炙甘草汤调控miR-26b-5p/SMAD4通路对心房颤动大鼠模型心房重构的影响

doi:10.11958/20252534

天津医药 ›› 2026, Vol. 54 ›› Issue (1): 14-22.doi: 10.11958/20252534

炙甘草汤调控miR-26b-5p/SMAD4通路对心房颤动大鼠模型心房重构的影响

刘魁智(), 宣学习, 周芃, 袁孝伟, 朱自强^△()

郑州市第七人民医院心血管内科（邮编450000）

收稿日期:2025-07-22 修回日期:2025-09-21 出版日期:2026-01-15 发布日期:2026-01-19
通讯作者: ^△ E-mail：1393946386@qq.com
作者简介:刘魁智（1991），男，主治医师，主要从事心血管疾病方面研究。E-mail：lkuizhi010407@163.com
基金资助:
河南省医学科技攻关计划联合共建项目(LHGJ20230738)

Effects of Zhigancao Decoction on atrial remodeling in atrial fibrillation rat model via modulating the miR-26b-5p/SMAD4 pathway

LIU Kuizhi(), XUAN Xuexi, ZHOU Peng, YUAN Xiaowei, ZHU Ziqiang^△()

Department of Cardiovascular Medicine, the 7th People's Hospital of Zhengzhou, Zhengzhou 450000, China

Received:2025-07-22 Revised:2025-09-21 Published:2026-01-15 Online:2026-01-19
Contact: ^△ E-mail：1393946386@qq.com

刘魁智, 宣学习, 周芃, 袁孝伟, 朱自强. 炙甘草汤调控miR-26b-5p/SMAD4通路对心房颤动大鼠模型心房重构的影响[J]. 天津医药, 2026, 54(1): 14-22.

LIU Kuizhi, XUAN Xuexi, ZHOU Peng, YUAN Xiaowei, ZHU Ziqiang. Effects of Zhigancao Decoction on atrial remodeling in atrial fibrillation rat model via modulating the miR-26b-5p/SMAD4 pathway[J]. Tianjin Medical Journal, 2026, 54(1): 14-22.

摘要/Abstract

摘要：

目的探讨炙甘草汤对心房颤动（AF）大鼠模型心房重构及微小RNA（miR）-26b-5p/母亲DPP同源物（SMAD）4通路的影响。方法构建AF大鼠模型，将造模成功的大鼠随机分为AF组、维拉帕米组、炙甘草汤低剂量（炙甘草汤-L）组、炙甘草汤高剂量（炙甘草汤-H）组、炙甘草汤-H+anti-NC组、炙甘草汤-H+miR-26b-5p抑制剂（anti-miR-26b-5p）组，每组18只；另取18只健康大鼠作为对照组。检测各组大鼠心功能、体外AF诱发率；苏木精-伊红（HE）染色及Masson染色观察心房组织病理变化；原位末端标记（TUNEL）染色检测心房组织心肌细胞凋亡情况；免疫组化检测心房组织Ⅰ型胶原（Col Ⅰ）、α-平滑肌肌动蛋白（α-SMA）表达；qRT-PCR检测miR-26b-5p相对表达水平；Western blot检测SMAD4、BCL-2相关X蛋白（BAX）、活化的胱天蛋白酶-3（C-caspase3）的表达；双萤光素酶实验验证miR-26b-5p与SMAD4的靶向关系。结果与对照组相比，AF组心房组织出现严重病理损伤，心肌细胞结构异常、肿胀稀疏排列紊乱、蓝色胶原沉积明显；左室收缩末期内径（LVESD）、左室舒张末期内径（LVEDD）升高，左室射血分数（LVEF）、左室缩短分数（LVFS）降低；AF诱发率、胶原面积百分数、细胞凋亡率增加；BAX、C-caspase3、 ColⅠ、α-SMA、SMAD4蛋白表达水平升高，miR-26b-5p水平降低（P<0.05）。与AF组相比，维拉帕米组、炙甘草汤-L组、炙甘草汤-H组治疗可逆转上述指标的变化趋势，减轻心房组织病理损伤，减少蓝色胶原沉积。炙甘草汤-H+anti-miR-26b-5p组可逆转炙甘草汤-H对AF大鼠模型心房重构的改善作用。Starbase网站及双萤光素酶基因报告结果显示，miR-26b-5p与SMAD4之间存在靶向关系。结论炙甘草汤可能通过上调miR-26b-5p进而抑制SMAD4表达，改善AF大鼠心房重构。

