WO2023159850A1 - 可调节雌激素信号通路的茶组合物及其在防治神经退行性疾病中的应用 - Google Patents
可调节雌激素信号通路的茶组合物及其在防治神经退行性疾病中的应用 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Definitions
- the invention relates to the field of preventing and treating neurodegenerative diseases, in particular to a tea composition capable of regulating estrogen signaling pathway and its application in preventing and treating neurodegenerative diseases.
- AD Alzheimer's disease
- a ⁇ ⁇ -amyloid protein
- tau protein phosphorylation tau protein phosphorylation
- AD pathogenesis of AD
- amyloid deposition apoptosis or loss of nerve cells
- oxidative stress oxidative stress
- endoplasmic reticulum stress mitochondrial dysfunction
- inflammation etc.
- pathogenesis of AD has not been reported from the aspect of gene expression
- the mechanism is related to the gene expression of the estrogen signaling pathway, and there is no report that regulating the gene expression of the estrogen signaling pathway can prevent and treat neurodegenerative diseases such as AD.
- Theaflavins are a class of benzotropolone compounds produced by enzymatic reaction of catechins through phenylpropanoid cyclization. They have significant cardiovascular protective effects and have the reputation of "soft gold”.
- Theaflavin digallate (theaflavin-3,3′-digallate, TFDG) is one of the main monomer components of theaflavin TFs, which contains more aromatic rings and hydroxyl groups than EGCG ( Figure 1), not only has antioxidant, Anti-inflammation, prevention and treatment of cardiovascular diseases, lowering blood fat, anti-cancer and anti-cancer effects, but the prior art has never reported that theaflavins are used as estrogen signal regulators to protect nerve cells and promote the growth of nerve cell axons , for the prevention and treatment of neurodegenerative diseases such as AD.
- the technical problem to be solved by the present invention is: to overcome the deficiencies of the prior art, to provide a tea composition that can regulate the estrogen signal pathway, which uses theaflavin as the main raw material and as a regulator of estrogen signal, and can protect nerve cells. function, and promote the growth of nerve cell axons, and its application in the prevention and treatment of neurodegenerative diseases.
- a tea composition that can regulate estrogen signaling pathways is theaflavin digallate (theaflavin-3,3′-digallate, TFDG), and an effective amount of the tea composition can up-regulate PC12 cells estrogen signaling pathway.
- Said effective amount refers to making cytokeratin gene (keratins, abbreviated as Krt), keratin-associated protein gene (keratin-associated protein, abbreviated as Krtap), type II keratin gene (type II keratin, abbreviated as Kb) )
- the expression of at least one gene in ) is effectively up-regulated by more than 40% of the amount of the tea composition.
- the effective amount refers to the concentration of theaflavin digallate TFDG as the main active ingredient of the tea composition ⁇ 10 ⁇ M (solvent type), or 10 mg/g (tablet).
- At least one Krt gene is selected from Krt1, Krt5, Krt14, Krt15, Krt16, Krt17, Krt25, Krt27, Krt28, Krt31, Krt32, Krt34, Krt35, Krt73, Krt75, Krt81, Krt83, Krt86, at least one Krtap gene is selected from Krtap7- 1.
- Krtap8-1, Krtap11-1, type II keratin gene is Kb23.
- the active ingredients of the tea composition include theaflavin digallate and derivatives thereof, pharmaceutically acceptable salts, hydrates or any combination thereof.
- the theaflavin digallate derivatives include theaflavin TF1, theaflavin-3-gallate (TF-3G), theaflavin-3-gallate (TF-3G), theaflavin-3 '-gallate (TF-3'G), thearubigin, theabrownin and other aqueous extracts of black tea.
- the tea composition also includes pharmaceutically acceptable excipients or carriers.
- tea composition capable of regulating estrogen signaling pathway in prevention and treatment of neurodegenerative diseases.
- the specific method of the application includes administering an effective amount of the tea composition to the patient, the effective amount refers to the amount of the tea composition that can effectively up-regulate the expression of at least one Krt gene or Krtap gene by at least 40%.
- the tea composition prevents or treats neurodegenerative diseases by up-regulating the expression of at least one Krt gene or Krtap gene by at least 40%, and promoting the growth of nerve cell axons.
- the tea composition prevents or treats neurodegenerative diseases by up-regulating the expression of two or more Krt genes or Krtap genes by at least 40%, and promoting the growth of nerve cell axons.
- the tea composition prevents or treats neurodegeneration by up-regulating the expression of two or more genes in Krt5, Krt14, Krt17, Krt25, Krt27, Krt31, and Krt35 genes by at least 40%, and promoting the growth of nerve cell axons. disease.
- the tea composition up-regulates estrogen signaling pathway in PC12 cells by binding at least one estrogen receptor.
