CN113058036B - SCGB3A2-PDPN-RhoA信号通路作为抑制肺部炎症因子风暴的药物靶点 - Google Patents

SCGB3A2-PDPN-RhoA信号通路作为抑制肺部炎症因子风暴的药物靶点 Download PDF

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CN113058036B
CN113058036B CN202110418539.9A CN202110418539A CN113058036B CN 113058036 B CN113058036 B CN 113058036B CN 202110418539 A CN202110418539 A CN 202110418539A CN 113058036 B CN113058036 B CN 113058036B
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韩磊
宋怀东
郑翠霞
韩兵
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Abstract

本发明通过研究发现肺气管支气管中的外分泌蛋白SCGB3A2通过与细胞表面的受体PDPN结合并激活RhoA蛋白增强了肺炎的强度和炎症因子的表达量，而通过抗体阻断SCGB3A2与PDPN结合则可以缓解肺部炎症，减少炎症因子的表达和肺部炎症细胞的浸润。因此本发明为控制细菌或病毒导致的肺部炎症因子风暴提供了一个全新靶点。由于该靶点有较明确的信号传递途径，针对该靶点所做的抑制炎症因子风暴的药物相比糖皮质激素的副作用应该更小。

Description

SCGB3A2-PDPN-RhoA信号通路作为抑制肺部炎症因子风暴的 药物靶点
技术领域
本发明涉及医药技术领域，尤其涉及SCGB3A2-PDPN-RhoA信号通路作为抑制肺部炎症因子风暴的药物靶点的应用。
背景技术
细菌或病毒感染肺部后可以被肺中巨噬细胞的模式识别受体识别，随后模式识别受体会被激活并进一步活化细胞内的转录因子NF-κB，从而激活炎症因子如IL-6、IL-1β和TNFα，这些炎症因子可以进一步激活其他免疫细胞，调控适应性免疫反应，从而帮助机体清除感染并修复损伤组织。尽管炎症反应对保护宿主抗感染至关重要，但炎症通路的过度活化会产生过量的炎症因子，产生“炎症因子风暴”并导致严重的疾病如肺炎和脓毒症休克，进而导致患者死亡。
目前临床中控制肺炎应用较多的药物是抗生素，抗生素可以直接杀死细菌，只能用于细菌感染引发的肺炎，糖皮质激素则可以直接控制炎症反应，可用于细菌或病毒性肺炎的控制。但是糖皮质激素的作用靶点非常广泛，在抑制炎症反应的同时也会带来一系列副作用，例如，使机体抵抗能力降低，长期使用糖皮质激素会导致血糖升高、胆固醇代谢紊乱、骨质疏松甚至白内障。
蛋白SCGB3A2是一种肺中的外分泌蛋白，全称secretoglobin family 3A member2，别名UGRP1，编码该蛋白的人源基因的NCBI Gene ID：117156，编码该蛋白的鼠源基因的NCBI Gene ID：117158。蛋白PDPN是细胞表面的一种单次跨膜受体，全称podoplanin，编码该蛋白的人源基因的NCBI Gene ID：10630，编码该蛋白的鼠源基因的NCBI Gene ID：14726。但是现有技术中未有将SCGB3A2-PDPN-RhoA信号通路作为抑制肺部炎症因子风暴的药物靶点的应用。
发明内容
我们通过研究发现肺气管支气管中的外分泌蛋白SCGB3A2通过与细胞表面的受体PDPN结合并激活RhoA蛋白增强了肺炎的强度和炎症因子的表达量，而通过抗体阻断SCGB3A2与PDPN结合则可以缓解肺部炎症，减少炎症因子的表达和肺部炎症细胞的浸润。因此我们在本发明中提供了SCGB3A2-PDPN-RhoA信号通路作为治疗肺部炎症因子风暴的靶点。
具体而言，本发明第一方面提供阻断SCGB3A2和/或PDPN信号的物质在制备治疗或预防感染性疾病引起炎症风暴的药物中的应用。
优选的，所述感染性疾病为病毒、衣原体、支原体、细菌或寄生虫引起的感染。
进一步优选的，所述炎症风暴为感染性疾病引起机体内多种细胞因子迅速大量产生的现象，优选的所述机体内多种细胞因子为TNFα、IL-1β、IL-6、MCP-1、IL-1α、IL12、IL27、GM-CSF、IFNα、IFN-γ。
进一步优选的，所述阻断SCGB3A2和/或PDPN信号的物质包括PDPN抗体(B-11)(产品编号：sc-166906)。
本发明还提供阻断SCGB3A2和/或PDPN信号的物质在制备治疗或预防病毒、衣原体、支原体、细菌或寄生虫引起的肺炎的药物中的应用。
优选的，所述阻断SCGB3A2和/或PDPN信号的物质包括PDPN抗体(B-11)(产品编号：sc-166906)。
优选的，前述任一项所述的应用中，所述阻断SCGB3A2和/或PDPN信号的物质和药学上常规的药用辅料制成药物制剂，进一步优选的，所述药物制剂是口服剂型、注射剂型或粉针剂。
本发明中所述PDPN抗体(B-11)(产品编号：sc-166906)为Santa Cruz公司产品，详细信息可参见https://datasheets.scbt.com/sc-166906.pdf，https://www.scbt.com/p/podoplanin-antibody-b-11？requestFrom＝search。
