PA19 was prepared at a purity of 99.2% as previously described (12), and dissolved in 0.5% sodium carboxylmethylcellulose (CMC-Na; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for in vivo experiments.

A total of 21 male C57BL/6 mice (8–10 weeks old, 18–22 g) and a total of 20 male 18–20 g ICR mice (8–10 weeks old) were obtained from the Animal Center of Wenzhou Medical University (Wenzhou, China). All animal care and experimental procedures complied with the Ordinance in Experimental Animal Management (order no. 1998-02; Ministry of Science and Technology, Beijing, China) and were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (Wenzhou, China). Mice were housed at 22°C, with 55±15% humidity and maintained under a 12-h light/dark cycle in an SPF environment. All animals were provided with free access to water. Following an acclimatization period of 1 week, mice were randomly assigned into three weight-matched groups for 20 weeks (n=7/group): Mice were fed a standard rodent diet (CON) or a high-fat diet (HFD), or fed a HFD and received intragastric administration of PA19 (10 mg/kg/day) via oral gavage on alternate days for the final 12 weeks of the study (HFD + PA19). The mice in the CON and HFD groups received vehicle treatment (0.5% CMC-Na solution) as a control. The body weights of the mice were monitored weekly during the 20-week period of the study. The mice were subsequently sacrificed under anesthesia, heart and kidney tissues were fixed in 4% paraformaldehyde at room temperature for 48 h for histological, and gene and protein expression analyses.

Total RNA was isolated from tissues using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); the concentration of RNA was determined using a SpectraMax M5 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) and the purity of the samples was estimated from the optical density ratio (A260/A280, 1.8–2.2). RT was performed using an M-MLV Platinum RT-qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) with the reaction parameters of incubation at 65°C for 5 min, 37°C for 52 min and 70°C for 15 min. qPCR was conducted with a total reaction volume of 20 µl using iQ™ SYBR^®-Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a MasterCycler Realplex⁴ (Eppendorf, Hamburg, Germany). A total of 3 parallel experiments of one sample were conducted. A total of 60 cycles of qPCR were performed. The thermocycling conditions were as follows: Denaturation at 95°C for 2 min; annealing at 95°C for 15 sec and extension at 60°C for 30 sec. Primers were obtained from Invitrogen (Thermo Fisher Scientific, Inc.); the sequences are presented in Table I. The relative expression of each gene normalized to β-actin was determined using the 2^−ΔΔCq method (17). mRNA expression levels were calculated as percentages of the expression in the median tissue from the HFD group.

Heart and kidney tissues from each mouse were homogenized in radioimmunoprecipitation assay (RIPA) buffer (P0013B; Beyotime Institute of Biotechnology, Haimen, China;) with protease inhibitor. The Bradford assay (Bio-Rad Laboratories, Inc.) was performed to determine the concentration of protein. Following boiling in loading buffer for 10 min, total protein samples (60 µg) were separated via 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% milk for 1.5 h at room temperature and then incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Vascular cell adhesion molecule 1 (VCAM-1; 1:1,000; ab134047; Abcam, Cambridge, UK), intercellular adhesion molecule 1 (ICAM-1; 1:1,000; ab171123, Abcam), monocyte chemoattractant protein 1 (MCP-1; 1:1,000; sc-52701, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), collagen 1 (Col-1; 1:1,000; ab34710, Abcam) and β-actin (1:1,000; ab8226, Abcam). The membranes were subsequently washed in TBST (TBS solution containing 0.2% Tween-20) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000; 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Blots were visualized using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.) and protein expression was quantified using ImageJ software (version 1.42q; National Institutes of Health, Bethesda, MD, USA).

Heart and kidney tissues from each mouse were homogenized in RIPA buffer with protease inhibitor, and the Bradford assay was performed to determine the concentration of protein. Following adjustment to equal concentrations, ELISAs were performed on the samples using mouse IL-6 (85-88-7064-76; eBioscience; Thermo Fisher Scientific, Inc.) and mouse IL-1β ELISA kits (70-EK201B/3-96; MultiSciences, Hangzhou, China) according to the manufacturer's protocols.

Blood samples were collected through the eyeball after sacrifice of the animals and centrifuged at 3,000 × g for 15 min at 4°C to isolate the serum. Heart and kidney injury and failure were determined by measuring the serum expression levels of various biochemical markers, including blood urea nitrogen (BUN), serum creatinine (Cr), creatine kinase (CK), CK-muscle/brain (CK-MB), total cholesterol (TCH), alkaline phosphatase (AKP), low density lipoprotein (LDL) and aspartate transaminase (AST) using respective commercial kits (Jiancheng Bioengineering Institute, Nanjing, China).

Kidney and heart tissues were fixed in 4% paraformaldehyde for 48 h at room temperature and embedded in paraffin. Samples were sectioned (5-µm thickness) and mounted on slides. Then, sections were stained with H&E and Sirius Red (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's protocols. To analyze the extent of damage, 5 fields of one specimen were observed under a light microscope (magnification, ×200; Nikon Corporation, Tokyo, Japan). The area of fibrosis in the heart and kidney tissues was determined using ImageJ software (version 1.42q, National Institutes of Health).

