黄芪丹参配伍调控ASIC1a/CaMKⅡδ信号抑制酸性微环境中心肌细胞焦亡

张蒙; 王新东

doi:10.14148/j.issn.1672-0482.2023.0138

黄芪丹参配伍调控ASIC1a/CaMKⅡδ信号抑制酸性微环境中心肌细胞焦亡

doi: 10.14148/j.issn.1672-0482.2023.0138

张蒙^{1, 2,},
王新东^1, ,

1.
南京中医药大学第三临床医学院, 江苏南京 210028
2.
上海中医药大学附属曙光医院，上海 201203

基金项目:

江苏省中医局科技项目 YB201922

董其美全国名老中医药专家传承工作室建设项目国中医药人教函〔2022〕75号

江苏省研究生科研与实践创新计划项目 SJCX22_0837

江苏省研究生科研与实践创新计划项目 SJCX22_0858

江苏省研究生科研与实践创新计划项目 SJCX22_0859

详细信息

作者简介:
张蒙, 女, 硕士研究生, E-mail：20201260@njucm.edu.cn

通讯作者:
王新东, 男, 主任中医师, 主要从事中医心系疾病研究, E-mail：wangxd@njucm.edu.cn

中图分类号: R285.5
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- 被引次数: 0
出版历程
- 收稿日期: 2022-08-11
- 网络出版日期: 2023-02-17
- 发布日期: 2023-02-10

Huangqi-Danshen in the Regulation of ASIC1a/CaMKⅡδ Signaling to Inhibit Cardiomyocytes from Pyroptosis in Acidic Microenvironment

ZHANG Meng^{1, 2
,},
WANG Xin-dong^{1
, ,}

1.
The Third School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, China
2.
Shuguang Hospital, Shanghai University of Chinese Medicine, Shanghai 201203, China

摘要

摘要: 目的探讨黄芪丹参配伍对乳酸诱导的酸性微环境中心肌细胞焦亡损伤的保护作用及基于酸敏感离子通道1a(ASIC1a)/钙离子-钙调蛋白依赖性激酶Ⅱδ(CaMKⅡδ)信号通路的调控机制。方法采用Lactate刺激H9C2心肌细胞建立pH 6.5酸性微环境细胞损伤模型。分为正常对照组、模型组、黄芪丹参提取物(HQ)组、HQ+ASIC激动剂Nocistatin组和ASIC抑制剂NS383阳性药对照组。采用CCK-8法测定心肌细胞活力, 流式细胞术检测心肌细胞凋亡, 免疫荧光检测ASIC1a表达, qPCR分析NLRP3、Caspase-1、IL-1β及GSDMD mRNA表达, Western blot检测ASIC1a、CaMKⅡδ、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD及GSDME-N蛋白的表达。结果在pH 6.5酸性环境中, 心肌细胞活力显著降低(P < 0.01), 细胞凋亡明显增加(P < 0.01), NLRP3、Caspase-1、IL-1β及GSDMD mRNA表达明显增加(P < 0.01), ASIC1a、CaMKⅡδ、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD及GSDME-N蛋白表达明显增加(P < 0.01)。60 μg · mL^-1HQ可显著促进pH6.5酸性微环境中心肌细胞活力(P < 0.01), 抑制心肌细胞凋亡(P < 0.01), 下调NLRP3、Caspase-1、IL-1β、GSDMD mRNA和ASIC1a、CaMKⅡδ、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD及GSDME-N蛋白表达(P < 0.01)。并且HQ的以上效应可被ASIC激动剂Nocistatin逆转。结论黄芪丹参配伍可下调ASIC1a/CaMKⅡδ信号抑制酸性微环境下NLRP3炎症小体介导的心肌细胞焦亡。
- 黄芪丹参配伍 /
- 冠心病 /
- ASIC1a /
- 酸性微环境 /
- 细胞焦亡
Abstract: OBJECTIVE To observe the protective effect of Huangqi-Danshen on cardiomyocytes pyroptosis or injury in acidic microenvironment induced by lactic acid and the regulatory mechanism based on the acid-sensitive ion channel 1a (ASIC1a) or calcium/calmodulin-dependent protein kinase Ⅱδ (Ca MKⅡδ) signaling pathway. METHODS Lactate was used to stimulate H9C2 cardiomyocytes to establish the cell injury model in the acidic environment of pH 6.5. The rats were divided into the normal control group, model group, Huangqi-Danshen extract (HQ) group, HQ + ASIC agonist Nocistatin group, as well as ASIC inhibitor NS383 positive drug control group. The viability of cardiomyocytes was measured by CCK-8 method. The apoptosis of cardiomyocytes was detected by flow cytometry. Besides, the expression of ASIC1a was detected by immunofluorescence. The mRNA expression of NLRP3, Caspase-1, IL-1β, and GSDMD was analyzed by qPCR. In addition, the protein expression of ASIC1a, CaMKⅡδ, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, and GSDME-N was detected by Western blot. RESULTS In the acidic environment of pH 6.5, the viability of cardiomyocytes was significantly reduced (P < 0.01), but the apoptosis was significantly increased (P < 0.01). The mRNA expressions of NLRP3, Caspase-1, IL-1β and GSDMD were significantly increased (P < 0.01), and the protein expressions of ASIC1a, CaMKⅡδ, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD and GSDME-N were also significantly increased (P < 0.01). In addition, we found that HQ (60 μg·mL^-1) could significantly promote the viability of cardiomyocytes in acidic microenvironment (P < 0.01), inhibit cardiomyocyte apoptosis (P < 0.01), and down-regulate the mRNA expressions (NLRP3, Caspase-1, IL-1β, GSDMD) combined with protein expressions (ASIC1a, CaMKⅡδ, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, GSDME-N) (P < 0.01). Besides, the above effects of HQ can be reversed by ASIC agonist Nocistatin. CONCLUSION Huangqi-Danshen can down-regulate ASIC1a/CaMKⅡδ signaling to inhibit NLRP3 inflammasome -mediated cardiomyocyte pyroptosis in acidic microenvironment.
- Huangqi-Danshen /
- coronary heart disease /
- ASIC1a /
- acidic microenvironment /
- pyroptosis

