CN116671630A - 储氢牡蛎钙在防治肥胖、胰岛素抵抗和脂肪肝的特殊医学用途食品、保健品及药品中的应用 - Google Patents
储氢牡蛎钙在防治肥胖、胰岛素抵抗和脂肪肝的特殊医学用途食品、保健品及药品中的应用 Download PDFInfo
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Abstract
本发明公开了储氢牡蛎钙在防治肥胖、胰岛素抵抗和脂肪肝的特殊医学用途食品、保健品及药品中的应用，涉及生物学和医药学技术领域，储氢牡蛎钙在防治肥胖、胰岛素抵抗和脂肪肝的特殊医学用途食品、保健品及药品中的应用，使用储氢牡蛎钙作为固态的载氢剂，能够与水反应产生氢气，可持续释放高浓度氢气，对防治肥胖、胰岛素抵抗和脂肪肝起到显著效果。
Description
技术领域
本发明涉及生物学和医药学技术领域，具体是储氢牡蛎钙在防治肥胖、胰岛素抵抗和脂肪肝的特殊医学用途食品、保健品及药品中的应用。
背景技术
随着社会经济的发展和人们生活方式的改变导致的肥胖已成为是一个全球性的健康问题：据估计，全世界约有15亿成年人超重，其中约2亿男性和3亿女性肥胖。世卫组织报告称，全球儿童肥胖人数突然增加，从1990年的3200万增加到2016年的4100万。肥胖导致代谢综合征(metabolic syndrome，MetS)和合并症的发展，包括Ⅱ型糖尿病(type2diabetes，T2DM)、非酒精性脂肪肝病(non-alcoholic fatty liver disease，NAFLD)、胰岛素抵抗、高血压、高脂血症、慢性肾病、心血管疾病、甚至恶性肿瘤(例如，乳腺、结肠和前列腺)等，增加肥胖个体的死亡率。肥胖者体内往往存在一种特殊的病理状态，叫做“胰岛素抵抗”，会导致胰岛素的生物效能下降，无法有效降低血糖。同时，非酒精性脂肪肝患病率和严重程度的增加也与肥胖的上升密切趋势有关，估计全球脂肪肝患病率为25-30％，病态肥胖患者的患病率高达90％。肥胖驱动的非酒精性脂肪肝的流行和随后的发病率被认为是未来十年的主要健康危机之一。肥胖给医疗保健系统带来巨大的经济负担，严重影响人们的生活质量和心理健康。因此，减肥干预措施已成为降低胰岛素抵抗及脂肪肝等相关的并发症发病率主要手段之一，目前认为最好的办法是控制饮食和增强运动。然而这种疗法难以持久，饮食控制和增强运动并不能适用于所有人群。同时，现有的药物治疗存在一定的药物毒副作用，主要表现在脂肪肝加重、肝脏出现纤维化、炎症的加重、甚至肾脏毒性。因此开发新型药物缓解和治疗饮食诱导的肥胖、胰岛素抵抗和非酒精性脂肪肝非常必要。
因此，我们应用储氢牡蛎钙来缓解和治疗饮食诱导的肥胖、胰岛素抵抗和非酒精性脂肪肝，储氢牡蛎钙作为固态的载氢剂，与水结合能够持续稳定的释放大量氢气，另一方面，牡蛎作为一种中药材，具有养阴潜阳作用，适用于肝阴不足、肝阳上亢之症。
发明内容
本发明的目的在于提供储氢牡蛎钙在防治肥胖、胰岛素抵抗和脂肪肝的特殊医学用途食品、保健品及药品中的应用，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：包括使用储氢牡蛎钙作为固态的载氢剂，能够与水反应产生氢气，可持续释放高浓度氢气。
进一步地，其储氢牡蛎钙可以有效的降低身体、肝脏、肾周脂肪和棕色脂肪的重量。
进一步地，其储氢牡蛎钙可以显著的改善葡萄糖耐受和胰岛素敏感性。
进一步地，其储氢牡蛎钙可以增强肝脏线粒体功能。
进一步地，其储氢牡蛎钙可以降低饮食诱导得到血脂和肝脏脂肪的累积，并抑制脂肪酸合成促进脂肪酸的β氧化。
进一步地，其储氢牡蛎钙可以改善抗氧化蛋白的表达降低氧化应激。
进一步地，其储氢牡蛎钙可以改善血清中炎症和关键代谢组织的炎症。
与现有技术相比，本发明的有益效果是：
