CN116370452A - 萝卜硫素在缓解糖尿病性心肌病中的应用 - Google Patents
萝卜硫素在缓解糖尿病性心肌病中的应用 Download PDFInfo
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- CN116370452A CN116370452A CN202211618730.9A CN202211618730A CN116370452A CN 116370452 A CN116370452 A CN 116370452A CN 202211618730 A CN202211618730 A CN 202211618730A CN 116370452 A CN116370452 A CN 116370452A
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- sulforaphane
- diabetic cardiomyopathy
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- alleviation
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Abstract
本发明属于生物医药技术领域,具体涉及萝卜硫素在缓解糖尿病性心肌病中的应用。本发明通过研究发现萝卜硫素可以有效缓解糖尿病性心肌病的心肌胰岛素抵抗、心肌重构、心脏功能紊乱、炎症、凋亡和氧化应激症状,表现出良好的治疗作用,因此具有重要的临床意义和社会价值。
Description
技术领域
本发明属于生物医药技术领域,具体涉及萝卜硫素在缓解糖尿病性心肌病中的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
糖尿病性心肌病(DCM)被定义为在没有高血压心脏病、冠状动脉疾病和瓣膜性心脏病的情况下发生的一种心功能障碍。通常,糖尿病发展为DCM是一个漫长而又隐匿的过程,一般从初始的亚临床阶段逐渐进展为伴有临床症状的心脏功能失常,最终发展为需要干预的难治性心力衰竭。心脏代谢、功能甚至结构改变等多种因素都可能导致DCM的发生。特别是已被证明是糖尿病主要标志的胰岛素抵抗(IR)参与了DCM的发病机制,即使它只发生在心脏局部。虽然心肌IR已得到广泛认可,但糖尿病心肌IR发病率高、危害大,临床仍没有减轻IR对心脏的损伤有效治疗方法。因此,寻找一种方法预防糖尿病诱导的心肌IR,并鉴定新的潜在治疗药物迫在眉睫。
萝卜硫素(SFN)作为一种潜在的抗心血管疾病的细胞抗氧化剂引起了人们的广泛关注。在一项前瞻性队列研究中,每天补充10克富含SFN的西兰花芽粉4周对2型糖尿病(T2DM)患者的血清胰岛素敏感性和改善IR有良好的影响,这与SFN的抗氧化特性密切相关。此外,一项Meta分析提出了使用SFN补充剂治疗代谢综合征的临床前策略的新见解。最近的研究还表明SFN可以通过调节脂质代谢途径和逆转氧化应激来改善DCM。然而,SFN在糖尿病背景下对心肌IR的影响及相关机制仍有待阐明。
发明内容
为了解决现有技术的不足,本发明的目的是提供萝卜硫素在缓解糖尿病性心肌病中的应用。本发明通过研究发现,萝卜硫素能够显著改善糖尿病所导致的糖尿病性心肌病的症状。
为了实现上述目的,本发明是通过如下的技术方案来实现:
第一方面,本发明提供了萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,所述糖尿病性心肌病的症状包括心肌胰岛素抵抗、心脏功能紊乱或心脏病理改变。
上述本发明的一种或多种技术方案取得的有益效果如下:
本发明通过研究发现萝卜硫素可以有效缓解糖尿病性心肌病的心肌胰岛素抵抗、心肌重构、心脏功能紊乱、炎症、凋亡和氧化应激症状,表现出良好的治疗作用,因此具有重要的临床意义和社会价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中(A)乳鼠原代心肌细胞和(B)大鼠胚胎心肌细胞的葡萄糖摄取实验结果图,其中①空白对照组、②Insulin(100nM)组、③PA(100μM)组、④Insulin(100nM)+PA(100μM)组、⑤SFN(10μM)组、⑥Insulin(100nM)+SFN(10μM)组、⑦PA(100μM)+SFN(10μM)组、⑧Insulin(100nM)+PA(100μM)+SFN(10μM)组;
