Hyaluronic acid/diminazene aceturate combination ameliorates osteoarthritic anomalies in a rodent model: a role of the ACE2/Ang1-7/MasR axis

Abstract

The implication of the tissue-localized renin-angiotensin system (RAS) in the pathogenesis of osteoarthritis (OA) has been documented in the last decades. A combination of intraarticular (IA) corticosteroid and hyaluronic acid (HYAL) is approved for pain relief in patients with mild to moderate OA. Combining HYAL with an activator of angiotensin-converting enzyme 2, diminazen aceturate (DIZE), was evaluated in this study for its therapeutic potential. Monosodium iodoacetate was used to induce OA. The effects of daily administration of DIZE versus once-per-week IA injection of HYAL and a combination of both drugs for 21 days on OA deformities in rats' knees were observed. Evaluation of motor activities, pain, and inflammatory response was done using rotarod, knee bend, and knee swelling tests. RAS components, inflammatory biomarkers, and oxidative stress mediators were measured in the knee joint. X-ray radiological examination and histopathological investigations were used to assess joint degeneration and regeneration. Levels of both inflammatory and oxidative markers in knee joint homogenate of OA rats rose, and these increments were mostly improved by the three therapies with a more prominent effect of the drug combination, an effect that was also reflected in the behavioral tests. RAS markers have shown better responsiveness to the combination therapy over both drugs individually, showing a pronounced increase in the angiotensin 1-7 amount. Both radiological and histopathology investigations came to confirm the biochemical results, nominating a combination of HYAL and DIZE as a possible therapeutic option for OA.

Keywords: Diminazene aceturate; Hyaluronic acid; Inflammatory mediators; Osteoarthritis; Oxidative stress; Renin-angiotensin system.

Fig. 1

The experimental design timeline

Fig. 2

Evaluation of osteoarthritis-associated behavioral changes. a, b Rotarod test showing the falling latency at the end of the treatment period on day 41 in (a) and % change from day zero in (b), c knee bend test and d knee swelling test on day 41. Data are shown as means ± SD (n = 10). One-way ANOVA was used for comparing the different groups, followed by the Tukey post hoc test. In the knee bend test data were analyzed using Kruskal–Wallis test, followed by Dunn's post-hoc-test. Data are compared to (*) CN, (#) OA and (–) HYAL at p < 0.05. CN, control; OA, osteoarthritis; HYAL, hyaluronic acid, DIZE, diminazene

Fig. 3

Evaluation of the osteoarthritis-associated radiological changes. Assessment of the right knee joint is shown in (a) as representative lateral view radiographs. The mean of the Kellgren–Lawrence radiographic score is shown in (b). White arrows indicate osteophytes and red arrows indicate joint space narrowing. Data are shown as means ± SD (n = 4). One-way ANOVA was used for comparing the different groups, followed by the Tukey post hoc test. Data are compared to (*) CN and (#) OA at p < 0.05. CN, control; OA, osteoarthritis; HYAL, hyaluronic acid, DIZE, diminazene

Fig. 4

Effect of drug treatments on the knee tissue biochemical analysis. Knee tissue levels of a angiotensin-converting enzyme 1 (ACE1), b angiotensin-converting enzyme 2 (ACE2), c ACE2/ACE1ratio %, d angiotensin 1–7 (Ang 1–7), e Ang II, f transforming growth factor beta 1 (TGF-β1) and g NADPH oxidases 4 (NOX-4). Data are shown as means ± SD (n = 6). The tissue was analyzed using ELISA according to the manufacturer’s instructions. One-way ANOVA was used for comparing the different groups, followed by the Tukey post hoc test. Data are compared to (*) CN, (#) OA, (–) HYAL and (+) DIZE at p < 0.05. CN, control; OA, osteoarthritis; HYAL, hyaluronic acid, DIZE, diminazene

Fig. 5

Effect of drug treatments on the knee tissue western blot analysis. Knee tissue levels of a tumor necrosis factor-alpha (TNF-α), b matrix metalloproteinases 13 (MMP-13), c mas receptor (MasR) and d representative western blot bands. Data are shown as means ± SD (n = 3). One-way ANOVA was used for comparing the different groups, followed by the Tukey post hoc test. Data are compared to (*) CN, (#) OA, (–) HYAL and (+) DIZE at p < 0.05. CN, control; OA, osteoarthritis; HYAL, hyaluronic acid, DIZE, diminazene

Fig. 6

Synovial tissue assessment in H&E-stained sections of studied groups. a Low-power image of the knee joint to highlight different parts of the patellofemoral joint and the anatomical site of synovial tissues (× 40). Synovium in the CN group (× 100) showed fat cells with no synovial hyperplasia, inflammation, or fibrosis (score 0). b Synovium assessment in other groups: OA group showing wide dense fibrosis with totally absent fat. Inflammation is seen and scored as (5). The different treated groups showed variable decline of both synovitis and fibrosis scores (low power × 100, inset × 400)

Fig. 7

Assessment of articular cartilage in different studied groups in H&E and Safranin O fast green (SOFG) stained sections. The low power view of the CN group shows the normal architecture of the knee joint, SOFG stain shows high proteoglycan content (seen as red color). The OA group shows rough articular cartilage, decreased thickness and pale SOFG stain. In HYAL and DIZE-treated groups, mild roughness is still seen in articular cartilage (arrowheads). SOFG stained section shows increased proteoglycan in articular cartilage and increased thickness. In the HYAL + DIZE group, articular cartilage is smooth. Yellow lines represent articular thickness in different groups

Fig. 8

Effect of drug treatments on osteoarthritis-associated histopathology changes. The H&E and safranin O fast green stained sections show articular cartilage thickening (a), OARSI grade (b), synovitis (c), fibrosis (d) and total infrapatellar fat pad, IPF (e). The sum scores of synovitis and fibrosis make the total IPF score. Data are shown as means ± SD (n = 4). One-way ANOVA was used for comparing the articular cartilage thickening, followed by the Tukey post hoc test. Kruskal–Wallis test was used to analyze the OARSI grade and IPF scores, followed by Dunn's post-hoc test. Data are compared to (*) CN, (#) OA and (–) HYAL at p < 0.05. CN, control; OA, osteoarthritis; HYAL, hyaluronic acid, DIZE, diminazene

Fig. 9

Regression analysis showing the possible synergistic effect of the combination therapy on the different parameters. a Angiotensin-converting enzyme 1 (ACE1), b angiotensin-converting enzyme 2 (ACE2), c angiotensin 1–7 (Ang 1–7), d Ang II, e transforming growth factor beta 1 (TGF-β1), f NADPH oxidases 4 (NOX-4), g tumor necrosis factor-alpha (TNF-α), h mas receptor (MasR) and i matrix metalloproteinases 13 (MMP-13). Data are compared at p < 0.05