技术领域Technical field
本发明涉及纳米生物医药领域，具体是指一种丙二酸修饰的富勒烯C70在制备治疗非酒精性脂肪肝病药物中的应用。The invention relates to the field of nano-biomedicine, and specifically refers to the application of malonate-modified fullerene C70 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.
背景技术Background technique
随着人们生活水平的提高和生活方式的改变，我国非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)的患者人数逐年增加，已经成为慢性肝病的重要病因。据统计，目前全球拥有超过10亿非酒精性脂肪肝患者，我国成人NAFLD的发病率也达20％左右[1-3]。NAFLD患者通常无过量饮酒史，以弥漫性肝细胞大泡性脂肪变和脂肪蓄积为病理特征。而且，如果非酒精性脂肪肝缺乏及时治疗，还可以发展为非酒精性脂肪肝炎(nonalcoholic steatohepatitis,NASH)、肝纤维化、肝硬化乃至肝癌[4]。然而目前临床并无理想的预防和治疗药物，形势非常严峻。With the improvement of people's living standards and changes in lifestyle, the number of patients with nonalcoholic fatty liver disease (NAFLD) in my country is increasing year by year, and it has become an important cause of chronic liver disease. According to statistics, there are currently more than 1 billion patients with non-alcoholic fatty liver disease in the world, and the incidence of NAFLD among adults in my country is about 20% [1-3]. Patients with NAFLD usually have no history of excessive drinking and are pathologically characterized by diffuse hepatocellular macrovesicular steatosis and fat accumulation. Moreover, if non-alcoholic fatty liver disease lacks timely treatment, it can develop into non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and even liver cancer [4]. However, there are currently no ideal preventive and therapeutic drugs in clinical practice, and the situation is very serious.
富勒烯是除石墨、金刚石和无定型碳之外碳元素的另一种同素异形体，是一种完全由碳组成的笼状中空分子。C70是最常见的富勒烯之一。富勒烯材料具有清除自由基、抗氧化、抗衰老、抑制细胞凋亡等丰富的生物活性，其水溶性衍生物具有良好的生物应用前景[5-7]。然而，目前水溶性富勒烯衍生物大多通过强酸强碱或双氧水，在富勒烯表面引入羟基、氨基或氨基酸等水溶性基团得到。这类制备方法条件苛刻，富勒烯本体结构破坏严重，容易导致富勒烯活性下降甚至产生毒性。此外，这种富勒烯衍生物一般无确定结构或分子式，导致产品批次有差异不易质控，难以成药，限制了其在医学领域的药用价值。Fullerene is another allotrope of the carbon element in addition to graphite, diamond and amorphous carbon. It is a cage-like hollow molecule composed entirely of carbon. C70 is one of the most common fullerenes. Fullerene materials have rich biological activities such as free radical scavenging, antioxidant, anti-aging, and inhibition of apoptosis. Its water-soluble derivatives have good biological application prospects [5-7]. However, at present, water-soluble fullerene derivatives are mostly obtained by introducing water-soluble groups such as hydroxyl, amino or amino acids on the surface of fullerene through strong acid, strong alkali or hydrogen peroxide. This type of preparation method has harsh conditions and severely damages the fullerene structure, which can easily lead to a decrease in fullerene activity or even toxicity. In addition, such fullerene derivatives generally do not have a definite structure or molecular formula, resulting in differences in product batches and difficult quality control, making it difficult to make drugs, which limits their medicinal value in the medical field.
发明内容Contents of the invention
针对现有技术的不足，本发明目的是提供一种具有确定结构的C70-丙二酸衍生物在制备治疗非酒精性脂肪肝病药物中的应用。In view of the shortcomings of the prior art, the purpose of the present invention is to provide a C70-malonic acid derivative with a definite structure for use in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.
为实现上述目的，本发明提供的丙二酸修饰的富勒烯C70在制备治疗非酒精性脂肪肝病药物中的应用。In order to achieve the above object, the invention provides the application of malonate-modified fullerene C70 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.
作为优选方案，所述C70-丙二酸衍生物的结构式如下：As a preferred embodiment, the structural formula of the C70-malonic acid derivative is as follows:
其中R选自H和Na，n取值为1-70。 Where R is selected from H and Na, and the value of n is 1-70.
