Results of osteoarthritis score

Following a 15-day injection of sodium iodoacetate solution, according to the score table (Figure 1),

Figure 1: Swelling degree of right limb and standing condition of rats.

A). Degree of leg stiffness and swelling in osteoarthritis rats.

B). Bone articular rats with stiff legs when standing

the swelling degree of the rightlimb of the rat was obvious, and the swelling score was set at 2 points; The right limb cannot touch the ground when standing and is accompanied by marked lameness when walking, and the classification score can be set at three; when the right knee of the rat was turned, the stiffness was apparent, the affected limb contracted violently and struggled, and the pain scale was set at two. This indicates that the modeling of intra-articular injection of sodium acetate is effective.T 

X-ray test results

According to the X-ray (Figure 2),

Figure 2: Radiographs of the right limb of rats

the distal end of the right femur was dissolved at the proximal end of the right tibia, the bone surface was not smooth, the boundary between the acetabulum and the femoral head was unclear, and the osteolysis of the right hip was initially diagnosed as arthritis. It is furthermore demonstrated that the modeling of intra-articular injection of sodium acetate is effective.

Effect of CBD treatment on arthritis score

The discomfort and claudication scores of rats treated with CBD were significantly decreased (Figure 3).

Figure 3: Changes in discomfort and lameness scores after CBD.

The degree of swelling of knee joint tissue of rats in each group was observed (Figure 4).

Figure 4: Changes in knee swelling after CBD administration.

A). Picture of knee anatomy in osteoarthritis rats.

B). Picture of knee anatomy in osteoarthritis rats after 50 mg/kg CBD treatment.

C). Picture of knee anatomy in osteoarthritis rats after 100 mg/kg CBD treatment.

D). Pictures of knee anatomy of PBS control group.

Significant swelling and hyperemia could be seen around the bone tissue of rats in model control group, but there was no obvious swelling of knee joint in PBS control group, 50 mg/kg CBD and 100 mg/kg CBD experimental groups. It is suggested that CBD can alleviate the pathological changes of OA induced by intra-articular injection of sodium iodoacetate.

Pathological changes of knee joint

According to the knee joint pathology of rats, there were no significant pathological changes in the knee in either the PBS control group, or the 100 mg/kg CBD PBS control group (Figures 5A,B).

Figure 5: Pathological findings of knee joint.

A). In PBS control group, there was no fibrosis around the bone tissue and no inflammatory cells invaded.

B). In the CBD control group, there was no fibrosis around the bone tissue and no inflammatory cell invasion.

C). In the solvent group, fibrosis and inflammatory cells were evident around the bone tissue.

D). In the model group, fibrosis and inflammatory cells were evident around the bone tissue.

E). After 50 mg/kg CBD treatment, inflammatory cells around bone tissue decreased relative to the model group.

F) After 100 mg/kg of CBD treatment, no fibrosis was observed in histological incisions of the knee cartilage.

While, significant fibrous tissue hyperplasia was seen around the bone tissue of rats in the model control group, and inflammatory cells were mixed in it; The quantity and size of chondrocytes are decreased, the chondrocyte partition is destroyed, sodium iodoacetate can substantially disrupt the structure of hyaline cartilage and damage its tissue structure, and the connective tissue totally replaces the cartilage surface [34]. The intramuscular administration of sodium iodoacetate solution has been shown to exacerbate pathological changes in OA (Figure 5D). 50mg/kg CBD has been suggested to alleviate these changes; fibrosis expression was reduced, the cartilage surface remained uniform, and the differentiation of chondrocyte partitions was partially preserved; however, local loss of cellular components on the surface and transition layer of the hyaline cartilage was still visible (Figure 5A). While 100 mg/kg of CBD can almost fully heal the pathological damage brought on by OA, no fibrosis was seen in the histological incision of the knee cartilage (Figure 5F) [34]. In the solvent group, fibrosis and inflammatory cells were evident around the bone tissue (Figure 5C).

Effect of CBD on the level of serum inflammatory factors in rats with osteoarthritis

In order to further determine the anti-inflammatory effect of CBD on OA rats, the concentrations of IFN-γ, IL-1β, TNF-α and IL-6 in the serum of rats in each group were detected according to the instructions of the ELISA kit. The results showed that compared with the OA model control group, the serum concentrations of IFN-γ, IL-1 β and TNF-α in the high and low dose CBD experimental groups decreased significantly, and the levels of inflammatory factors in the high dose group were lower than those in the low dose group (P< 0.001), but there was basically no difference between the high dose group and the PBS control group (Figure 6).

Figure 6: Cytokine levels in serum.

A). The bar graphs shows the IFN-γ content in the model group (n = 6), 50 mg/kg CBD-experimental group (n = 6), 100 mg/kg CBD-experimental group (n = 6), and PBS control group (n = 6). There was a statistically significant difference between groups, p > 0.05.