图2为实施例2中(A)超声心动图和(B)左心室射血分数、缩短分数对比图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图3为实施例2的(A)苏木精-伊红染色检测小鼠心肌结构图,(B)马松染色显示心脏组织中血管纤维化程度图,(C)天狼星红染色显示心脏组织中间质纤维化程度图,(D)马松染色和天狼星红染色的纤维化数据图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图4为实施例2的(A)心脏宏观照片,(B)WGA染色图,(C)心肌细胞面积图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图5为实施例2的纤维化标志物转化生长因子-β(A)和结缔组织生长因子(B)、心脏肥大标志物心房利钠肽(C)和脑钠肽(D)的mRNA水平的倍数变化图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图6为实施例3中(A)免疫组化(IHC)染色图像和(B)小鼠肿瘤坏死因子-α蛋白表达水平图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图7为实施例3中逆转录定量PCR检测心脏组织中炎症标志物小鼠肿瘤坏死因子-α(A)和小鼠白细胞介素-6(B)的相对mRNA水平数据图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图8为实施例3中以甘油醛-3-磷酸脱氢酶为内参进行蛋白质印迹法检测小鼠肿瘤坏死因子-α和CD68在心肌中的蛋白表达水平的(A)蛋白质印迹法显示心脏组织中蛋白表达,甘油醛-3-磷酸脱氢酶作为内参对照,,(B)表达数据图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图9为实施例3中免疫荧光(IF)染色图像显示NF-κB p65蛋白表达水平,其中①空白组、②SFN组、③STZ组和④STZ+SFN组,Merge表示复合图像,DAPI为4',6-二脒基-2-苯基吲哚,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图10为实施例4中(A)在心脏组织中通过TUNEL染色检测心脏细胞凋亡,(B)蛋白质印迹法显示心脏组织中活化半胱氨酸蛋白酶-3和B淋巴细胞瘤-2的蛋白表达,甘油醛-3-磷酸脱氢酶作为内参对照,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图11为实施例5中(A)超氧化物阴离子荧光探针染色检测心脏组织中的超氧阴离子水平的染色图,(B)荧光强度对比图,其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图12为实施例5中免疫组化染色图像(A)显示3-硝基酪氨酸和4-羟基壬烯酸蛋白表达水平,3-硝基酪氨酸和4-羟基壬烯酸蛋白表达水平对比图(B),其中①空白组、②SFN组、③STZ组和④STZ+SFN组;
图13为采用qRT-PCR检测心脏组织中氧化应激标志物过氧化氢酶(A)和超氧化物歧化酶(B)的相对mRNA水平,其中①空白组、②SFN组、③STZ组和④STZ+SFN组。
具体实施方式
本发明的第一种典型实施方式,萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,所述糖尿病性心肌病的症状包括心肌胰岛素抵抗、心脏功能紊乱或心脏病理改变。
该实施方式的一种或多种实施例中,所述产品针对心肌胰岛素抵抗的作用具体为减轻糖尿病削弱的心肌葡萄糖摄取。
该实施方式的一种或多种实施例中,所述产品针对心脏功能紊乱的作用具体为:
减轻糖尿病诱导的心腔扩大、室壁变薄;
减轻糖尿病诱导的左心室射血分数和缩短分数降低。
该实施方式的一种或多种实施例中,所述产品针对心脏病理改变的作用具体为:
减轻糖尿病诱导的心肌重构;
减轻糖尿病诱导的心肌炎症;
减轻糖尿病诱导的心肌细胞凋亡;
减轻糖尿病诱导的心脏氧化应激。
该实施方式的一种或多种实施例中,所述产品包括药物、食品或保健品。
该实施方式的一种或多种实施例中,所述药物包括萝卜硫素及至少一种药物非活性组分。
该实施方式的一种或多种实施例中,所述药物非活性成分为载体、赋形剂或稀释剂中的一种。
该实施方式的一种或多种实施例中,所述载体、赋形剂及稀释剂包括但不限于乳糖、葡萄糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁或矿物油。
该实施方式的一种或多种实施例中,所述药物为粉剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、喷雾剂等的口服剂、外用剂、栓剂或无菌注射溶液形式的剂型。
该实施方式的一种或多种实施例中,所述药物可通过已知的方式施用。例如通过静脉全身递送。可选地经由静脉内、经皮、鼻内、粘膜或其他递送方法进行施用。这样的施用可以经由单剂量或多剂量来进行。本领域技术人员理解的是,本发明中有待施用的实际剂量可以在很大程度上取决于多种因素而变化,如靶细胞、生物类型或其组织、待治疗受试者的一般状况、给药途径、给药方式等等。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例与对比例详细说明本发明的技术方案。
实施例1SFN可以显著抑制心肌细胞胰岛素抵抗
一.材料
大鼠胚胎心肌细胞(H9c2)购自ATCC细胞库;低糖、高糖DMEM培养基、胎牛血清(fetal bovine serum,FBS)和青链霉素购自美国Gibco公司;胶原酶、5-溴-2'-脱氧尿苷(BrdU)购自美国Sigma-Aldrich公司;马血清、牛血清、棕榈酸酯(PA)购自中国鲲创公司;葡萄糖摄取试剂盒购自英国Abcam有限公司;胰岛素(Insulin)购自中国上海碧云天生物技术有限公司;萝卜硫素(SFN)购自美国MCE公司。
二.方法
1.细胞培养以及分组:乳鼠原代心肌细胞于含10%新生牛血清、6%马血清、1%BrdU、100U/ml青霉素和100μg/ml链霉素的DMEM培养液中,H9c2细胞于含10%胎牛血清、100U/ml青霉素和100μg/ml链霉素的DMEM培养液中,37℃、5%CO2饱和湿度的培养箱内培养。选取处于对数生长期的细胞,制成单个细胞悬液,以合适的密度铺板。设①空白对照组、②Insulin(100nM)组、③PA(100μM)组、④Insulin(100nM)+PA(100μM)组、⑤SFN(10μM)组、⑥Insulin(100nM)+SFN(10μM)组、⑦PA(100μM)+SFN(10μM)组、⑧Insulin(100nM)+PA(100μM)+SFN(10μM)组。
2.葡萄糖摄取实验:按照葡萄糖摄取测定试剂盒的方法进行葡萄糖摄取测定。选取处于对数生长期的乳鼠原代心肌细胞和H9c2细胞分别制成单细胞悬液,以合适的密度铺96孔板。细胞经SFN(10μM)处理2h后,直接暴露于PA(100μM)处理24h,然后用葡萄糖摄取试剂盒对细胞进行测定。在96孔板中加入Krebs-Ringer-Phosphate-Hepes(KRPH)缓冲液,预孵育细胞40min,随后加入100nM胰岛素继续孵育20min,接下来加入2-脱氧葡萄糖(2-DG)孵育20min,随后用荧光酶标仪在412nm波长下测量荧光强度。
三.结果
葡萄糖摄取实验:以2-脱氧-D-葡萄糖6-磷酸(2-DG6P)作为葡萄糖摄取标志物,如图1所示,胰岛素可刺激心肌细胞葡萄糖摄取增加,且PA暴露显著抑制心肌细胞基础和胰岛素刺激下的葡萄糖摄取,SFN治疗可以在很大程度上逆转PA抑制的胰岛素刺激的葡萄糖摄取。
实施例2SFN治疗可减轻糖尿病诱导的小鼠的心肌重构和功能障碍
一.材料