进一步地，所述C70-丙二酸衍生物在调控肝细胞的脂质代谢中具有重要作用：通过C70-丙二酸衍生物处理小鼠原代肝细胞以及C57BL/6小鼠NASH模型，发现C70-丙二酸衍生物能够显著地抑制肝脏细胞的脂质堆积。Furthermore, the C70-malonic acid derivative plays an important role in regulating lipid metabolism of hepatocytes: by treating primary mouse hepatocytes and the C57BL/6 mouse NASH model with C70-malonic acid derivatives, it was found that C70-malonic acid derivatives can significantly inhibit lipid accumulation in liver cells.
更进一步地，以所述C70-丙二酸衍生物为活性成分制备预防、缓解和/或治疗非酒精性脂肪肝病的药物。Furthermore, the C70-malonic acid derivative is used as an active ingredient to prepare a medicine for preventing, alleviating and/or treating non-alcoholic fatty liver disease.
更进一步地，所述非酒精性脂肪肝病包括：单纯性肝脏脂肪肝变性、非酒精性脂肪性肝炎，肝纤维化、肝硬化和肝癌。Furthermore, the non-alcoholic fatty liver disease includes: simple fatty liver degeneration, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis and liver cancer.
本发明所提供的以C70-丙二酸衍生物为活性成分的预防、缓解和/或治疗非酒精性脂肪肝病及其相关疾病药物具有以下优点：The medicine for preventing, alleviating and/or treating non-alcoholic fatty liver disease and related diseases using C70-malonic acid derivatives as active ingredients provided by the present invention has the following advantages:
1、药效明显，C70-丙二酸衍生物可以显著抑制小鼠原代肝细胞中的脂质堆积，10μM就能看到明显的变化。此外，C70-丙二酸衍生物对HFHC饮食诱导产生的肝脏脂质堆积、纤维化也具有明显的改善作用。1. The drug has obvious efficacy. C70-malonic acid derivatives can significantly inhibit lipid accumulation in mouse primary liver cells. Obvious changes can be seen at 10 μM. In addition, C70-malonic acid derivatives also have a significant improvement effect on liver lipid accumulation and fibrosis induced by HFHC diet.
2、丙二酸修饰富勒烯C70能有效降低富勒烯改性过程中碳笼的破坏，从而更好保留富勒烯本体的药效活性。2. Malonic acid-modified fullerene C70 can effectively reduce the damage of the carbon cage during the fullerene modification process, thereby better retaining the pharmacodynamic activity of the fullerene itself.
3、C70-丙二酸衍生物具有确定的分子结构，成功解决了其它水溶性富勒烯衍生物无确定结构、质控难的问题，有利于成药。3. C70-malonic acid derivatives have a definite molecular structure, which successfully solves the problems of other water-soluble fullerene derivatives with no definite structure and difficult quality control, which is beneficial to pharmaceutical production.
附图说明Description of the drawings
附图1实施例一制备得到的C70-丙二酸衍生物水解前体质谱图Figure 1: Mass spectrum of hydrolysis precursor of C70-malonic acid derivative prepared in Example 1
附图2不同浓度的C70-丙二酸衍生物预处理小鼠原代肝细胞再加入PA/OA处理12h后的油红染色结果图Figure 2. Oil red staining results of mouse primary liver cells pretreated with different concentrations of C70-malonic acid derivatives and then treated with PA/OA for 12 hours.
附图3对照组小鼠以及给以C70-丙二酸衍生物组小鼠体重检测结果(*：p≤0.05)。Figure 3: Body weight test results of mice in the control group and mice in the C70-malonic acid derivative group (*: p≤0.05).
附图4对照组小鼠以及给以C70-丙二酸衍生物组小鼠肝重检测结果(*：p≤0.05)。Figure 4: Liver weight detection results of mice in the control group and mice in the C70-malonic acid derivative group (*: p≤0.05).
附图5对照组小鼠以及给以C70-丙二酸衍生物组小鼠脂肪重量检测结果(**：p≤0.01)。Figure 5: Fat weight detection results of mice in the control group and mice in the group given C70-malonic acid derivatives (**: p≤0.01).
附图6对照组小鼠以及给以C70-丙二酸衍生物组小鼠肝脏的H&E染色图(×20)。Figure 6: H&E staining pictures of livers of mice in the control group and mice in the group given C70-malonic acid derivatives (×20).
附图7对照组小鼠以及给以C70-丙二酸衍生物组小鼠组肝脏的马松染色图(×20)。Figure 7 shows Masson staining of the livers of the mice in the control group and the mouse group given C70-malonic acid derivatives (×20).