- the tea composition stimulates the transcription levels of keratin family genes such as Krt14 and Krt15, thereby making cytokeratin genes (keratins, abbreviated as Krt) or/and keratin-associated protein genes (keratin-associated protein, abbreviated as Krtap) ) has a significant regulatory effect on estrogen signaling pathway at the gene level, and the upregulation trend of keratin family genes can protect PC12 cells from oxidative shock caused by glutathione depletion and increase NGF-induced axonal Therefore, the estrogen signaling pathway plays an important role in the protection of nerve cells and the promotion of axon growth.
- Keratin intermediate filaments are the third skeleton component of cells, which are related to important life processes such as cell differentiation and intracellular information transmission.
- keratin can also promote the extension of neuronal axons and can be used as a conduit filler for peripheral nerve regeneration.
- Fig. 1 - is the molecular formula structural diagram of TFDG in the tea composition of a kind of adjustable estrogen signaling pathway of the present invention
- Fig. 2 is the ELISA detection analysis diagram of the axon growth factor GAP43 of PC12 cells cultivated by a tea composition capable of regulating the estrogen signaling pathway in Example 1;
- Fig. 3 is the energy currency ATP level analysis figure in the PC12 cell of a kind of tea composition that can regulate estrogen signaling pathway culture in embodiment 1;
- Figure 6 is a differential gene volcano map for inhibiting A ⁇ 25-35- induced PC12 damage by a tea composition that can regulate the estrogen signaling pathway in Example 1;
- Figure 7 a tea composition that can regulate the estrogen signaling pathway in Example 1 is used to inhibit the differential gene PPI network analysis of PC12 damage induced by A ⁇ 25-35 , red represents up-regulation, green represents down-regulation, and the size of the point represents the relative expression level;
- Fig. 8 is a tea composition that can regulate estrogen signaling pathway in Example 1 and is used to inhibit the differential gene GO enrichment analysis of PC12 damage induced by A ⁇ 25-35 ;
- Fig. 9 KEGG enrichment analysis of differential genes for inhibiting A ⁇ 25-35- induced PC12 damage by a tea composition capable of regulating estrogen signaling pathway in Example 1;
- FIG. 10 a tea composition that can regulate the estrogen signaling pathway in Example 1 is used to inhibit the differential gene heat map of the estrogen signaling pathway for PC12 damage induced by A ⁇ 25-35 ;
- Figure 11 a tea composition that can regulate the estrogen signaling pathway in Example 1 is used to inhibit the expression of mRNA in the estrogen signaling pathway A ⁇ 25-35 and A ⁇ 25-35 /TFDG induced by A ⁇ 25-35 in PC12 damage Relative expression analysis graph.
- the active ingredient of the tea composition is theaflavin digallate (theaflavin-3,3'-digallate, TFDG), and the concentration of the TFDG is 50 ⁇ M, and an effective amount of the tea composition can up-regulate the estrogen signaling pathway in PC12 cells.
- the effective amount refers to making cytokeratin gene (keratins, abbreviated as Krt), keratin-associated protein gene (keratin-associated protein, abbreviated as Krtap) and type II keratin gene (type II keratin, abbreviated as Kb) )
- the expression of at least one gene in ) is effectively up-regulated by more than 40% of the amount of the tea composition.
- Krt genes include Krt1, Krt5, Krt14, Krt15, Krt16, Krt17, Krt25, Krt27, Krt28, Krt31, Krt32, Krt34, Krt35, Krt73, Krt75, Krt81, Krt83, Krt86, Krtap genes include Krtap7-1, Krtap8-1, Krtap11-1, the type II keratin gene is Kb23.
- the tea composition also includes pharmaceutically acceptable excipients or carriers.
- the applicant used the above composition to treat rat adrenal pheochromocytoma cell line PC12 cells, and analyzed its effect on the growth of PC12 cell nerve cells.
- the specific operation is as follows:
- the cells of the control group (Control) were added with the same amount of sterile water.
- EGCG ( ⁇ 99%) and TFDG ( ⁇ 99%) were ordered from Shanghai Tongtian Biological Company (Shanghai, China).
- the rat adrenal pheochromocytoma cell line PC12 was ordered from Xiehe Cell Bank (Beijing, China).
- Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Biological Industries (Cromwell, CT, USA).
- Adenosine triphosphate (ATP) detection kit and anti-fluorescence quencher (including DAPI) were purchased from Beyontien Institute of Biotechnology (Shanghai, China).
- Rat nerve growth-associated protein-43 (GAP43) ELISA kit was purchased from Jiangsu Feiya Biotechnology Co., Ltd.
- GAP43 Growth-associated protein-43
- the DAPI staining results shown in Figure 5 showed that, compared with the Control group, the blue fluorescence intensity of the A ⁇ 25-35 group nuclei was enhanced; The blue fluorescent staining intensity of the nuclei in the TFDG group decreased, which was basically the same as that of the cells in the Control group.
- RNA samples were collected from the experimental group and the control group, and 3 biological replicates were set up in each group. Subsequently, all samples were sent to BGI Company (Shenzhen, China) for further RNA-seq detection and analysis by BGISEQ-500 sequencer.