1.8周龄C57BL/6J清洁级雄性小鼠,购自北京维通利华实验室,在无病原体(SPF)设施中的受控环境中饲养(22℃,睡眠-觉醒周期12h),并可自由获得充足的食物和自来水,所有动物在实验前适应性喂养1周。马松染色试剂盒购自中国塞维尔公司;天狼星红染色试剂盒购自中国Leagene生物技术公司;WGA染色试剂盒购自美国Sigma-Aldrich公司;TRIzol、HiFiScript cDNA合成试剂盒购自中国康为世纪公司;转化生长因子β(Tgfβ)、结缔组织生长因子(Ctgf)、心房利钠肽(Anp)、脑钠肽(Bnp)和小鼠甘油醛-3-磷酸脱氢酶(Gapdh)引物购自中国上海生工生物技术;萝卜硫素(SFN)、链脲佐菌素(STZ)购自美国MCE公司。
二.方法
1.动物模型的建立以及分组:24只C57BL/6J雄性小鼠随机分为4组。分别为①空白组、②SFN组、③STZ组和④STZ+SFN组,每组6只小鼠。各组小鼠给药方案为:①空白组小鼠每天腹腔注射柠檬酸钠缓冲液,连续5天;②SFN组小鼠每天腹腔注射柠檬酸钠缓冲液,连续5天,然后给于小鼠腹腔注射SFN(0.5mg/kg),每周5天,连续3个月;③STZ组小鼠每天腹腔注射STZ(50mg/kg),连续5天;④STZ+SFN组小鼠每天腹腔注射STZ(50mg/kg),连续5天,最后一次注射1周后,空腹3h血糖高于250mg/dL的小鼠为糖尿病小鼠,然后给于小鼠腹腔注射SFN(0.5mg/kg),每周5天,连续3个月。治疗结束后,通过超声心动图评估小鼠心功能,随后处死动物,取心脏组织进行进一步研究。所有小鼠实验方案均经山东大学动物护理与伦理委员会批准。
2.逆转录定量PCR(qRT-PCR)实验:取组织10~20mg放于2ml EP管中,置于冰上。使用RNA抽提试剂Trizol提取小鼠心脏组织RNA,使用SMA100软件,测定样本RNA浓度以及纯度。逆转录试剂盒,逆转1μg RNA至cDNA(反应液配制在冰上进行)反应体系为上游引物0.5μl、下游引物0.5μl、2×UHraSYBR Mixture 12.5μl、cDNA1μl和ddH2O 10.5μl,总体积为25μl。应用CFX Connect Real-Time PCR Detect System,以甘油醛-3-磷酸脱氢酶(GAPDH)为内参进行qRT-PCR。
3.苏木精-伊红(H&E)染色:小鼠心脏组织分离,10%福尔马林固定,石蜡包埋,将4μm厚的石蜡切片进行H&E染色,用于心脏组织学形态学评估。
4.天狼星红染色:小鼠心脏组织分离,10%福尔马林固定,石蜡包埋,常规脱蜡至水,天狼星红染液滴染1h,流水冲洗,苏木素染液染细胞核9min,常规脱水透明,中性树胶封片固定。
5.马松染色:小鼠心脏组织分离,10%福尔马林固定,石蜡包埋,将4μm厚的石蜡切片依次进行用马松三色染色液染色,用于心脏组织学形态学评估。
6.WGA染色:将OCT包埋的心脏组织切片(4μm)进行WGA染色。将冰冻切片置于丙酮中,避光固定10min。3% BSA室温封闭30min,在37℃条件下,避光孵育60min,抗荧光衰减封片剂和指甲油封片。
三.结果
1.心脏彩超结果:如图2A所示,与空白组相比,STZ组出现明显心腔扩大、室壁变薄。如图2B所示,STZ组左心室射血分数(EF)和缩短分数(FS)明显降低。而STZ+SFN组相比于STZ组明显改善了心功能。
2.H&E、天狼星红、马松和WGA染色结果:与空白组相比,STZ组心肌结构出现紊乱,如图3所示血管和间质纤维化程度增加,如图4所示心肌细胞明显增大,表明STZ引起了心脏病理改变。与STZ组相比,STZ+SFN组明显改善以上心脏病理改变。
3.qRT-PCR结果:如图5所示,与空白组相比,STZ显著增加纤维化标志物转化生长因子-β(Tgfb)和结缔组织生长因子(Ctgf)、心脏肥大标志物心房利钠肽(Anp)和脑钠肽(Bnp)的mRNA水平,而STZ+SFN组的结果表明SFN可以明显降低STZ诱导的以上标志物表达。
实施例3.SFN治疗可减轻糖尿病诱导的小鼠心肌炎症
一.材料