关键词: 心房颤动, 心房重构, 炙甘草汤, miR-26b-5p, SMAD4

Abstract:

Objective To explore the effect of Zhigancao Decoction on atrial remodeling and the microrNA (miR)-26b-5p/ maternal DPP homologous product (SMAD) 4 pathway in rat model of atrial fibrillation (AF). Methods The AF rat model was constructed. The successfully modeled rats were randomly divided into the AF group, the verapamil group, the low-dose Zhigancao Decoction (Zhigancao Decoction-L) group, the high-dose Zhigancao Decoction (Zhigancao Decoction-H) group, the Zhigancao Decoction-H+anti-NC group and the Zhigancao Decoction-H+miR-26b-5p inhibitor (anti-miR-26b-5p) group, with 18 rats in each group. Another 18 healthy rats were taken as the control group. Cardiac function of rats and induction rate of AF in vitro were detected in each group. Hematoxylin-eosin (HE) staining and Masson staining were used to observe the pathological changes of atrial tissue. TdT-mediated dUTP nick-end labeling (TUNEL) staining was used to observe cardiomyocyte apoptosis in atrial tissue. Immunohistochemistry was used to detect expressions of type Ⅰ collagen (ColⅠ) and α-smooth muscle actin (α-SMA) in atrial tissue. qRT-PCR was used to explore the relative expression level of miR-26b-5p. Western blot assay was used to detect the expression levels of SMAD4, BCL-2 associated X protein (BAX) and activated caspase-3 (C-caspase3). Dual-luciferase assay was performed to verify the targeting relationship between miR-26b-5p and SMAD4. Results Compared with the control group, the atrial tissue in the AF group exhibited severe pathological damage, including abnormal myocardial cell structure, swelling, sparse and disordered arrangement, and significant blue collagen deposition (P<0.05). In cardiac function indexes of AF group, left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were increased, while left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were decreased (P<0.05). Furthermore, the induction rate of AF, collagen area percentage and cell apoptosis rate of atrial fibrillation were increased. The protein expression levels of BAX, C-caspase3, ColⅠ, α-SMA and SMAD4 were enhanced, and the level of miR-26b-5p was decreased (P<0.05). Compared with the AF group, treatment with verapamil and Zhigancao Decoction reversed the trends of the above indicators, alleviated the pathological damage in atrial tissue and reduced blue collagen deposition. Zhigancao Decoction-H+anti-miR-26b-5p reversed the ameliorative effects of Zhigancao Decoction-H on atrial remodeling in the AF rat model. Starbase website and the dual-luciferase gene report indicated a targeting relationship between miR-26b-5p and SMAD4. Conclusion Zhigancao Decoction may improve atrial remodeling in AF rats by up-regulating miR-26b-5p and thereby inhibiting the expression of SMAD4.