1.与普通的富氢水相比，储氢牡蛎钙在水中溶解后稳定持续高效的释放大量的氢气。
2.储氢牡蛎钙治疗可以显著的降低高脂饮食诱导的肥胖、体重、肝脏、肾周和附睾脂肪的重量，在防治饮食导致的肥胖和脂肪肝中应用。
3.储氢牡蛎钙治疗可以显著的改善高脂饮食诱导的肥胖、脂肪肝患体胰岛素明显和葡萄糖耐受，在防治饮食导致的胰岛素抵抗和糖尿病中的应用。
4.储氢牡蛎钙的治疗可以显著的改善高脂饮食诱导的肥胖、脂肪肝患体的肝脏脂质代谢异常和脂肪肝，在防治饮食诱导的脂肪肝中的应用。
5.储氢牡蛎钙的治疗可以显著的改善高脂饮食诱导的肥胖、脂肪肝患体的血清中炎性因子、肝脏、肌肉和心脏炎性因子的表达，在防治饮食诱导的系统性炎症中的应用。
附图说明
图1为储氢牡蛎钙(HOP)体外持续释放氢气情况；
图2为储氢牡蛎钙(HOP)治疗对HFD模型小鼠过程数据；
图3为模型小鼠改善高脂饮食诱导小鼠胰岛素抵抗和葡萄糖的耐量；
图4为储氢牡蛎钙(HOP)治疗改善HFD模型小鼠肝脏线粒体功能；
图5为模型小鼠的血脂和肝脏脂质的累积及促进肝脏的脂质代谢表；
图6为模型小鼠高脂饮食诱导关键代谢组织中活性氧(reactive oxygenspecies，ROS)水平及氧化应激表；
图7为模型小鼠改善血清中炎性因子的水平和关键代谢组织中炎性因子的mRNA表达水平
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
为了阐明HOP在体内的治疗效果，设计的实验方案为，给动物模型小鼠灌胃HOP，通过检测血糖，血脂，肝脏组织中脂质的累积，脂质代谢，关键代谢组织中活性氧水平来评价HOP在治疗肥胖、胰岛素抵抗和脂肪肝中的应用。
请参阅图1～7，本发明实施例中，准确称取不同剂量的储氢牡蛎钙粉末，用水分别配制浓度为30mg/mL，100mg/mL，300mg/mL的储氢牡蛎钙(HOP)水溶液5mL置于20mL的小烧饼中，轻轻搅拌混合均匀后，密封烧杯口，待检测时打开烧杯，用氢电极检测水溶液中氢气的浓度。分别检测1h，4h，8h，24h，48h，72h，96h，120h，144h，168h，192h储氢牡蛎钙释放氢气的浓度，同时测定溶液的PH。
动物模型：高脂饮食诱导的肥胖、胰岛素抵抗和脂肪肝模型
选取6周龄的C57小鼠，在适应环境后，给予60％高脂饮食，同时给予储氢牡蛎钙，观察储氢牡蛎钙对高脂饮食诱导的代谢综合征的缓解作用,具体分组如下：
A.正常组(Chow diet,CD) 11
B.高脂组(high fat diet,HFD) 12
C.高脂+低剂量储氢牡蛎钙(L-HOP,30mg/kg) 12
D.高脂+高剂量的储氢牡蛎钙(H-HOP,300mg/kg) 10
E.高脂组((高脂模型小鼠饲喂两个月后)+高剂量的储氢牡蛎钙(HOP-T，300mg/kg))12
动物适应环境后，喂食高脂,高脂饲喂1周，收集粪便,在高脂饲喂4周，老鼠体重和进食量每周记录2次，实验持续时间为3个月(根据小鼠口服糖耐量实验(oral glucosetolerance tests,GTT)结果，直至出现稳定的胰岛素抵抗现象为止)。每4个周进行OGTT检测一次，小鼠禁食过夜后，小鼠注射葡萄糖(1g/kg体重)后，尾部取血检测0,15,30,60,120分钟时血液中葡萄糖含量。小鼠实验结束前，测一次小鼠胰岛素敏感性实验(Insulintolerance test，ITT)，ITT和OGTT检测之间至少间隔5天。小鼠注射胰岛素(0.75U/kg)后，尾部取血检测0,15,30,60,120分钟时血液中葡萄糖含量。在实验结束时，老鼠禁食过夜后处死小鼠，取组织，脑、心脏、肝脏，胫骨前肌，腓肠肌，肾周脂肪，附睾脂肪，背部棕色脂肪，空肠，乙状结肠，回肠采集，其中肝脏，脂肪(肾脏，附睾，棕色脂肪)组织拍照保存。其中肝脏和脂肪称重，所有组织冻存于液氮中，-80℃储存。收集老鼠血液样本，3000g，15min，血清于-80℃冰箱保存。