具体实施方式Detailed ways
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容The features and advantages of the present invention can be further understood from the following detailed description in conjunction with the accompanying drawings. The provided embodiments are only illustrative of the method of the present invention and do not limit the rest of the disclosure of the present invention in any way.
【实施例1】C70-丙二酸衍生物C70[C(COONa)2]4的制备[Example 1] Preparation of C70-malonic acid derivative C70 [C(COONa)2 ]4
50mg C70溶于10ml甲苯，氮气保护下加入0.8ml溴代丙二酸二乙酯和0.7ml DBU。室温下搅拌反应约2h。硅胶柱层析分离提纯(乙酸乙酯：甲苯＝1：99)，取第4色带，减压浓缩。将所得产品于氮气保护下加NaH水解，60℃下搅拌反应约6h。反应结束后加入甲醇淬灭过量的NaH，并离心得到沉淀。乙醇洗涤沉淀三次及即可得到棕褐色C70[C(COONa)2]4产品，其质谱结果如图1所示。Dissolve 50mg C70 in 10ml toluene, add 0.8ml diethyl bromomalonate and 0.7ml DBU under nitrogen protection. The reaction was stirred at room temperature for about 2 hours. Separate and purify by silica gel column chromatography (ethyl acetate:toluene=1:99), take the fourth color band, and concentrate under reduced pressure. The obtained product was hydrolyzed by adding NaH under nitrogen protection, and the reaction was stirred at 60°C for about 6 hours. After the reaction, methanol was added to quench excess NaH, and centrifuged to obtain precipitate. Wash the precipitate three times with ethanol to obtain the tan C70 [C(COONa)2 ]4 product. The mass spectrometry results are shown in Figure 1.
【实施例2】C70-丙二酸衍生物C70[C(COONa)2]4预处理减轻肝细胞脂肪积累[Example 2] Pretreatment of C70-malonic acid derivative C70 [C(COONa)2 ]4 reduces fat accumulation in liver cells
小鼠原代肝细胞的分离和培养：Isolation and culture of mouse primary hepatocytes:
采用Ⅳ型胶原酶消化分离小鼠原代肝细胞。小鼠乙醚吸入麻醉，留置针穿刺门静脉，肝脏原位灌流(SC-1和SC-2轮流灌注)至肝脏消化完全，取下肝脏，反复吹打并过滤，收集肝细胞悬液。Primary mouse hepatocytes were isolated by digestion with type IV collagenase. The mice were anesthetized by ether inhalation, an indwelling needle was inserted into the portal vein, and the liver was perfused in situ (SC-1 and SC-2 were perfused alternately) until the liver was completely digested. The liver was removed, piped and filtered repeatedly, and the hepatocyte suspension was collected.
培养皿包被：用灭菌的超纯水将无水乙醇稀释为30％乙醇后，0.22μM滤器过滤，然后将100×的鼠尾胶稀释为1×。在六孔板中每孔加入稀释后的鼠尾胶200μl，旋转六孔板，使胶铺满整个板底。开盖，在超净台吹风过夜。Petri dish coating: Dilute absolute ethanol to 30% ethanol with sterile ultrapure water, filter with a 0.22 μM filter, and then dilute 100× rat tail glue to 1×. Add 200 μl of diluted rat tail glue to each well of a six-well plate, and rotate the six-well plate so that the glue covers the entire bottom of the plate. Open the lid and let it air dry overnight.
铺板：分离的原代肝细胞经台盼蓝计数，之后铺6孔板，每孔细胞数目为1×106个，细胞大约6-8h贴壁。Plating: The isolated primary hepatocytes were counted with trypan blue, and then plated on a 6-well plate. The number of cells in each well was 1×106 . The cells adhered in about 6-8 hours.