- HISAT2 http://www.ccb.jhu.edu/software/hisat ) was used to map clean reads to the rat genome.
- Bioinformatics analysis mainly relies on R language and online software for analysis and visualization. Differentially expressed genes were screened using the Limma package of R,
- a protein-protein interaction (PPI) network analysis was performed on the differentially expressed genes of A ⁇ 25-35 vs A ⁇ 25-35 /TFDG to identify key biological signaling pathways and perform functional annotation of module-related genes, as shown in Figure 7
- the results showed that the interacting genes were enriched in modules such as estrogen signaling pathway, cell cycle, ubiquitination-mediated proteolysis, and metabolic pathways.
- the annotations mainly show up-regulation in the estrogen signaling pathway and metabolic pathway network
- the annotations mainly show down-regulation in the cell cycle and ubiquitination-mediated proteolysis pathway network.
- the GO enrichment results of differential genes shown in Figure 8 show that: A ⁇ 25-35 vs A ⁇ 25-35 /TFDG differentially expressed genes are involved in transcription regulation, mRNA processing, microtubule-based Significantly enriched in biological processes such as motility and mitotic cell cycle; the KEGG functional enrichment results of differential genes shown in Figure 9 show that among all the enriched signaling pathways that undergo significant changes, most of the differential genes are in neuroprotection and axon Growth-related pathways were significantly enriched, involving estrogen signaling pathway, cell cycle, regulation of actin cytoskeleton, focal adhesions, GAP junction, etc. Changes in these pathways indicate that TFDG is closely related to neuroprotective and axonal growth-promoting biological activities.
- SERMs Selective estrogen receptor modulators
- 1 triphenylethylene compounds 2 benzothiophene compounds
- 3 naphthalene compounds 3 naphthalene compounds
- 4 benzopyran compounds 5 other compounds.
- the heat map of differential genes related to the estrogen signaling pathway shows that the active ingredient TFDG of the tea composition in this application acts as an estrogen agonist during the culture of PC12 cells, promoting the combination of estrogen and estrogen receptors ER ⁇ and ER ⁇ , Activation of target gene transcription is one of the classic estrogen signaling pathways; the estrogen signaling pathway plays an important role in nerve cell protection and promoting nerve axon growth, which may be the reason why theaflavin as an active ingredient in the tea composition protects nerve cells and One of the important potential mechanisms for promoting axon growth, based on this mechanism, the applicant has developed a new application of the tea composition with TFDG as the main active component.
- Theaflavin monomers or compositions such as TFDG all contain benzopyran chemical groups, and the proportion of this chemical group is relatively high, which is different from the benzopyran compounds in the selective estrogen receptor modulators in the prior art. It has the same functional group, and in addition, TFDG contains more antioxidant structure conjugation effects and phenol-quinone balance than ordinary catechins, and has a resting state repair effect on aging and hand-damaged nerve cells, by activating signals such as estrogen pathway significantly promotes the expression of keratin that favors axon outgrowth.
- tea composition capable of regulating estrogen signaling pathway in prevention and treatment of neurodegenerative diseases.
- the specific method of the application includes administering an effective amount of the tea composition to the patient, the effective amount refers to the amount of the tea composition that can effectively increase the expression of at least one of the Krt gene, Krtap gene, and Kb gene by more than 40%.
- the tea composition can prevent or treat neurodegenerative diseases by up-regulating the expression of at least one Krt gene or Krtap gene by more than 40%, and promoting the growth of nerve cell axons.
- the tea composition prevents or treats neurodegenerative diseases by up-regulating the expression of two or more Krt genes or Krtap genes by at least 40%, and promoting the growth of nerve cell axons.
- the tea composition prevents or treats neurodegenerative diseases by up-regulating the expression of two or more Krt genes or Krtap genes or Kb23 genes by at least 40%, and promoting the growth of nerve cell axons.
- the tea composition prevents or treats neurodegeneration by up-regulating the expression of two or more genes in Krt5, Krt14, Krt17, Krt25, Krt27, Krt31, and Krt35 genes by at least 40%, and promoting the growth of nerve cell axons. disease.
- the tea composition up-regulates estrogen signaling pathway in PC12 cells by binding at least one estrogen receptor.
- the active ingredients of the tea composition include theaflavin digallate and its derivative theaflavin-3-gallate (TF-3G),
- the concentration of the TFDG is 25 ⁇ M
- the concentration of TF-3G is 25 ⁇ M
- the effective amount of the tea composition can up-regulate the estrogen signaling pathway in PC12 cells.
- Said effective amount refers to making cytokeratin gene (keratins, abbreviated as Krt), keratin-associated protein gene (keratin-associated protein, abbreviated as Krtap) and type II keratin gene (ype II keratin, abbreviated as Kb) )
- the expression of at least one gene in ) is effectively up-regulated by more than 40% of the amount of the tea composition.