HTML全文

图 1 CCK8检测细胞活力

]注: 与Blank组相比, ^{* *}P < 0.01;与Control组相比, ^##P < 0.01; 与HQ 60 μg · mL^-1组相比, ^▲P < 0.05。x±s, n=6。]> HQ抑制pH 6.5酸性环境中心肌细胞凋亡本研究进一步采用流式细胞术检测pH 6.5酸性环境中心肌细胞凋亡, 结果显示: pH 6.5酸性环境可诱导细胞凋亡明显增加(P < 0.01), HQ和ASIC1a抑制剂NS383均可减少酸性环境中的心肌细胞凋亡(P < 0.01), 且HQ的这种保护效应可被ASIC1a激动剂Nocistatin逆转, 提示HQ对酸性环境中的心肌细胞凋亡的保护效应与调控ASIC1a酸敏感Ca²⁺通道有关(图 2)。流式细胞术检测各组pH 6.5酸性环境中心肌细胞凋亡 Flow cytometry detection of cardiomyocyte apoptosis in the acidic environment of pH 6.5 in each group 注: Q1-UL.坏死细胞；Q1-UR.晚期凋亡细胞；Q1-LL.正常细胞；Q1-LR.早期凋亡细胞。与Control组相比, ^{* *}P < 0.01;与Model组相比, ^##P < 0.01;与HQ组相比, ^▲▲P < 0.01。x±s, n=3。

Figure 1. CCK8 assay for cell viability

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图 3 各组pH 6.5酸性环境中心肌细胞ASIC1a表达情况比较

注: 比例尺=50 μm。与Control组相比, ^{* *}P < 0.01;与Model组相比, ^##P < 0.01;与HQ组相比, ^▲P < 0.05。x±s, n=3。

Figure 3. Comparison of ASIC1a expression in cardiomyocytes in the acidic environment of pH 6.5 among groups

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图 4 pH 6.5酸性环境中心肌细胞NLRP3、Caspase-1、IL-1β及GSDMD mRNA表达比较

注: 与Control组相比, ^{* *}P < 0.01;与Model组相比, ^##P < 0.01;与HQ组相比, ^▲P < 0.05，^▲▲P < 0.01。x±s, n=3。

Figure 4. Comparison of cardiomyocytes mRNA expression of NLRP3, Caspase-1, IL-1β and GSDMD in the acidic environment of pH 6.5

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图 5 pH 6.5酸性环境中心肌细胞ASIC1a、CaMKⅡδ、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD及GSDME-N蛋白表达比较

注: 与Control组相比, ^{* *}P < 0.01;与Model组相比, ^#P < 0.05, ^##P < 0.01;与HQ组相比, ^▲P < 0.05, ^▲▲P < 0.01。x±s, n=3。

Figure 5. Comparison of cardiomyocytes protein expression of ASIC1a, CaMKⅡδ, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD and GSDME-N in the acidic environment of pH 6.5

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表 1 特定引物序列

Table 1. Specific primer sequences

引物		序列(5′→3′)
NLRP3	Forward Reverse	5′-GTGTTTCGAATCCCACTGTG-3′ 5′-TCTGCTTCTCACGTACTTTCTG-3′
Caspase-1	Forward Reverse	5′-ACGCCTTGCCCTCATAAT-3′ 5′-TCTAATACATCTGGGACTTCTT-3′
IL-1β	Forward Reverse	5′-ATGCACCTGTACGATCACTG-3′ 5′-ACAAAGGACATGGAGAACACC-3′
GSDMD	Forward Reverse	5′-CTACCTTACCTCATGCCGTTCGT-3′ 5′-ACCTCCGTCCGAGAAGTCTGAA-3′
β-actin	Forward Reverse	5′-TACAACTCCTTGCAGCTCC-3′ 5′-ATCTTCATGAGGTAGTCAGTC-3′

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