本发明的有益效果在于，本发明通过研究发现肺气管支气管中的外分泌蛋白SCGB3A2通过与细胞表面的受体PDPN结合并激活RhoA蛋白增强了肺炎的强度和炎症因子的表达量，而通过抗体阻断SCGB3A2与PDPN结合则可以缓解肺部炎症，减少炎症因子的表达和肺部炎症细胞的浸润。因此本发明为控制细菌或病毒导致的肺部炎症因子风暴提供了一个全新靶点，由于该靶点有较明确的信号传递途径，针对该靶点所做的抑制炎症因子风暴的药物相比糖皮质激素的副作用应该更小。
附图说明：
图1.PDPN抗体B-11抑制了SCGB3A2促进的IL-6表达升高。
图2.对照IgG和PDPN B-11抗体通过气管注射的方式连续三天打入小鼠肺中，第三天通过气管注射的方式同时将肺炎链球菌注射到小鼠肺中引发肺炎。
图3.PDPN抗体B-11延长了小鼠注射肺炎链球菌后的生存时间。
图4.PDPN抗体B-11抑制了肺炎小鼠肺中IL-6表达。
图5.PDPN抗体B-11抑制了肺炎小鼠肺中IL-6、IL-1β和TNFα的mRNA水平。
图6.PDPN抗体B-11抑制了肺炎小鼠肺中细胞浸润。
具体实施方式
下面结合具体的实施例对本发明作进一步地说明，以更好地理解本发明。
实施例1
在SCGB3A2存在条件下，Santa Cruz公司的PDPN抗体(B-11)阻止了原代巨噬细胞中过量炎症因子的产生。
实验材料：野生型C57BL/6小鼠购买于上海南方模式生物科技股份有限公司，PDPN抗体(B-11)(产品编号：sc-166906)购买于Santa Cruz公司，TLR2受体激活剂Pam3CSK4购买于InvivoGen公司(产品编号tlrl-pms)，DMEM培养液购买于Gibico公司，胎牛血清购买于Hyclone公司。Trizol总RNA提取试剂、RNA反转录酶和qRT-PCR试剂盒购买于Takara公司。
实验方法：从野生型C57BL/6小鼠分离出原代腹腔巨噬细胞PEM，以每孔100万个细胞的数量接种到12孔板中，同时每孔加入1mL含有10％FBS的DMEM培液，在37摄氏度5％CO2的培养箱中培养过夜。第二天用浓度为2μg/mL的对照IgG或PDPN抗体B-11预处理巨噬细胞1小时，然后使用浓度为0.5μg/mL重组人源SCGB3A2蛋白联合0.1μg/mL Pam3CSK4共同刺激巨噬细胞6小时，之后每孔加入1mL Trizol，按照说明书提取RNA，并对RNA进行定量，再按照说明书将mRNA反转录为cDNA，并使用ABI QuantStudio 6Flex荧光定量PCR仪对反转录得到的cDNA进行荧光定量扩增，以检测IL-6的mRNA水平。
实验结果：PDPN抗体B-11可以很好的抑制SCGB3A2促进IL-6表达增强的作用，如图1所示。
实施例2
在野生型的C57BL/6小鼠中使用肺炎链球菌诱导肺部炎症因子风暴，Santa Cruz公司的PDPN抗体(B-11)通过气管注射到小鼠肺部可以减少小鼠肺中炎症因子的产生，延长小鼠的存活时间，并且减少小鼠肺部免疫细胞的浸润。
实验材料：野生型C57BL/6小鼠购买于上海南方模式生物科技股份有限公司，PDPN抗体(B-11)(产品编号：sc-166906)购买于Santa Cruz公司，肺炎链球菌S.pneumoniae购买于ATCC(产品编号：6303)，IL-6ELISA试剂盒购买于Invitrogen公司(产品编号：88-7064-22)。Trizol总RNA提取试剂、RNA反转录酶和qRT-PCR试剂盒购买于Takara公司。
实验方法：分别将对照IgG和PDPN B-11抗体通过气管注射的方式连续三天打入小鼠肺中，第三天通过气管注射的方式同时将肺炎链球菌注射到小鼠肺中引发肺炎(如图2)，注射肺炎链球菌24小时后，杀掉小鼠使用PBS灌洗小鼠肺部拿到小鼠肺泡灌洗液，按照说明书进行ELISA检测肺泡灌洗液中IL-6蛋白水平。同时使用Trizol提取肺组织中的RNA，按照说明书将mRNA反转录为cDNA，并使用ABI QuantStudio 6Flex荧光定量PCR仪对反转录得到的cDNA进行荧光定量扩增，以检测肺中IL-6、IL-1β和TNFα的mRNA水平。同时观察小鼠诱导肺炎后的生存时间。
实验结果：PDPN抗体B-11可以很好的抑制肺炎链球菌导致的肺部炎症因子水平升高(如图4,5)和肺部细胞的浸润(如图6)，并延长肺炎小鼠的生存时长(如图3)。
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (2)

1.阻断SCGB3A2和/或PDPN信号的物质及其药物制剂在制备治疗或预防肺炎链球菌诱导的肺部炎症因子风暴的药物中的应用，其特征在于，所述阻断SCGB3A2和/或PDPN信号的物质为产品编号为sc-166906的PDPN抗体B-11，所述肺部炎症因子风暴为多种细胞因子迅速大量产生的现象，所述多种细胞因子为TNFα、IL-1β和IL-6。
2.根据权利要求1所述的应用，其特征在于，所述药物制剂是口服剂型、注射剂型或粉针剂。
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