L-Carnitine potentiates the anti-inflammatory and antinociceptive effects of diclofenac sodium in an experimentally-induced knee osteoarthritis rat model

Abstract

Objectives: The aim of the present research is to investigate the efficacy of L-carnitine (LC) as a complementary therapy to diclofenac sodium (Dic) treatment in a mono-iodoacetate (MIA) induced knee osteoarthritis (OA) rat model, with respect to pain relief and the underlying pathology.

Materials and methods: Fifty adult male albino rats were randomly divided into five groups (n=10): Control, OA, OA/Dic, OA/LC, and OA/Dic+LC. Knee diameter and pain assessment tests were done weekly. After four weeks, serum malondialdehyde, reduced glutathione, interleukin 1-β, tumor necrosis factor-alpha, prostaglandin E2, and bone-specific alkaline phosphatase were measured. The injected knees were removed and processed for the histological and immunohistological study of matrix metalloproteinase-13 (MMP-13) and cyclooxygenase 2 (COX-2). Also, histological examination of dorsal root ganglia and calcitonin gene-related peptide (CGRP) expression in the spinal cord were assessed.

Results: Treatment with Dic and/or LC significantly reduced knee swelling, improved pain-related behaviors, inflammatory and oxidative stress markers, attenuated the MIA-mediated histopathological alteration in the knee joint, and down-regulated expression of MMP-13 and COX-2 in the knee joint. It, also, significantly reduced CGRP expression, compared with the OA group. Dic+LC showed a better effect in improving some parameters than each treatment alone.

Conclusion: LC plus Dic is a more effective therapy than Dic alone for OA treatment.

Keywords: CGRP; COX-2; L-carnitine; MMP-13; Osteoarthritis.

Figure 1

Effect of diclofenac sodium and/or L-carnitine on knee diameter (left-right) in mono-iodoacetate induced osteoarthritis rat model. * P<0.05, ** P<0.001 vs C group; # P<0.05, ## P<0.001 vs OA group

Figure 2

Effect of diclofenac sodium and/or L-carnitine on knee bend and gait scores in mono-iodoacetate induced osteoarthritis rat model

Figure 3

Effect of diclofenac sodium and/or L-carnitine on rotarod and exercise endurance tests in mono-iodoacetate induced osteoarthritis rat model. * P<0.05, ** P<0.001 vs C group; # P<0.05, ## P<0.001 vs OA group; $ P<0.05, $$ P<0.001 vs OA/Dic; † P<0.05, †† P<0.001 vs OA/ LC group

Figure 4

Effect of diclofenac sodium and/or L-carnitine on mRNA expression of calcitonin gene-related peptide. ** P<0.001 vs C group; ## P<0.001 vs OA group; † P<0.05 vs OA/ LC

Figure 5

Representative H&E staining of rat synovial membrane of different groups: the membrane of C group consists of thin synovial intima (black arrow), and subintima with predominance of adipocytes (AD). The synovial membrane of OA group appeared moderately thickened with a disorganized intima (black arrow) and, the subintima, excessive fibrous tissue (asterisk) with few adipocytes (AD) and blood vessels full of red blood cells (HE). The synovial membranes of both OA/Dic and OA/LC groups, showed intimal tissue organization (black arrow), although the subintima appeared moderately fibrous (asterisk). OA/Dic+LC group, histopathological changes were considerably ameliorated with mild irregularity of the synovial intimal layer (black arrow) and remarkable reduction in the subintimal fibrous tissue with appearance of abundant adipocytes (AD). Scale bar 50 µM, ×200

Figure 6

Representative H&E staining of the knee joint of different groups. In the C group, smooth articular surface (black arrow), normal parallel rows of chondrocytes (asterisk) and intact tidemark (black arrowhead) were detected. The subchondral bone showing bone marrow (®) filled with blood-forming elements surrounded by bone trabeculae (white arrow). Note, bony spicules fixing it to cartilage (white arrowhead). In the OA group, irregularity of articular surface (black arrow), hypocellularity (asterisk) with chondrocytes disorganization of articular cartilage and ill-defined tidemark line were detected. Subchondral bone showed cyst formation (curved arrow), fragmentation of the bony trabeculae (crossed arrows) and cracks (arrow with rounded end). Disappeared bony spicules and fibrous tissue formation in the bone marrow space (®) can also be detected. In OA/Dic, OA/LC, and OA/Dic+LC groups, articular cartilage surface was regular (black arrow). Chondrocyte number was increased (asterisk) with intact tidemark (black arrowhead). Few trabecular cracks (arrow with rounded end) were still detected in the subchondral bone. Subchondral cysts (curved arrow) were detected in OA/Dic and OA/LC groups. Scale bar=50 μm×200

Figure 7

Modified Mankin’s score. ** P<0.001 vs C group; ## P<0.001 vs OA group

Figure 8

Representative H&E staining of rat dorsal root ganglia of different groups. The dorsal root ganglia of C group showing multiple large and small neurons (arrow heads) with rounded open face nuclei surrounded by a satellite glial cell sheath (thin arrows) and enclosed by a thin connective tissue capsule (notched arrow). Note, few capillaries (©) in the interstitial stroma and nerve fibers close to the ganglion (F) could be detected. The OA group shows shrunken neurons (black arrows) with wide perineural spaces, some neurons show peripheral chromatolysis (white arrows) others show central chromatolysis (white arrow heads). There is empty neural spaces (stars), satellite cell proliferation (thin black arrows) and dilated capillaries (©) in the interstitial stroma. Both OA/Dic and OA/LC groups show multiple cells that appear normal (black arrowhead). A few cells appear distorted and shrunken (thick arrow) and others show central chromatolysis (white arrowhead). In OA/LC group, proliferated satellite cells (thin black arrows) could be noticed in the interstitial stroma. OA/Dic+LC group appears similar to the control group except for some dilated capillaries (©). The number of neurons (F) were dramatically decreased in OA group; ** P<0.001 vs C group. This decrease was significantly increased in the OA/Dic and OA/Dic+LC treated groups; # P<0.05, ## P<0.001, respectively vs OA group. Scale bar 20 µM, ×400

Figure 9

Representative MMP-13 and COX-2 staining of rat knee joint of different groups: MIA injection up-regulated expression of both MMP-13 and COX-2, ** P<0.001 vs control group. This increase was significantly decreased in OA/Dic, OA/LC, and OA/Dic+LC groups; ## P<0.001 vs OA group. Scale bar 20 µM, ×400