Geraniol Suppresses Oxidative Stress, Inflammation, and Interstitial Collagenase to Protect against Inflammatory Arthritis

Abstract

Geraniol (GER) is a plant-derived acyclic isoprenoid monoterpene that has displayed anti-inflammatory effects in numerous in vivo and in vitro models. This study was therefore designed to evaluate the antiarthritic potential of GER in complete Freund's adjuvant (CFA)-induced inflammatory arthritis (IA) model in rats. IA was induced by intraplantar injection of CFA (0.1 mL), and a week after CFA administration, rats were treated with various doses of methotrexate (MTX; 1 mg/kg) or GER (25, 50, and 100 mg/kg). Treatments were given on every alternate day, and animals were sacrificed on the 35th day. Paw volume, histopathological, hematological, radiographic, and qPCR analyses were performed to analyze the severity of the disease. GER significantly reduced paw edema after 35 days of treatment, and these results were comparable to the MTX-treated group. GER-treated animals displayed a perfect joint structure with minimal inflammation and no signs of cartilage or bone damage. Moreover, GER restored red blood cell and hemoglobin levels, normalized erythrocyte sedimentation rate, platelet, and c-reactive protein values, and also attenuated the levels of rheumatoid factor. RT-qPCR analysis demonstrated that GER decreased mRNA expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta. GER also down-regulated the transcript levels of cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1, prostaglandin D2 synthase, and interstitial collagenase (MMP-1). Molecular docking of GER with COX-2, TNF-α, and MMP-1 also revealed that the antiarthritic effects of GER could be due to its direct interactions with these mediators. Based on our findings, it is conceivable that the antiarthritic effects of GER could be attributed to downregulation of pro-inflammatory mediators and protease like MMP-1.

Figure 1

GER abated CFA-induced paw inflammation. IA was induced by injecting 100 μL of CFA in the right hind paw of the rats. After 7 days, treatments were given with the mentioned doses of MTX and GER. Paw volume was measured with a digital plethysmometer to check the edema at the mentioned intervals of time. GER at 25 and 50 mg/kg reduced paw edema, which was comparable to the MTX-treated group. # ≤ 0.001 (two-way ANOVA followed by a Bonferroni multiple comparison test); significant acronyms (#) represent the comparison of treatment groups with the disease group (CFA); n = 7.

Figure 2

X-ray images of the right hind paws of the rats exhibiting a reduction in the severity of IA by MTX and GER. IA was induced by injecting 0.1 mL of CFA, and a week after CFA administration, rats were treated with the mentioned doses of MTX or GER. On every other day, treatments were repeated until day 35 and animals were sacrificed. The hind paws were cut and fixed in 10% (buffered) solution of formaldehyde. X-ray films of the right hind paws were developed, which revealed a clear reduction in inflammation and erosion of bone and cartilage by MTX and GER (n = 3). “Photographs courtesy of Ishtiaq Ahmed, University of Veterinary and Animal Sciences, Lahore; Copyright 2022”.

Figure 3

Hematological and biochemical analysis revealed protective effects of GER against CFA-induced IA damage. IA was induced by injecting 100 μL of CFA, and after a week of CFA administration, rats were treated with the mentioned doses of MTX or GER. Treatments were given every other day until day 35, and later, animals were sacrificed. Biochemical and hematological parameters were analyzed using an automatic hematology and biochemical analyzer machine. GER successfully restored (A) RBC, (B) Hb, (C) ESR, (D) WBC, (E) PLT, and (F) CRP levels. GER (100 mg/kg) induced (G) AST, (H) ALT, and (I) ALP, displaying its hepatotoxic potential at higher doses. # ≤ 0.001 (one-way ANOVA followed by the Tukey’s multiple comparison test); significant acronyms (#) represent the comparison of treatment groups with the disease group (CFA); n = 7.

Figure 4

GER displayed an antioxidant effect. GER increased serum levels of SOD and GSH and reduced the levels of MDA, illustrating its beneficial antioxidant effects. # ≤ 0.001 (one-way ANOVA followed by the Tukey’s multiple comparison test); significant acronyms (#) represent the comparison of treatment groups with the disease group (CFA); n = 6.

Figure 5

GER attenuated the mRNA levels of CFA-induced pro-inflammatory mediators. IA was induced by injecting 0.1 mL of CFA, and after 7 days of CFA administration, rats were treated with the abovementioned doses of MTX or tested compound (GER). On alternative days, treatments were repeated until day 35. Relative mRNA expressions of indicated genes were measured from liver homogenates. GER significantly reduced (A) NF-kB1, (B) IL-1β, (C) TNF-α, (D) COX-2, (E) mPGES-1, (F) PTGDS, and (G) MMP-1. # ≤ 0.001 (one-way ANOVA followed by the Tukey’s multiple comparison test); RT-qPCR. Significant acronyms (#) represent the comparison of treatment groups with the disease group (CFA); n = 6.

Figure 6

Binding interactions of GER with COX-2, TNF-α, and MMP-1. 2D and 3D presentation of binding interactions of GER with the amino acid residues of the binding site of (A, B) COX-2, (C, D) TNF-α, and (E, F) MMP-1.

Figure 7

Binding interactions of MTX with COX-2, TNF-α, and MMP-1. 2D and 3D presentation of binding interactions of GER with the amino acid residues of the binding site of (A, B) COX-2, (C, D) TNF-α, and (E, F) MMP-1.