实验方法
1)活性氧(ROS)水平测定
(1)H2DCF-DA储存液配制：H2DCF-DA试剂配制成10mmol/L的储存液，配置过程中需注意避光，配制好的储备液分装成小份，置于-20℃避光保存。
(2)组织中活性氧的测定：称取20mg组织，用剪刀剪碎，然后使用预定的PBS清洗一遍，重新加入200μL的预冷PBS，使用组织研磨器进行研磨，研磨匀浆后，4℃，1000g离心10min，取上清，然后重复离心一次，收取上清到心的1.5mL离心管，取10μL加入到96孔板，然后将H2DCF-DA试剂用PBS稀释1000倍，每孔加入100μL，将96孔板避光37℃孵育30min,放入酶标仪进行荧光检测，设定激发光和发射光为485nm和538nm；同时将测定上清样品进行BCA蛋白定量；组织中ROS含量为荧光OD值与相应蛋白含量的比值。
2).蛋白质印记
从细胞裂解液中提取总蛋白，在SDS-PAGE胶进行蛋白质分离，并印染至PVDF薄膜上。1％牛血清白蛋白封闭1小时后，用特定的一抗在4oC孵育过夜。第二天，用辣根过氧化物酶交联的二抗室温孵育1小时后，将条带在加强的化学发光仪上进行显影(Bio-RadLaboratories,Hercules,CA,USA)。
Anti-TFAM(D5C8,#8076s)抗体，Anti-β-Actin(8H10D10，#3700S)抗体和Anti-GAPDH(14C10，#2118S)抗体，PPARγ(2435S)抗体，FAS(3189S)抗体，α-Tubulin(3873S)抗体，Anti-P-ACC(#3661s)抗体，Anti-ACC(3676S)抗体购自Cell Signaling Technology(Danvers,MA)。Anti-NDUFS3(ComplexⅠ，#459130)抗体，Anti-SDHB(ComplexⅡ，#459230)抗体，Anti-UQCRC1(ComplexⅢ，#45914)抗体，Anti-MTCO1(ComplexⅣ，#459600)抗体，Anti-ATP Synthase Subunit Alpha(ComplexⅤ，#459240)抗体购自Invitrogen(Meridian,USA)。Anti-DRP1(611113)抗体和Anti-OPA1(612607)抗体购自BD Biosciences(Mexico,US)。Anti-MFN1(D-10，sc-166644)抗体，Anti-MFN2(F-5，sc-515647)抗体，Anti-NQO1(H-9，Sc-376023)抗体，Anti-SOD1(24，Sc-101523)抗体，Anti-SOD2(E-10，Sc-137254)抗体和Anti-catalase(F-17，Sc-34285)抗体，PPARα(sc-9000)抗体，SREBP1(Sc13551)抗体购自Santa Cruz Biotechnology(Dallas,TX)。Anti-KEAP1(#60027-1-Ig)抗体和Anti-NRF2(#66504-1-Ig)抗体购自Proteintech(Rosemont,IL)。CPT1A(A5307)抗体,UCP1(A5857)抗体，UCP3(A16996)抗体购自ABclonal(Wuhan,China)。
3).实时定量PCR
使用TriPure Isolation Reagent(Roche,Basel,Switzerland)从细胞中提取总RNA，然后利用试剂盒(BioRad,Hercules,CA,USA)反转录成cDNA。使用iQ SYBR GreenSupermix(BioRad)进行PCR反应，并利用CFX Connect real-time PCR detection system(BioRad)进行数据分析。