- Krt genes include Krt1, Krt5, Krt14, Krt15, Krt16, Krt17, Krt25, Krt27, Krt28, Krt31, Krt32, Krt34, Krt35, Krt73, Krt75, Krt81, Krt83, Krt86, Krtap genes include Krtap7-1, Krtap8-1, Krtap11-1, the type II keratin gene is Kb23.
- the tea composition also includes pharmaceutically acceptable excipients or carriers.
- the specific method of the application includes administering an effective amount of the tea composition to the patient, the effective amount refers to the amount of the tea composition that can effectively increase the expression of at least one of the Krt gene, Krtap gene, and Kb gene by more than 40%.
- the tea composition prevents or treats neurodegeneration by up-regulating the expression of two or more genes in Krt5, Krt14, Krt17, Krt25, Krt27, Krt31, and Krt35 genes by at least 40%, and promoting the growth of nerve cell axons. disease.
- the tea composition up-regulates estrogen signaling pathway in PC12 cells by binding at least one estrogen receptor.
- the present invention provides a tea composition capable of regulating estrogen signaling pathways, wherein the theaflavin digallate derivatives include theaflavin TF1, theaflavin-3-gallate (TF-3G), tea Flavin-3'-gallate (TF-3'G), thearubigin, theabrownin or other aqueous extracts of black tea.
- the theaflavin digallate derivatives include theaflavin TF1, theaflavin-3-gallate (TF-3G), tea Flavin-3'-gallate (TF-3'G), thearubigin, theabrownin or other aqueous extracts of black tea.
- a tea composition that can regulate the estrogen signaling pathway of the present invention can replace the active ingredient TFDG in Example 1 with other TFDG derivatives according to the difficulty of raw material acquisition, such as using TFDG and TF-3'G is combined at a mass ratio of 2:1, or TFDG and theaflavin TF1 are combined at a mass ratio of 3:1.
- TFDG and TF-3'G is combined at a mass ratio of 2:1
- TFDG and theaflavin TF1 are combined at a mass ratio of 3:1.
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Abstract
可调节雌激素信号通路的茶组合物及其在防治神经退行性疾病中的应用，其可调节雌激素信号通路的茶组合物的主要活性成分为茶黄素双没食子酸酯(theaflavin-3,3'-digallate,TFDG)，且有效量的茶组合物能够通过上调PC12细胞中雌激素信号通路。有效量是使细胞角蛋白基因Krt、角蛋白相关蛋白基因Krtap、II型角蛋白基因中至少一个基因的表达有效上调40％以上的茶组合物的用量。该可调节雌激素信号通路的茶组合物，通过上调雌激素信号通路中的角蛋白基因表达，促进PC12细胞的轴突生长，起到保护神经的作用。本发明还提供了可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用。