Key words: atrial fibrillation, atrial remodeling, Zhigancao Decoction, miR-26b-5p, SMAD4

中图分类号:

R285

图/表 14

表1 引物序列

Tab.1 Primer sequence

基因名称	引物序列（5′→3′）	产物大小/bp
miR-26b-5p	上游：GCGCGTTCAAGTAATTCAGG	116
	下游：AGTGCAGGGTCCGAGGTATT	116
U6	上游：CTCGCTTCGGCAGCACA	98
	下游：AACGCTTCACGAATTTGCGT	98

表2 各组大鼠心功能比较

Tab.2 Comparison of cardiac function between different groups of rats （n=18，$\bar{x}±s$）

组别		LVEF		LVESD/mm
对照组		0.725±0.075		3.11±0.32
AF组		0.481±0.049^a		7.38±0.75^a
维拉帕米组		0.683±0.070^b		4.11±0.44^b
炙甘草汤-L组		0.574±0.058^b		5.76±0.59^b
炙甘草汤-H组		0.705±0.071^bc		3.75±0.38^bc
炙甘草汤-H+anti-NC组		0.698±0.071		3.84±0.40
炙甘草汤-H+anti-miR-26b-5p组		0.582±0.059^d		5.61±0.58^d
F		34.884^＊		154.484^＊
组别	LVEDD/mm		LVFS
对照组	6.18±0.63		0.497±0.051
AF组	9.94±1.05^a		0.258±0.028^a
维拉帕米组	6.54±0.68^b		0.372±0.040^b
炙甘草汤-L组	8.36±0.87^b		0.310±0.032^b
炙甘草汤-H组	6.37±0.65^bc		0.411±0.043^bc
炙甘草汤-H+anti-NC组	6.41±0.66		0.401±0.041
炙甘草汤-H+anti-miR-26b-5p组	8.19±0.84^d		0.315±0.033^d
F	60.126^＊		74.737^＊

表3 各组大鼠AF诱发率和胶原面积百分数比较

Tab.3 Comparison of AF induction rate and collagen area percentage between different groups of rats （n=6，$\bar{x}±s$）

组别	AF诱发率/%	胶原面积百分数/%
对照组	24.00±2.06	0.64±0.08
AF组	36.59±2.81^a	5.11±0.54^a
维拉帕米组	28.16±1.95^b	1.23±0.15^b
炙甘草汤-L组	32.25±2.47^b	2.76±0.31^b
炙甘草汤-H组	25.34±1.72^bc	0.87±0.11^bc
炙甘草汤-H+anti-NC组	26.05±1.79	0.92±0.12
炙甘草汤-H+anti-miR-26b-5p组	31.92±2.28^d	2.65±0.29^d
F	26.039^＊	206.513^＊

图1 HE染色观察心房组织病理学变化（×200）

Fig.1 Pathological changes of atrial tissue observed by HE staining (×200)

图2 Masson染色观察心房组织纤维化情况（×200）

Fig.2 Fibrosis of atrial tissue observed by Masson staining (×200)

图3 TUNEL染色观察各组心房组织心肌细胞凋亡情况（×200） A—G分别为对照组、AF组、维拉帕米组、炙甘草汤-L组、炙甘草汤-H组、炙甘草汤-H+anti-NC组、炙甘草汤-H+anti-miR-26b-5p组。

Fig.3 Apoptosis of cardiomyocytes in atrial tissue observed by TUNEL staining (×200)

图4 Western blot检测心房组织中BAX、C-caspase3蛋白的表达 1：对照组；2：AF组；3：维拉帕米组；4：炙甘草汤-L组；5：炙甘草汤-H组；6：炙甘草汤-H+anti-NC组；7：炙甘草汤-H+anti-miR-26b-5p组。

Fig.4 Detection of BAX and C-caspase3 expression by Western blot assay

表4 各组大鼠心房组织心肌细胞凋亡情况比较

Tab.4 Comparison of cardiomyocyte apoptosis in atrial tissue between different groups of rats （n=6，$\bar{x}±s$）

