Probenecid and/or l-carnitine mitigated the MIA-provoked OA knee size

The % change in OA (right) knee size from the normal knee (left) size on days 0,1,7,14, 21,28, and 33 is illustrated in Fig. 2. The intra-articular injection of MIA in the OA knee of all osteoarthritic untreated rats caused a significant continuous increase in the knee size revealing induction of inflammation and development of edema. All treatments given in this study caused a significant decline in the OA knee size on days 28 and 33. On day 33, there were no significant differences between the effects of probenecid- and l-carnitine-treated groups. The most significant reduction in the OA knee size was observed in the combination therapy that was not significantly different from the normal control rats at days 28 and 33.

Fig. 2

The % change in the OA (right) knee edema size from the normal knee (left) size on days 0,1,7,14,28,33 following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats. Values are means ± S.D. of 6 rats. P < 0.05 vs. control rats (a), OA rats (b), Probenecid-treated rats (c), and L-Carnitine-treated rats (d)

Probenecid and/or l-carnitine reversed the MIA-induced aberration in motor coordination and joint mobility

Figure 3 depicts the percentage change in latency to fall on day 33 with reference to day 0. On day 33, the rotarod performance of the OA untreated rats was 47.6% ± 2.7 lower than on day 0. Furthermore, the drop-in performances were 14.6% ± 2.5 and 16.4% ± 2.5 in the probenecid and l-carnitine-treated groups, respectively, when compared to day 0. There were no significant differences between the probenecid and l-carnitine groups. Furthermore, as compared to day 0, the combination treatment group exhibited a 2.5% ± 2.9 improvement in performance, which was not substantially different from the normal control group’s performance rise 3.5% ± 2.3.

Fig. 3

The % change in latency to fall on day 33 as compared to day 0 following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats. Values are means ± S.D. of 6 rats. P < 0.05 vs. control rats (a), OA rats (b), Probenecid-treated rats (c), and L-Carnitine-treated rats (d)

Probenecid and/or l-carnitine attenuated the MIA-provoked P2X7R/NLRP3/cleaved caspase-1/IL-1β/IL-18 cue

As illustrated in Fig. 4, the OA rats showed increased protein expression of P2X7R (5.88 folds), NLRP3 (7.11 folds), and cleaved caspase-1 (5.48 folds) with a decreased expression of procaspase-1 (3.71 folds) in the knee joint cartilage as compared to control rats. In addition, OA induction significantly increased serum IL-1β (8.46 folds), and IL-18 (3.83 folds) as compared to the control group. Probenecid, L-carnitine treatments significantly declined protein expression of P2X7R (2.51, 2.52 folds), NLRP3 (2.00, 1.95 folds), and cleaved caspase-1 (2.02, 2.08 folds) as well as elevated the expression of procaspase-1 (2.11, 2.18 folds) in the knee joint cartilage as compared to OA rats, respectively. Also, serum IL-1β and IL-18 were significantly mitigated by probenecid treatment (1.57, 1.35 folds) as compared to the OA group, respectively. L-carnitine treatment possessed comparable results to that obtained by probenecid.

Meanwhile, the combination-treated group showed the best results in the aforementioned parameters, as there was a significant reduction in the protein expression of P2X7R (5.9 folds), NLRP3 (6.76 folds), and cleaved caspase-1 (5.58 folds) with augmented expression of procaspase-1 (3.45 folds) in the knee joint cartilage. The combination therapy significantly reduced serum IL-1β and IL-18 (5.79, 2.80 folds), as compared to OA rats, respectively.

Fig. 4

Change in knee cartilage expression of P2X7R (A), NLRP3 (B), procaspase-1 (C), cleaved caspase-1 (D) and serum levels of IL-1β (E), and IL-18 (F) following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats. Values are means ± S.D. of 6 rats. P < 0.05 vs. control rats (a), OA rats (b), probenecid-treated rats (c), and L-Carnitine-treated rats (d). Western blot images shown are representative of one rat per group

Probenecid and/or l-carnitine counteracted the MIA-augmented NF-κB activation and its downstream inflammatory cytokines

As shown in Fig. 5, OA induction significantly decreased the expression of IκB (7.49 folds) as well as increased the expression of NF-κB (5.67 folds) in the knee joint cartilage of OA rats as compared to the control rats. Moreover, the OA model significantly increased serum IL-6 (2.74 folds) and TNF-α (3.49 folds) as compared to normal rats.

Treatment with probenecid or l-carnitine significantly elevated the IκB expression (4.99, 4.71 folds) as well as declined the expression of NF-κB (1.89, 1.98 folds) in the knee joint cartilage of treated rats as compared to OA rats. The combination therapy of both drugs caused the most significant elevation of IκB expression (7.08 folds) and reduction of NF-κB expression (5.75 folds) as compared to OA rats.

