丹参酮Ⅰ调控巨噬细胞非经典焦亡通路机制研究

郑逸豪; 陈月; 赵凯迪; 杨宇; 马重阳; 张秋云

doi:10.16766/j.cnki.issn.1674-4152.004159

丹参酮Ⅰ调控巨噬细胞非经典焦亡通路机制研究

doi: 10.16766/j.cnki.issn.1674-4152.004159

首都医科大学中医药学院中医络病研究北京市重点实验室，北京 100069

基金项目:

国家自然科学基金项目 82074237

详细信息

通讯作者:
张秋云，E-mail: zhangqiuyun8202@aliyun.com

中图分类号: R-332
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- 文章访问数: 3
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- 被引次数: 0
出版历程
- 收稿日期: 2025-03-24
- 网络出版日期: 2025-11-17

The mechanistic study of tanshinone Ⅰ Regulating non-canonical pyroptosis pathway in macrophages

School of Traditional Chinese Medicine, Capital Medical University, Beijing Key Lab of Traditional Chinese Medicine Collateral Disease Theory Research, Beijing 100069, China

摘要

摘要: 目的探讨丹参酮Ⅰ对脂多糖(LPS)诱导的小鼠巨噬细胞J774.1非经典细胞焦亡的干预作用及其潜在机制。方法通过Lipofiter转染LPS建立J774.1细胞非经典焦亡模型，采用不同浓度丹参酮Ⅰ干预，筛选40 μmol/L为后续实验浓度。通过LDH释放实验检测细胞毒性；qPCR和Western blotting分析Caspase-11、GSDMD-N、NLRP3、cleaved-Caspase-1及IL-1β的表达；免疫荧光观察细胞焦亡；联合AMPK抑制剂(Dorsomorphin)验证通路机制。结果丹参酮Ⅰ在40 μmol/L浓度下对J774.1细胞无明显毒性(P>0.05)，干预后细胞毒性从(29.540±2.123)%降至(5.887±1.219)%(P=0.018)。与模型组相比，丹参酮Ⅰ显著抑制Caspase-11、GSDMD-N、NLRP3、Caspase-1、cleaved-Caspase-1及IL-1β表达(P < 0.05)，减少焦亡细胞比例，显示其抑制非经典细胞焦亡的作用。同时，丹参酮Ⅰ上调p-AMPK表达，AMPK抑制剂Dorsomorphin可逆转其效应，提示其可能通过激活AMPK通路发挥保护作用。结论丹参酮Ⅰ可通过激活AMPK通路抑制LPS诱导的J774.1细胞非经典细胞焦亡，减轻炎症反应，具有潜在的抗炎及保护作用。
- 丹参酮Ⅰ /
- 非经典细胞焦亡 /
- 巨噬细胞
Abstract: Objective To investigate the inhibitory effect and mechanism of Tanshinone Ⅰ on non-canonical pyroptosis in LPS-induced murine macrophage J774.1 cells. Methods A non-canonical pyroptosis model was induced in J774.1 cells by LPS transfection using Lipofecter. Cells were treated with various concentrations of Tanshinone Ⅰ, and 40 μmol/L was selected for subsequent experiments based on cytotoxicity screening. LDH release assay was used to assess cell toxicity. The expression levels of Caspase-11, GSDMD-N, NLRP3, cleaved-Caspase-1, and IL-1β were evaluated by qPCR and Western blot. Pyroptotic cells were observed by immunofluorescence. To explore the signaling pathway, the AMPK inhibitor dorsomorphin was co-administered with Tanshinone Ⅰ. Results Tanshinone Ⅰ at a concentration of 40 μmol/L showed no significant toxicity to J774.1 cells (P>0.05). After treatment, cell toxicity decreased from (29.540±2.123) % to (5.887±1.219) % (P=0.018). Compared with the model group, Tanshinone Ⅰ significantly inhibited the expression of Caspase-11, GSDMD-N, NLRP3, Caspase-1, cleaved-Caspase-1, and IL-1β (P < 0.05), and reduced the proportion of pyroptotic cells, indicating its ability to suppress non-canonical pyroptosis. Additionally, Tanshinone Ⅰ upregulated p-AMPK expression, whereas the AMPK inhibitor dorsomorphin reversed this effect, suggesting that its protective role may be mediated through activation of the AMPK pathway. Conclusion Tanshinone Ⅰ can inhibit LPS-induced non-canonical pyroptosis in J774.1 cells by activating the AMPK pathway, thereby alleviating inflammatory responses and exerting anti-inflammatory and cytoprotective effects.
- Tanshinone Ⅰ /
- Non-canonical pyroptosis /
- Macrophage