1.RIPA裂解缓冲液、磷酸酶抑制剂、BCA蛋白检测试剂盒购自中国上海碧云天生物技术有限公司;硝化纤维素膜购自中国GE Healthcare Life Sciences公司;化学发光检测试剂盒购自Biosharp公司;TRIzol、HiFiScript cDNA合成试剂盒购自中国康为世纪公司;小鼠肿瘤坏死因子-α(Tnfα)、小鼠白细胞介素-6(Il6)和小鼠甘油醛-3-磷酸脱氢酶(Gapdh)引物购自中国上海生工生物技术;4',6-二脒基-2-苯基吲哚(DAPI)染液购自美国Abcam公司。
二.方法
动物模型的建立以及分组与实施例2中一致。
1.免疫组化(IHC)染色:心脏组织用10%福尔马林固定过夜,经脱水、包埋、切片处理。将石蜡切片置于65℃烘箱中,烘片30min。脱腊至水后将组织切片置于枸橼酸抗原修复缓冲液(pH=6.0)中于微波炉内进行抗原修复。自然冷却后将切片置于PBS中漂洗三次,每次5min。于心脏组织上滴加3%过氧化氢溶液,室温避光孵育25min,阻断内源性过氧化物酶,甩掉过氧化氢,将切片置于PBS中漂洗三次,每次5min。3% BSA室温封闭30min。甩掉封闭液,在切片上滴加TNF-α一抗,4℃孵育过夜。随后切片置于PBS中漂洗三次,每次5min。滴加二抗覆盖组织,室温孵育90min,DAB显色后用苏木素复染细胞核。脱水后,用中性树胶封片。染色切片用光学显微镜(尼康)成像,显微图像用Image J软件定量。
2.免疫荧光(IF)染色:心脏组织用10%福尔马林固定过夜,经脱水、包埋、切片处理。将石蜡切片置于65℃烘箱中,烘片30min。脱腊至水后将组织切片置于枸橼酸抗原修复缓冲液(pH=6.0)中于微波炉内进行抗原修复。自然冷却后将切片置于PBS中漂洗三次,每次5min。于心脏组织上滴加3%过氧化氢溶液,室温避光孵育25min,阻断内源性过氧化物酶,甩掉过氧化氢,将切片置于PBS中漂洗三次,每次5min。3% BSA室温封闭30min。甩掉封闭液,在切片上滴加NF-κB p65一抗,4℃孵育过夜。随后切片置于PBS中漂洗三次,每次5min。滴加荧光二抗覆盖组织,室温孵育90min,PBS洗3次,每次5min。切片稍甩干后,滴加DAPI,盖玻片封片。染色切片用光学显微镜(尼康)成像。
3.逆转录定量PCR(qRT-PCR)实验:取组织10~20mg放于2ml EP管中,置于冰上。使用RNA抽提试剂Trizol提取小鼠心脏组织RNA,使用SMA100软件,测定样本RNA浓度以及纯度。逆转录试剂盒,逆转1μg RNA至cDNA(反应液配制在冰上进行)反应体系为上游引物0.5μl、下游引物0.5μl、2×UHraSYBR Mixture 12.5μl、cDNA1μl和ddH2O 10.5μl,总体积为25μl。应用CFX Connect Real-Time PCR Detect System,以GAPDH为内参进行qRT-PCR。
4.蛋白质印迹法(Western blot):使用添加蛋白酶和磷酸酶抑制剂的RIPA裂解缓冲液在冰上分离心脏组织蛋白。使用BCA蛋白检测试剂盒测定蛋白浓度。样品与上样缓冲液混合,在95℃金属浴中加热10min,用10% SDS-PAGE凝胶进行电泳,然后电转移到硝化纤维素膜上。用5%脱脂牛奶封闭2h后,在4℃下用一抗孵育过夜。第二天,用封闭液稀释二抗,室温孵育1h。使用增强化学发光检测试剂盒对探针蛋白进行可视化。采用Image J软件进行密度分析。
三.结果
1.IHC染色结果:与空白组相比,如图6A所示,STZ组的肿瘤坏死因子-α(TNF-α)阳性区域明显增加,表明STZ引起了心肌炎症。如图6B所示,使用SFN处理后,均可逆转STZ引起的肿瘤坏死因子-α(TNF-α)水平的增加,减轻心肌炎症。
2.qRT-PCR结果:与空白组相比,如图7所示,STZ显著增加促炎细胞因子肿瘤坏死因子-α(Tnfa),小鼠白细胞介素-6(Il6)的mRNA水平,而SFN可以明显降低STZ引起的炎症因子的表达。
3.Western blot结果:与空白组相比,如图8所示,STZ显著提高了肿瘤坏死因子-α(TNF-α)和CD68的表达,使用SFN可明显抑制STZ引起的TNF-α和CD68的升高。
4.IF染色结果:如图9所示,红染代表炎症因子NF-κB p65的表达水平,蓝染代表细胞核。结果表明,STZ显著促进了NF-κB p65炎症因子的表达,而SFN可以明显阻止这一进程。
