Proprotein convertase subtilisin/kexin type 9 inhibitors in peripheral artery disease:A review of efficacy,safety,and outcomes

Peripheral artery disease(PAD)is a common condition characterized by atherosclerosis in the peripheral arteries,associated with concomitant coronary and cerebrovascular diseases.Proprotein convertase subtilisin/kexin type 9(PCSK9)inhibitors are a class of drugs that have shown potential in hypercholesterolemic patients.This review focuses on the efficacy,safety,and clinical outcomes of PCSK9 inhibitors in PAD based on the literature indexed by PubMed.Trials such as FOURIER and ODYSSEY demonstrate the efficacy of evolocumab and alirocumab in reducing cardiovascular events,offering a potential treatment option for PAD patients.Safety evaluations from trials show few adverse events,most of which are injection-site reactions,indicating the overall safety profile of PCSK9 inhibitors.Clinical outcomes show a reduction in cardiovascular events,ischemic strokes,and major adverse limb events.However,despite these positive findings,PCSK9 inhibitors are still underutilized in clinical practice,possibly due to a lack of awareness among care providers and cost concerns.Further research is needed to establish the long-term effects and cost-effectiveness of PCSK9 inhibitors in PAD patients.

豆豉纤溶酶Subtilisin FS33的溶栓作用及其机制的研究

目的:通过动物血栓模型研究豆豉纤溶酶Subtilisin FS33对机体凝血系统和纤溶系统的影响及其作用机制。方法:Subtilisin FS33与不同处理的血凝块37℃恒温孵育,测定血凝块溶解时间(CLT);利用FeCl3氧化损伤动脉内膜法制造大鼠血栓模型,不同剂量的Subtilisin FS33、尿激酶和生理盐水分别给于受试大鼠。结果:Subtilisin FS33对80℃加热处理血凝块的水解明显慢于未经加热处理组,提示Subtilisin FS33在缺乏内源性纤溶因子的情况下,仍能溶解血栓纤维蛋白。Subtilisin FS33高剂量组和尿激酶组,静脉注射15min和1h后,与机体凝血功能相关的血液学指标活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)和凝血时间(CT)明显延长,机体的纤溶酶活力显著增高,血栓降解产物FDP含量升高,D-二聚体均呈阳性,而机体优球蛋白溶解时间(ELT)值明显降低;Subtilisin FS33低剂量组的APTT、PT、TT也比生理盐水组相应延长,但差异性大多未达到显著水平。病理组织切片结果表明,静注Subtilisin FS33后,大鼠肺静脉和肾小球毛细血管等无明显血栓,而生理盐水对照组的大鼠肺静脉等仍存在少量丝状纤维物。结论:Subtilisin FS33在体内和体外均具有明显的降解血栓纤维蛋白的能力,增强机体的纤溶活性、提高抗凝能力是其溶栓作用的主要途径。

Subtilisin FS33 RGDS-载酶纳米脂质体的制备与效果评价

研究评价了Subtilisin FS33 RGDS-载酶纳米脂质体的制备及其效果。按照正交设计试验确定硫酸铵梯度法制备载酶脂质体的工艺条件为:胆脂比1∶2,硫酸铵浓度为0.15 mol/L,孵化温度为45℃,酶脂比1∶1。制得的载酶脂质体粒径在50～150 nm左右,属于纳米级单室脂质体。在制备RGDS-载酶脂质体的工艺过程中,制备开始时就加入氨基酰化修饰的RGDS衍生物,其成品脂质体中RGDS含量可达到93μg/mL,并有利于分布于脂质体表面。RGDS-纳米脂质体中酶对于高温、极端pH、模拟胃肠道环境等条件的稳定性都有明显提高;酯酶存在时,脂质体中FS33释放速度明显加快,并可使血凝块完全溶解,表现出较好的溶栓效果。

