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

doi:10.11958/20252534

Abstract

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

CLC Number:

R285

Figures/Tables 14

References 33

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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

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

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^＊

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^＊

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^＊