目标基因的引物经设计、合成后，用灭菌超纯水溶解成100μM的储备液，-20℃保存。临用前将上、下游引物混合，稀释10倍后得终浓度为10μM的应用液。
4)TG和TC水平的测定
根据购买的南京建成公司的检测试剂盒中的操作说明书，检测血清中和肝脏组织中TG和TC水平，具体操作步骤按照说明书进行操作。
5)口服糖耐量实验(OGTT)
小鼠口服葡萄糖耐受量(OGTT)检测。小鼠禁食过夜(12小时)之后尾静脉取血并测量小鼠的血糖水平，按照1.5g/kg的剂量腹腔注射葡萄糖溶液。在葡萄糖给药前后，在规定时间点(0、15、30、60和120min)从尾静脉抽取血样，血糖仪测量血糖水平并记录实验数据。
6)胰岛素敏感性实验(ITT)
小鼠胰岛素耐受量(ITT)检测，小鼠禁食6小时之后，测量小鼠血糖记录为0点，腹腔注射胰岛素(0.75U/kg)。在胰岛素给药之后15、30、60和120min从尾静脉抽取血样，使用血糖仪测量血糖水平并记录实验数据。
7)组织HE切片染色
首先将组织样本使用4％的多聚甲醛固定，然后石蜡包埋。包埋完成后切片厚度在4-8μm左右；样本脱蜡和水化；切片苏木素染色、分化与反蓝；切片伊红染色与脱水；HE染色样本切片风干封片；最后在显微镜下观察并拍照
8)统计分析
使用Graphpad Prism8软件进行统计分析。首先采用Shapiro-Wilk正态性检验检验样本的正态分布。如果符合正态分布，则进一步检验方差齐性。如果数据也通过了方差齐性检验，则使用双尾Student t-text或One-way ANOVA(Tukey事后检验)计算p值；否则，使用Welch t检验或Kruskal-Wallis检验计算p值。对于不符合正态分布的样本，使用Mann-Whitney或Kruskal-Wallis非参数检验。数据以平均值±SEM表示。显著统计学意义为*p<0.05,**p<0.01,***p<0.001。
图1为HOP体外持续释放氢气情况。A为浓度为30mg/mL，100mg/mL，300mg/mL的储氢牡蛎钙水溶液在192h内持续释放氢气浓度的情况。B为浓度为30mg/mL，100mg/mL，300mg/mL的储氢牡蛎钙水溶液在192h内随氢气释放，溶液的PH监测情况(n＝3，***p<0.001)。
图2为HOP治疗对HFD模型小鼠过程中，可减轻体重、肝脏、肾周脂肪、附睾脂肪、棕色脂肪及肾脏的重量。A为HOP治疗过程中，对小鼠体重的监测。B为HOP治疗结束后，对小鼠肝脏增量的监测。C为HOP治疗过程中，对小鼠附睾的监测。D为HOP治疗过程中，对小鼠肾周的监测。E为HOP治疗结束后，对小鼠的棕色脂肪称重。F为HOP治疗结束后，对小鼠的肾脏称重(CD,n＝11；HFD，n＝12；HFD+LHOP,n＝12；HFD+HOP-P,n＝10；HFP+HOP-T,n＝12；**p<0.01，***p<0.001)。
图3为HOP治疗HFD模型小鼠，改善改善高脂饮食诱导小鼠胰岛素抵抗和葡萄糖耐量。A为HOP治疗8周后的OGTT结果，n＝5。B为HOP治疗12周后的OGTT结果,n＝5。C为HOP治疗12周后的ITT结果,n＝5。D为HOP治疗16周后的OGTT结果。E为HOP治疗16周后的ITT结果,n＝5。F为肝脏组织中P-AKT/AKT的结果，n＝4。G为肌肉组织中P-AKT/AKT结果,n＝4。(*p<0.05，**p<0.01，***p<0.001)。
图4为HOP治疗改善HFD模型小鼠肝脏线粒体功能。A肝脏组织线粒体膜电位检测，n＝4。B肝脏组织线粒体的呼吸功能，n＝4。C肝脏组织解偶联能力测定，n＝4。D为HOP治疗后，肝脏线粒体蛋白表达情况。E为HOP治疗结束后，肝脏组织线粒体相关转录因子及线粒体相关基因的表达情况。(*p<0.05，**p<0.01，***p<0.001)。