ABO血型与血脂和PCSK9相关性研究

目的 探讨ABO血型和前蛋白转化酶枯草杆菌蛋白酶9（PCSK9）水平之间的联系.方法 人选507例行诊断性或治疗性冠状动脉造影检查的患者,收集基线数据并用ELISA法评价血浆PCSK9水平.结果 非O型受试者与O型受试者相比,总胆固醇（TC）[（5.07± 1.14） mmol/L比（4.78±1.01）mmol/L]、低密度脂蛋白胆固醇（LDL-C）[（3.35± 1.08） mmol/L比（3.12±0.91）mmol/L]、非高密度脂蛋白胆固醇（NHDL-C）[（3.94±1.10）mmol/L比（3.68±1.01）mmol/L]、载脂蛋白B（ApoB）[（1.0±0.28）g/L比（0.96±0.27）g/L]和PCSK9[226.32（118.91～279.28）ng/ml比202.33（171.27～254.31）ng/ml]水平更高（P均＜0.05）.PCSK9水平明显与TC、LDL-C 、NHDL-C和apoB呈正相关（r=0.253,P＜0.01;r=0.262,P＜0.01;r=0.215,P＜0.01;r=0.187,P＜0.01）.多变量回归分析显示,ABO血型与PCSK9水平显著而且独立相关（β=7.91,P=0.009）.中介分析显示,ABO血型对PCSK9的作用8％～19％是通过TC、LDL-C或NHDL-C水平介导的.结论 本研究首次指出ABO血型可能是PSCK9的重要决定因素.PCSK9与ABO血型之间的联系也可能在一定程度上参与了冠心病的发病.

酶解褐藻寡糖的分离制备及其脂蛋白调节作用

采用Isoptericola halotolerans CGMCC 5336褐藻胶裂解酶制备褐藻寡糖,通过液质联用技术(liquid chromatograph-mass spectrometer,LC-MS)对其组成进行初步分析,后经过凝胶色谱柱对其进行分离、纯化,得到4种组分,分别命名为Fr1、Fr2、Fr3、Fr4。通过LC-MS分析各组分纯度并确定分子量,使用G-25凝胶柱进行脱盐。通过RT-PCR和Western Blot技术,研究所获得的4个组分对低密度脂蛋白受体(low-density-lipoprotein receptor,LDLR)基因和蛋白水平的调节作用以及对前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type9,PCSK9)蛋白水平的调节作用。结果表明,Fr1中含有2种聚合度的寡糖,为五糖和六糖; Fr2、Fr3和Fr4均为聚合度均一的寡糖,分别为四糖、三糖和二糖。4个组分中,Fr1、Fr3、Fr4均能显著上调LDLR的蛋白水平,其中Fr3和Fr4的活性尤为显著。Fr3能下调PCSK9的蛋白水平,推测Fr3对LDLR蛋白表达的上调作用可能与PCSK9有关。而Fr4对LDLR的上调作用与PCSK9无关,可能受固醇反应原件结合蛋白-2(SREBP-2)的调节。结果表明,Fr3和Fr4可开发作为食品添加剂用于降低血浆胆固醇。

从药物多肽到蛋白质全合成:酶促拼接的方法原理与前沿应用

蛋白质是生命活动的基础功能元件,其化学合成与定点修饰已成为合成生物学领域探索复杂生物大分子“结构-功能”关系的重要前沿方向。近年来,以多肽固相合成与特异性拼接为核心的蛋白质合成和修饰技术蓬勃发展,打破了生命合成系统仅能使用天然及少数非天然氨基酸的瓶颈,为制备含有数百个氨基酸残基的非天然蛋白质提供了技术平台,让原子水平的蛋白质人工设计成为现实。作为一类广受关注的多肽拼接策略,基于天然或人工改造多肽连接酶的技术方法不仅在基础研究领域拓展了人们对蛋白质这一生命核心元件的理解,还在工业领域崭露头角,被应用于多种多肽类药物的生产。针对蛋白质合成领域中酶促多肽拼接技术平台,本文介绍了Sortase A转肽酶、Butelase 1转肽酶以及Subtilisin人工连接酶的来源以及催化过程,探讨了各自的优势以及局限性,并综述了三种酶在蛋白质修饰、蛋白质合成、多肽药物环化等方面的应用。通过计算机辅助设计、定向进化等技术对转肽酶、连接酶进行改造来提升其在底物谱、催化活性等方面的特性,将化学方法与酶促方法联用来建立多样的生物大分子从头设计与合成路线是目前的主要发展趋势。

Effects of proprotein convertase subtilisin/kexin type-9 inhibitors on fatty liver