Description
本发明涉及预防和治疗神经性退行疾病领域，尤其是涉及可调节雌激素信号通路的茶组合物及其在防治神经退行性疾病中的应用。
阿尔茨海默病(AD)是一种临床表现为进行性认知衰退的神经系统退行性疾病，近年来，AD是全球内最为常见的一种痴呆性疾病。目前，AD的起因及发病机制尚不太清楚，AD患者大脑中出现的由β-淀粉样蛋白(Aβ)组成的老年斑和tau蛋白磷酸化形成的神经纤维缠结，成为AD发病机制中两种典型的病理特征。现存在众多解释AD发病机制的理论，如淀粉样蛋白沉积，神经细胞凋亡或丢失，氧化应激，内质网应激，线粒体功能障碍，炎症等，但尚未从基因表达方面报道AD的发病机制与雌激素信号通路上的基因表达相关，也没有报道通过调节雌激素信号通路的基因表达，可以防治AD等神经退行性疾病。
茶黄素是儿茶素经过酶促反应发生苯丙环化作用后生成的一类苯并卓酚酮化合物，具有显著的保护心血管作用，具有“软黄金”的美誉。茶黄素双没食子酸酯(theaflavin-3,3′-digallate,TFDG)是茶黄素TFs的主要单体成分之一，含有比EGCG更多的芳香环和羟基(图1)，不仅具有抗氧化、抗炎、防治心血管疾病、降血脂、抗癌防癌等功效，但现有技术未曾报道将茶黄素作为雌激素信号调节剂，对神经细胞的起保护作用，并促进神经细胞轴突生长，用于AD等神经性退行性疾病的预防与治疗。
发明内容
本发明要解决的技术问题是：克服现有技术的不足，提供以茶黄素为主要原料并作为雌激素信号的调节剂的可调节雌激素信号通路的茶组合物，对神经细胞的起保护作用，并促进神经细胞轴突生长，及其在防治神经退行性疾病中的应用。
本发明解决其技术问题所采用的技术方案之一是：
可调节雌激素信号通路的茶组合物，所述茶组合物的主要活性成分为茶黄素双没食子酸酯(theaflavin-3,3′-digallate,TFDG)，且有效量的茶组合物能够上调PC12细胞中雌激素信号通路。
所述有效量是指使细胞角蛋白基因(keratins，简记为Krt)、角蛋白相关蛋白基因(keratin-associated protein，简记为Krtap)、II型角蛋白基因(type II keratin，简记为Kb)中至少一个基因的表达有效上调40％以上的茶组合物的用量。
所述有效量是指茶黄素双没食子酸酯TFDG作为茶组合物的主要活性成分的浓度≥10μM(溶剂型)，或者10mg/g(片剂)。
至少一个Krt基因选自Krt1、Krt5、Krt14、Krt15、Krt16、Krt17、Krt25、Krt27、Krt28、Krt31、Krt32、Krt34、Krt35、Krt73、Krt75、Krt81、Krt83、Krt86，至少一个Krtap基因选自Krtap7-1、Krtap8-1、Krtap11-1，II型角蛋白基因为Kb23。
所述茶组合物的活性成分包括茶黄素双没食子酸酯及其衍生物，及药学上可接受的盐、水合物或其任意组合。
所述茶黄素双没食子酸酯衍生物包括茶黄素TF1、茶黄素-3-没食子酸酯(TF-3G)、茶黄素-3-没食子酸酯(TF-3G)、茶黄素-3'-没食子酸酯(TF-3'G)、茶红素、茶褐素及其他红茶的水提取物。
所述茶组合物还包括药用的赋形剂或载体。
本发明解决其技术问题所采用的另一技术方案是：
可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用。
所述应用的具体方法包括向患者施用有效量的茶组合物，所述有效量是指使至少一个Krt基因或Krtap基因的表达有效上调至少40％以上的茶组合物的用量。
所述茶组合物通过上调至少一个Krt基因或Krtap基因表达至少40％以上，促进神经细胞轴突生长的方式达到预防或治疗神经退行性疾病。
所述茶组合物通过上调两个或多个Krt基因或Krtap基因表达至少40％以上，促进神经细胞轴突生长的方式达到预防或治疗神经退行性疾病。
所述茶组合物通过上调Krt5、Krt14、Krt17、Krt25、Krt27、Krt31、Krt35基因中的2个或多个基因表达至少40％以上，促进神经细胞轴突生长的方式达到预防或治疗神经退行性疾病。
所述茶组合物通过结合至少一种雌激素受体，上调PC12细胞中雌激素信号通路。
本发明一种可调节雌激素信号通路的茶组合物的有益效果：
该茶组合物通过刺激Krt14、Krt15等角蛋白家族基因的转录水平，进而使细胞角蛋白基因(keratins，简记为Krt)或/和角蛋白相关蛋白基因(keratin-associated protein，简记为Krtap)的上调表达，在基因层面对雌激素信号通路具有显著的调节作用，角蛋白家族基因的上调趋势，可保护PC12细胞免受谷胱甘肽消耗引起的氧化休克，并增加NGF诱导的轴突生，故，雌激素信号通路在神经细胞保护和促进神经轴突生长方面具有重要作用。
在PC12细胞中，细胞角蛋白和神经丝蛋白共表达，形成角蛋白中间丝和神经丝。角蛋白中间丝是细胞的第三种骨架成分，与细胞分化，细胞内信息传递等重要生命活动过程有关。此外，角蛋白还可以促进神经元轴突的延伸，可作为外周神经再生的导管填充剂。
图1—为本发明一种可调节雌激素信号通路的茶组合物中TFDG的分子式结构图；
图2—为实施例1中一种可调节雌激素信号通路的茶组合物培养的PC12细胞的轴突生长因子GAP43的ELISA检测分析图；
