2.1.Experimental animals and drugs
36 SPF-grade 6-week-old SD female rats, weighing190 g±10 g, 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).
2.2.Methods
Grouping and treatment methods. 36 female SD rats were randomly divided into 6 groups: model control group, CBD high dose experimental group, CBD low dose experimental group, solvent control group and PBS control group. 5mg sodium iodoacetate was dissolved in 500 μL PBS. 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 or not. After 15 days, the rats in the low-dose and high-dose CBD experimental groups were given 10mg/kgCBD and 20mg/kgCBD respectively by intraperitoneal injection, while the solvent control group was injected with 100 μL (20% anhydrous ethanol + 80% normal saline) every day. The rats in each group were fed and drank normally and moved freely.
Osteoarthritis score. After the rat model was made, the rats were scored according to the score scale.
Gait: divided into 4 levels according to the gait of the affected limb when walking and running.
Grade I: no limp in the affected limb, normal running, strong pedal.
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0
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Class II: mild claudication and strong pedal when the affected limb is running.
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1
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Grade Ⅲ: the affected limb takes part in walking, but the limp is obvious.
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2
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Grade IV: the affected limb can not participate in walking, can not touch or pedal the ground.
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3
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Local pain response: touch or squeeze the affected area with fingers or cotton swabs, which can be divided into 4 grades according to the degree of stimulation response.
Grade I: no abnormal pain response
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0
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Grade II: contraction of the affected limb
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1
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GradeⅢ: limb contraction, spasm, with mild systemic reaction
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2
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Grade IV: severe contraction, spasm, tremor and struggle of the affected limb
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3
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Joint swelling: divided into 3 grades according to the degree of joint swelling.
Grade I: no swelling, bone marks clearly visible
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0
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GradeⅡ: mild swelling, shallower bone marks
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1
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Grade Ⅲ: osseous markers disappear
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2
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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[24-26]. 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 Detection of tumor necrosis factor α (TNF-α), interleukinin (IL-1 β), interferon γ (IFN-γ) and IL-6 Detection kit in rats was detected by forest-forming ELISA, and the content of inflammatory cytokines in serum of each group was detected 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).
2.3 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.