WO2022007284A1 - 一种钙通道抑制剂氧海罂粟碱在骨关节炎中的应用 - Google Patents
一种钙通道抑制剂氧海罂粟碱在骨关节炎中的应用 Download PDFInfo
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- WO2022007284A1 WO2022007284A1 PCT/CN2020/127493 CN2020127493W WO2022007284A1 WO 2022007284 A1 WO2022007284 A1 WO 2022007284A1 CN 2020127493 W CN2020127493 W CN 2020127493W WO 2022007284 A1 WO2022007284 A1 WO 2022007284A1
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- oxoglaucine
- osteoarthritis
- oxysapaverine
- calcium channel
- chondrocytes
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Definitions
- the invention relates to the technical field of biomedicine, in particular to the application of a calcium channel inhibitor oxysapaverine in osteoarthritis.
- Osteoarthritis is a chronic joint disease characterized by degeneration, destruction of articular cartilage and bone hyperplasia.
- the disease is common in middle-aged and elderly people, and the most common clinical symptoms include joint swelling and pain, bone hyperplasia, and limited mobility.
- Age, obesity, inflammation, trauma and genetic factors are associated with the occurrence of this disease.
- the most common symptoms are: joint pain and stiffness. The symptoms are obvious when standing up after sitting for a long time, and the symptoms are relieved slightly after the activity, but they will increase after excessive activity.
- the main risk factors for osteoarthritis include obesity, articular cartilage damage, knee joint deformity, prolonged cold and damp environment, etc. The older you are, the more likely you are to develop the disease.
- OA is the most common joint disease affecting 10%-20% of the world's population. As the population ages now, the incidence of OA will soar further.
- non-steroidal anti-inflammatory drugs are used for symptomatic treatment of OA such as analgesia and anti-inflammatory.
- this kind of treatment cannot fundamentally solve the problem. Long-term use may also bring some serious side effects to the digestive system. Society brings a heavy economic burden.
- Hyaluronic acid (HA) is also widely used in the treatment of OA, but current studies have found that HA does not significantly improve the clinical symptoms of OA. With the in-depth study of OA in recent years, current studies have confirmed that the stimulation of calcium influx and the inhibition of autophagy play an important role in the pathogenesis of osteoarthritis (OA).
- the molecular formula of oxopapaverine is C20H17NO5, the molecular weight is 351.35268g/mol, and the structural formula is as follows:
- Papaverine is a typical oxidized apophine, which is widely found in Chinese medicinal plants such as Magnoliaceae, Annuaceae, Papaveraceae, and Fangchiaceae.
- Oxyaporphine alkaloids are a series of alkaloids with significant pharmacological activity.
- Previous studies have shown that oxidative sea papaverine has significant antitumor, antiviral, and antiplatelet aggregation, accelerated tissue relaxation, and immunosuppressive activities. Due to the lack of in-depth understanding of the biological function of this alkaloid, its application in the biological field has been limited.
- One of the objectives of the present invention is to provide the application of oxopapaverine in calcium channel inhibitors in view of the deficiencies in the prior art.
- Another object of the present invention is to provide the application of oxysapaverine in the preparation of medicaments for treating osteoarthritis.
- the present invention provides the application of oxysapaverine in calcium channel inhibitor.
- the calcium channel inhibitor is a voltage-gated calcium channel TRPV5 inhibitor.
- the invention also provides the application of oxysapaverine in the preparation of medicaments for treating osteoarthritis.
- octopapaverine is a novel calcium channel TRPV5 inhibitor, and it is confirmed through cell experiments that octopapaverine can significantly inhibit the expression of TRPV5 and its downstream key indicators, and further confirmed through animal experiments.
- Oxypavine can significantly inhibit the apoptosis of osteoarthritis (OA) chondrocytes and the expression of inflammatory mediators.
- OA osteoarthritis
- the articular cartilage surface of rats treated with Oxapaverine is smoother, and the articular cartilage fiber hyperplasia is significantly reduced, and its cartilage layer is smoother.
- oxysapaverine can be used to prepare medicines for treating osteoarthritis.
- the present invention provides a new scientific research idea and clinical feasibility for treating osteoarthritis, and has great significance for exerting the therapeutic effect of oxysapaverine on osteoarthritis. It has broad application prospects.