BACKGROUND Many studies have investigated the progression of nonalcoholic fatty liver disease(NAFLD)and its predisposing risk factors,but the conclusions from these studies have been conflicting.More challenging is the fact that no effective treatment is currently available for NAFLD.AIM To determine the effects of proprotein convertase subtilisin/kexin type-9(PCSK9)inhibitors on fatty infiltration of the liver.METHODS This retrospective,chart review-based study was conducted on patients,18-yearold and above,who were currently on PCSK9 inhibitor drug therapy.Patients were excluded from the study according to missing pre-or post-treatment imaging or laboratory values,presence of cirrhosis or rhabdomyolysis,or development of acute liver injury during the PCSK9 inhibitor treatment period;the latter being due to false elevation of liver function markers,alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Radiographic improvement was assessed by a single radiologist,who read both the pre-and post-treatment images to minimize reading bias.Fatty infiltration of the liver was also assessed by changes in ALT and AST,with pre-and post-treatment levels compared by paired t-test(alpha criterion:0.05).RESULTS Of the 29 patients included in the study,8 were male(27.6%)and 21 were female(72.4%).Essential hypertension was present in 25(86.2%)of the patients,diabetes mellitus in 18(62.1%)and obesity in 15(51.7%).In all,patients were on PCSK9 inhibitors for a mean duration of 23.69±11.18 mo until the most recent ALT and AST measures were obtained.Of the 11 patients who received the radiologic diagnosis of hepatic steatosis,8(72.73%)achieved complete radiologic resolution upon use of PCSK9 inhibitors(mean duration of 17.6 mo).On average,the ALT level(IU/L)decreased from 21.83±11.89 at pretreatment to 17.69±8.00 at posttreatment(2-tailed P=0.042)and AST level(IU/L)decreased from 22.48±9.00 pretreatment to 20.59±5.47 post-treatment(2-tailed P=0.201).CONCLUSION PCSK9 inhibitors can slow down or even completely resolve NAFLD.

PCSK9 and carbohydrate metabolism:A double-edged sword

Proprotein convertase subtilisin/kexin type 9(PCSK9) plays a paramount role in the degradation of lowdensity lipoprotein(LDL) receptors(LDLR) on the hepatic cells surface and subsequently affects LDL particles catabolism and LDL cholesterol(LDL-c) levels. The anti-PCSK9 monoclonal antibodies lead to substantial decrease of LDL-c concentration. PCSK9(which is also expressed in pancreatic delta-cells) can decrease LDLR and subsequently decrease cholesterol accumulation in pancreatic beta-cells, which impairs glucose metabolism and reduces insulin secretion. Thus, a possible adverse effect of PCSK9 inhibitors on carbohydrate metabolism may be expected by this mechanism, which has been supported by the mendelian studies results. On the other hand, clinical data have suggested a detrimental association of PCSK9 with glucose metabolism. So, the inhibition of PCSK9 may be seen as a double-edged sword regarding carbohydrate metabolism. Completed clinical trials have not shown a detrimental effect of PCSK9 inhibitors on diabetes risk, but their short-term duration does not allow definite conclusions.

The proprotein convertase subtilisin/kexin type 9 geneE670G polymorphism and serum lipid levels in the Guangxi Bai Ku Yao and Han populations

Background The association of E670G polymorphism in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene and serum lipid profiles is inconsistent in dif- ferent ethnic groups.Bai Ku Yao is a special subgroup of the Yao minority in China.The present study was undertaken association of PCSK9 E670G polymorphism and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations.Methods A total of 649 subjects of Bai Ku Yao and 646 participants of Han Chinese were randomly selected from our previous stratified randomized cluster samples.Genotyping of the PCSK9 E670G polymorphism was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis,and then confirmed by direct sequencing. Results The levels of serum total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C) and apolipoprotein(Apo) AI were lower in Bai Ku Yao than in Han(P【0.01 for all).The frequency of A and G alleles was 98.00%and 2.00%in Bai Ku Yao,and 95.20%and 4.80%in Han(P【0.01);respectively. The frequency of AA,AG and GG genotypes was 95.99%,4.01%and 0%in Bai Ku Yao,and 91.02%, 8.36%and 0.62%in Han(P【0.01);respectively.There were also significant differences in the genotypic and allelic frequencies between n and the ratio of ApoAI to ApoB in Han Chinese but not in Bai Ku Yao were different between the AA and AG/GG genotypes(P【0.05 for all).The G allele carriers had higher serum HDL-C and higher ApoAI to ApoB ratio than the G allele noncarriers.When serum lipid parameters in Han were analyzed according to sex,the G allele carriers had higher serum HDL and ApoAI levels in males (P【0.05),and lower ApoB level and higher ApoAI to ApoB ratio in females(P【0.05 for all).Multiple linear regression analysis showed that serum HDL-C levels were correlated with genotypes in both ethnic groups(P【0.05 each).Serum lipid parameters were also correlated with sex,age,body massindex,alcohol consumption,cigarette smoking,and blood pressure in both ethnic groups(P【0.05-0.001).Conclusions These results suggest that the PCSK9 E670G polymorphism is mainly associated with some serum lipid parameters in the Han population,both gender show different relations to different serum lipid parameters.The G allele carriers might have higher serum lipid profiles than the G allele noncarriers. ormal LDL-C(≤3.20 mmol/L) and high LDL-C subgroups (】 3.20 mmol/L,P【0.01;respectively) in Bai Ku Yao, and between normal ApoB(≤1.14 g/L) and high ApoB subgroups(】 1.14 g/L,P 【 0.01;respectively) in Han.