将小鼠原代肝细胞分为2组，分别为PA/OA组，C70-丙二酸衍生物10μM PA/OA组，C70-丙二酸衍生物20μM PA/OA组。待细胞贴壁后，分别加入PBS、10μM C70-丙二酸衍生物、20μM C70-丙二酸衍生物处理3h，然后用0.2/0.4mM PA/OA处理12h后，进行油红O染色。染色结果如图2所示，与PA/OA组相比，C70-丙二酸衍生物预处理组红色脂肪滴的量明显降低。该结果说明，经C70-丙二酸衍生物预处理可显著抑制肝细胞脂质积累。Mouse primary hepatocytes were divided into 2 groups, namely PA/OA group, C70-malonic acid derivative 10 μM PA/OA group, and C70-malonic acid derivative 20 μM PA/OA group. After the cells adhered, PBS, 10 μM C70-malonic acid derivatives, and 20 μM C70-malonic acid derivatives were added for 3 hours, and then treated with 0.2/0.4mM PA/OA for 12 hours before Oil Red O staining. The staining results are shown in Figure 2. Compared with the PA/OA group, the amount of red fat droplets in the C70-malonic acid derivative pretreatment group was significantly reduced. This result shows that pretreatment with C70-malonic acid derivatives can significantly inhibit lipid accumulation in hepatocytes.
【实施例3】C70-丙二酸衍生物C70[C(COONa)2]4抑制非酒精性脂肪肝病的进程[Example 3] C70-malonic acid derivative C70 [C(COONa)2 ]4 inhibits the progression of non-alcoholic fatty liver disease
1实验动物及饲养1Experimental animals and feeding
实验动物：选用8-10周龄，体重在23.5-27.5g，雄性C57BL/6小鼠为实验对象。Experimental animals: Male C57BL/6 mice aged 8-10 weeks and weighing 23.5-27.5g were selected as experimental subjects.
饲养环境：SPF级实验动物中心，SPF级小鼠饲料购自北京华阜康生物科技有限公司。饲养条件：室温在22-24℃之间，湿度在40-70％之间，明暗交替照明时间为12h，自由饮水摄食。Breeding environment: SPF grade experimental animal center, SPF grade mouse feed was purchased from Beijing Huafukang Biotechnology Co., Ltd. Raising conditions: room temperature between 22-24°C, humidity between 40-70%, alternating light and dark lighting time of 12 hours, free access to water and food.
2模型构建及给药2 Model construction and drug administration
C57小鼠(共20只)利用高脂高胆固醇(HFHC)喂养10周，建立NASH模型。之后，小鼠被分为两组(每组10只，分别为给药组和对照组)，对照组通过腹腔注射生理盐水，给药组通过腹腔注射C70-丙二酸衍生物C70[C(COONa)2]4(10mg/kg)(给药频率：每天一次)，同时继续HFHC喂养4周后取材。测定两组小鼠的肝重、白脂重量，并对小鼠肝脏进行HE染色及马松染色。C57 mice (20 mice in total) were fed high-fat and high-cholesterol (HFHC) for 10 weeks to establish a NASH model. After that, the mice were divided into two groups (10 mice in each group, namely the administration group and the control group). The control group was injected intraperitoneally with normal saline, and the administration group was intraperitoneally injected with C70-malonic acid derivative C70 [C (COONa)2 ]4 (10 mg/kg) (administration frequency: once a day), while continuing HFHC feeding for 4 weeks and then taking samples. The liver weight and white lipid weight of the two groups of mice were measured, and the mouse livers were stained with HE and Masson staining.
3病理学检测3Pathological testing
3.1制备石蜡标本切片3.1 Preparing paraffin specimen sections
主要操作程序包括：修剪肝脏→包埋框处理→流水冲洗→脱水→透明→浸蜡→包埋→切片→摊片→晾干或烘烤后备用。The main operating procedures include: trimming the liver → processing the embedding frame → rinsing with running water → dehydration → transparency → dipping in wax → embedding → slicing → spreading → drying or baking for later use.
3.2苏木精-伊红(H&E)染色3.2 Hematoxylin-eosin (H&E) staining
主要步骤为：取石蜡标本切片55℃烘烤30min→二甲苯5min，3次→100％酒精1min→95％酒精1min→70％酒精1min→双蒸水1min→苏木精溶液5min→水洗1min→1％盐酸酒精1-3s→水洗1min→Scott液1min→水洗1min→伊红溶液3-5min→蒸馏水洗去浮色→70％酒精1s→95％酒精1s→100％酒精30s，3次→二甲苯2min，3次→趁二甲苯未干立即封片→通风橱内吹干，显微镜拍照。The main steps are: take paraffin specimen slices and bake at 55°C for 30 min → xylene for 5 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → double distilled water for 1 min → hematoxylin solution for 5 min → wash with water for 1 min → 1% hydrochloric acid alcohol 1-3s→Water 1min→Scott solution 1min→Water 1min→Eosin solution 3-5min→Distilled water to wash away floating color→70% alcohol 1s→95% alcohol 1s→100% alcohol 30s, 3 times→Two Toluene for 2 minutes, 3 times → Immediately seal the slide while the xylene is still wet → Blow dry in a fume hood and take pictures under a microscope.