- Fig. 1 is a graph showing the effect of the oxysapaverine of Example 1 on the inhibitory effect of calcium channel TRPV5.
- FIG. 2 is an effect diagram of the ability of oxysapaverine of Example 2 to significantly activate autophagy.
- Fig. 3 is a graph showing the effect that the oxysapaverine of Example 3 can significantly inhibit the apoptosis of OA chondrocytes and the expression of inflammatory mediators.
- FIG. 4 is a graph showing the effect that the autophagy inhibitor 3-MA of Example 4 can reverse the therapeutic effect of octopapine on OA chondrocytes.
- Fig. 5 is the effect diagram of the therapeutic effect of oxysepapaverine of Example 5 on OA.
- test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
- Example 1 Oxapaverine is an excellent inhibitor of the voltage-gated calcium channel TRPV5.
- Cell viability detection The chondrocytes of three generations of OA patients were seeded in 96-well plates at 5,000 cells/well, and added after 24 hours at concentrations of 0, 1.25ng/ml, 2.5ng/ml, 5ng/ml, and 10ng/ml, respectively. ml, 20ng/ml, 40ng/ml, 80ng/ml and 160ng/ml of octopapaverine solution. After culturing for 24 hours, add 20 ⁇ L of CCK-8 kit and incubate at 37°C for 3 hours to detect the OD value with a fluorescence microplate reader.
- Half-inhibitory concentration The chondrocytes of three generations of SD rats were seeded in 96-well plates at a number of 5000/well. 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml and 160ng/ml oxymipapine solutions. After culturing for 24 hours, add 20 ⁇ L of CCK-8 kit and incubate at 37°C for 3 hours to detect the OD value with a fluorescence microplate reader.
- the chondrocytes of three generations of OA patients were seeded in 6-well plates at 50,000 cells/well, and after 24 hours of culture, oxysapaverine (the concentration was determined by the cell viability test and the half-inhibitory concentration test) was added after 24 hours of intervention.
- the cell protein extraction kit was used to extract the total protein of the cells, and the concentration of the protein solution was determined.
- the sample loaded with 50ug was used for electrophoresis and mold transfer. After blocking with a blocking solution, the module was used to incubate the primary antibody (TRPV5, Calmodulin, CAMK-II). ,LC3-2/1) for 24 hours, after incubating the fluorescent secondary antibody for two hours, the ODYSSEY two-color infrared laser imaging system scans the membrane.
- chondrocytes of three generations of OA patients were inoculated in 6-well plates at the number of 50,000/well, and after 24 hours of culture, oxysapaverine (the concentration was determined by the cell viability test and the half-inhibitory concentration test) was added after 24 hours of intervention.
- Fluo-4AM (calcium channel fluorescent probe) working solution was added to the cells and incubated at 37°C for 20 minutes. After washing three times with PBS, fluorescence pictures were taken under the excitation wavelength of 494 nm under a fluorescence microscope.
- Figure 1c shows that octopapaverine can significantly inhibit the expression of TRPV5 and its downstream key indicators; the application of TRPV5 agonist D3 can reverse the inhibitory effect of octopapaverine on calcium channels;
- Figure 1d shows a concentration-dependent inhibition of calcium channels by oxysapaverine.
- Oxapaverine has the ability to significantly activate autophagy.
- chondrocytes of three generations of OA patients were inoculated in 6-well plates at the number of 50,000/well, and after 24 hours of culture, oxysapaverine (the concentration was determined by the cell viability test and the half-inhibitory concentration test) was added after 24 hours of intervention.
- the chondrocytes of three generations of OA patients were seeded in a laser confocal culture dish with a diameter of 30mm at a number of 100,000/well. After culturing for 24 hours, an appropriate amount of GFP-mRFP-LC3 lentivirus was used. After eight hours, it was replaced with oxygen-containing In the complete medium of sea papaverine (the concentration was determined by the cell viability test and the half-inhibitory concentration test), after 24 hours of intervention, the fluorescence picture was taken under the excitation wavelength of 488nm under the fluorescence microscope. The fluorescence intensity was quantitatively detected by Image J.