Influence of Single Base Change in Shine-Dalgarno Sequence on the Stability of B.Subtilis Plasmid PSM604

B.Subtilis expression plasmids generally require a stringent Shine Dalgarno Sequence(SDS). Site directed mutagenesis was explored to change the Shine Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.

Intensive lipid-lowering therapy,time to think beyond low-density lipoprotein cholesterol

Statins have been shown to be effective in reducing cardiovascular events.Their magnitude of benefits has been proportionate to the reduction in low-density lipoprotein cholesterol(LDL-c).Intensive lipid-lowering therapies using ezetimibe and more recently proprotein convertase subtilisin kexin 9 inhibitors have further improved clinical outcomes.Unselective application of these treatments is undesirable and unaffordable and,therefore,has been guided by LDL-c level.Nonetheless,the residual risk in the post-statin era is markedly heterogeneous,including thrombosis and inflammation risks.Moreover,the lipoprotein related risk is increasingly recognised to be related to other non-LDL-c markers such as Lp(a).Emerging data show that intensive lipid-lowering therapy produce larger absolute risk reduction in patients with polyvascular disease,post coronary artery bypass graft and diabetes.Notably,these clinical entities share similar phenotype of large burden of atherosclerotic plaques.Novel plaque imaging may aid decision making by identifying patients with propensity to develop lipid rich plagues at multi-vascular sites.Those patients may be suitable candidates for intensive lipid lowering treatment.

Proprotein convertase subtilisin/kexin type 9 inhibitor non responses in an adult with a history of coronary revascularization:A case report

BACKGROUND Familial hypercholesterolemia(FH)is an autosomal dominant disorder that is characterized by severely increased low-density lipoprotein(LDL)cholesterol levels.At the same time,elevated LDL levels accelerated the development of coronary heart disease.Several classes of drugs are currently in use to treat FH.Proprotein convertase subtilisin/kexin type 9 inhibitor(PCSK9i)is novel one of these.CASE SUMMARY This manuscript reports a case of FH that responded modestly after treatment with PCSK9i and statin drugs.Of even more concern is that the patient frequently admitted to the hospital during a 12-year follow-up period.Subsequently,we identified a heterozygous mutation,1448G>A(W483X)of the LDL receptor(LDLR)in this patient.The serum levels of PCSK9(proprotein convertase subtilisin/kexin type 9)in the patient was 71.30±26.66 ng/mL,which is close the average level reported in the literature.This LDLR mutation affects LDLR metabolism or structure,which may make it unsuitable for use of PCSK9i.CONCLUSION Our outcome demonstrates that LDLR-W483X represents a partial loss-of-function LDLR and may contribute to PCSK9i ineffective. In the meanwhile, additional measures aretherefore required (particularly with gene sequencing or change the treatment plan) must beinitiated as early as possible. Genetic testing for clinically challenging cases who do not respond toPCSK9i therapy is very helpful.

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15~C and 30~C, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35~C and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride （PMSF） and hydrochloride 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF）, indicating that it was a serine protease. The presence of Cae＋ and Mnz＋ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid （AA） residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.

Changes in proprotein convertase subtilisin/kexin type 9 mRNA expression in rat cortex after cerebral ischemia

Oxidized low density lipoprotein is a risk factor for cerebrovascular disease. Proprotein convertase subtilisin/kexin type 9 （PCSK9） can increase the level of low density lipoprotein. Therefore, this study assumed that PCSK9 plays important roles in ischemic cerebrovascular disease. The present study established transient focal cerebral ischemia models after 100 minutes of middle cerebral artery occlusion. In situ hybridization demonstrated that PCSK9 mRNA expression increased gradually with prolonged reperfusion time in ischemic cortices. This indicated that transient focal cerebral ischemia upregulated PCSK9 mRNA expression in ischemic cortices.

