Animal experiments were performed as previously described (23). A total of 40 male Wistar rats (age, 6-7 weeks; weight, 160-180 g) were obtained from Nanjing Jiancheng Bioengineering Institute, and maintained in a 12 h light/dark cycle, with 50-60% humidity at 22-26˚C. All rats were provided ad libitum access to a standard diet and water. Rats were randomly divided into two groups; a control (n=20) and CRF (n=20) groups after 1 week of adaptive feeding. Adenine (2.5 g; Sigma-Aldrich; Merck KGaA) was added to 100 ml normal saline to prepare a 2.5% adenine suspension. Rats in the CRF group received 250 mg/kg adenine once a day via oral gavage for a total of 14 days, and adenine was administrated every other day for the next 14 days. Rats in the control group received the same amount of normal saline. All rats were fasted for 12 h prior to the last administration. Rats were anesthetized with 2% sodium pentobarbital (50 mg/kg) 1 h after the final administration, and cervical dislocation of the spine was immediately performed following collection of 4-6 ml blood from the abdominal aorta. All animal experiments were approved by the Experimental Animal Center of Lianyungang Hospital of Traditional Chinese Medicine (Lianyungang, China; approval. no. IACUC-20200312-07).

All experiments were performed as previously described (23-26). Blood samples were centrifuged at 1,000 x g for 10 min to collect serum. The concentrations of serum creatinine (SCr) and blood urea nitrogen (BUN) were measured using commercial kits (cat. nos. C011-2-1 and C013-2-1, respectively; Nanjing Jiancheng Bioengineering Institute), and measured using a biochemical autoanalyzer (ROCHE Modular P800; Roche Diagnostics GmbH).

Human umbilical vein smooth muscle cells (HUVSMCs; cat. no. CP-H084) were purchased from Procell Life Science & Technology Co., Ltd., with the approval of Ethics Committee of Lianyungang Hospital of Traditional Chinese Medicine (Lianyungang, China. Approval no. IACUC-20200611-03), and maintained in DMEM supplemented with 10% FBS (both purchased from Gibco; Thermo Fisher Scientific, Inc.), at 37˚C with 5% CO₂.

The rat whole blood was collected from the control (control serum) and CRF (CRF serum) groups into coagulation tubes and allowed to clot on ice for 50 min. Subsequently, centrifugation at 1,000 x g for 15 min was used for separating serum. Rat serum was maintained in DMEM at 37˚C for 48 h at dilutions of 2.5:100, 5:100 or 10:100. HUVSMCs were pretreated with 1, 5 or 25 µM HSYA (27-30) (purity >98%; Beijing Solarbio Science & Technology Co., Ltd.) prior to stimulation of rat serum. AMG 511 (5 nM; MedChemExpress) was used to inhibit PI3K.

A Cell Counting Kit-8 (CCK-8) assay was performed to assess the effect of rat serum and/or HSYA on cell viability. HUVSMCs were seeded in 96-well plates at a density of 5x10³ cells/well and treated with rat serum and/or HSYA at 37˚C for 48 h. Cells were subsequently incubated with 10 µl CCK-8 reagent (cat. no. C0037; Beyotime Institute of Biotechnology) for 1 h at 37˚C. Cell viability was analyzed at a wavelength of 450 nm, using a microplate spectrophotometer (BioTek Instruments, Inc.).

HUVSMCs were seeded into 6-well plates at a density of 1x10⁶ cells/well. Sterile 200 µl pipette tips were used to scratch the cell monolayers. Cell medium was replaced with fresh serum-free DMEM. The migratory ability of cells characterized by wound width was observed at 0 and 48 h under a light microscope (Olympus Corporation; magnification, x100).

HUVSMCs (5x10⁵) were collected by centrifugation at 37˚C and 300 x g for 3 min and re-suspended in 200 µl Annexin V binding buffer (cat. no. C1062M; Beyotime Institute of Biotechnology). After resuspension, cells were incubated with 10 µl PI (cat. no. C1062M; Beyotime Institute of Biotechnology) for 15 min at room temperature in the dark. Apoptotic cells were subsequently analyzed using a flow cytometer (Beckman Coulter, Inc.).

HUVSMCs were seeded in 12-well plates at a density of 2x10⁵ cells/well and treated with rat serum and/or HSYA. HUVSMCs were subsequently lysed using Lysis Buffer (cat. no. S3090; Beyotime Institute of Biotechnology). The Nitrate/Nitrite assay kit (cat. no. S0023; Beyotime Institute of Biotechnology) was used to determine NO concentration in HUVSMCs, according to the manufacturer's protocol. NO concentration was measured at a wavelength of 540 nm, using a microplate spectrophotometer (BioTek Instruments, Inc.).