实施例4.SFN治疗可减轻糖尿病诱导的小鼠心肌细胞凋亡一、材料
脱氧核苷酸转移酶末端标记(TUNEL)染色试剂盒购自上海罗氏生物科技有限公司;4',6-二脒基-2-苯基吲哚(DAPI)染液购自美国Abcam公司。RIPA裂解缓冲液、磷酸酶抑制剂、BCA蛋白检测试剂盒购自中国上海碧云天生物技术有限公司;硝化纤维素膜购自中国GE Healthcare Life Sciences公司;化学发光检测试剂盒购自Biosharp公司。
二、方法
动物模型的建立以及分组与实施例2中一致。
1.TUNEL染色:将OCT包埋的心脏组织切片(4μm)进行TUNEL染色检测细胞凋亡。采用Sigma-Aldrich公司的原位细胞凋亡检测试剂盒(In Situ Cell Death Detection Kit)在组织切片上检测TUNEL染色。组织切片在室温下用4%多聚甲醛中固定20min,并用0.1%Triton X-100和0.1%柠檬酸钠的混合物孵育打孔2min。将组织与末端脱氧核苷酸转移酶反应混合物在37℃条件下避光孵育60min,随后,用DAPI进行核染色。染色切片用荧光显微镜(尼康)成像,显微图像用Image J软件定量。
2.Western blot:使用添加蛋白酶和磷酸酶抑制剂的RIPA裂解缓冲液在冰上分离心脏组织蛋白。使用BCA蛋白检测试剂盒测定蛋白浓度。样品与上样缓冲液混合,在95℃金属浴中加热10min,用10%SDS-PAGE凝胶进行电泳,然后电转移到硝化纤维素膜上。用5%脱脂牛奶封闭2h后,在4℃下用一抗孵育过夜。第二天,用封闭液稀释二抗,室温孵育1h。使用增强化学发光检测试剂盒对探针蛋白进行可视化。采用Image J软件进行密度分析。
三、结果
1.TUNEL染色结果:与空白组相比,如图10A所示,STZ组TUNEL阳性细胞明显增加,表明STZ引起了心肌细胞凋亡。而使用SFN治疗后,逆转STZ引起的心肌细胞凋亡。
2.Western blot结果:与空白组相比,如图10B所示,STZ明显加重了心肌细胞的凋亡(活化半胱氨酸蛋白酶-3cleaved caspase-3表达增加,B淋巴细胞瘤-2BCL2表达减少)。而使用SFN处理后,可逆转STZ引起的心肌细胞凋亡。
实施例5.SFN治疗可减轻糖尿病诱导的小鼠心脏氧化应激一、材料:
超氧化物阴离子荧光探针(DHE)染色试剂盒购自中国上海碧云天生物技术有限公司,TRIzol、HiFiScript cDNA合成试剂盒购自中国康为世纪公司;过氧化氢酶(Cat)、超氧化物歧化酶(Sod)和小鼠甘油醛-3-磷酸脱氢酶(Gapdh)引物购自中国上海生工生物技术。
二、方法:
动物模型的建立以及分组与实施例2中一致。
1.超氧化物阴离子荧光探针(DHE)染色:将OCT包埋的心脏组织切片(4μm)进行DHE染色。将冰冻切片置于丙酮中,避光固定10min。滴加DHE染液,在37℃条件下,避光孵育60min,滴加抗荧光衰减剂封片,染色切片用尼康荧光显微镜成像,显微图像用Image J软件定量。
2.IHC:心脏组织用10%福尔马林固定过夜,经脱水、包埋、切片处理。将石蜡切片置于65℃烘箱中,烘片30min。脱腊至水后将组织切片置于枸橼酸抗原修复缓冲液(pH=6.0)中于微波炉内进行抗原修复。自然冷却后将切片置于PBS中漂洗三次,每次5min。于心脏组织上滴加3%过氧化氢溶液,室温避光孵育25min,阻断内源性过氧化物酶,甩掉过氧化氢,将切片置于PBS中漂洗三次,每次5min。3%BSA室温封闭30min。甩掉封闭液,在切片上滴加3-硝基酪氨酸(3-NT)、4-羟基壬烯酸(4-HNE)一抗,4℃孵育过夜。随后切片置于PBS中漂洗三次,每次5min。滴加二抗覆盖组织,室温孵育90min,DAB显色后用苏木素复染细胞核。脱水后,用中性树胶封片。染色切片用光学显微镜(尼康)成像,显微图像用Image J软件定量。