组别	细胞凋亡率/%	BAX/β-actin	C-caspase3/β-actin
对照组	3.11±0.37	0.33±0.04	0.28±0.04
AF组	29.84±3.15^a	0.89±0.10^a	0.83±0.09^a
维拉帕米组	10.17±1.21^b	0.42±0.05^b	0.35±0.05^b
炙甘草汤-L组	20.49±2.24^b	0.69±0.08^b	0.63±0.07^b
炙甘草汤-H组	7.65±0.83^bc	0.37±0.05^bc	0.31±0.04^bc
炙甘草汤-H+anti-NC组	8.33±0.89	0.39±0.05	0.32±0.04
炙甘草汤-H+ anti-miR-26b-5p组	16.21±1.75^d	0.65±0.08^d	0.59±0.07^d
F	166.978^＊	58.539^＊	74.484^＊

表5 各组大鼠心房组织ColⅠ、α-SMA蛋白表达水平比较

Tab.5 Comparison of mean optical density values of ColⅠand α-SMA in atrial tissue between different groups of rats （n=6，$\bar{x}±s$）

组别	ColⅠ	α-SMA
对照组	0.25±0.04	0.20±0.03
AF组	0.83±0.09^a	0.76±0.09^a
维拉帕米组	0.31±0.04^b	0.26±0.04^b
炙甘草汤-L组	0.64±0.07^b	0.57±0.07^b
炙甘草汤-H组	0.28±0.04^bc	0.23±0.03^bc
炙甘草汤-H+anti-NC组	0.30±0.04	0.24±0.03
炙甘草汤-H+anti-miR-26b-5p组	0.59±0.07^d	0.52±0.06^d
F	89.514^＊	96.105^＊

图5 免疫组化检测各组心房组织ColⅠ、α-SMA蛋白的表达（×200） A—G分别为对照组、AF组、维拉帕米组、炙甘草汤-L组、炙甘草汤-H组、炙甘草汤-H+anti-NC组、炙甘草汤-H+anti-miR-26b-5p组。

Fig.5 Detection of ColⅠand α-SMA expression in atrial tissue detected by immunohistochemistry (×200)

图6 Western blot检测心房组织中SMAD4蛋白的表达 1：对照组；2：AF组；3：维拉帕米组；4：炙甘草汤-L组；5：炙甘草汤-H组；6：炙甘草汤-H+anti-NC组；7：炙甘草汤-H+anti-miR-26b-5p组。

Fig.6 SMAD4 expression deteced by Western blot assay

表6 各组大鼠心房组织miR-26b-5p及SMAD4表达比较

Tab.6 Comparison of miR-26b-5p levels and SMAD4 expression in atrial tissue between different groups of rats （n=6，$\bar{x}±s$）

组别	miR-26b-5p	SMAD4/ β-actin
对照组	0.96±0.11	0.37±0.05
AF组	0.42±0.05^a	0.88±0.10^a
维拉帕米组	0.85±0.09^b	0.45±0.06^b
炙甘草汤-L组	0.60±0.07^b	0.69±0.08^b
炙甘草汤-H组	0.92±0.10^bc	0.41±0.05^bc
炙甘草汤-H+anti-NC组	0.90±0.10	0.42±0.05
炙甘草汤-H+anti-miR-26b-5p组	0.64±0.07^d	0.67±0.08^d
F	33.170^＊	45.752^＊

图7 Starbase网站预测miR-26b-5p与SMAD4的靶向关系

Fig.7 Prediction of the target relationship between miR-26b-5p and SMAD4 by Starbase

表7 2组相对萤光素酶活性比较

Tab.7 Comparison of relative luciferase activity between the two groups （n=6，$\bar{x}±s$）

组别	WT-SMAD4	MUT-SMAD4
miR-NC组	0.99±0.11	1.02±0.11
miR-26b-5p mimic组	0.56±0.07	1.00±0.11
t	8.078^＊	0.315

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炙甘草汤调控miR-26b-5p/SMAD4通路对心房颤动大鼠模型心房重构的影响

Effects of Zhigancao Decoction on atrial remodeling in atrial fibrillation rat model via modulating the miR-26b-5p/SMAD4 pathway

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