Protocols for animal experiments
Seven-week-old male Dahl salt-sensitive (DS) rats (n = 44) (Japan SLC, Shizuoka, Japan) were fed a normal diet (ND; 0.3% NaCl and 4.5% fat) (CE-2, CLEA Japan, Inc., Tokyo, Japan) or a high-salt and high-fat diet (HD; 8% NaCl and 29.4% fat) (CLEA Japan), as previously described [26], and they were treated with a vehicle (0.5% carboxy methyl cellulose and 0.5% methyl cellulose), pitavastatin (0.3 mg/kg/day) (Kowa Co., Ltd., Tokyo, Japan), pemafibrate (K-877) (0.5 mg/kg/day) (Kowa Co., Ltd.) or a combination of pitavastatin (0.3 mg/kg/day) and pemafibrate (K-877) (0.5 mg/kg/day) (Figure 1) by oral gavage for a period of 12 weeks (Figure 1). Body weight was measured once a week for a period of 12 weeks. Rats were divided into the following five groups: (1) ND-vehicle group (n = 5) fed an ND and treated with vehicle; (2) HD-vehicle group (n = 9) fed an HD and treated with vehicle; (3) HD-pitavastatin group (n = 10) fed an HD and treated with pitavastatin; (4) HD-pemafibrate group (n = 10) fed an HD and treated with pemafibrate; and (5) HD-combination group (n = 10) fed an HD and treated with combination of pitavastatin and pemafibrate. All experimental protocols were approved by and conducted in accordance with the recommendations of the Okayama University Animal Care and Use Committee (permit number OKU-2019349).
Blood pressure and pulse rate measurement
Systolic blood pressure and pulse rate were measured at 12 and 19 weeks using a tail-cuff plethysmography (MK-2000 Muromachi, Tokyo, Japan or BP-2000, Visitech Systems, Inc., Apex, NC, USA). An average of three measurements was used.
Blood collection and measurements
At 19 weeks, rats were anesthetized with isoflurane. Non-fasting whole blood was collected from the abdominal aorta into a chilled tube. After centrifugation at 3000 x g for 10 minutes at 4°C, plasma was collected and stored at −80°C. Plasma total bilirubin was measured using the vanadate oxidase method. Plasma glucose was measured using the hexokinase/glucose-6-phosphate dehydrogenase method. Plasma cholesterol and TG content in lipoprotein fractions including chylomicron (CM), very low-density lipoprotein (VLDL), LDL, and HDL were analyzed using high-performance liquid chromatography by Skylight Biotech (Akita, Japan), as described [27, 28].
Vascular relaxation studies
Endothelium-dependent relaxation in response to acetylcholine was evaluated. At 19 weeks, rats were anesthetized with isoflurane. The thoracic aorta was rapidly removed, gently cleaned taking care not to damage the endothelium, and it was cut into 3-mm rings. The rings were then cut open. Open aortic rings were placed in a 10-mL organ bath containing Krebs−Henseleit solution (KHS; in mmol/L: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 11.1 glucose). One end of the open ring was connected to a tissue holder and the other end was connected to a force displacement transducer (AD-611J, Nihon Kohden, Tokyo, Japan). The bathing solution was gassed with 95% O2 and 5% CO2 at 37°C (pH 7.4). The tissue was equilibrated for 60 min under a resting tension of 1 g. During this time, Krebs−Henseleit solution was replaced every 15 min with fresh solution. The tissues were pre-contracted with phenylephrine (0.3 µmol/L). Tissues were then re-washed and pre-contracted with phenylephrine (0.3 µmol/L). After the phenylephrine-induced contraction had reached a plateau, the concentration–response relationships for acetylcholine (1–10000 nmol/L) were obtained by adding acetylcholine to the bath in a cumulative manner. Finally, papaverine (100 µmol/L) relaxation responses were obtained. The relaxation responses obtained were expressed as a percentage of the maximal relaxation that was evoked by papaverine (100 µmol/L).
Western blot analysis
Protein samples from the abdominal aortas of 4 or 6 randomly selected rats in each group were prepared using a BEAD crusher (µT-12, Taitec, Koshigaya, Japan). Tissue lysates were extracted in radioimmunoprecipitation (RIPA) buffer with 2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, and 10 mmol/L sodium fluoride (sc-24948, Santa Cruz, Dallas, Texas, USA) and 20 µg of lysates was subjected to SDS-PAGE. Rabbit anti-eNOS antibody (#610297, BD Biosciences, Franklin lakes, New Jersey, USA), rabbit anti-phospho-eNOS (Ser1177) antibody (#612393, BD Biosciences, Franklin lakes, New Jersey, USA), and mouse anti-beta actin antibody (ab6276, Abcam, Cambridge, UK) were used. All primary antibodies were used at a dilution of 1:1000. The second antibody was horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (NA934 and NA931, GE Healthcare Bio-Sciences, Buckinghamshire, England). Positive signals were detected using a chemiluminescence system (ECL plus, GE Healthcare Bio-Sciences).
Quantitative real-time polymerase chain reaction (qPCR) analysis
For reverse transcription (RT)- PCR analysis, RNA was extracted from the abdominal aortas of 4 or 6 randomly selected rats in each group with RNeasy Mini Kit (Qiagen). The total RNA (2 μg) from each tissue sample was used to generate complementary DNA (cDNA) with ReverTra Ace (TOYOBO, Osaka, Japan). The cDNA was subjected to PCR with TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) and predesigned gene specific primer and probe sets (TaqMan Gene Expresso in Assays; Applied Biosystems). Quantitative real-time PCR was performed using the Applied Biosystems 7300 realtime PCR System (Applied Biosystems). The PCR primers used were the following: NADPH oxidase-4 (NOX4), Rn00585380 and actin beta (ACTB), Rn00667869. ACTB was used as the internal control.
Statistical analysis
Statistical analysis was performed using SPSS version 24 (IBM, New York, USA). All results are expressed as the mean ± standard deviation (SD). For comparison between different treatment groups, statistical analysis was performed using a one-way analysis of variance (ANOVA) with a Bonferroni post-hoc test. Vascular relaxation studies were analyzed using a mixed effect model with a Bonferroni post-hoc test. P-values < 0.05 were considered to be significant.