As 4

Figure 1 shows the timeline of this animal experimental design. All mice were randomly allotted into the following three groups: control (n=15), model (n=20), and AS-IV (n=20). Mice in the AS-IV groups were orally administered AS-IV (100 mg/kg/d) for 14 days (Wan et al., 2018 (link)). AS-IV (HPLC purity> 98%) was bought from Chengdu Chroma-Biotechnology Co., Ltd., Chengdu, China, and dissolved in carboxymethyl cellulose (CMC) aqueous solution (0.5%). Mice in the remaining two groups were given 0.5% CMC aqueous solution by gavage. Concurrently, mice in the model and AS-IV groups received subcutaneous isoprenaline (ISO) (25 mg/kg, Sigma, USA) injection once a day for 5 days and then were left for a further 9 days to develop cardiac fibrosis (Samuel et al., 2014 (link); Cáceres et al., 2019 (link)), while the control group was injected with 0.9% sterile saline. On the 14th day of the experiments, feces samples were collected aseptically and stored at -80°C until further processing. After the experiments, the animals were weighed. Subsequently, echocardiography detection was performed in mice anesthetized with isoflurane. The blood was immediately collected, the heart tissue samples were isolated, and their heart weights were measured.

GC cell lines (HGC-27 and MKN-45) were used for the current research, with GES cells as negative control. All cells were acquired from the JCRB cell bank (Osaka, Japan) and cultured with 5% CO₂. HGC-27 and MKN-45 cells were exposed to AS-IV (0, 10, 20, 40 μg/mL, Sigma-Aldrich, St Louis, MO, USA). The circDLST vector (circDLST), the control (vector); miR-489-3p mimic (miR-489-3p, miR10002805-1-5), mimic control (miR-NC, miR1N0000001-1-5), miR-489-3p inhibitor (in-miR-489-3p, miR20002805-1-5), inhibitor control (in-miR-NC, miR2N0000001-1-5); Bio-miR-NC, Bio-miR-489-3p; small interfering RNA (siRNA) of EIF4A1 (si-EIF4A1) and siRNA control (si-NC) were offered by Ribobio (Guangzhou, China). Transfection was carried out by Lipofectamine 2000 (11,668,019; Invitrogen, Carlsbad, CA, USA).

Min6 cells were treated with UA (5 mg/dl; Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 24 h to induce cell injury. To study the effects of AS-IV (ChengDu Conbon Biotech Co., LTD, Chengdu, China) on UA-induced Min6 cell injury, Min6 cells were pretreated with various concentrations of AS-IV (0, 12.5, 25, and 50 μmol/l) for 2 h followed by 5 mg/dl UA for another 24 h. To inhibit the PI3K/AKT pathway in Min6 cells, 100 nM of wortmannin (a PI3K/AKT pathway inhibitor; Sigma-Aldrich, St. Louis, MO, USA) was used.

NP cells were cultured to passages 2-3 by DMEM complete medium that had been added with 10% fetal bovine serum (FBS) (121000061, Gibco, Shanghai, CHN), 100 U/ml penicillin (Sigma-Aldrich Chemical Company, St-Louis MO, USA), and 100 mg/ml streptomycin (Sigma-Aldrich Chemical Company, St-Louis MO, USA). TS IIA (B20257) and AS IV (B20564) which purity is greater than 98% were purchased from Shanghai Yuanye Bio-Technology Co. To make the concentration of DMSO 0.1%, TS IIA and AS IV were deliquesced in dimethyl sulfoxide (DMSO; Sigma-Aldrich). After that, they were intermixed with the culture medium. Cells of experimental group were given a variety of doses of TS IIA and AS IV. The negative control (NC) cells were treated with 0.1% DMSO.

Bap and AS-IV were of high purity (98%) and were purchased from Sigma-Aldrich (St. Louis, MO, United States) and the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), respectively. For cell culture experiments, they were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 50 mg/ml and diluted to final concentration in culture medium.