图5为HOP治疗HFD模型小鼠，改善血脂和肝脏脂质的累积及促进肝脏的脂质代谢功能。A为肝脏的HE染色切片，n＝3。B肝脏组织中TG和TC水平，n＝3。C为HOP治疗后，小鼠血清中TG、TC、FFA及L-HDL水平的变化，n＝3。D血清中ALT/AST的水平,n＝3。E为HOP治疗后，小鼠肝脏组织中脂质代谢相关蛋白的表达水平,n＝4。F血为HOP治疗后，小鼠肝脏组织中脂质代谢相关基因的表达水平,n＝4。(*p<0.05，**p<0.01，***p<0.001)。
图6为HOP治疗HFD模型小鼠，改善降低高脂饮食诱导关键代谢组织中ROS水平及氧化应激。A肝脏组织ROS水平检测，n＝4。B肌肉组织ROS水平检测，n＝4。C脑组织ROS水平检测，n＝4。D为HOP治疗后，肌肉组织中抗氧化相关蛋白的表达水平血清中ALT/AST的水平,n＝3。E为HOP治疗后，小鼠肝脏组织中脂质代谢相关蛋白的表达水平,n＝4。F为HOP治疗后，小鼠肝脏组织中脂质代谢相关基因的表达水平,n＝4。(*p<0.05，**p<0.01，***p<0.001)。
图7为HOP治疗HFD模型小鼠，改善血清中炎性因子的水平和关键代谢组织中炎性因子的mRNA表达水平。A血清中炎性因子的表达水平,n＝3。B肝脏组织炎性因子的mRNA水平，n＝4。C心脏组织炎性因子的mRNA水平，n＝4。D肌肉组织炎性因子的mRNA水平，n＝4。(*p<0.05，**p<0.01，***p<0.001)。
(1)HOP体外持续高效缓慢释放氢气
为了明确储氢牡蛎钙在体外释放氢气的情况，用水分别配制浓度为30mg/mL，100mg/mL，300mg/mL的储氢牡蛎钙水溶液5mL置于20mL的小烧饼中，轻轻搅拌混合均匀后，密封烧杯口，待检测时打开烧杯，用氢电极检测水溶液中氢气的浓度。分别检测1h，4h，8h，24h，48h，72h，96h，120h，144h，168h，192h储氢牡蛎钙释放氢气的浓度，同时测定溶液的PH，检测结果如图1所示，图1(A)中我们发现30mg/mL，100mg/mL，300mg/mL的储氢牡蛎钙水溶液中氢气的浓度随着时间的增加先升高后降低，并且，30mg/mL，100mg/mL，300mg/mL的储氢牡蛎钙水溶液在8h氢气的浓度达到巅峰(氢气的峰值浓度分别为520ppb，600ppb，740ppb)，随着储氢牡蛎钙浓度的增加，释放氢气的浓度也是增加的，300mg/mL储氢牡蛎钙水溶液释放氢气的浓度显著高于30mg/mL和100mg/mL，随着时间的延长，氢气的释放逐渐下降，30mg/mL储氢牡蛎钙水溶液在96h后氢气释放为0，100mg/mL的储氢牡蛎钙水溶液在120h后氢气释放为0，300mg/mL的储氢牡蛎钙水溶液在192h后氢气释放为0；PH检测检测结果如图1(B)所示，储氢牡蛎钙水溶液均呈现碱性(PH>8),且随着浓度的升高，储氢牡蛎钙水溶液的PH呈现显著性的升高，高剂量300mg/mL的储氢牡蛎钙水溶液PH大于13可以维持96h，然后开始降低，100mg/mL的储氢牡蛎钙水溶液PH大于12维持24h，之后开始逐渐降低，30mg/mL储氢牡蛎钙水溶液PH大于12维持8h，然后开始逐渐降低。
(2)HOP降低高脂饮食诱导的肥胖小鼠的体重以及脂肪和肝脏的重量
为了探究HOP治疗对高脂饮食诱导肥胖小鼠的减肥作用，我们评价了小鼠体重和各组织的重量。检测发现，L-HOP、H-HOP及HOP-T治疗可以显著的降低高脂饮食诱导小鼠体重增加、肝脏重量、肾周脂肪和棕色脂肪的重量(图2A，B，D-E)，还发现H-HOP治疗可以显著的降低附睾脂肪和肾脏的重量(图2C和F)。以上结果显示，HOP治疗可以显著改善高脂饮食诱导肥胖小鼠的肥胖。
(3)HOP改善高脂饮食诱导的小鼠胰岛素抵抗和葡萄糖耐量受损
