Jack Allicia, Mohd Muzaida Aminah, Kamaruddin Nurjannatul Naim, Mohd Din Lukman Hakim, Hajri Nor Azwin, Tengku Muhammad Tengku Sifzizul
Nutrition & Food Safety Programme, Food Science & Technology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI), Persiaran MARDI-UPM, 43400 Serdang, Selangor, Malaysia.
Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia.
Saudi J Biol Sci. 2021 Dec;28(12):7105-7116. doi: 10.1016/j.sjbs.2021.08.003. Epub 2021 Aug 8.
has been long used as traditional medicinal resources and reported to demonstrate various important bioactivities such as anticoagulation, antithrombosis, anti-hyperglycemia and anticancer. However, its lipid lowering activity is yet to be fully explored. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that enhances the lysosomal degradation of hepatic low density lipoprotein receptor (LDLR) resulting in excessive accumulation of the plasma levels of LDL-cholesterols (LDL-C) which subsequently accelerate atherosclerosis. In the present study, fractions were subjected to promoter-reporter luciferase assay to determine its role as PCSK9 inhibitors. It was found both fractions (EFA and EFB) reduced the transcriptional activity of PCSK9 promoter. Among the seven 5'end deletion constructs of PCSK9 promoter, fragments D1 (-1,711/-94), D3 (-709/-94) and D4 (-440/-94), were suppressed in the presence of both fractions whereas D2 (-1,214/-94), and, D6 (-351/-94) as well as D7 (-335/-94) were inhibited only by EFA and EFB, respectively. Further transcription factor binding sites prediction using MatInspector software discovered various potential -regulatory elements namely, PPAR, KLFs, RBPJ-kappa and SREBP that may potentially be involved in ameliorating the transcriptional activity of PCSK9. Immunofluorescence staining was used to evaluate the effects of both fractions on LDL-C and LDLR. Results showed that levels of LDL-C uptake in EFA-treated cells were 69.1% followed by EFB at 32.6%, as compared to untreated control after 24 h treatment. The LDLR protein distribution was induced by 62.41% and 32.2%, which corresponded to an increase in LDL-C uptake in both EFA and EFB treatment, respectively. Hence, the inhibition of PCSK9 by bioactive compounds in EFA and EFB could be another promising therapeutic agent in reducing the cholesterol levels and atherosclerosis by targeting PCSK9.
长期以来一直被用作传统药用资源，并据报道具有多种重要的生物活性，如抗凝、抗血栓形成、抗高血糖和抗癌作用。然而，其降脂活性尚未得到充分探索。前蛋白转化酶枯草溶菌素/kexin 9型（PCSK9）是一种酶，它可增强肝脏低密度脂蛋白受体（LDLR）的溶酶体降解，导致血浆中低密度脂蛋白胆固醇（LDL-C）水平过度积累，进而加速动脉粥样硬化。在本研究中，对各组分进行启动子-报告基因荧光素酶测定，以确定其作为PCSK9抑制剂的作用。结果发现，两个组分（EFA和EFB）均降低了PCSK9启动子的转录活性。在PCSK9启动子的七个5'端缺失构建体中，片段D1（-1,711/-94）、D3（-709/-94）和D4（-440/-94）在两个组分存在的情况下均受到抑制，而D2（-1,214/-94）、D6（-351/-94）以及D7（-335/-94）分别仅被EFA和EFB抑制。使用MatInspector软件进一步预测转录因子结合位点，发现了各种潜在的调控元件，即PPAR、KLFs、RBPJ-κ和SREBP，它们可能参与改善PCSK9的转录活性。采用免疫荧光染色来评估两个组分对LDL-C和LDLR的影响。结果显示，处理24小时后，与未处理的对照相比，EFA处理的细胞中LDL-C摄取水平为69.1%，其次是EFB处理的细胞，为32.6%。LDLR蛋白分布分别被诱导了62.41%和32.2%，这分别对应于EFA和EFB处理中LDL-C摄取的增加。因此，EFA和EFB中的生物活性化合物对PCSK9的抑制作用可能是另一种有前景的治疗剂，通过靶向PCSK9来降低胆固醇水平和治疗动脉粥样硬化。