B). The bar graphs shows the IL-1β content in the model group (n = 6), 50 mg/kg CBD-experimental group (n = 6), 100 mg/kg CBD-experimental group (n = 6), and PBS control group (n=6). There was a statistically significant difference between groups, p > 0.05.

C). The bar graphs shows the TNF-α content in the model group (n = 6), 50 mg/kg CBD-experimental group (n = 6), 100 mg/kg CBD-experimental group (n = 6), and PBS control group (n = 6). There was a statistically significant difference between groups, p > 0.05.

In osteoarthritis-affected rats, levels of inflammatory cytokines were lower after 50 mg/kg CBD treatment than after 100 mg/kg CBD treatment. Neither the PBS control group nor the 100 mg/kg CBD control group had any appreciable serum inflammatory factors. Furthermore, it is suggested that CBD has anti-inflammatory effect on osteoarthritis rats, the higher dose group had better treatment effect.

OA is a chronic degenerative joint disease characterized by destruction of cartilage and loss of extracellular matrix, which can lead to chronic joint pain, deformity and dyskinesia [35]. Chronic pain is one of the factors that severely affect the quality of life of patients. In this study, SD rats were injected with sodium acetate into the knee joint cavity to induce OA model. The clinical signs are swollen joints, stiffness, pain and even tingling. X-rays additionally diagnosed arthritis, indicating the successful fabrication of the OA rat model.

The specific pathogenesis of arthritis is not clear. In recent years, several authors have suggested that if dysregulation of the gut microbiome is closely related to the pathogenesis of several metabolic and inflammatory diseases, it may also be involved in the progression of OA [36,37]. Korotkyi, et al. [38]. showed negative changes in the number and species composition of the faecal microbiome in mice induced by OA, which may indicate that the development of intestinal dysbiosis is due to the presence of chronic systemic inflammation associated with local degenerative changes in cartilage. There is no clear means of prevention of OA, the existing drugs are still in the exploratory stage [39].At present, the clinical treatment of OA is mostly focused on symptomatic treatment and pain relief, and the end point of OA can not be intervened, but can only delay its development. Therefore, the search for ingredients in natural materials with minor side effects in the treatment of arthritis is certainly a hot topic in this field. Korotkyi, et al found that the levels of IL-1β, TNF-α, IL-6, IL-8, IFN-γ, IL-10, TGF-β were increased, IGF-1 levels were decreased, and IL-4 levels were unchanged in the MIA-OA model. Compared with MIA-OA, probiotic during MIA-OA increased IGF-1 levels and decreased IL-1β, TNF-α, IL-6, IL-8, IFN-γ, and TGF-β levels, but did not reach control values (same as IL-4 and IL-10). They suggest that MIA-OA can cause significant changes in the levels of cartilage cytokines in the knee studied. Probiotics had an active local anti-inflammatory effect on cartilage tissue in MIA-OA rats [40,41].

CBD is one of the main components of the cannabis plant’s cannabinoids. It has anticonvulsant, anti-inflammatory, neuroprotective, and analgesic effects, and has no psychiatric activity. CBD participates in the regulation of pain and inflammation and plays an influential role in relieving pain and disease progression in OA. Verrico et al [26]. found that CBD could significantly reduce the production of proinflammatory cytokines IL-6 and TNF- α in in vitro experiments and mouse inflammatory models. CBD therapy in dogs with spontaneous OA is safe and relieves pain and increases range of motion in a dose-dependent manner. Philpott et al [30]. Established an OA rat model induced by sodium acetate. Local application of CBD around the joint can eliminate inflammation, prevent the loss of nerve myelin sheath, and slow down the process of central sensitization and neuropathic pain of OA. In this trial, CBD was found to significantly reduce pain, lameness, swelling and histopathological changes in affected limbs in sodium acetate OA rats by intramuscular injection. At the same time, the contents of serum inflammatory factors IFN- γ, IL-1 β and TNF- α also decreased significantly, but had no effect on the expression level of IL-6, indicating that CBD can alleviate the further development of osteoarthritis by inhibiting inflammatory cytokines. Of course, whether in the established rat OA model or in the analysis of the therapeutic effect of CBD, it is not enough to detect and analyze only four pro-inflammatory factors to confirm inflammation, and more inflammatory factors need to be detected and analyzed in this study [36]. In summary, CBD has better anti-inflammatory effect on OA model induced by sodium acetate in rats, and the effect of 100 mg/kg CBD is superior to that of 50 mg/kg CBD. Although different experimental results may be caused by different model species, modeling methods, and administration doses, it has been established that CBD has a precise effect on pain and inflammation relief in OA, and the corresponding mechanism is still unclear and thus needs to be explored further.