Characterizing proteases in an Antarctic Janthinobacterium sp. isolate: Evidence of a protease horizontal gene transfer event

We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium （named AU 11）, from a water sample collected in Lake Uruguay （King George Island, South Shetlands）. AUI 1 （growth between 4℃ and 30℃） produces a single cold-active extracellular protease （ExPAU11）, differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-ToF MS） as an alkaline metallo-protease （70% coverage with an extracellular protease of Janthinobacterium sp. PI12）, and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene （named JSP8A） showing 60% identity （98% query coverage） to subtilisin peptidases belonging to the $8 family （S8A subfamily） of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla （including its own）. An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer （HGT） from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

Value of serum PCSK9 for evaluating the severity in patients with type 2 diabetes mellitus and atherosclerosis

Objective:To study the value of serum PCSK9 for evaluating the severity in patients with type 2 diabetes mellitus and atherosclerosis. Methods:Type 2 diabetic patients with carotid atherosclerosis who were treated in our hospital between May 2014 and September 2016 were selected as group A (n=69), type 2 diabetic patients without carotid atherosclerosis were selected as group B (n=79) and healthy subjects were selected as group C (n=55). Serum levels of SPCSK9, lipid metabolism indexes and oxidative stress indexes of three groups of subjects were detected. Results:Serum proprotein convertase subtilisin kexin 9 (PCSK9), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), apolipoprotein B (ApoB), CyPA, paraoxonase-1 (PON-1), PON-3, malondialdehyde (MDA) and 8-iso-prostaglandin F-2α(8-iso-PGF2α) levels of group A and group B were significantly higher than those of group C (P<0.05) while high-density lipoprotein cholesterol (HDL-C) and apolipoprotein AI (ApoAI) levels were significantly lower than those of group C (P<0.05);serum PCSK9, TG, TC, LDL-C, ApoB, CyPA, PON-1, PON-3, MDA and 8-iso-PGF2αlevels of group A were significantly higher than those of group B (P<0.05) while HDL-C and ApoAI levels were significantly lower than those of group B (P<0.05);serum TG, TC, LDL-C, ApoB, CyPA, PON-1, PON-3, MDA and 8-iso-PGF2αlevels of high PCSK9 level subgroup in group A were significantly higher than those of low PCSK9 level subgroup (P<0.05) while HDL-C and ApoAI levels were significantly lower than those of low PCSK9 level subgroup (P<0.05). Conclusions:Abnormally elevated serum PCSK9 can affect lipid metabolism and enhance oxidative stress in patients with type 2 diabetes mellitus and atherosclerosis.

Research on Hepatocyte Regulation of PCSK9-LDLR and Its Related Drug Targets

The prevalence of hyperlipidemia has increased significantly due to genetic, dietary, nutritional and pharmacological factors, and has become one of the most common pathological conditions in humans. Hyperlipidemia can lead to a range of diseases such as atherosclerosis, stroke, coronary heart disease, myocardial infarction, diabetes, and kidney failure, etc. High circulating low-density lipoprotein cholesterol (LDL-C) is one of the causes of hyperlipidemia. LDL-C in the blood binds to LDL receptor (LDLR) and regulates cholesterol homeostasis through endocytosis. In contrast, proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates LDLR degradation via the intracellular and extracellular pathways, leading to hyperlipidemia. Targeting PCSK9-synthesizing transcription factors and downstream molecules are important for development of new lipid-lowering drugs. Clinical trials regarding PCSK9 inhibitors have demonstrated a reduction in atherosclerotic cardiovascular disease events. The purpose of this review was to explore the target and mechanism of intracellular and extracellular pathways in degradation of LDLR and related drugs by PCSK9 in order to open up a new pathway for the development of new lipid-lowering drugs.

反胶团萃取分离纯化纳豆激酶

纳豆激酶是一新型溶栓药物,它和SubtilisinCarlsberg为同源蛋白.以SubtilisinCarlsberg为模拟蛋白,以AOT/isooctane反胶团体系为有机相,研究了各种萃取条件和反萃取条件对SubtilisinCarlsberg萃取过程的影响.在对模拟体系研究的基础上,利用AOT/isooctane反胶团体系从发酵液中分离纯化纳豆激酶.结果表明,纳豆激酶的萃取行为和SubtilisinCarlsberg非常类似,以目标蛋白的同源蛋白作为模拟蛋白研究萃取规律是合理可行的.纳豆激酶经过一次萃取循环,蛋白质回收率约为33.25%,酶活力回收率达到80.2%,纯化因子约为2.5左右.