Total protein was extracted from HUVSMCs using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology). Cell supernatants were collected by centrifugation at 12,000 x g for 15 min at 4˚C. Total protein was quantified using the BCA protein assay kit (cat. no. P0012s; Beyotime Institute of Biotechnology) and 20 µg protein/lane was separated by 12% SDS-PAGE. The separated proteins were subsequently transferred to PVDF membranes (EMD Millipore) and blocked with 5% skimmed milk for 2 h at room temperature. The membranes were incubated with primary antibodies against PI3K (1:1,000), Akt (1:1,000), phosphorylated (p)-Akt (cat. no. Ser473, 1:1,000), endothelial NO synthase (eNOS; 1:1,000), p-eNOS (1:1,000) and GAPDH (1:10,000) overnight at 4˚C (all purchased from Affinity Biosciences). Following the primary incubation, membranes were incubated with goat anti-rabbit/mouse IgG (H+L) HRP-conjugated secondary antibodies (1:10,000; Affinity Biosciences) for 2 h at room temperature. Protein bands were semi-quantified using Image Lab version 4.1 software (Bio-Rad Laboratories, Inc.).

All experiments were performed in triplicate and data are presented as the mean ± standard deviation. A Student's t-test was used to compare differences between two groups, and a one-way ANOVA followed by a Tukey's post hoc test was used to compare differences between multiple groups in GraphPad Prism version 5.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Rat serum from the control and CRF groups was collected to assess changes in the parameters of renal function. As presented in Fig. 1A, the BUN concentration was significantly higher in the CRF group compared with the control group. Similarly, SCr concentration was higher in the CRF group compared with the control group (Fig. 1B). These results suggest that the renal failure model was established successfully (24-26).

HUVSMCs were treated with rat serum to assess the effect of CRF serum on HUVSMCs. The results demonstrated that 10% CRF serum significantly promoted cell viability compared with 10% control serum, whereas low concentrations of CRF serum (2.5 and 5.0%) slightly enhanced cell viability (Fig. 1C). In addition, the migratory ability of HUVSMCs was inhibited following stimulation of CRF serum compared with the same concentration of control serum. Furthermore, the results of the wound healing assay demonstrated that CRF serum promoted cell migration in a concentration-dependent manner, whereas control serum had little effect on cell migration (Fig. 1D and E). Collectively, these results suggest that serum from CRF rats promotes the viability and migration of HUVSMCs.

The results demonstrated that the apoptotic rate increased following treatment of HUVSMCs with rat serum. Notably, 10% CRF serum markedly promoted cell apoptosis compared with 10% control serum (Fig. 2A). This may be explained by the presence of toxic substances in the serum of rats with CRF, which affects cell survival and promotes apoptosis.

HUVSMCs were treated with HSYA or AMG 511 in the presence of 10% CRF serum. As presented in Fig. 3A, HSYA inhibited cell viability induced by 10% CRF serum in a concentration-dependent manner. Similarly, AMG 511 suppressed the viability of HUVSMCs. Notably, pretreatment with HSYA or AMG 511 alleviated 10% CRF serum-induced cell migration (Fig. 3B), suggesting that HSYA reverses the effect of CRF serum on cell migration. In addition, HSYA partially alleviated 10% CRF serum-induced apoptosis, the effects of which were reversed following pretreatment with AMG 511 (Fig. 3C). Taken together, these results suggest that HSYA counteracts the effects of CRF serum on the viability, migration and apoptosis of HUVSMCs.

NO inhibits neointimal hyperplasia (31); thus, the NO concentration in HUVSMCs was detected. The NO concentration was notably decreased in the presence of 10% CRF serum compared with the control group; however, treatment with HSYA elevated NO levels in a concentration-dependent manner. Similarly, treatment with AMG 511 promoted NO production (Fig. 4A).

Given that HSYA exhibited similar inhibitory effects to AMG 511 on the viability and migration of HUVSMCs, it was next investigated whether HSYA can exert these effects by inhibiting the PI3K/Akt pathway. As presented in Fig. 4B, 10% CRF serum upregulated PI3K expression and phosphorylation of Akt, suggesting that serum from CRF rats can activate PI3K/Akt signaling in HUVSMCs. Notably, the expression levels of PI3K and p-Akt decreased following treatment with AMG 511. In addition, treatment with HSYA markedly decreased the protein expression levels of PI3K and p-Akt. Treatment with both HSYA and AMG 511 upregulated p-eNOS expression, respectively, which explains the increase in NO production (Fig. 4A).