In conclusion, we have achieved remarkable results in treating OA rats with CBD. CBD alleviated the discomfort symptoms such as pain and lameness in the affected limbs of rats, and effectively inhibited the inflammatory cytokine IFN-γ,IL-β and TNF-α. It has significant effect on the treatment of OA. The results of this study can promote the treatment of OA. At the same time, it has a positive impact on the clinical application of CBD. However, the specific mechanism of CBD in the treatment of OA still needs further study.

Not applicable.

The authors declare that there is no conflict of interest regarding the publication of this article.

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Objective: To investigate the therapeutic effect of CannaBiDiol (CBD) on OsteoArthritis (OA) in rats.

Methods: We randomly divided 36 female SD rats with SPF values of 7 weeks into model control, CBD at elevated doses, CBD at low doses, solvent control, and PBS control. Sodium acetate OA was modeled in the model, experimental and solvent control groups. After modeling, the experimental group was injected daily with CBD and solvent, and the control group was injected with PBS. After 12 days of treatment, the OA grade was scored, and the rats were anesthetized, and the rats were sacrificed by neck dislocation, blood was collected from the heart, knee joints were collected, the degree of joint swelling was recorded, and serum inflammatory cytokines IFN-γ, IL-1β, TFN-α and IL-6 concentration were and pathological changes of knee joint.

Results: Compared with the model group and the solvent control group, the pain and claudication of the rats in the CBD experimental group were effectively relieved, and the levels of inflammatory cytokines (IFN-γ, IL-1β, TFN-α) in serum and synovial fluid were significantly reduced. Arthritic pathologic changes in the knee were significantly reduced.

Conclusion: CBD is effective in reducing blood inflammatory factors in OA rats, relieving pain and pathological changes, and has significant therapeutic effects in osteoarthritis.

• Rats; CBD; Osteoarthritis; Inflammatory

Yin HC, Zhang XY, Chen Z, Yin NN, Zhao M, et al. (2024) Cannabidiol Inhibits Inflammation in Rat Osteoarthritis. JSM Trop Med Res 5(1): 1020.

Osteoarthritis (OA) is a common joint degenerative disease, which commonly occurs in the elderly. Joint pain, swelling, bone rubbing and limited movement are the main clinical manifestations of osteoarthritis [1]. According to the study, there are about 250 million OA patients worldwide. Arthritis is a more common disease in the United States, with approximately 52.5 million (22.7%) of adults ≥ 18 diagnosed with arthritis, Over 22.7 million people reporting arthritis-attributable activity limitations [2-4]. In China, the incidence of OA is 8.1%, and in people over 65, the incidence of OA is as high as 50% [5]. Such a large number of patients has brought huge burdens and economic losses to the whole society. It is estimated that the medical expenses of OA account for 1% to 2.5% of GDP in developed countries [6]. With the increasing aging of the world, it is reasonable to believe that the medical cost of OA will be higher and higher. Therefore, the research on OA has increasingly become a current hot spot in the medical industry and social research. At present, the clinical treatment of OA is mostly focused on symptomatic treatment and pain relief, and the end point of OA cannot be intervened, but can only delay its development. Oral or topical non-steroidal anti-inflammatory drugs are the most commonly used in clinics, including oral glucosamine, intra-articular injection of sodium hyaluronate or glucocorticoid. In recent years, we have also frequently used anxiolytic drugs such as duloxetine in clinical practice [7]. However, the above treatments are symptomatic and the effect is limited [8,9]. Artificial joint replacement is the only effective treatment for patients with terminal OA. Exploration of drugs that can effectively relieve pain and delay the progression of OA is therefore an urgent issue.