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图 1 各组J774.1细胞Caspase-11、GSDMD、GSDMD-N蛋白表达免疫印迹

Figure 1. Western blot analysis of Caspase-11, GSDMD, and GSDMD-N protein expression in J774.1 cells of each group

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图 2 各组J774.1细胞NLRP3、Caspase-1、cleaved-Caspase-1、IL-1β蛋白表达免疫印迹

Figure 2. Western blot analysis of NLRP3, Caspase-1, cleaved-Caspase-1, and IL-1β protein expression in J774.1 cells of each group

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图 3 各组J774.1细胞焦亡百分比(放大倍数200×)

Figure 3. Percentage of pyroptosis in J774.1 cells in each group. Magnification: 200×

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图 4 各组J774.1细胞AMPK、P-AMPK蛋白表达免疫印迹

Figure 4. Immunoblotting of AMPK and P-AMPK protein expression in J774.1 cells in each group

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图 5 各组J774.1细胞AMPK、P-AMPK蛋白表达免疫印迹

Figure 5. Immunoblotting of AMPK and P-AMPK protein expression in J774.1 cells in each group

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表 1 qRT-PCR所用引物序列

Table 1. Primer sequences used for qRT-PCR

引物	序列(5’-3’)
Caspase-11	F：GACTTAGGCTACGATGTGGTGGT
	R：TGATGACTTTGGGTTTGTCTCGT
β-actin	F：CTACCTCATGAAGATCCTGACC
	R：CACAGCTTCTCTTTGATGTCAC

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表 2 丹参酮Ⅰ浓度筛选(x ± s)

Table 2. Screening of Tanshinone Ⅰ concentrations(x ± s)

组别	n	浓度(μm/L)	细胞毒性百分比(%)
正常组	3		5.873±0.076
丹参酮Ⅰ	3	10	7.157±0.395
	3	20	5.893±0.045
	3	40	5.640±0.221
	3	60	7.227±0.051^a
	3	80	6.737±0.121^a
	3	100	7.777±0.189^a
F值			15.781
P值			＜0.001
注：与正常组比较，^aP < 0.05。

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表 3 造模后丹参酮Ⅰ浓度筛选(x ± s)

Table 3. Screening of Tanshinone Ⅰ concentrations after modeling(x ± s)

组别	n	浓度(μm/L)	细胞毒性百分比(%)
正常组	3		2.177±0.296
模型组	3		29.540±2.123^a
丹参酮Ⅰ	3	10	14.190±6.297
	3	20	9.637±2.385^b
	3	40	5.887±1.219^b
	3	60	9.540±3.740^c
	3	80	9.545±2.499^b
	3	100	9.801±4.080^c
F值			20.452
P值			< 0.001
注：与正常组比较，^aP < 0.01；与模型组比较，^bP < 0.01，^cP < 0.05。

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表 4 造模后不同组细胞毒性分析(x ± s)

Table 4. Analysis of cytotoxicity in different groups after modeling(x ± s)