However, serum IL-6 and TNF-α levels were significantly ameliorated by probenecid and l-carnitine when compared to the untreated OA group to a comparable level respectively, but still significant when compared to the normal one, whereas the best results were witnessed in the combination-treated group to reach normal levels. The serum IL-6 and TNF-α levels were significantly declined by probenecid treatment (2.00, 1.67 folds), while l-carnitine declined their levels by (1.89, 1.61 folds), respectively. The combination therapy showed the most significant mitigation in the serum levels of IL-6 and TNF-α levels (2.74, 3.15 folds) as compared to OA rats.

Fig. 5

Change in knee cartilage expression of IκB (A), and NF-κB (B) and serum levels of IL-6 (C), and TNF-α (D) following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats. Values are means ± S.D. of 6 rats. P < 0.05 vs. control rats (a), OA rats (b), Probenecid-treated rats (c), and L-Carnitine-treated rats (d). Western blot images shown are representative of one rat per group

Probenecid and/or l-carnitine augmented MIA-attenuated miR-373 expression

MiR-373 expression was significantly decreased in the OA rats (0.35 ± 0.08 fold change) as compared to control rats (0.97 ± 0.06 fold change), Fig. 6. Upon different treatments, its expression was significantly increased, whereas the effect of probenecid (0.65 ± 0.05 fold change) was comparable to that of l-carnitine (0.63 ± 0.07 fold change). The best effect was observed in the combination group (0.99 ± 0.02 fold change) to reach normal value; an effect that was more significant than either treatment alone.

Fig. 6

Change in knee cartilage miRNA-373 level following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats. Values are means ± S.D. of 6 rats. P < 0.05 vs. control rats (a), OA rats (b), Probenecid-treated rats (c), and L-Carnitine-treated rats (d)

Probenecid and/or l-carnitine reversed the MIA-induced knee joints histopathological changes observed in H&E stained sections

The histopathological assessment done for H&E stained sections of knee joints of different experimental groups is illustrated in Fig. 7A. The synovial membrane scoring method, which included (synovial membrane hyperplasia, lymphocytic/plasmacytic inflammation, sub-synovial fibrosis, and vascularity), was examined and scored (0–3) individually, yielding a total score of 12 as shown in Fig. 7B.

Fig. 7

A Synovial membrane assessment in knee joint H&E stained sections following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats, (× 40, scale bar = 500 microns, high power of squared area: × 100, scale bar = 200 microns). S  synovium, i  inflammation, v  vascularization, f  fibrosis, *meniscal sclerosis. CTRL GP shows thin synovium (s) with absent inflammation, vascularization, or fibrosis in sub-synovial tissue. OA GP shows synovial hyperplasia (s) with multiple foci of lymphoplasmacytic infiltrate (i) as well as fibrosis (f). also noted multiple dilated vascular spaces (v) indicating a high degree of synovitis. Osteosclerosis of the meniscus is seen (*) in the form of disfigurement of the meniscus and ossification. Prob GP shows improvement of three parameters with only mild synovial hyperplasia (s), inflammation (i) in addition to focal vascularization (v). L Car GP shows less improvement with residual meniscal sclerosis (*). Meanwhile, Prob + L CAR GP showed the best effect. Synovium is thin (s) and fibrosis is minimal (f). Occasional inflammation (i) is seen, and no vascular spaces are detected. Histology images shown are representative of one rat per group. B Total score of synovial membrane assessment in knee joint H&E stained Sects. (0–12) following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats. The synovial membrane scoring method, which included (synovial membrane hyperplasia, lymphocytic/plasmacytic inflammation, sub-synovial fibrosis, and vascularity), was examined and scored (0–3) individually, yielding a total score of 12 (6 rats/group). P < 0.05 vs. control rats (a), OA rats (b), probenecid-treated rats (c), and l-carnitine-treated rats (d)

The control rats showed a preserved anatomical structure of the knee joint. Both femoral and tibial condyles were covered by smooth cartilage. The meniscus was regular in shape. The synovium was covered by one to two layers of synovial cells. Sub-synovial fat was free of inflammation, fibrosis, or vascular proliferation.

In contrast to the osteoarthritic untreated group, knee joints showed evident OA. The joint space was narrowed with irregular surfaces of both tibia and femur condyles. The meniscus showed disfigurement and sclerosis. Synovial hyperplasia was seen in addition to sub-synovial tissue fibrosis. Collections of mixed lymphoplasmacytic infiltrate and Large congested vascular spaces were noted.

Those changes were improved in treated groups. Probenecid-treated rats regained the anatomical integrity of knee joints. Mild synovial hyperplasia was seen with less fibrosis. Vascular spaces were only focally seen. L-carnitine group which showed residual meniscal sclerosis with mild fibrosis and congestion.