Heart and kidney tissues were sectioned as aforementioned, deparaffinized in xylene and rehydrated in a graded alcohol series. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.5) by microwaving (10 min). Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 30 min at room temperature. Sections were then blocked for 30 min at 37°C in 1% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) dissolved in phosphate buffer solution, prior to incubation with primary anti-epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80) antibody (1:200; sc-377009; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Then, sections were incubated with HRP-conjugated secondary antibodies (1:200; 33201ES60; Yeasen, Shanghai, China) at 37°C for 30 min, prior to immersion in 3′3-diaminobenzidine solution (OriGene Technologies, Inc., Beijing, China) for 1 min. Finally, sections were stained with hematoxylin for 5 min at room temperature, dehydrated and mounted, and images were captured using a light microscope (5 fields of one specimen were randomly selected; magnification, ×200, Nikon Corporation).

The 20 male 18–20 g ICR mice were randomly divided into two experimental groups: CON group and PA19 group. CON and PA group mice were received 0.5% CMC-Na and 500 mg/kg PA, respectively. The treatments were administered once by oral gavage. Animals were closely observed every 12 h following administration on general behavior; mortality and body weights were also measured.

All data were obtained from three independent experiments. The results were presented as the mean ± standard error of the mean. All data were analyzed using GraphPad Pro Prism 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). One-way analysis of variance followed by a Dunnett's post-hoc test was employed to analyze the differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Heart and kidney tissues and serum were collected from mice following CON or HFD feeding for 20 weeks. As presented in Table II, animals fed with the HFD exhibited significantly increased serum expression levels of lipid accumulation markers (TCH and LDL), renal function markers (BUN and Cr) and cardiac function markers (CK, CK-MB, AKP and AST) compared with the CON group; however, treatment with PA19 (10 mg/kg) suppressed the increased expression of these markers. An acute toxicity assay was conducted to determine the effects of chronic PA19 treatment in vivo; it was demonstrated that intragastric administration of PA19 (500 mg/kg) did not significantly alter body weight (Fig. S1) or the serum expression levels of the aforementioned biochemical markers (Table SI) compared with treatment with 0.5% CMC-Na. The results indicated that PA19 treatment at the selected dose was safe for mice and attenuated the increased serum expression levels of functional biomarkers following exposure to an HFD.

The cardioprotective effects of PA19 were investigated by determining the effects of PA19 treatment on obesity-induced alterations in the morphology of cardiac tissue. H&E staining revealed that the heart tissue of mice in the HFD group presented structural abnormalities that were not observed in the PA19-treated group, including broken fibers and irregular cellular structures (Fig. 2A). Additionally, Sirius Red staining revealed that PA19 treatment attenuated HFD-induced collagen deposition in heart tissues and significantly reduced fibrosis compared with the control (Fig. 2B and C). It was demonstrated that feeding with the HFD induced significant increases in the levels of Col-1 mRNA and protein expression in heart tissue compared with the CON group, but were suppressed by PA19 treatment (Fig. 2D, E and S2A).

H&E staining of kidney tissue revealed that the HFD group exhibited glomerular shrinkage and expansion of the mesangial matrix compared with the CON group (Fig. 3A). Conversely, treatment with PA19 markedly improved obesity-induced renal injury. The extent of fibrosis in the kidneys induced by HFD was also evaluated. Sirius Red staining revealed notable collagen deposition in the kidney tissues of HFD mice (Fig. 3B and C), in addition to significant increases in the expression of Col-1 at the mRNA and protein levels compared with the CON group (Fig. 3D, E and S2B). PA19 treatment significantly reduced the extent of HFD-induced fibrosis and decreased the expression of Col-1 in kidney tissue. The results suggested that treatment with PA19 protects heart and kidney tissue from obesity-induced injury and fibrosis.

The association between the potential cardio- and renoprotective effects of PA19 against obesity-induced damage and its anti-inflammatory properties was investigated. Immunohistochemical staining of F4/80 expression revealed that there was marked HFD-induced inflammatory cell infiltration of cardiac tissue; however, the percentage of F4/80⁺ cells in tissue samples from the HFD + PA19 group was significantly lower compared with in the HFD group (Fig. 4A and B). Additionally, RT-qPCR, ELISA and western blot analyses demonstrated that HFD exposure significantly increased the mRNA and protein expression levels of inflammatory cytokines (IL-6 and IL-1β), chemokines (MCP-1) and adhesion molecules (VCAM-1 and ICAM-1) in heart tissue compared with in the CON group (Fig. 4C-J). Conversely, PA19 treatment significantly suppressed the HFD-induced upregulation of these markers.

As presented in Fig. 5A and B, feeding with the HFD induced a marked increase in the number of F4/80⁺ inflammatory cells in the renal sections; however, inflammatory cell infiltration was significantly attenuated by treatment with PA19. As presented in Fig. 5C-J, the expression levels of inflammatory cytokines, chemokines and adhesion molecules were significantly increased in kidney tissues from HFD mice compared with the CON group, but were suppressed by PA19 treatment. Of note, the protein expression of IL-1β was not significantly reduced following PA19 administration. Collectively, these results indicated that PA19 exhibits anti-inflammatory properties in cardiac and renal tissues in vivo.