为了探究HOP治疗对高脂饮食诱导肥胖、胰岛素抵抗小鼠的葡萄糖耐量和胰岛素敏感性的影响，我们检测了OGTT和ITT。在葡萄糖耐量方面，我们发现8周的L-HOP和H-HOP治疗可以显著改善葡萄糖耐量。随着治疗时间的延长，检测发现L-HOP、H-HOP治疗12和16周及HOP-T治疗4周和8周，均可以显著的降低高脂饮食小鼠的空腹血糖，并且12周和16周H-HOP及16周L-HOP治疗可以显著的降低葡萄糖耐量的曲线积分面积(图3B和3D)。在胰岛素敏感性方面，检测发现，12周和16周H-HOP及12周L-HOP治疗可以显著降低高脂饮食小鼠的空腹血糖，且12周和16周H-HOP、12周L-HOP治疗和4周及8周HOP-T治疗可以显著降低高脂饮食小鼠的胰岛素敏感性的曲线积分面积(图3C和3E)。高脂饮食诱导情况下，无insulin刺激肝脏和肌肉组织中P-AKT/AKT比率是下降的；在给insulin刺激条件下，HOP治疗可以提高P-AKT/AKT比率(图3F和3G)。上述的研究结果表明HOP的治疗可以显著的改善高脂饮食诱导肥胖、胰岛素抵抗小鼠的葡萄糖耐受和胰岛素敏感性。
(4)HOP改善高脂饮食诱导的肝脏组织线粒体功能紊乱
为了探究HOP治疗对高脂饮食诱导肥胖小鼠线粒体功能的影响，我们检测了小鼠肝脏组织中线粒体膜电位和线粒体的呼吸能力及肝脏组织线粒体蛋白和基因的表达水平。在肝脏组织中，与对照组CD小鼠相比，高脂饮食组小鼠肝脏组织膜电位下降，HOP治疗有恢复趋势(图4A)，同时检测线粒体的有氧呼吸，发现给予HOP治疗后线粒体的基础呼吸能力增强(图4B)及解偶联能力增强(图4C)。蛋白水平检测发现DRP1蛋白表达水平升高，线粒体生成转录因子(Peroxisome proliferator-activated receptor-γcoactivator-1α,PGC1α)及融合相关基因(mitofusin 1,MFN1)和(optic atrophy 1,OPA1)表达水平增加(图4D，E)。这些结果显示，HOP治疗可以增强高脂饮食小鼠肝脏线粒体功能。
(5)HOP降低高脂饮食诱导血脂水平升高、改善高脂饮食诱导的脂肪肝，其机制是在关键代谢组织中促进脂肪酸β氧化及抑制脂质合成。
为了探究HOP治疗对高脂饮食小鼠血清中血脂水平和脂肪肝的缓解的影响，我们检测了血清和肝脏中TG和TC水平及评价肝脏脂代谢功能。发现L-HOP、H-HOP、HOP-T治疗高脂饮食诱导的肥胖小鼠均可以显著的降低肝脏脂质的TG和TC的累积及血清中TG、TC、FFA及LDL的水平(图5A-C)。同时也发现L-HOP、H-HOP、HOP-T治疗可以有效降低ALT/AST的比率(图D)，这表明HOP治疗可以有效改善脂肪肝。HOP-T治疗表现出显著抑制脂质合成因子(phosphate-acetyl-coA carboxylase,P-ACC)及(sterol regulatory element bindingprotein 1-cleaved,SREBP1-cleaved)蛋白表达水平及脂肪酸合酶(fatty acidsynthase，Fasn)转录水平，增强脂肪酸β氧化相关基因(carnitine palmitoyltransferase 1,Cpt1),(peroxisome proliferator activated receptor alpha,Pparaα)，(acyl-CoA oxidase 1,Acox1)，(acyl-CoA dehydrogenase medium chain,Acadm)基因表达水平(图5E-F)。这些结果显示，HOP治疗对于改善饮食诱导肥胖小鼠血脂和肝脏脂肪的累积，并抑制脂肪酸合成促进脂肪酸的β氧化。
(6)HOP在关键代谢组织中降低高脂饮食诱导的ROS水平增加及氧化应激
