE-/-mice.Summaries:?1?KAL up-regulates the expression of ABCA1,increases the level of plasma HDL-C,promotes RCT,and alleviates atherosclerosis in apoE-/-mice;?2?KAL increases the expression of ABCA1 in apoE-/-mice through mi R-29c-3p/BACH2 pathway.Conclusions 1.The content of KAL in CHD patients is significantly decreases;KALconcentration is positively correlates with the level of HDL-C.2.KAL up-regulates ABCA1 expression through mi R-29c-3p/BACH2pathway,promotes cholesterol efflux and inhibits lipid accumulation inmacrophages.3.KAL up-regulates the expression of ABCA1 through mi R-29c-3p/BACH2 pathway in apoE-/-mice,and increases the level of plasmaHDL-C,promotes RCT,alleviates atherosclerosis. |
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