CN115120603A - Parishin A在制备巨噬细胞极化功能调控剂中的应用 - Google Patents
Parishin A在制备巨噬细胞极化功能调控剂中的应用 Download PDFInfo
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Abstract
本发明提供了Parishin A在制备巨噬细胞极化功能调控剂中的应用。本发明对Prishin A的免疫调节功能进行体内外实验验证，证明具有很好的免疫调节功能。本发明的Prishin A在一定浓度条件下具有抑制巨噬细胞向促炎型巨噬细胞(M1型)转化，下调相关促炎性细胞因子的水平，加速炎症的缓解。Prishin A具有良好的抗炎免疫抑制活性和较高的安全性，可用于制备缓解炎症性疾病的药物从而提高机体抵御炎症反应的能力。
Description
技术领域
本发明属于Parishin A新用途技术领域，特别涉及Parishin A在制备巨噬细胞极化功能调控剂中的应用。
背景技术
随着免疫系统知识的进步，巨噬细胞在免疫反应过程中表现出的特殊功能正在逐渐被大家所认知。巨噬细胞可以在特定组织内从血液来源的单核细胞中最终分化，或者从胚胎发育期进入常驻组织中巨噬细胞而来。组织稳态收到破坏，促炎趋化因子释放，将会触发骨髓来源的循环单核细胞迁移到炎症部位，分化成巨噬细胞，以维持免疫和炎症消退和组织重塑。它们起到免疫监视作用，巨噬细胞感知不同的刺激并以复杂的激活机制做出反应，分化为促炎M1或抗炎M2型巨噬细胞。经典的M1极化可通过脂多糖(LPS)激活。极化程序通过特定转录因子的激活和核转位发生，例如B细胞的核因子kappa-轻链增强子(NF-κ)、信号转导和转录激活因子STAT1和STAT3等。这种转录激活导致特定细胞标志物的表达，例如CD80、CD86、环氧合酶2(COX-2)，以及诱导型一氧化氮合酶(iNOS),并伴随促炎细胞因子的释放，例如TNF-α、IL1-β、IL-6、IL-12等。
M2巨噬细胞通常以抗炎基因表达谱为特征，有利于炎症消退和组织修复。M2巨噬细胞可由白细胞介素4(IL-4)或白细胞介素13(IL-13)诱导，常见激活分子机制为STAT6,GATA结合蛋白3(GATA3)以及细胞因子信号1抑制因子(SOCS1)等。与M2相关的典型标志物是甘露糖受体(CD206)、IL-1R以及IL-1R拮抗剂。这些细胞通过促纤维化因子的释放，例如转化生长因子β(TGF-β)，胰岛素样生长因子1(IGF-1)和精氨酸酶1(ARG1)，有助于缓解炎症、帮助组织愈合和修复。
小分子药物是热门的话题，在肿瘤治疗、干细胞调控、再生医学领域逐渐兴起。其分子量<1000KD，可以跨越生物屏障，通过简单的扩散进入细胞，甚至细胞器。天然小分子化合物是从食品成分、植物或中草药中提取的，具有多种药理活性，包括抗炎、抗氧化、抗寄生虫和抗病毒作用。
Parishin A是一种从天麻中分离得到的酚类葡萄糖苷类中药。天麻是一种名贵中药,用于治疗头痛、头昏、神经衰弱等。研究表明，PA在抗衰老，抗精神病，抗癌中起到一定作用，但PA是否具有抗炎作用，是否调节巨噬细胞极化功能，从而阻止炎症病变的进展尚不清楚。
发明内容
发明目的：为了解决现有技术的缺陷，本发明提供了Parishin A在制备巨噬细胞极化功能调控剂中的应用。
技术方案：为达到上述发明目的，本发明采用以下技术方案：
Parishin A在制备巨噬细胞极化功能调控剂中的应用。
更具体的，所述巨噬细胞极化功能调控剂，能够抑制巨噬细胞向促炎型巨噬细胞M1型转化。
更具体的，所述巨噬细胞极化功能调控剂，能够促进巨噬细胞向抗炎型巨噬细胞M2型转化。
包含Parishin A的组合物在制备巨噬细胞极化功能调控剂中的应用。
Parishin A、或包含Parishin A的组合物在制备JAK1抑制剂中的应用。
Parishin A、或包含Parishin A的组合物在制备STAT1抑制剂中的应用。
本发明最后提供了Parishin A在制备抗炎药物中的应用。
更具体的，所述抗炎药物，能够治疗急性炎症。
更具体的，所述抗炎药物，能够治疗慢炎症。
本发明中所述Parishin A，又称巴利森苷A或派立辛，其CAS号为62499-28-9，分子式为C45H56O25。
有益效果：本发明首次提出Prishin A在制备抗炎药物，治疗炎症性疾病中的应用，其能够通过调控巨噬细胞极化有效抑制炎症反应，可作为炎症反应的临床治疗中较为有效且毒副作用较小的候选药物。此外，本发明还提出了活性成分包括Prishin A的药物，其具有较高的抗炎作用，具备良好的市场潜力和价值。