- oxysapaverine significantly increased LC3-2 /1, the expression of Beclin-1, ATG5 and ATG7; similar results were presented in the autophagic flux assay, the oxoglaucine treatment group (oxoglaucine group) significantly increased the level of autophagic flux compared with the control group (OA group), The level of autophagic flux was reversed in chondrocytes treated with 3-MA, an inhibitor of autophagy.
- Example 3 Papaverine can significantly inhibit the apoptosis of OA chondrocytes and the expression of inflammatory mediators.
- the chondrocytes of three generations of OA patients were seeded in 6-well plates at a number of 50,000/well, and after 24 hours of culture, oxysapaverine (the concentration was determined by the cell viability test and the half-inhibitory concentration test) was added after 24 hours of intervention.
- the mRNA extraction kit extracts the mRNA of OA chondrocytes, and detects inflammation (IL-6, IL-1 ⁇ , TNF- ⁇ and MMP-13) and apoptosis genes (BAX and CASP-1) in chondrocytes according to the standard qPCR procedure. 3) expression.
- Step 3 Western blotting detection in Example 1, the detection of receiving or not receiving different concentrations of oxygen sea papaverine to interfere with inflammation (IL-6, IL-1 ⁇ , TNF- ⁇ and MMP-13) in OA chondrocytes and Apoptosis (BAX and CASP-3) protein expression.
- oxysapaverine significantly inhibited the gene expression and protein expression of inflammatory mediators (IL-6, IL-1 ⁇ , TNF- ⁇ and MMP-13), and oxysapaverine also inhibited apoptosis-related Gene and protein expression of indicators (BAX and CASP-3).
- the autophagy inhibitor 3-MA can reverse the therapeutic effect of oxysapaverine on OA chondrocytes.
- the chondrocytes of three generations of OA patients were inoculated in 6-well plates at the number of 50,000 cells/well, and after 24 hours of culture, they were divided into a normal group, an oxysapaverine group, and an oxysapaverine+3-MA group.
- the mRNA of OA chondrocytes was extracted with an mRNA extraction kit after 24 hours of intervention by adding oxopapaverine (40ng/ml) and/or 3-MA (autophagy inhibitor, 5mM/L), and detected according to the standard qPCR procedure. Inflammation (IL-6, TNF- ⁇ and MMP-13) and expression of apoptotic genes (BAX and CASP-3) in chondrocytes.
- Step 3. Western blotting of embodiment 1, it is divided into normal group, oxysapaverine group and oxysepapaverine+3-MA group. After 24 hours of intervention with oxysapaverine (40ng/ml) and/or 3-MA (autophagy inhibitor, 5mM/L), inflammation (IL-6, TNF- ⁇ and MMP-13) and Apoptosis (BAX and CASP-3) protein expression.
- oxysapaverine 40ng/ml
- 3-MA autophagy inhibitor, 5mM/L
- inflammation IL-6, TNF- ⁇ and MMP-13
- BAX and CASP-3 Apoptosis
- oxopapaverine can significantly reduce the gene expression and protein expression of apoptosis (BAX and CASP-3) and inflammation-related indicators (IL-6, TNF- ⁇ and MMP-13) in chondrocytes of OA patients expression, and 3-MA could significantly reverse the therapeutic effect of oxysapaverine on OA chondrocytes. This indicates that autophagy is of great significance in the role of oxymipapine in the treatment of OA.
- Example 5 In vivo experiment to verify the therapeutic effect of oxysapaverine on OA and its mechanism research.
- the animals were injected with 0.1 ml of normal saline into the joint cavity, and the synovial fluid was withdrawn 1 minute later, and the secretion of BAX, CASP-3, IL-6 and TNF- ⁇ in the synovial fluid was detected by standard ELISA procedure.
- the isolated joint tissue was decalcified with EDTA decalcification solution for 1 month, dehydrated and sectioned by paraffin embedding. HE staining and safranin fast green staining were performed.
- the isolated joint tissue was decalcified with EDTA decalcification solution for 1 month, dehydrated and sectioned by paraffin embedding. After deparaffinization, antigen retrieval was performed by high score, and immunohistochemical staining (TNF- ⁇ , MMP-13, TRPV5 and Beclin-1) was performed. Finally, photographs were taken by an inverted microscope, and the quantitative analysis of expression was performed by Image J.
