Wanlan Fu,1 Yunxiang Cao,2 Jian Liu,2 Chuanbing Huang,2 Kaiyan Shu,1 Nanfei Zhu1 

1First Clinical Medical College, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, People’s Republic of China; 2Department of Rheumatology, First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui, 230031, People’s Republic of China

Correspondence: Yunxiang Cao, Department of Rheumatology, First Affiliated Hospital of Anhui University of Chinese Medicine, 117 Meishan Road, Shushan District, Hefei, Anhui, 230031, People’s Republic of China, Tel +86-13514984395 ; +0551-62838591, Email cyx800805@163.com

Purpose: This study probed the mechanism of action of Xinfeng Capsule (XFC) in myocardial injury in rats with adjuvant arthritis (AA) via the growth arrest-specific transcript 5 (GAS5)/microRNA-21 (miR-21)/Toll-like receptor 4 (TLR4) axis.
Methods: Rats were injected with Freund’s complete adjuvant to establish a rat model of AA. Then, some modeled rats were given normal saline or drugs only, and some modeled rats were injected with adeno-associated viruses or necrosulfonamide (NSA; a pyroptosis inhibitor) before drug administration. Toe swelling and arthritis index (AI) were calculated. Pathological and morphological changes in synovial and myocardial tissues were analyzed with hematoxylin-eosin staining, and pyroptotic vesicles and the ultrastructural changes of myocardial tissues were observed with transmission electron microscopy. The serum levels of interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α were detected, and lactate dehydrogenase (LDH) release was measured in myocardial tissues, accompanied by the examination of GAS5, miR-21, TLR4, nuclear factor-kB (NF-κB) p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), Caspase-1, and Gasdermin D (GSDMD) expression in myocardial tissues.
Results: After AA modeling, rats presented with significantly increased toe swelling and AI scores, synovial and myocardial tissue damage, elevated pyroptotic vesicles, and markedly enhanced serum levels of IL-1β, IL-18, IL-6, and TNF-α, accompanied by significantly diminished GAS5 expression, substantially augmented miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression, greatly increased LDH release in myocardial tissues. XFC treatment significantly declined toe swelling, AI scores, synovial and myocardial tissue damage, and the serum levels of IL-1β, IL-18, IL-6, and TNF-α in AA rats. Additionally, XFC treatment markedly elevated GAS5 expression and substantially lowered LDH release and miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression in myocardial tissues of AA rats. Moreover, the above effects of XFC in AA rats were further promoted by GAS5 overexpression or NSA treatment.
Conclusion: XFC alleviated myocardial injury in AA rats by regulating the GAS5/miR-21/TLR4 axis and inhibiting pyroptosis and pro-inflammatory cytokine secretion.

Keywords: adjuvant arthritis, myocardial injury, GAS5/miR-21/TLR4 axis, pyroptosis, Xinfeng Capsule