3.qRT-PCR实验:取组织10~20mg于2ml EP管中,置于冰上。使用RNA抽提试剂Trizol提取小鼠心脏组织RNA,使用SMA100软件,测定样本RNA浓度以及纯度。逆转录试剂盒,逆转1μg RNA至cDNA(反应液配制在冰上进行)反应体系为上游引物0.5μl、下游引物0.5μl、2×UHraSYBR Mixture 12.5μl、cDNA 1μl和ddH2O 10.5μl,总体积为25μl。应用CFXConnect Real-Time PCR Detect System,以GAPDH为内参进行qRT-PCR。
三、结果
1.DHE染色结果:如图11A所示,使用DHE染剂所导致的红染表示组织ROS水平。如图11B所示,在STZ处理的小鼠心脏中,ROS水平明显增高,SFN可减轻ROS的产生。
2.IHC染色结果:与空白组相比,如图12A-B所示,STZ组的3-硝基酪氨酸(3-NT)和4-羟基壬烯酸(4-HNE)阳性区域明显增加,表明STZ引起了心脏氧化应激。使用SFN处理后,均可逆转STZ引起的心脏氧化应激水平。
3.qRT-PCR结果:与空白组相比,如图13所示,STZ显著降低抗氧化酶过氧化氢酶(Cat)和超氧化物歧化酶(Sod)的mRNA水平,而SFN可以明显改善STZ诱导的过氧化氢酶(Cat)和超氧化物歧化酶(Sod)表达。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述糖尿病性心肌病的症状包括心肌胰岛素抵抗、心脏功能紊乱或心脏病理改变。
2.如权利要求1所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述产品针对心肌胰岛素抵抗的作用具体为减轻糖尿病削弱的心肌葡萄糖摄取。
3.如权利要求1所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述产品针对心脏功能紊乱的作用具体为:
减轻糖尿病诱导的心腔扩大、室壁变薄;
减轻糖尿病诱导的左心室射血分数和缩短分数降低。
4.如权利要求1所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述产品针对心脏病理改变的作用具体为:
减轻糖尿病诱导的心肌重构;
减轻糖尿病诱导的心肌炎症;
减轻糖尿病诱导的心肌细胞凋亡;
减轻糖尿病诱导的心脏氧化应激。
5.如权利要求1所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述产品包括药物、食品或保健品。
6.如权利要求5所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述药物包括萝卜硫素及至少一种药物非活性组分。
7.如权利要求6所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述药物非活性成分为载体、赋形剂或稀释剂中的一种。
8.如权利要求7所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述载体、赋形剂及稀释剂包括但不限于乳糖、葡萄糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁或矿物油。
9.如权利要求5所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述药物为粉剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、喷雾剂等的口服剂、外用剂、栓剂或无菌注射溶液形式的剂型。
10.如权利要求5所述的萝卜硫素在制备缓解糖尿病性心肌病产品中的应用,其特征在于,所述药物可通过已知的方式施用至体内。
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CN111315730A (zh) * | 2017-10-19 | 2020-06-19 | 艾伊莱布 | 包含新型衍生物作为有效成分的用于预防或治疗肿瘤坏死因子相关疾病的组合物及利用其的肿瘤坏死因子活性抑制方法 |
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