Abstract
发现氧海罂粟碱是一种新型的钙通道TRPV5抑制剂,并通过细胞实验证实了氧海罂粟碱能够显著抑制TRPV5及其下游的关键指标的表达,并进一步通过动物实验证实了氧海罂粟碱能显著抑制骨关节炎软骨细胞凋亡和炎症介质的表达。因此,氧海罂粟碱可用于制备治疗骨关节炎的药物,对发挥氧海罂粟在骨关节炎的治疗作用上具有重大意义,具有广阔的应用前景。
Description
本发明涉及生物医药技术领域,具体涉及一种钙通道抑制剂氧海罂粟碱在骨关节炎中的应用。
骨关节炎(osteoarthritis,OA)是一种以关节软骨的变性、破坏及骨质增生为特征的慢性关节病。该疾病在中老年人多发,临床上以关节肿痛、骨质增生及活动受限最为常见。年龄、肥胖、炎症、创伤及遗传因素与本病的发生有关。最常见的症状是:关节疼痛和僵硬,久坐后起立活动时症状明显,活动后稍缓解,但活动过量后又会再加重。骨关节炎主要危险因素:包括肥胖、关节软骨损伤、膝关节畸形、长时间寒冷阴湿环境等。年龄越大患病可能性越大,女性患病风险是男性的2~3倍,多累及手指关节,膝关节、髋关节、脊柱等,是影响老年人活动的最常见关节疾病。采取健康生活方式:积极治疗,有助于身体康复、改善生活质量。OA是影响世界10%-20%人口的最常见的关节疾病。随着现在人口老龄化的日益严重,OA的发病率将进一步飙升。
目前,OA多用非甾体类抗炎药实施镇痛抗炎等对症治疗,但是这种治疗不能从根本上解决问题,长期服用还可能给消化系统等带来一些严重的副作用,另外给患者和社会带来了沉重的经济负担。透明质酸(HA)也被广泛的应用于OA的治疗,但是目前研究发现HA对OA的临床症状没有明显的改善。随着近年对OA研究的深入,目前研究证实钙内流的刺激和自噬的抑制在骨关节炎(OA)的发病机制中起重要作用。
氧海罂粟碱分子式为C20H17NO5,分子量为351.35268g/mol,结构式如下所示:
氧海罂粟碱是一种典型的氧化阿朴啡碱,广泛存在于木兰科、番荔枝科、罂粟科、防己科等中药植物中。氧化阿朴啡类生物碱是一类具有显著药理活性的系列生物碱。以前的研究表明,氧化海罂粟碱具有显著的抗肿瘤、抗病毒,以及抗血小板凝聚、加速组织舒张以及免疫抑制活性等。由于目前对该生物碱生物学功能认识不够深入,一直限制了其在生物领域的应用。
发明内容
本发明的目的之一在于针对现有技术中的不足,而提供氧海罂粟碱在钙通道抑制剂中的应用。
本发明的另一目的在于提供氧海罂粟碱在制备治疗骨关节炎药物中的应用。
本发明的目的通过以下技术方案实现:
本发明提供氧海罂粟碱在钙通道抑制剂中的应用。
优选的,所述钙通道抑制剂为电压门控钙通道TRPV5抑制剂。
本发明还提供氧海罂粟碱在制备治疗骨关节炎药物中的应用。
本发明的有益效果:
本发明首次发现了氧海罂粟碱是一种新型的钙通道TRPV5抑制剂,并通过细胞实验证实了氧海罂粟碱能够显著抑制TRPV5及其下游的关键指标的表达,并进一步通过动物实验,证实了氧海罂粟碱能显著抑制骨关节炎(OA)软骨细胞凋亡和炎症介质的表达,通过氧海罂粟碱治疗的大鼠关节软骨面更加光滑,显著减少了关节软骨纤维增生,其软骨层的厚度相对于模型组(OA+PBS)大幅增加,更重要的是,从大体观和关节的组织学评分来看,氧海罂粟碱对关节的保护作用甚至比临床的常用药透明质酸还要好。因此,氧海罂粟碱可用于制备治疗骨关节炎的药物,本发明为治疗骨关节炎提供了一条新的科研思路与临床可行性,对发挥氧海罂粟在骨关节炎的治疗作用上具有重大意义,其具有广阔的应用前景。
图1为实施例1的氧海罂粟碱对钙通道TRPV5抑制作用的效果图。
图2为实施例2的氧海罂粟碱具有显著激活自噬的能力的效果图。
图3为实施例3的氧海罂粟碱能显著抑制OA软骨细胞凋亡和炎症介质表达的效果图。