研究报道，肥胖、脂肪肝引起代谢异常引起氧化应激，导致代谢组织发生氧化还原失衡，氧化损伤进而导致功能损伤。图A-C结果表明，L-HOP，HOP-P，HOP-T治疗可以显著的降低肝脏、肌肉及心脏组织中ROS的水平(图6A-C)。抗氧化相关蛋白和分子检测发现，HOP治疗可以恢复肌肉组织中SOD2蛋白的表达水平，增强心脏组织中超氧化物歧化酶(superoxidedismutase 2，SOD2)的蛋白水平(图6D-E)，通过改善抗氧化蛋白的表达降低氧化应激。
(7)HOP降低高脂饮食诱导的血清中炎性因子水平升高以及关键代谢组织中炎性因子mRNA表达水平升高
为了探究HOP治疗对高脂饮食小鼠血清中炎症和代谢组织炎症水平影响，我们检测了血清中炎症因子白介素1β(interleukin 1beta，IL1β)、白介素6(interleukin 6，IL6)、肿瘤坏死因子-α(tumornecrosis factor alpha，TNFα)的水平及肝脏、心脏和肌肉等关键代谢组织中炎症因子的水平。发现L-HOP、H-HOP、HOP-T治疗高脂饮食诱导的肥胖小鼠均可以显著的降低血清中TNFα的水平，H-HOP、HOP-T治疗可以显著的降低血清中IL1β的水平及L-HOP、H-HOP治疗可以显著的降低血清中IL6的水平(图7A)。同时检测肝脏、心脏及肌肉等重要代谢器官中炎性因子的水平，在肝脏组织中检测发现，H-HOP、HOP-T治疗高脂饮食诱导的肥胖小鼠显著的降低肝脏组织中IL1β的mRNA水平及L-HOP、H-HOP、HOP-T治疗显著降低肝脏组织中IL6的mRNA水平(图7B)；在心脏组织中检测发现，L-HOP治疗高脂饮食诱导的肥胖小鼠显著的降低肝脏组织中IL1β的mRNA水平(图7C)；在肌肉组织中检测发现，L-HOP、H-HOP治疗高脂饮食诱导的肥胖小鼠显著的降低肝脏组织中Mcp1(chemokine(C-C motif)ligand 2，参与免疫调节和炎症过程的分泌蛋白超家族)的mRNA水平(图7D)。综上结果显示，HOP治疗可以改善高脂饮食诱导肥胖小鼠血清中炎症和关键代谢组织的炎症。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内，不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
Claims (7)
1.储氢牡蛎钙在防治肥胖、胰岛素抵抗和脂肪肝的特殊医学用途食品、保健品及药品中的应用，其特征在于，所述肥胖导致的疾病选自胰岛素抵抗和脂肪肝。
2.根据权利要求1的应用，其特征在于，其储氢牡蛎钙可以有效的降低身体、肝脏、肾周脂肪和棕色脂肪的重量。
3.根据权利要求1的应用，其特征在于，其储氢牡蛎钙可以显著的改善葡萄糖耐受和胰岛素敏感性。
4.根据权利要求1的应用，其特征在于，其储氢牡蛎钙可以增强肝脏线粒体功能。
5.根据权利要求1的应用，其特征在于，其储氢牡蛎钙可以降低饮食诱导得到血脂和肝脏脂肪的累积，并抑制脂肪酸合成，促进脂肪酸的β氧化。
6.根据权利要求1的应用，其特征在于，其储氢牡蛎钙可以改善抗氧化蛋白的表达降低氧化应激。
7.根据权利要求1的应用，其特征在于，其储氢牡蛎钙可以改善血清中炎症和关键代谢组织的炎症。
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| CN103710028A (zh) * | 2013-12-31 | 2014-04-09 | 王绪珍 | 担载负氢离子的粉体及其制备方法 |
| CN106085437A (zh) * | 2016-08-08 | 2016-11-09 | 钱卫群 | 一种固体负氢离子制品及其制备方法和应用 |
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