CB2-mediated Attenuation Of Nucleus Pulposus Degeneration Via The Amelioration Of Oxidative Stress In Vivo And In Vitro

ObjectiveTo investigate the mechanism of cannabinoid type II receptor(CB2)in nucleus pulposus degenerationMethodsThe study was conducted in vitro and in vivo.Eighty 3-month rats(weighing 400±20g)were selected for the in vivo experiment and divided into four groups:control group,vehicle group,CB2 agonist(JWH133)treatment group and CB2 inhibitor(AM630)treatment group.IVDD model was established by needle puncture.The vehicle group was treated with PBS by a micro syringe after puncture,and the treatment group was given CB2-specific agonist JWH133 and inhibitor AM630,respectively.After modeling,X-ray and MRI examinations were performed at each week.After the sacrifice,intervertebral discs were collected and made into paraffin sections.Morphological changes were assessed by Hematoxylin-eosin(H&E)staining and Safranin O Fast Green staining.In vitro experiments,8-week-old male Sprague-Dawley rats(weight 250±25g)were selected and sacrifice,their tails were soaked in 75%alcohol for 15 minutes.Skin was removed with a sharp knife,and intervertebral disc tissue was exposed.The experimental groups were also divided into four groups,including control group,H2O2 group,JWH133 and AM630 group.The H2O2 group was treated with 100 ?MH2O2 for 12 hours to induce nucleus pulposus degeneration,and the treatment group was respectively treated with H2O2 for 3 hours after the pretreatment of JWH133 or Am630.After cell samples were collected,changes in mRNA expression were detected by RT-PCR,protein expression was detected by Western Blot,and changes in oxidative stress factors and related indicators of degeneration were detected by immunocytochemical staining.ResultsThe immunohistochemical staining results of the collected nucleus pulposus tissues with different degrees of degeneration collected from IVDD patients showed that the expression of CB2 was negatively correlated with the degree of nucleus pulposus degeneration.The 21G needle was used to established rat IVDD model.The height of intervertebral disc collapsed obviously,and the tendency of the nucleus pulposus changed to fibrous tissue was obvious.MRI revealed significant decrease in intervertebral disc signal.The CB2 expression in degenerative rat NP decreased significantly,which was consistent with human.The JWH133 treatment group could significantly attenuate the deterioration of disc collapse and the process of fibrosis of the nucleus pulposus.The results of Disc Height Index(DHI)showed that the value of vehicle group decreased to 0.05±0.005 from the normal 0.08±0.005,while the value of DHI increased by 1.4 times after JWH133 treatment compared with the vehicle group(*p<0.05),and there was no significant changes in the AM630 treatment group(*p>0.05).MRI showed that the intervertebral disc signal in the JWH133 treatment group was significantly higher than that in the vehicle group,with statistically significant difference in quantitative analysis(*p<0.05).H&E staining and Safranin O Fast Green staining suggested that JWH133 significantly improved the morphological structure of the intervertebral disc after acupuncture,and the morphological score showed that the JWH133 treatment group decreased from 9.2 to 5.5 compared with the vehicle group.The results of immunohistochemical staining showed that the positive expression of Collagen ? in the vehicle group decreased significantly,while the expression of MMP3 increased significantly,indicating the establishment of the degenerate model.However,Collagen ?recovered after JWH133 treatment,while the expression of MMP3 decreased.The results of in vitro experiments showed that H2O2 intervention induced degeneration of nucleus pulposus cells.CCK-8 results showed no significant cytotoxicity on cell proliferation and differentiation within concentrations of 100?M H2O2(*p>0.05).The results of RT-PCR,Western Blot and immunocytochemical staining showed that Collagen ? expression decreased and MMP3 expression significantly increased after H2O2(100?M,12h)intervention.The expression levels of reactive oxygen species(ROS),induced nitric oxide synthase(iNOS)and NADPH oxidase-4(NOX4)were increased,while the expression levels of superoxide dismutase-2(SOD-2)were significantly decreased after H2O2 intervention,suggesting that the level of oxidative stress in nucleus pulposus cells was significantly increased.CCK-8 results showed that the concentrations of JWH133 and AM630 within 10?M had no toxicity on NPC(*p>0.05).RT-PCR results showed that JWH133 could reverse the expression change of Collagen ? induced by H2O2 and MMP3.Western Blot results showed that JWH133 had a similar effect on protein expression.Further cellular immunocytochemical assay showed that the expression was consistent with the RT-PCR.Furthermore,JWH133 intervention can obviously relieve after H2O2-induced high-level oxidative stress,reducing the expression of ROS,iNOS and NOX4 mRNA and protein expression,increasing the expression of SOD-2.Quantitative analysis show that the different between JWH133 and H2O2 group was statistically significant(*p<0.05),AM630 was found no significant differences with H2O2 group(*p<0.05).ConclusionActivation of CB2 can improve the degeneration of nucleus pulposus tissues by regulating the oxidative stress.These studies indicate that CB2 may develop into a promising target of IVDD therapy.