附图说明
图1为0，10μM，20μM，50μM以及100μM的Prishin A处理大鼠骨髓源性巨噬细胞24h后的活死染色荧光结果图，及相应的统计图。
图2为0，10μM，20μM，50μM以及100μM的Prishin A处理大鼠骨髓源性巨噬细胞24h后的细胞凋亡的流式结果图。
图3为Prishin A处理LPS激活的大鼠骨髓源性巨噬细胞12h后的炎症因子及巨噬细胞标志物的PCR结果。
图4为Prishin A处理LPS激活的大鼠骨髓源性巨噬细胞24h后的炎症因子及巨噬细胞标志物的WB结果。
图5为Prishin A处理LPS激活的大鼠骨髓源性巨噬细胞48h后的巨噬细胞标志物免疫荧光结果及阳性细胞比例统计图。
图6为Prishin A处理LPS激活的人来源巨噬细胞系THP-1在12h后的炎症因子及巨噬细胞标志物的PCR结果。
图7为Prishin A处理LPS激活的人来源巨噬细胞系THP-1在24h后的的炎症因子及巨噬细胞标志物的WB结果。
图8为Prishin A处理LPS激活的人来源巨噬细胞系THP-1在24h后巨噬细胞标志物免疫荧光结果。
图9为Prishin A处理LPS激活的大鼠骨髓源性巨噬细胞1h后JA1K，STAT1变化的WB结果，及统计图。
图10为Prishin A处理LPS激活的大鼠骨髓源性巨噬细胞1h后STAT1磷酸化水平变化的免疫荧光图，及阳性细胞比例统计图。
图11为Prishin A处理LPS激活的人来源巨噬细胞系THP-1在1h后JAK-STAT通路的JA1K，STAT1变化的WB结果图。
图12为Prishin A处理LPS激活的人来源巨噬细胞系THP-1在1h后STAT1磷酸化水平变化的免疫荧光图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
(1)获取3-4周龄大鼠股骨，尽可能靠近关节处剪去骨头的两端。1ml注射器中插入骨头，用含双抗的PBS将骨髓冲出到离心管中。冲洗2-3次，直至骨头完全变白。用移液管吸打骨髓液几次，以分离所有的团块，并使用70μm的滤器过筛。加入淋巴细胞分离液1800rpm，30min密度梯度离心，取中间单核细胞层加RPMI培养基重悬，加20ng/ml的rM-CSF铺板，放入37℃，5％CO2培养箱中培养(6孔板，1盘/每鼠)，每3d更换新鲜培养基，7d后获得成熟的大鼠骨髓源性巨噬细胞(BMDM)。加入0，10μM，20μM，50μM以及100μM的Parishin A，在加药24h后对BMDM进行活死染色，并在荧光显微镜下进行观察。
(2)将人来源单核细胞系THP-1细胞按9×105个/孔的密度种于6孔板，加入佛波醇12-十四酸酯13-乙酸酯(PMA)贴壁过夜极化为巨噬细胞后，用0、10μM、20μM、50μM以及100μM的Parishin A处理24后，收集细胞进行7-AAD和Annexin V凋亡检测。
结果显示：
1)镜下观察BMDM在0-100μM浓度范围的Parishin A处理后未见大量BMDM细胞死亡，见图1；
2)流式结果显示在0-100μM浓度范围的Parishin A处理后THP-1巨噬细胞未出现明显凋亡，见图2；
以上结果表明：Parishin A对巨噬细胞在一定剂量范围内没有显著的细胞毒性，因此这种抗炎作用不是通过细胞毒性而实现。
实施例2
为了探究Parishin A对BMDM巨噬细胞极化的影响，收集成熟的BMDM，加入100ng/ml的LPS刺激12h后，实验组BMDM加入20μM的PA溶液，对照组加入相同体积的DMSO。
(1)在相同条件下连续培养12h后，收集细胞的mRNA进行RT-PCR，分析IL-6、IL-1β、iNOS、CD206及CD163的表达情况；
(2)相同条件下连续培养24h天后，进行Western blot实验检测BMDM细胞中IL-6、iNOS、CD206及Arg-1的蛋白表达水平。
(3)相同条件下连续培养48h天后，进行免疫荧光染色实验检测BMDM细胞中巨噬细胞marker中CD68、iNOS及CD206蛋白表达水平。
结果显示：
1)实验组的M1标志物iNOS及分泌的促炎因子IL-6和IL-1β在转录水平相较对照组明显下降，M2标志分子CD206和CD163表达上升，实验组和对照组相比具有统计学意义。通过RT-PCR实验在基因水平初步证明：20μm的PA能够抑制BMDM巨噬细胞的M1型极化，下调促炎因子的表达，诱导M2极化。见图3。