图3—为实施例1中一种可调节雌激素信号通路的茶组合物培养的PC12细胞内能量货币ATP水平分析图；
图4—为实施例1中一种可调节雌激素信号通路的茶组合物用于PC细胞培养处理后的倒置显微镜观察细胞形态观察图(bar＝50μm)；
图5—为实施例1中一种可调节雌激素信号通路的茶组合物用于PC细胞培养处理后的DAPI染色图(bar＝50μm)；
图6—为实施例1中一种可调节雌激素信号通路的茶组合物用于抑制Aβ
25-35诱导的PC12损伤的差异基因火山图；
图7—为实施例1中一种可调节雌激素信号通路的茶组合物用于抑制Aβ
25-35诱导的PC12损伤的差异基因PPI网络图分析，红色代表上调，绿色代表下调，点的大小代表相对表达量；
图8—为实施例1中一种可调节雌激素信号通路的茶组合物用于抑制Aβ
25-35诱导的PC12损伤的差异基因GO富集分析；
图9—为实施例1中一种可调节雌激素信号通路的茶组合物用于抑制Aβ
25-35诱导的PC12损伤的差异基因KEGG富集分析；
图10—为实施例1中一种可调节雌激素信号通路的茶组合物用于抑制Aβ
25-35诱导的PC12损伤的雌激素信号通路差异基因热图；
图11—为实施例1中一种可调节雌激素信号通路的茶组合物用于抑制Aβ
25-35诱导的PC12损伤的雌激素信号通路Aβ
25-35和Aβ
25-35/TFDG中mRNA的相对表达量分析图。
以下结合附图及实施例对本发明作进一步说明。
实施例1
本实施例的一种可调节雌激素信号通路的茶组合物，所述茶组合物的活性成分为茶黄素双没食子酸酯(theaflavin-3,3′-digallate,TFDG)，所述TFDG的浓度为50μM，且有效量的茶组合物能够上调PC12细胞中雌激素信号通路。
所述有效量是指使细胞角蛋白基因(keratins，简记为Krt)、角蛋白相关蛋白基因(keratin-associated protein，简记为Krtap)及II型角蛋白基因(type II keratin，简记为Kb)中至少一个基因的表达有效上调40％以上的茶组合物的用量。
Krt基因包括Krt1、Krt5、Krt14、Krt15、Krt16、Krt17、Krt25、Krt27、Krt28、Krt31、Krt32、Krt34、Krt35、Krt73、Krt75、Krt81、Krt83、Krt86，Krtap基因包括Krtap7-1、Krtap8-1、Krtap11-1，II型角蛋白基因为Kb23。
所述茶组合物还包括药用的赋形剂或载体。
申请人将上述组合物用于大鼠肾上腺嗜铬细胞瘤细胞系PC12细胞的处理， 分析其对PC12细胞神经细胞的生长影响。具体操作如下：
1)细胞培养与药物处理
将PC12细胞以1×10
5/mL接种于含有10％FBS的DMEM培养基的培养皿中，将其置于含有5％CO
2的37℃恒温培养箱中培养。24h后，分别用50μM Aβ
25-35、50μM Aβ
25-35+50μM EGCG、50μM Aβ
25-35+50μM TFDG处理24h。对照组(Control)细胞则加入等量的无菌水。
2)实验材料
EGCG(≥99％)和TFDG(≥99％)订购自上海同田生物公司(Shanghai,China)。大鼠肾上腺嗜铬细胞瘤细胞系PC12细胞订购自协和细胞库(Beijing,China)。Dulbecco的改良Eagle培养基(DMEM)、胎牛血清(FBS)和胰蛋白酶购自Biological Industries(Cromwell,CT,USA)。Adenosine triphosphate(ATP)检测试剂盒、防荧光淬灭剂(含DAPI)购自碧云天生物技术研究所(Shanghai,China)。大鼠神经生长相关蛋白-43(GAP43)ELISA试剂盒购自江苏菲亚生物科技有限公司。
3)茶组合物对轴突的影响分析
GAP43在神经元中的表达与发育过程中与轴突伸长、突触形成和神经发芽密切相关。ELISA试剂盒检测生长相关蛋白-43(GAP43)，结果如图2所示，与Control组相比，Aβ
25-35组细胞的GAP43含量显著下降(p<0.01)。与Aβ
25-35组相比，Aβ
25-35/TFDG组细胞的GAP43含量显著上升(p<0.01)。结果表明，TFDG能够促进轴突的形成和发育。
轴突中含有丰富的线粒体，线粒体功能障碍会导致细胞轴突退化。图4所示的细胞形态白光拍照结果表明，与Control组比较，Aβ
25-35组的细胞轴突变细 并出现断裂；加入50μM的EGCG和TFDG干预后，细胞轴突变粗，且数量增加，其中TFDG的效果要好于EGCG，图3所示的ATP水平检测结果也印证了EGCG和TFDG干预有利于细胞轴突生长。
图5所示的DAPI染色结果表明，与Control组相比，Aβ
25-35组细胞核的蓝色荧光强度增强；与Aβ
25-35组相比，Aβ
25-35/EGCG与Aβ
25-35/TFDG组细胞核的蓝色荧光染色强度降低，与Control组细胞基本一致，染色均匀，其中TFDG效果更明显，说明Aβ
25-35孵育促进细胞核的异染色质集聚化，而EGCG与TFDG具有抑制细胞核的异染色质集聚化的作用。
4)茶组合物抑制神经细胞退化、促进轴突生长的转录组分析
收集实验组和对照组的细胞样本，每组设置3个生物学重复。随后，所有样本被送往BGI公司(中国深圳)，通过BGISEQ-500测序仪进行进一步的RNA-seq检测和分析。HISAT2(