3.3马松(Masson)染色3.3 Masson staining
主要步骤为：取石蜡标本切片55℃烘烤30min→二甲苯5min，3次100％酒精1min→95％酒精1min→70％酒精1min→双蒸水1min→苏木精溶液7min→自来水5min→1％盐酸酒精分化1-3s→流水冲洗5min→双蒸水1min→丽春红酸性品红溶液7min→双蒸水5min→磷钼酸水溶液分化5min→苯胺蓝染色5min→1％冰醋酸1min×2次→70％酒精1s→95％酒精1s→100％酒精30s，3次→二甲苯2min，3次→趁二甲苯未干立即封片→通风橱内吹干，显微镜拍照。The main steps are: take paraffin specimen slices and bake at 55°C for 30 min → xylene for 5 min, 3 times 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → double distilled water for 1 min → hematoxylin solution for 7 min → tap water for 5 min → 1 Differentiation with % hydrochloric acid and alcohol for 1-3 seconds→Rinse with running water for 5 min→Double-distilled water for 1 min→Ponceau acid fuchsin solution for 7 min→Double-distilled water for 5 min→Differentiate with phosphomolybdic acid aqueous solution for 5 min→Aniline blue staining for 5 min→1% glacial acetic acid 1 min×2 times → 70% alcohol for 1 s → 95% alcohol for 1 s → 100% alcohol for 30 s, 3 times → xylene for 2 min, 3 times → seal the slide immediately while the xylene is still wet → blow dry in the fume hood and take pictures under the microscope.
两组小鼠体重、肝重及白脂重量的变化结果如图3、4、5所示，给药组小鼠的体重、肝重及白脂重量显著低于对照组。H&E染色及马松染色结果如图6、7所述，可以观察到对照组小鼠的肝细胞发生脂肪变性，空泡化且融合连成片状，肝脏细胞形态受到严重破坏，而给药组中小鼠肝细胞形态得以显著改善，此外肝脏纤维化情况同样得到减轻。以上结果说明C70-丙二酸衍生物C70[C(COONa)2]4对非酒精性脂肪肝病具有良好的治疗效果。The results of the changes in body weight, liver weight and white fat weight of the two groups of mice are shown in Figures 3, 4 and 5. The body weight, liver weight and white fat weight of the mice in the administration group were significantly lower than those in the control group. The results of H&E staining and Masson staining are shown in Figures 6 and 7. It can be observed that the liver cells of mice in the control group undergo fatty degeneration, vacuolation and fusion into sheets, and the morphology of liver cells is severely damaged, while in the drug group The morphology of liver cells in mice was significantly improved, and liver fibrosis was also reduced. The above results indicate that C70-malonic acid derivative C70 [C(COONa)2 ]4 has a good therapeutic effect on non-alcoholic fatty liver disease.
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| CN201911380288.9ACN110960553B (en) | 2019-12-27 | 2019-12-27 | Application of malonic acid modified fullerene C70 in preparation of medicine for treating non-alcoholic fatty liver disease |
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| CN118576621A (en)* | 2023-03-01 | 2024-09-03 | 中国科学院化学研究所 | Application of fullerene nanomaterials targeting mannose receptors in the preparation of drugs for the treatment of hepatic steatosis |
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| CN108201543A (en)* | 2016-12-19 | 2018-06-26 | 北京福纳康生物技术有限公司 | Application of water-soluble fullerene structure in preparation of medicine for treating fatty liver |
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009114087A2 (en)* | 2008-03-03 | 2009-09-17 | Luna Innovations Incorporated | A method for treating pruritus by administering fullerenes |
| CN108201543A (en)* | 2016-12-19 | 2018-06-26 | 北京福纳康生物技术有限公司 | Application of water-soluble fullerene structure in preparation of medicine for treating fatty liver |
| WO2018113686A1 (en)* | 2016-12-19 | 2018-06-28 | 北京福纳康生物技术有限公司 | Use of water-soluble fullerene structure in preparation of drug for treating fatty liver |
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| CN110960553A (en) | 2020-04-07 |
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