Study On The Expression And Regulation Of IL-10 And CNTF After Spinal Cord Contusion In Rats

Research background and purposeSpinal cord contusion(SCC)is a kind of complex and serious central nervous system injury.Because central nervous system has poor ability of regenerating,SCC causes a patient’s permanent paralysis,leading to very adverse effect to the individual,family and society.Neurons and neuron axons can hardly be repaired after spinal cord contusion due to the internal mechanism of neurons and the influence of extracellular environmental obstacles.Inflammation and limited regeneration of neurons are important factors affecting spinal cord contusion repair in the process of SCC.Interleukin-10(IL-10)is an important anti-inflammatory factor,which plays a very important role in reducing the inflammatory response,excitotoxicity injury and neuronal apoptosis after spinal cord contusion and IL-10 may be as the potential treatment of gene therapy targeting.Ciliary neurotrophic factor(CNTF)is an important nerve growth factor for repairing spinal cord contusion,which can prevent the degeneration and death of damaged neurons and can protect the damaged neurons,especially the motor neurons.However,whether the inhibition of IL-10 expression after SCC would affect the expression of CNTF and further affect the repair of motor function in the spinal cord contusion was still unclear.Therefore,the purpose of this study was to investigate the correlation between IL-10 and CNTF after SCC and the effect on repair of spinal cord contusion in rats.Materials and MethodsPart 1 The Sprague-Dawley(SD)rats were randomly divided into the spinal cord contusion group and the sham group.The animal models of spinal cord contusion were prepared by Allen’s percussion method,and the motor function of the rats were evaluated by Basso,Beattie,Bresnahan(BBB)locomotor rating scale.The gene and protein localization expression of IL-10 and CNTF in the spinal cord were detected by the real-time quantitative polymerase chain reaction technology(qPCR),the immunofluorescence technology(IF)and the immunohistochemistry technology(IHC).Part 2 The SD rats were randomly divided into the lentivirus inhibition(IL-10-RNAi-LV)group and the negative plasmid control(Vector)group.Lentivirus-mediated gene interference(RNAi)technology was used to inhibit the expression of IL-10 mRNA 48 h before spinal cord contusion.Bioinformatics method GeneMANIA was used to predict the relationship between IL-10 and CNTF.The BBB rating scale was used to evaluate the changes of motor function in rats after IL-10 inhibition.The qPCR and IHC were used to detect the gene and protein localization expression of CNTF in spinal cord after IL-10 inhibition.Results1.Changes in the expression of IL-10 and CNTF1.1 Evaluation of experimental animal models: After SCC,the hind limb of the rats were paralyzed,and the BBB score was 0.After that,it gradually increased,and recovered significantly on the 14 th day,but it could not recover to the score before injury,indicating that the model was successful.1.2 Gene expression of IL-10 to CNTF: The results of qPCR indicated that IL-10 mRNA was significantly up-regulated at 6 h and 12 h(P <0.05),and significantly down-regulated at 7 d and 14 d(P <0.05),compared with the sham operation group.In addition,the expression of CNTF and CNTF receptor(CNTFR)mRNA were also significantly down-regulated in the spinal cord contusion group(P <0.05).1.3 Protein localization of IL-10 and CNTF: IF and IHC results suggested that IL-10 and CNTF were localized at the neurons of the spinal cord anterior horn after spinal cord contusion.2.Regulation mechanism of IL-10 to CNTF2.1 Bioinformatics prediction: GeneMANIA analysis shows that IL-10 has a protein molecule interaction domain with CNTF suggestting that IL-10 and CNTF have similar function.2.2 Effect of inhibiting the expression of IL-10 on motor function after spinal cord contusion in rats: After IL-10 was inhibited,compared with the vector group,BBB score decreased significantly at 5 d and 7 d(P<0.05).Therefore,after inhibiting the expression of IL-10,the motor function of rats after spinal cord contusion reduced.2.3 Effect of inhibiting IL-10 expression on CNTF gene expression: The qPCR results showed that compared with the vector group,the expression of CNTF mRNAwas significantly up-regulated after IL-10 inhibition in the virus group(P <0.05).2.4 Effect of inhibiting IL-10 expression on CNTF protein localization: the results of Immunohistochemistry showed that CNTF was localized in the neurons of the gray matter in the spinal cord.Conclusion1.The IL-10 expression increased in the early stage of SCC significantly,and then decreased gradually.Meanwhile,the expression of CNTF and CNTFR also decreased significantly after spinal cord contusion.The experimental results suggest that the expression of IL-10 and CNTF were involved in the recovery of the hind limb motor function after spinal cord contusion.2.After inhibiting IL-10 expression by lentivirus-mediated RNAi technology,CNTF expression increased significantly.The experimental results suggest that the inhibition of IL-10 leads to the increase of CNTF expression,and CNTF affects the repair of spinal cord contusion through the increased compensation mode,which provides a new perspective of gene-targeted therapy in spinal cord contusion.