http://www.ccb.jhu.edu/software/hisat)用于将干净读数映射到大鼠基因组。生物信息学分析主要依靠R语言和在线软件进行分析和可视化。使用R的Limma软件包筛选差异表达基因，|fold changes|≥2 and adjusted p value≤0.05。基于GO和KEGG数据库进行DEGs的通路分析。除热图(https://github.com/CJChen/TBtools)，所有分析均使用BGI提供的在线生物信息平台Dr.Tom(biosys.bgi.com/)进行。
我们收集不同处理组的细胞样品进行转录组测序分析。经过严格的筛选和质量控制筛选显著差异表达基因(discover differentially expressed genes,DEGs)(差异倍数≥2倍，p<0.05)，结果如图6所示：Aβ
25-35 vs Aβ
25-35/EGCG组筛选出67个，上调基因为45个，下调基因为22个；Aβ
25-35 vs Aβ
25-35/TFDG组筛选出2012个，上调基因为199个，下调基因为1813个，结果表明，TFDG孵育可 显著调节(下调/上调)基因表达。
对Aβ
25-35 vs Aβ
25-35/TFDG差异表达基因进行蛋白质-蛋白质互作(PPI)网络分析，以识别关键作用的生物信号通路，并对模块相关的基因进行功能注释，图7所示的结果表明：相互作用的基因在雌激素信号通路、细胞周期、泛素化介导的蛋白质水解及代谢途径等模块中富集。且注释在雌激素信号通路和代谢途径网络主要表现为上调作用，注释在细胞周期和泛素化介导的蛋白质水解通路网络主要表现为下调作用。
以Q值<0.05作为显著富集的标准，图8所示的差异基因GO富集结果表明：Aβ
25-35 vs Aβ
25-35/TFDG差异表达基因在转录调控、mRNA加工、基于微管的运动及有丝分裂细胞周期等生物学过程中显著富集；图9所示的差异基因KEGG功能富集结果表明，在所有发生显著变化的富集信号通路中，大部分差异基因在神经保护及轴突生长相关的通路显著富集，涉及雌激素信号通路、细胞周期、肌动蛋白细胞骨架的调节、黏着斑、GAP连接等。这些途径的变化说明了TFDG对神经保护及促轴突生长的生物活性密切相关。
对图6中Aβ
25-35 vs Aβ
25-35/TFDG PPI网络图中的雌激素信号通路模块进一步分析，结果如图10所示，在该模块中，共有22个差异表达基因DEGs，其中包括Krt15、Krt16、Krt83等18个角蛋白基因家族成员；3个角蛋白相关蛋白，一个II型角蛋白23，且这些基因的表达均呈上调趋势，其中Krt5、Krt17、Krt25、Krt27、Krt31和Krt35共6个角蛋白基因的上调表达幅度超过100％。
选择性雌激素受体调节剂(SERMs)是一类非甾体化合物，能与ER结合，依据靶组织和激素内环境的不同，它们表现为雌激素激动剂和(或)雌激素拮抗剂，根据化学结构分为五大类：①三苯乙烯类化合物；②苯并噻吩类化合物； ③萘类化合物；④苯并吡喃类化合物；⑤其他类化合物。本申请中的茶组合物的活性成分TFDG，与现有的选择性雌激素受体调节剂中的苯并吡喃类化合物，具有共同的化学基团——苯并吡喃，由图10所示的雌激素信号通路相关的差异基因热图可知，本申请中的茶组合物的活性成分TFDG在PC12细胞培养过程中作为雌激素激动剂，促进雌激素与雌激素受体ERα和ERβ结合，活化靶基因转录是经典的雌激素信号通路之一；雌激素信号通路在神经细胞保护和促神经轴突生长方面具有重要作用，这可能是茶黄素作为活性成分的茶组合物保护神经细胞和促轴突生长的重要潜在机制之一，申请人基于该作用机制，开发出以TFDG为主要活性组分的茶组合物的新用途。
TFDG等茶黄素单体或组合物均含苯并吡喃化学基团，且该化学基团的占比较高，与具有现有技术中选择性雌激素受体调节剂中的苯并吡喃类化合物有相同的功能基团，另外TFDG含有比一般儿茶素更多的抗氧化结构共轭效应和酚醌平衡，对衰老和手损伤的神经细胞具有静息态修复作用，通过激活雌激素等信号通路显著促进有利于轴突生长的角蛋白的表达。
可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用。
所述应用的具体方法包括向患者施用有效量的茶组合物，所述有效量是指使Krt基因、Krtap基因、Kb基因中的至少一个表达有效上调40％以上的茶组合物的用量。
其中，所述茶组合物通过上调至少一个Krt基因或Krtap基因表达40％以上，促进神经细胞轴突生长的方式，达到预防或治疗神经退行性疾病。
优选，所述茶组合物通过上调两个或多个Krt基因或Krtap基因表达至少40％以上，促进神经细胞轴突生长的方式，达到预防或治疗神经退行性疾病。
进一步优选，所述茶组合物通过上调两个或多个Krt基因或Krtap基因或Kb23基因表达至少40％以上，促进神经细胞轴突生长的方式达到预防或治疗神经退行性疾病。
所述茶组合物通过上调Krt5、Krt14、Krt17、Krt25、Krt27、Krt31、Krt35基因中的2个或多个基因表达至少40％以上，促进神经细胞轴突生长的方式达到预防或治疗神经退行性疾病。
所述茶组合物通过结合至少一种雌激素受体，上调PC12细胞中雌激素信号通路。
实施例2
