| Objective: The aim is to study the effect of Hyperbaric Oxygen Therapy(HBOT)on paclitaxel(PTX)-induced peripheral neuropathic pain(PIPNP),and further relation on Cannabinoid-dependent neuroinflammation.Methods: 125 healthy adult SD rats were divided into 5 groups,which were Control group,Model group(PTX),Treatment group(PTX+HBOT),AM630 group(PTX+HBOT+AM630),and AM251 group(PTX+HBOT+AM251).Except the Control group,the other groups were intraperitoneally injected with paclitaxel 2 mg/kg on the first,third,fifth and seventh days of the experiment,and the total injection dose was up to 8mg/kg.A stable peripheral neuralgia model was established on the 14 th day of the experiment.The Treatment group was treated with HBOT on 2.5ATA for consecutive 7days after modeling.The AM630 and AM251 groups were intraperitoneally injected with Cannabinoid receptor type I antagonist AM251(1 mg/kg)and Cannabinoid receptor type II antagonist AM630(3 mg/kg)half an hour before HBOT.The behavioral tests were conducted using UP and Down method to test the Mechanical Withdraw Threshold(MWT)before,after,during,and within two weeks after treatment to observe the short and long-term effects of HBOT.Another 15 rats in each group were modeled and treated as the same protocol above.After the treatment,the rats in each group were sacrificed to take the L4~L6 of the spinal cord for subsequent molecular biology tests.The expression of cannabinoid receptor type I(CBR1),cannabinoid receptor type II(CBR2),spinal astrocytes(GFAP),and spinal microglia(CD11b)were examined by Western blot.The contents of inflammatory factors IL-1β and TNF-α in the spinal cord were measured using an enzyme-linked immunosorbent assay(Elisa)kit.HBOT was found to have long-term analgesic effects,and closely related to activation of CBR2 and downstream neuroinflammation.Therefore,The m RNA expression levels of CBR2,GFAP,CD11 b,IL-1β and TNF-α were observed by Real-time RT-PCR.Results: Through behavioral tests the MWT of the Treatment group showed an increasing trend during the treatment of HBOT,and were significantly higher than that of the model group since the fifth day of treatment(p<0.05).Although,downward trend of MWT was observed within 2 weeks after the end of treatment,it was still higher than that of the Model group(p <0.05).AM630 reversed this effect,while AM251 did not.Western blot results showed that compared with the model group,the protein levels of CBR2 was significantly increased in the treatment group(p<0.05);the protein levels of CBR1 has no statistical differences(p>0.05);the protein levels of GFAP and CD11 b were significantly decreased(p<0.05).AM630 reversed the changes of protein levels above,while AM251 did not.The results of Elisa showed that compared with the model group,the contents of IL-1β and TNF-α in the treatment group decreased significantly(p<0.05).AM630 reversed the content changes of inflammatory cytokines,while AM251 did not.Real-time RT-PCR results showed that compared with the model group,the m RNA level of CBR2 in the treatment group was significantly increased(p<0.05),and the m RNA levels of IL-1β and TNF-α were significantly decreased(p<0.05);Compared with the control group,the m RNA levels of GFAP and CD11 b in the model group were slightly increased(p=0.049,p=0.040).There were no statistical differences as size of test is 0.01 in RT-PCR.The m RNA level changes of CBR2,IL-1β,and TNF-α were reversed by AM630,while were not by AM251.Conclusion: HBOT can alleviate PIPNP and has long-term analgesic effects via regulation of CBR2-dependent neuroinflammation. |
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