The combination of both therapies showed notable improvement. The smooth surface of both the tibia and femur as well as the meniscus was seen. The synovium was thin and formed of 2 layers only. Sub-synovial tissues showed only light occasional fibrosis with minimal inflammation. No vascular spaces were detected.

Probenecid and/or l-carnitine reversed the MIA-induced knee joints histopathological changes observed in Safranin O-Fast green stained section

Results of assessments of Safranin O-Fast green-stained sections are shown in Fig. 8A. Total Modified Mankin scores given for the safranin O stained sections of different experimental groups (0–13) are illustrated in Fig. 8B. Control group showed thick articular cartilage with smooth surface. On high-power examination, the cartilage layers were organized. It showed a superficial thin layer composed of flat cells and then a mid and deep zones of chondrocytes are present. All were embedded in a cartilaginous matrix showing a deep red colour indicating high proteoglycan content. The chondrocytes were viable. They were located within lacunae and showed large vesicular nuclei.

Fig. 8

A Articular cartilage assessment in knee joint Safranin O-fast green stained sections following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats, (× 40, scale bar = 500 microns, high power: × 400). Control GP shows a smooth surface of all bones (arrowheads) with deep red color indicating high proteoglycan content. High power view shows normal organization of chondrocytes into three superficial (I), mid (II) and deep zones (III) with intact tidemark (t). Black arrow is pointing at one viable chondrocyte. OA GP shows irregularities in articular surfaces (arrowheads) with sever proteoglycan depletion. High power shows evident hypocellularity and clustering of chondrocytes. Degenerated chondrocytes are noted (yellow arrow) and irregular tidemark (t). Prob GP shows a smooth articular surface and increased proteoglycan in the matrix (arrowheads). High power shows hypercellularity of cartilage with viable chondrocytes. L Car GP shows less improvement with residual focal surface irregularities (arrowhead). High power shows slightly irregular tidemark (t) and focal degenerated chondrocytes (yellow arrow). Meanwhile, Prob + L CAR GP showed the best effect. Articular cartilage is smooth (arrow head) with high proteoglycan content. In high power normal architecture and intact tide marks (t) are seen. Histology images shown are representative of one rat per group. arrowhead = articular surface, black arrow = viable chondrocyte, yellow arrow = degenerated chondrocyte, t = tidemark. I,II,III: represent different layers of articular cartilage. B Total Modified Mankin score in knee joint safranin O stained Sects. (0–13) following treatment with probenecid at a dose of 50 mg/kg/day, l-carnitine at a dose of 100 mg/kg/day, and their combination for 14 days in all experimental groups after MIA induction of osteoarthritis in rats. The scoring system includes 4 different parameters (cartilage structure, chondrocytes architecture, matrix staining, and tidemark integrity) with a total score out of 13 (6 rats/group). P < 0.05 vs. control rats (a), OA rats (b), probenecid-treated rats (c), and l-carnitine-treated rats (d)

The OA untreated group showed thinned-out articular cartilage. The surface showed irregularities and abrasions. On high-power examination, the chondrocytes exhibited evident disorganization with areas of hypocellularity and clustering. The background showed marked proteoglycan depletion. Degenerated chondrocytes were seen in the form of dark-stained irregular nuclei or empty lacunae. Tidemark destruction was seen. The modified Mankin score was (10.5) indicating severe cartilage injury.

In the probenecid-treated group, the intra-articular injection of probenecid had modified Mankin score (Chow and Chin 2020). The cartilage surface showed only mild irregularities. The chondrocytes partially regained their architecture however focal degenerated nuclei and hypercellularity were detected in some areas. Increased proteoglycan content was noted with intact tidemark. L-carnitine group showed also slight improvement in knee joint histopathology. The surface was smooth in some areas and slightly irregular in others. However, some disorganization of architecture and degenerated chondrocytes were still noted. Proteoglycan content showed a mild increase reaching a Mankin score of about [5.3].

Meanwhile, the combination of both treatments showed notable improvement in histopathology. The articular cartilage was smooth. The chondrocytes were of normal architecture with no clustering or degeneration. The matrix showed increased proteoglycan content. Tidemark integrity was retained. Those changes were evident in the modified Mankin score of this group (Yao et al. 2023).

Statistical correlations

The Pearson coefficient test was used to analyze the link between miRNA-373 and P2X7R in all experimental groups, as well as correlations between P2X7R, NLRP3, and NF-B p65 in the Probenecid/L-carnitine treatment group. In all experimental groups, the statistical correlations demonstrated a negative association between miRNA-373 and P2X7R. In the Probenecid/L-carnitine-treated group, there was a positive correlation between P2X7R and NLRP3, as well as NF-B p65 Table 3.

Table 3 A Statistical correlations between miRNA-373 and P2X7R in all experimental groups; B Statistical correlations between P2X7R, NLRP3, and NF-κB p65 in the Probenecid/L-carnitine-treated group