本实施例的一种可调节雌激素信号通路的茶组合物，所述茶组合物的活性成分包括茶黄素双没食子酸酯及其衍生物茶黄素-3-没食子酸酯(TF-3G)，所述TFDG的浓度为25μM，TF-3G的浓度为25μM，且有效量的茶组合物能够上调PC12细胞中雌激素信号通路。
所述有效量是指使细胞角蛋白基因(keratins，简记为Krt)、角蛋白相关蛋白基因(keratin-associated protein，简记为Krtap)及II型角蛋白基因(ype II keratin，简记为Kb)中至少一个基因的表达有效上调40％以上的茶组合物的用量。
Krt基因包括Krt1、Krt5、Krt14、Krt15、Krt16、Krt17、Krt25、Krt27、Krt28、Krt31、Krt32、Krt34、Krt35、Krt73、Krt75、Krt81、Krt83、Krt86，Krtap基因包括Krtap7-1、Krtap8-1、Krtap11-1，II型角蛋白基因为Kb23。
所述茶组合物还包括药用的赋形剂或载体。
本实施例的一种可调节雌激素信号通路的茶组合物，在防治神经退行性疾病中的应用。
所述应用的具体方法包括向患者施用有效量的茶组合物，所述有效量是指使Krt基因、Krtap基因、Kb基因中的至少一个表达有效上调40％以上的茶组合物的用量。所述茶组合物通过上调Krt5、Krt14、Krt17、Krt25、Krt27、Krt31、Krt35基因中的2个或多个基因表达至少40％以上，促进神经细胞轴突生长的方式达到预防或治疗神经退行性疾病。
所述茶组合物通过结合至少一种雌激素受体，上调PC12细胞中雌激素信号通路。
此外，本发明一种可调节雌激素信号通路的茶组合物，其中，所述茶黄素双没食子酸酯衍生物包括茶黄素TF1、茶黄素-3-没食子酸酯(TF-3G)、茶黄素-3'-没食子酸酯(TF-3'G)、茶红素、茶褐素或其他红茶的水提取物。
本发明一种可调节雌激素信号通路的茶组合物，本领域技术人员可以根据原料获取的难易程度，将实施例1中的活性成分TFDG部分替换为其他的TFDG衍生物，如采用TFDG与TF-3'G按质量比为2：1进行组合，或者，采用TFDG与茶黄素TF1按质量比为3:1进行组合，以上技术特征的改变，本领域的技术人员通过文字描述可以理解并实施，故不再另作附图加以说明。
在本发明的描述中，需要理解的是，术语“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
Claims (10)
- 可调节雌激素信号通路的茶组合物，其特征在于，所述茶组合物的主要活性成分为茶黄素双没食子酸酯(theaflavin-3,3′-digallate,TFDG)，且有效量的茶组合物能够上调PC12细胞中雌激素信号通路。
- 如权利要求1所述可调节雌激素信号通路的茶组合物，其特征在于，所述有效量是使细胞角蛋白基因Krt、角蛋白相关蛋白基因Krtap、II型角蛋白基因中至少一个基因的表达有效上调40％以上的茶组合物的用量。
- 如权利要求3所述可调节雌激素信号通路的茶组合物，其特征在于，至少一个Krt基因选自Krt1、Krt5、Krt14、Krt15、Krt16、Krt17、Krt25、Krt27、Krt28、Krt31、Krt32、Krt34、Krt35、Krt73、Krt75、Krt81、Krt83、Krt86，至少一个Krtap基因选自Krtap7-1、Krtap8-1、Krtap11-1，II型角蛋白基因为Kb23。
- 如权利要求1～3任一项所述可调节雌激素信号通路的茶组合物，其特征在于，所述茶组合物的活性成分包括茶黄素双没食子酸酯及其衍生物，及药学上可接受的盐、水合物或其任意组合。
- 如权利要求4所述可调节雌激素信号通路的茶组合物，其特征在于，所述茶黄素双没食子酸酯衍生物包括茶黄素TF1、茶黄素-3-没食子酸酯(TF-3G)、茶黄素-3'-没食子酸酯(TF-3'G)、茶红素、茶褐素。
- 如权利要求1～5任一项所述可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用。
- 如权利要求6所述的可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用，其特征在于，所述应用的具体方法包括向患者施用有效量的茶组合物，所述有效量是指使至少一个Krt基因或Krtap基因的表达有效上调至 少40％以上的茶组合物的用量。
- 如权利要求7所述的可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用，其特征在于，所述茶组合物通过上调两个或多个Krt基因表达至少40％以上，促进神经细胞轴突生长的方式，达到预防或治疗神经退行性疾病。
- 如权利要求7所述的可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用，其特征在于，所述茶组合物通过上调Krt5、Krt14、Krt17、Krt25、Krt27、Krt31、Krt35基因中的2个或多个基因表达至少40％以上，促进神经细胞轴突生长的方式达到预防或治疗神经退行性疾病。
- 如权利要求7所述的可调节雌激素信号通路的茶组合物在防治神经退行性疾病中的应用，其特征在于，所述茶组合物通过结合至少一种雌激素受体，上调PC12细胞中雌激素信号通路。
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