组别	n	细胞毒性百分比(%)
正常组	3	5.87±0.08
模型组	3	29.54±2.12^a
丹参酮Ⅰ	3	14.19±1.22
阳性对照	3	15.01±1.35
AMPK抑制剂	3	28.76±1.98^b
丹参酮Ⅰ+AMPK抑制剂	3	20.45±1.67^c
F值		25.612
P值		< 0.001
注：与正常组比较，^aP < 0.01；与丹参酮Ⅰ组比较，^bP < 0.01，^cP < 0.05。

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表 5 各组J774.1细胞Caspase-11 mRNA表达比较(x ± s)

Table 5. Comparison of Caspase-11 mRNA expression among different groups of J774.1 cells(x ± s)

组别	n	Caspase-11 mRNA
正常组	3	1.000±0.018
模型组	3	1.944±0.135^a
丹参酮Ⅰ	3	1.432±0.059^b
F值		12.345
P值		< 0.001
注：与正常组比较，^aP < 0.05；与模型组比较，^bP < 0.05。

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表 6 各组J774.1细胞Caspase-11、GSDMD、GSDMD-N蛋白表达比较(x ± s)

Table 6. Comparison of Caspase-11, GSDMD, and GSDMD-N protein expression among different groups of J774.1 cells(x ± s)

组别	n	Caspase-11	GSDMD	GSDMD-N
正常组	3	1.000±0.223	1.000±0.254	1.000±0.171
模型组	3	1.673±0.137^a	1.316±0.137^a	1.888±0.122^a
丹参酮Ⅰ	3	1.188±0.115^b	1.233±0.073^b	1.466±0.147^b
阳性对照组	3	1.120±0.113	1.131±0.124	1.124±0.232
F值		18.245	12.561	22.783
P值		< 0.001	< 0.005	< 0.001
注：与正常组比较，^aP < 0.05；与模型组比较，^bP < 0.05。

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表 7 各组J774.1细胞NLRP3、Caspase-1、cleaved-Caspase-1、IL-1β蛋白表达比较(x ± s)

Table 7. Comparison of protein expression of NLRP3, Caspase-1, cleaved-Caspase-1, and IL-1β among different groups of J774.1 cells (x ± s)

组别	n	NLRP3	Caspase-1	cleaved-Caspase-1	IL-1β
正常组	3	1.000±0.160	1.000±0.119	1.000±0.164	1.000±0.118
模型组	3	1.836±0.186^a	1.429±0.057^a	1.565±0.109^a	1.492±0.061^a
丹参酮Ⅰ	3	1.012±0.327^b	1.097±0.088^b	1.063±0.069^b	1.261±0.029^b
阳性对照组	3	1.120±0.131	1.093±0.039	1.084±0.224	1.187±0.213
F值		16.892	28.454	35.675	24.327
P值		< 0.001	< 0.001	< 0.001	< 0.001
注：与正常组比较，^aP < 0.05；与模型组比较，^bP < 0.05。

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表 8 各组J774.1细胞焦亡百分比(x ± s)

Table 8. The percentage of pyroptosis among different groups of J774.1 cells(x ± s)

组别	n	细胞焦亡百分比(%)
正常组	3	0.02±0.01
模型组	3	0.83±0.05^a
丹参酮Ⅰ	3	0.38±0.04^b
阳性对照	3	0.22±0.03^b
F值		279.529
P值		< 0.001
注：与正常组比较，^aP < 0.001；与模型组比较，^bP < 0.01。

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表 9 各组J774.1细胞AMPK、P-AMPK蛋白表达比较(x ± s)

Table 9. Comparison of AMPK and P-AMPK protein expression among different groups of J774.1 cells(x ± s)

组别	n	AMPK	p-AMPK
正常组	3	1.000±0.056	1.000±0.065
模型组	3	0.936±0.024	0.815±0.028^a
丹参酮Ⅰ	3	1.047±0.026^b	1.035±0.043^b
F值		3.451	42.786
P值		0.046	< 0.001
注：与正常组比较，^aP < 0.05；与模型组比较，^bP < 0.05。

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