新型米糠活性肽调控胆固醇代谢作用机制的研究

Hypothetical protein OsJ_13801, which has the bile acid binding ability in rice bran protein, is taken as the study object and the active peptides are separated and purified by gel filtration chromatography to explain the mechanism of cholesterol metabolism regulation by novel rice bran active peptides at molecular level. First, the Hypothetical protein OsJ_13801 is separated and purified by HiLoad 26/60 Superdex 200 p. g column, followed by pepsin-restricted hydrolysis, and then the active peptides group for cholesterol metabolism regulation is screened by in vitro bile acid binding ability experiment, cholesterol micelle solubility inhibition experiment and in vivo animal experiment. Secondly, the active peptides are separated and purified by SuperdexPeptidePC3.2/30 column, and the molecular weight and definite structure of active peptides are identified. The effect of novel rice bran active peptides on intestinal cholesterol absorption is investigated by in vitro bile acid binding ability experiment and cholesterol micelle solubility inhibition experiment. The in vivo experiment is performed to detect rat’s serum TC, TG and HDL-C level, liver TC, TG, fecal bile acids and cholesterol level, and total mRNA expression levels of liver and intestinal cholesterol metabolism related genes HMGR, LDLR, CYP7A1, NPC1L1, and ABCA1 to explain the mechanism of novel rice bran active peptides in improving cholesterol metabolism at molecular level. The study results will provide a theoretical basis for improvement cholesterol metabolism by bioactive peptides and create greater economic and social benefits by improving the development and utilization of rice bran active peptides.

以米糠（RB）蛋白中有胆汁酸结合能力的Hypothetical protein OsJ_13801为研究对象，用凝胶过滤层析（SEC）分离活性肽，从分子水平阐明新型RB活性肽调控胆固醇(CHOL)代谢作用机制。①SEC分离Hypothetical protein OsJ_13801，再胃蛋白酶水解，通过in vitro 胆汁酸结合能力实验、CHOL胶束溶解度抑制实验及in vivo 动物实验筛选调控CHOL代谢作用的肽群。用SEC分离肽群，鉴定其结构。②通过in vitro 胆汁酸结合能力实验、CHOL胶束溶解度抑制实验探究新型RB活性肽对小肠CHOL吸收的影响，in vivo 中检测大鼠血清及肝脏脂质水平，粪便胆汁酸、CHOL及其肝脏和小肠CHOL代谢相关基因表达，从分子水平阐明新型RB活性肽调控CHOL代谢作用机制。本成果将活性肽调控CHOL代谢做理论依据，提高RB活性肽的利用价值。

本课题以米糠蛋白中具有胆汁酸结合能力的Hypothetical protein OsJ_13801（HPO）为研究对象，（1）利用凝胶过滤层析法分离提纯并采用胃蛋白酶进行适度酶解（1h、3h、6h和12h），基于in vitro 胆固醇胶束溶解度抑制率、胰脂肪酶抑制率和胆固醇脂肪酶抑制率实验，筛选其多肽组分，LC-MS/MS法检测其氨基酸序。（2）采用在线酶水解模拟软件ExPASy PeptideCutter对以上蛋白进行虚拟水解，以疏水性为指标，合成了8种多肽。（3）基于in vitro 胆固醇胶束溶解度抑制率实验结果，采用高脂模型动物实验评价多肽VAYVL的降血脂作用及其分子机制。（4）探究米糠源8种多肽对HepG2细胞脂肪堆积的影响，基于反向对接、网络药理学和分子对接等生物信息学分析方法，分析其调控胆固醇代谢作用的分子机制。研究结果表明，米糠HPO胃蛋白酶酶解产物中F3-6和F3-12的体外降血脂作用最好；通过水解软件获得196种不同的多肽和氨基酸，疏水性较高的多肽LPPF、PLPPF、FFFAPGG、VAYVL、VFVAPAG、IVTK、8EQQK、GQ6EQVGQG中FFFAPGG和VAYVL显著抑制了胆固醇胶束溶解度-28.90%和-98.30%，能与考来烯胺作用相媲美；VAYVL(500 mg/kg B.W/d)的摄入与高脂模型组大鼠相比较对进食量及体重增加量无显著影响，但是能显著降低血清TC（P<0.05）和LDL+VLDL-C(P<0.05），同时对肝脏LDL-R、CYP7A1、HMGCR等的酶活性无影响；8种多肽中FFFAPGG显著降低油酸诱导的HepG2细胞脂肪堆积，且能降低FAS、SREBP1c、HMGCR和ACC mRNA表达。在HepG2细胞中，FFFAPGG减少了油红O染色，细胞内TC和TG的积累，而加入AMPK抑制剂（Compound C）后，HMGCR显著逆转了ACC的下调。分子对接和网络药理分析发现，FFFAPGG具有极高的蛋白亲和力，能够在AMPK通路中发挥降血脂活性，并可直接与AMPK相互作用。米糠源多肽FFFAPGG可以通过AMPK/HMGCR/ACC轴显著提高细胞内脂质积累水平，从而发挥其降血脂作用。以上结果为米糠源多肽在降血脂作用中的应用提供了理论依据。