图4为实施例4的自噬抑制剂3-MA能够逆转氧海罂粟碱对OA软骨细胞的治疗作用的效果图。
图5为实施例5的氧海罂粟碱对OA的治疗作用的效果图。
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1.氧海罂粟碱是一种优异的电压门控钙通道TRPV5抑制剂。
一、实验方法:
1、提取骨关节炎患者的软骨细胞:无菌条件下,收集行关节置换的骨关节炎患者的软骨组织,把软骨组织剪碎成1mm^3见方(即1立方毫米)的组织块;用含青霉素和链霉素混合液冲洗软骨3次。加入软骨体积10-15倍的0.25%胰蛋白酶,37℃消化半小时后终止消化。然后0.02%的二型胶原酶酶消化3小时。液体充分吹打混合、离心1000r/分钟5分钟,去上清液,重悬后将上清液移至10CM的培养皿中,加入10毫升完全培养基,置于37℃、5%CO
2及饱和湿度条件下培养,24小时可见细胞贴壁,此时首次换液,然后每3天换液1次。约7天细胞长满瓶底,此时进行传代培养,传代用0.25%胰蛋白酶消化5分钟,胎牛血清终止消化,按此法反复传代培养增殖。
同样的方法提取SD大鼠的软骨细胞
2、细胞活力检测和半量抑制浓度:
细胞活力检测:将三代的OA患者的软骨细胞以5000个/孔数量接种在96孔板中,24小时后分别加入浓度为0,1.25ng/ml,2.5ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml和160ng/ml的氧海罂粟碱溶液。培养24小时后,加入20μL CCK-8试剂盒37℃孵育3小时后用荧光酶标仪检测OD值。
半量抑制浓度:将三代的SD大鼠的软骨细胞以5000个/孔的数量接种在96孔板中,24小时后分别加入浓度为0,1.25ng/ml,2.5ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml和160ng/ml的氧海罂粟碱溶液。培养24小时后,加入20μL CCK-8试剂盒37℃孵育3小时后用荧光酶标仪检测OD值。
3、Western blotting检测:
将三代的OA患者的软骨细胞以50000个/孔的数量接种于6孔板中,培养24小时后加入氧海罂粟碱(浓度由细胞活力检测和半量抑制浓度检测实验确定)干预24小时后使用细胞蛋白提取试剂盒提取细胞总蛋白,测定蛋白液的浓度,使用上样量为50ug样品进行电泳和转模,使用封闭液封闭以后,模块被用以孵育一抗(TRPV5,Calmodulin,CAMK-II,LC3-2/1)24小时,孵育荧光二抗两小时后,ODYSSEY双色红外激光成像系统扫膜。
4、钙通道荧光检测:
将三代的OA患者的软骨细胞以50000个/孔的数量接种于6孔板中,培养24小时后加入氧海罂粟碱(浓度由细胞活力检测和半量抑制浓度检测实验确定)干预24小时后,Fluo-4AM(钙通道荧光探针)工作液加入细胞,在37℃培养20分钟。用PBS清洗三次,荧光显微镜下激发波长494nm下拍摄荧光图片。
二、实验结果:
结果见图1:如图1a和1b所示,40ng/mL是氧海罂粟碱作用于OA患者的软骨细胞的最适浓度,10,20,和40ng/mL被用于进一步实验;
图1c显示氧海罂粟碱能够显著抑制TRPV5及其下游的关键指标的表达;TRPV5激动剂D3应用可以逆转氧海罂粟碱对钙通道的抑制作用;
图1d显示氧海罂粟碱对钙通道的抑制呈现一种浓度依赖。
实施例2.氧海罂粟碱具有显著激活自噬的能力。
一、实验方法:
1、透射电镜检测:
将三代的OA患者的软骨细胞以50000个/孔的数量接种于6孔板中,培养24小时后加入氧海罂粟碱(浓度由细胞活力检测和半量抑制浓度检测实验确定)干预24小时后,用胰蛋白酶消化离心至1.5毫升的PE管中,使用3%的戊二醛固定软骨细胞24小时,PBS清洗三遍,用四氧化锇固定细胞和细胞器的生物膜15分钟,接着用浓度为50%,70%,80%,90%,100%的梯度酒精脱水(各脱15分钟),接着将细胞样本包埋在树脂中进行切片,最后在透射电镜下观察拍照。
2、Western Blotting检测:
方法参照实施例1的步骤3.Western blotting检测的方法,检测接收或者不接受不同浓度氧海罂粟碱干预OA软骨细胞中自噬相关指标(LC3-2/1,Beclin-1,ATG5和ATG7)的蛋白表达。
3、自噬流检测:
将三代的OA患者的软骨细胞以十万个/孔的数量接种于直径30mm的激光共聚焦培养皿中,培养24小时后适量的GFP-mRFP-LC3慢病毒,作用八小时后换成含有氧海罂粟碱的完全培养基(浓度由细胞活力检测和半量抑制浓度检测实验确定),干预24小时后,荧光显微镜下激发波长488nm下拍摄荧光图片。并用Image J定量检测荧光强度。
二、实验结果:
如图2所示,软骨细胞的透射电镜检测发现被氧海罂粟碱治疗后的OA细胞内含有大量的自噬小体和和自噬溶酶体,这表明氧海罂粟碱能够激活自噬,为了进一步证实这一结论,通过Westernbloting检测了自噬指标(LC3-2/1,Beclin-1,ATG5,ATG7)的蛋白表达,如图2b所示,氧海罂粟碱显著升高了LC3-2/1、Beclin-1、ATG5和ATG7的表达;相似的结果呈现在自噬流检测中,氧海罂粟碱治疗组(oxoglaucine组)相对于Control组(OA组)显著提高了自噬流水平,而自噬的抑制剂3-MA治疗后的软骨细胞自噬流的水平得到 了逆转。
综上研究表明,氧海罂粟碱具有确切的激活自噬的能力。
实施例3.氧海罂粟碱能显著抑制OA软骨细胞凋亡和炎症介质的表达。
一、实验方法:
1、qPCR检测:
将三代的OA患者的软骨细胞以50000个/孔的数量接种于6孔板中,培养24小时后加入氧海罂粟碱(浓度由细胞活力检测和半量抑制浓度检测实验确定)干预24小时后用mRNA提取试剂盒提取OA软骨细胞的mRNA,在按照标准的qPCR操作流程检测软骨细胞内的炎症(IL-6,IL-1β,TNF-α和MMP-13)和凋亡基因(BAX和CASP-3)的表达。
2、Western Blotting检测:
方法参照实施例1的步骤3.Western blotting检测的方法,检测接收或者不接受不同浓度氧海罂粟碱干预OA软骨细胞中炎症(IL-6,IL-1β,TNF-α和MMP-13)和凋亡(BAX和CASP-3)蛋白表达。
二、实验结果:
如图3所示,氧海罂粟碱显著抑制了炎症介质(IL-6,IL-1β,TNF-α和MMP-13)的基因表达和蛋白表达,同时氧海罂粟碱也抑制了凋亡相关指标(BAX和CASP-3)的基因和蛋白表达。
实施例4.自噬抑制剂3-MA能够逆转氧海罂粟碱对OA软骨细胞的治疗作用。
一、实验方法:
1、qPCR检测:
将三代的OA患者的软骨细胞以50000个/孔的数量接种于6孔板中,培养24小时后,分为正常组、氧海罂粟碱组和氧海罂粟碱+3-MA组。加入氧海罂粟碱(40ng/ml)和/或3-MA(自噬抑制剂,5mM/L)干预24小时后用mRNA提取试剂盒提取OA软骨细胞的mRNA,在按照标准的qPCR操作流程检测软骨细胞内的炎症(IL-6,TNF-α和MMP-13)和凋亡基因(BAX和CASP-3)的表达。
2、Western Blotting检测:
方法参照实施例1的步骤3.Western blotting检测的方法,分为正常组、氧海罂粟碱组和氧海罂粟碱+3-MA组。加入氧海罂粟碱(40ng/ml)和/或3-MA(自噬抑制剂,5mM/L)干预24小时后,OA软骨细胞中炎症(IL-6,TNF-α和MMP-13)和凋亡(BAX和CASP-3)蛋白表达。
二、实验结果:
如图4所示,氧海罂粟碱能够显著降低OA病人软骨细胞中的凋亡(BAX和CASP-3)和炎症相关指标(IL-6,TNF-α和MMP-13)的基因表达和蛋白表达,而3-MA可以显著的逆转氧海罂粟碱对OA软骨细胞的治疗作用。这表明自噬在氧海罂粟碱发挥对OA治疗作用中意义重大。
实施例5.体内实验验证氧海罂粟碱对OA的治疗作用及其机制研究。
一、实验方法:
1、动物分组和OA模型制作:
购买36只7周龄的SD大鼠,饲养一周后将所有大鼠分为6组(n=6),分别是Normal组、OA+PBS组、OA+HA组、OA+oxogluacine组、OA+oxoglaucine+3-MA组和OA+oxogluacine+D3(维生素D3)组,将其中30只大鼠(后五组)通过内侧半月板切除术制造外伤的骨关节炎模型,四周后通过关节腔给药注射对应的药物。连续注射四周,每次0.1毫升。
2、关节液的ELISA检测:
动物接收连续治疗四周后,注射0.1毫升生理盐水进入关节腔,1分钟后抽出关节液,通过标准的ELISA操作流程,检测关节液中BAX,CASP-3,IL-6和TNF-α的分泌。
3、大体观测及评分:
动物接收连续治疗四周后,安乐死所有的大鼠,截取关节组织,分离股骨髁进行拍照,并分析关节面的损伤情况和组织评分。
4、组织染色:
将分离出来的关节组织使用EDTA脱钙液脱钙1个月,脱水和进行石蜡包埋切片。行HE染色和番红固绿染色。
5、免疫组化染色:
将分离出来的关节组织使用EDTA脱钙液脱钙1个月,脱水和进行石蜡包埋切片。脱蜡后通过高分进行抗原修复,行免疫组化染色(TNF-α、MMP-13,TRPV5和Beclin-1)。最后通过倒置显微镜拍照,通过Image J进行表达量的定量分析。
6、Western Blotting检测
取SD大鼠股骨髁的软骨组织用钢珠在-4℃环境下研磨成匀浆,参照实施例1的步骤3.Western blotting检测的方法,检测软骨组织中TRPV5,CAMK-II,Calmodulin,Beclin-1和LC3-2/1的蛋白表达。
二、实验结果:
结果见图5,如图5a、5b和5c所示:通过氧海罂粟碱治疗的关节软骨面更加光滑, 显著减少了软骨面的纤维组织的增生,其软骨层的厚度相对于模型组(OA+PBS)大幅增加,更重要的是,从大体观和关节的组织学染色来看,氧海罂粟碱对关节的保护作用甚至比临床的常用药透明质酸还要好。另外免疫组化染色显示氧海罂粟碱抑制了电压门控钙通道受体TRPV5的表达,同时显著提高了自噬相关蛋白Beclin-1的表达,实现对炎症介质(MMP-13和TNF-a)的抑制。这个结论也得到了Westernbloting检测和Elisa的证实(见图5g、5h)。
以上所举实施例为本发明的较佳实施方式,仅用来方便说明本发明,并非对本发明作任何形式上的限制,任何所属技术领域中具有通常知识者,若在不脱离本发明所提技术特征的范围内,利用本发明所揭示技术内容所作出局部更动或修饰的等效实施例,并且未脱离本发明的技术特征内容,均仍属于本发明技术特征的范围内。
Claims (3)
- 氧海罂粟碱在钙通道抑制剂中的应用。
- 根据权利要求1所述的应用,其特征在于:所述钙通道抑制剂为电压门控钙通道TRPV5抑制剂。
- 氧海罂粟碱在制备治疗骨关节炎药物中的应用。
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