Cannabidiol (CBD) is the main non-psychoactive component in marijuana. Cannabis medications contain a variety of compounds, including the psychoactive cannabinoid delta9-Tetrahydrocannabinol (THC) and non-psychoactive CBD. In recent years, cannabidiol has become a major hot spot for analgesia, anxiolytic, antidepressant, and anti-inflammatory [10-13]. CBD has a wide range of pharmacological effects, such as anticonvulsant, hypnotic, anti-inflammatory, anti-anxiety, antipsychotic and neuroprotective effects [14]. In 2018, the FDA approved it as a drug for the treatment of seizures of Dravet syndrome and Lennox-Gastaut syndrome, indicating that it has some safety in clinical use. CBD has demonstrated analgesic effects in several animal disease models. It has been reported that CBD has analgesic effect in plantar inflammatory pain model [15], and it also has a significant relieving effect on maxillofacial inflammatory pain [16]. Cannabinoid (CBD) can exert a variety of biological effects through several different receptors and signal pathways, including anti-inflammatory effects on acute and chronic diseases [17-19]. In the rodent CFA model, CBD and its modified derivatives can relieve chronic pain [20]. In the mouse model of acetone Croton oil-induced inflammatory pain and LPS-induced inflammatory pain, CBD could reduce the levels of pro-inflammatory cytokines IL-6 and TNF- α in the serum and increase the level of anti-inflammatory IL-10 [21]. CBD dissolved in corn oil enhanced LPS-induced pulmonary inflammation and increased levels of pro-inflammatory factors and granulocyte colony-stimulating factor [22]. CBD has a wide range of targets, including eCBs, G Protein Receptor 55 (GPR55), 5-hydroxytryptamine 1A Receptor, dopamine Receptor, opioid Receptor, peroxisome proliferator-activated Receptor gamma and transient Receptor potential cation channel TRPV1 plasma channel [23], etc., which makes the explanation of the analgesic mechanism of CBD unclear. CBD reduces inflammation-related neurodegeneration and its antioxidant properties, has no psychoactive properties, and has a wide range of potential beneficial effects, suggesting that CBD may be a potential new drug for the treatment of inflammatory diseases. In fact, CBD has shown the potential to treat musculoskeletal disorders in humans, dogs, cats, Amazona amazonica and rabbits [24-29].

In this study, the OA rat model was induced by injection of sodium acetate and different doses of CBD were given to the OA model rats. We analyzed the efficacy of CBD by combining clinical scores, serum inflammatory factor analysis, histopathology analysis, and other indicators. The aim of this project is to explore the pain relief and therapeutic effects of CBD on OA, to provide a modern approach to the treatment of animal OA and pain relief, and to provide data to support the clinical application of CBD.

Experimental animals and drugs

36 SPF-grade 6-week-old SD female rats, weighing 190g±10g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: SCXK (Liao) 2020-0001. Each group of experimental rats were raised in a single cage, normal diet and drinking water, free exercise, feeding and adaptation for 7 days.

Sodium iodoacetate (source leaf, S30680), CBD (National Market Supervision Technology Innovation Center (industrial marijuana). Rat IFN gamma ELISA Kit (ab239425), Rat IL-1 beta ELISA Kit (ab255730), Rat TNF alpha ELISA Kit (ab236712) were purchased from Abcam.

Methods

Grouping and treatment methods. 36 female SD rats were randomly assigned to six groups: model control group, CBD high dose experimental group, CBD low dose experimental group, solvent control group, PBS control group, and CBD high dose PBS control group. 5 mg sodium iodoacetate was dissolved in 500 μL PBS. 7-week-old rats in the model group, experimental group and solvent control group were injected with 100 μL sodium iodoacetate solution after 45°puncture of the right knee joint cavity, and the same amount of PBS was injected into the PBS control group. Each group of experimental rats were raised in a single cage, normal diet and drinking water, free exercise, and analyzed whether the OA model was successful at 15 day. Then, the model rats in the low-dose and high-dose CBD experimental groups were given 50 mg/kg CBD and 100 mg/ kg CBD respectively by intraperitoneal injection [30], while the solvent control group was injected with 100 μL (20% anhydrous ethanol + 80% normal saline). The CBD high dose PBS control group means that the PBS control group was given 100 mg/kg of CBD by intraperitoneal injection. 12 days After CBD or solvent treatment, each group of rats were anesthetized by inhaling diethyl ether before sacrificed [30].

Osteoarthritis score. After the rat model was made, the rats were scored according to the score scale.

X-ray detection of knee joint injury. Osteoarthritis can lead to joint stiffness, limited mobility, and severe parolysis, and degenerative changes in the joints. Despite the continuous advancement of medical technology, X-rays are still the most suitable way to observe changes in bones and joints. X-rays require a scan of the side and front of the area with arthritis [31-33]. The rats were fixed to the X-ray board in the position of lying on the side for X-ray scanning, and the scanning results were imported into the computer to generate X-rays (Qihuo pet diagnosis and treatment).

Detection of serum inflammatory cytokines. After anesthesia, the cervical vertebra is removed and the blood is placed in a 1.5 mL centrifuge tube to separate the serum. In this experiment, the tumor necrosis factor α (TNF-α), interleukinin (IL-1 β), and interferon γ (IFN-γ) was detected by ELISA Kit according to the instructions.

Pathological analysis. At the end of the experiment, the right hindlimb knee of each rat was fixed at room temperature with 10% formalin, decalcified with 5% formic acid, and embedded in paraffin. The tissue section was 5 μm thick and stained with hematoxylin-eosin (hematoxylinandeosin, H&E). The histopathological features were observed under microscope (magnification 100 times and 200 times).

Statistical analysis

The data were analyzed by Prism9 software, the measurement data were expressed by mean ±standard deviation, and the statistical analysis was made by multivariate analysis of variance.