2)实验组CD68+iNOS+阳性细胞数较少，分泌的促炎细胞因子IL-6表达下调，而CD68+CD206+高表达，分泌Arg-1表达上升。通过Western blot和免疫荧光实验在蛋白水平进一步证明：PA可调控BMDM巨噬细胞的极化，起到抗炎作用，见图4,5。
结论：20μM的Parishin可调控BMDM巨噬细胞极化，抑制其M1向极化，促进M2向极化，有一定抗炎作用。
实施例3
为了进一步验证Parishin A对巨噬细胞极化的影响，推进临床转化，我们在人来源THP-1巨噬细胞进行了相似的研究，将人来源巨噬细胞系THP-1细胞按9×105个/孔的密度种于6孔板，加入佛波醇12-十四酸酯13-乙酸酯(PMA)贴壁过夜，用20μM的Parishin A处理12h后，实验组THP-1巨噬细胞加入20μM的PA溶液，对照组加入相同体积的DMSO。
(1)在相同条件下连续培养12h后，收集细胞的mRNA进行RT-PCR，分析IL-6、IL-1β、TNF-α和Arg-1的转录水平的变化；
(2)相同条件下连续培养24h天后，进行Western blot实验检测THP-1细胞中IL-6、iNOS和Arg-1的蛋白表达水平。
(3)相同条件下连续培养48h天后，进行免疫荧光染色实验检测THP-1细胞中巨噬细胞marker中CD68、iNOS和CD206蛋白表达水平。
结果显示：
1)实验组的巨噬细胞促炎因子IL-6、IL-1β和TNF-α在转录水平相较对照组明显下降，抗炎分子Arg-1表达上升，实验组和对照组相比具有统计学意义。通过RT-PCR实验在基因水平初步证明：20μm的PA能够下调THP-1巨噬细胞的促炎因子的表达，使抑炎因子表达上升，见图6。
2)实验组CD68+iNOS+阳性细胞数较少，分泌的促炎因子IL-6和iNOS表达下调，而CD68+CD206+高表达，分泌Arg-1表达上升。通过Western blot和免疫荧光实验在蛋白水平进一步证明：PA可调控THP-1巨噬细胞的极化，起到抗炎作用，见图7,8。
结论：20μM的Parishin可调控THP-1巨噬细胞极化，抑制其M1向极化，促进M2向极化，起到一定的抗炎作用。
实施例4
为了探究PA调控大鼠骨髓源性巨噬细胞BMDM及人来源巨噬细胞系THP-1极化的作用机制，我们通过Western blot和免疫荧光染色实验进行证明。对照组和实验组BMDM及THP-1巨噬细胞在LPS刺激15/30min后，对照组加入PBS溶液，实验组加入20μM的Parishin A处理30min/1h后，进行以下处理：
(1)使用Western blot实验检测BMDM细胞中炎性信号通路JAK-1、p-STAT1和STAT1的表达情况，见图9；
(2)对BMDM细胞进行p-STAT1免疫荧光染色，镜下随机拍照并分别统计p-STAT1的阳性细胞数目，见图10；
(3)使用Western blot实验检测THP-1细胞中炎性信号通路JAK-1、p-STAT1和STAT1的表达情况，见图11；
(4)对THP-1细胞进行p-STAT1免疫荧光染色，镜下随机拍照并分别统计p-STAT1的阳性细胞数目，见图12；
结果显示：对照组stat1磷酸化水平显著升高，PA可通过抑制JAK激活，下调STAT1的磷酸化，初步证实PA可通过抑制JAK/STAT1信号通路的活化影响巨噬细胞极化。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (9)
1.Parishin A在制备巨噬细胞极化功能调控剂中的应用。
2.根据权利要求1所述的应用，其特征在于，所述巨噬细胞极化功能调控剂，能够抑制巨噬细胞向促炎型巨噬细胞M1型转化。
3.根据权利要求1所述的应用，其特征在于，所述巨噬细胞极化功能调控剂，能够促进巨噬细胞向抗炎型巨噬细胞M2型转化。
4.包含Parishin A的组合物在制备巨噬细胞极化功能调控剂中的应用。
5.Parishin A、或包含Parishin A的组合物在制备JAK1抑制剂中的应用。
6.Parishin A、或包含Parishin A的组合物在制备STAT1抑制剂中的应用。
7.Parishin A在制备抗炎药物中的应用。
8.根据权利要求8所述的应用，其特征在于，所述抗炎药物，能够治疗急性炎症。
9.根据权利要求8所述的应用，其特征在于，所述抗炎药物，能够治疗慢炎症。
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