Effect of Quercetin on Atherosclerosis Based on Expressions of ABCA1, LXR-α and PCSK9 in ApoE-/- Mice

Objective:To investigate the effect of quercetin on ATP binding cassette transporter A1(ABCA1),liver X receptor(LXR),and proprotein convertase subtilisin/kexin type 9(PCSK9)expressions in apoE-knockout(ApoE-/-)mice.Methods:The high-fat diet-induced atherosclerosis(AS)in ApoE-/-mice was established.Thirtysix mice were divided into 3 groups using random number table method:model group(n=12),quercetin group(n=12),and atorvastatin group(n=12),with C57BL/6J mice of the same strain and age as the control group(n=12).Quercetin group and atorvastatin group were administrated with quercetin and atorvastatin by oral gavage,with doses of 12.5 and 4 mg/(kg・d),respectively.Animals in the control and model groups were given an equal volume of distilled water by oral gavage once per day for a total of 12 weeks.Western blot and immunohistochemical methods were employed to determine the aortic ABCA1,LXR-a and PCSK9 protein expressi on.En zyme linked imm uno sorbent assay method was used to detect the expressi on of serum total cholesterol(TC),triglyceride(TG),high density lipoprotein-cholesterol(HDL-C),low density lipoproteincholesterol(LDL-C),tumor necrosis factor-a(TNF-a),interleukin-6(IL-6),and IL-10,combined with tissue pathological exami nation.Results:ApoE-/-mice fed with a high-fat diet had no table atherosclerosis lesions,with reduced ABCA1,LXR-a and IL-10 levels(all P<0.01),elevated PCSK9,TNF-a and IL-6 expression,and increased TC and LDL-C con tents(all P<0.01).After querceti n in terventi on,the areas of AS plaques and the expressions of PCSK9,TNF-a and IL-6 were significantly reduced(all P<0.01),while the expressions of ABCA1 and LXR-a were increased significantly(all P<0.01).Conclusion:Quercetin effectively interfered with AS development by regulating the expressions of ABCA1,LXR-a and PCSK9 in ApoE,mice.

A novel mutation in proprotein convertase subtilisin/kexin type 9 gene leads to familial hypercholesterolemia in a Chinese family

Background Familial hypercholesterolemia （FH） is an autosomal disorder associated with elevated plasma low density lipoprotein （LDL） levels leading to premature coronary heart disease （CHD）. As a result of long-term hyperlipemia, FH patients will present endarterium thickening and atherosclerosis. In the present study we scanned the related gene of a clinically diagnosed autosomal genetic hypercholesterolemia family for the possible mutations and established eukaryotic expression vector of mutation of proprotein convertase subtilisin/kexin type 9 （PCSK9） gene with gene recombination technique to investigate the contributions of the variation on low density lipoprotein receptor （LDL-R） metabolism and function alternation.Methods Mutation detection was conducted for LDL-R, apolipoprotein B100 （apoB100） and PCSK9 gene with nucleotide sequencing in a Chinese FH family. The full-length cDNA of wild type PCSK9 gene （WT-PCSK9） was obtained from Bel-7402. Site mutagenesis was used to establish the recombinant eukaryotic expression vector carrying pathogenic type of PCSK9 gene and the inserted fragment was sequenced. With the blank vector as control, liposome transfection method was used to transfect the Bel-7402 cells with recombinant plasmid. The expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of LDL-R protein were determined by Western blotting. Results The G→T mutation at the 918 nucleotide of PCSK9 gene resulted in the substitution of the arginine by a serine at the codon 306 of exon 6. After sequencing, it was confirmed that the inserted fragment of established expression vector had correct size and sequence and the mutant was highly expressed in Bel-7402 cells. There was no significant variation in the levels of LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12% after the cells were transfected by R306S mutant as evidenced by gray scale scanning, suggesting that the new mutant R306S can significantly decrease the expression of mature LDL-R protein.Conclusions A novel missense mutation of PCSK9 gene, R306S, was found and the eukaryotic expression vectors of mutant and wild-type of PCSK9 gene were established. There was no significant variation in the levels of LDL-R mRNA. The R306S mutation could significantly lead to the decrease of LDL-R mature protein expression, which might be the pathogenic gene of the FH family.