| Background and purpose of the study:Spinal Cord Injury(SCI)is a serious disease of the central nervous system.SCI is mainly divided into two stages:primary and secondary injury,and inflammation is the key link in secondary injury.In a previous study,Iridoids Rich Fraction Valeriana(IRFV)were found to reduce the inflammatory response in rats with spinal cord injury,and their specific mechanisms of action need to be further explored.Microglia are resident immune cells in the CNS,which can be activated and polarized into two cell subtypes,namely pro-inflammatory M1 type and anti-inflammatory M2 type.In summary,whether IRFV attenuates the inflammatory response in SCI rats by affecting the polarization of microglia(reducing pro-inflammatory M1 type and promoting anti-inflammatory M2 type)deserves our further investigation.In the present study,we established a rat T10 spinal cord injury model with IRFV intervention,and observed the effect of the drug on microglia polarization by WB,fluorescent staining,and PCR,as well as ELISA to detect inflammation,to investigate whether IRFV attenuates the level of inflammation in SCI rats by affecting the polarization of microglia to play a role in improving the spinal cord of rats for IRFV injury pathology and motor function in rats to provide more basic theoretical support.Research methods:Fifty-four SD male rats were selected and randomly divided into sham-operated group,model group,and IRFV-treated group,with 18 rats in each group.Thoracic 10 spinal cord injury models were prepared for the model and treatment groups.Blood and spinal cord tissue specimens were collected after 7 days of intervention to observe the serum inflammatory changes and microglia-related protein expression in each group,and the motor function of lower limbs in each group was evaluated at 1d,7d,14d and 28d after modeling.Results of the study:1、Rat hind limb motor functionThe behavioral assessment of BBB suggested that:the model group was significantly lower than the sham-operated group at all time points,and the difference was statistically significant(P<0.05).The BBB scores of rats in the IRFV-treated group increased compared with the model group at 7 and 14 days after surgery,and the difference was not statistically significant(P>0.05),and at 28 days after surgery,the scores increased significantly(P<0.05).The results of the oblique plate experiment showed that the angle of the model group was significantly lower than that of the sham-operated group at all time points,and the difference was statistically significant(P<0.05);the angle values of the IRFV treatment group were slightly higher than those of the model group,and the difference was not statistically significant(P>0.05).2.Spinal cord histopathologyThe results of HE staining showed that the SCI model group had significantly damaged spinal cord tissue structure,disorganized cell arrangement and unclear gray-white matter demarcation compared with the sham-operated group,while the IRFV treatment group had significantly relieved spinal cord tissue lesions,restored structural integrity,clear gray-white matter demarcation and clearer neuronal cell structure compared with the model group.The percentage of cavity area of spinal cord tissue in the model group was significantly increased compared with the sham operation(P<0.001),and the percentage of cavity area in the IRFV-treated group was significantly reduced compared with the model group(P<0.01).3,InflammationELISA results suggested that serum IL-1β level was significantly increased(P<0.05)and IL-4 level was increased without statistical significance(P>0.05)in SCI model group compared with sham surgery;serum IL-1β level was significantly decreased(P<0.05)and IL-4 level was significantly increased(P<0.05)in IRFV treatment group compared with model group.4.Phenotypic polarization of microgliaImmunofluorescence staining suggested that:compared with the sham-operated group,the positive rate of microglia in the model group increased,with M1-type(iNOS)microglia predominating;the proportion of M1-type microglia in the IRFV-treated group significantly decreased(P<0.01)and the proportion of M2-type microglia significantly increased(P<0.01).The WB test results suggested that the expression of iNOS and CD86 proteins came significantly increased in the model group rats compared with the sham-operated group(P<0.01,P<0.05),and the expression of CD206 and Arg-1 proteins increased slightly,the difference was not statistically significant;the expression of iNOS and CD86 proteins decreased significantly in the IRFV-treated group compared with the model group(P<0.05),and the expression of CD206 and Arg-1 proteins decreased significantly in the IRFV-treated group compared with the model group(P<0.05).CD206,Arg-1 protein expression significantly increased(P<0.05),which was consistent with the immunofluorescence results;The PCR results suggested that the relative expression of iNOS and CD86 mRNA was significantly higher(P<0.001,P<0.01)and Arg-1 mRNA was higher(P<0.05)in the spinal cord tissue of rats in the model group compared with the sham-operated group;compared with the model group,the relative expression of CD86 and iNOS mRNA in the IRFV-treated group was significantly lower than that in the model group(P<0.05),and the relative expression of CD206 and Arg-1 mRNA was significantly higher in the IRFV-treated group(P<0.01,P<0.05).Conclusion:IRFV may play a role in reducing the inflammatory response and improving spinal cord pathological injury and motor function in SCI rats by reducing the polarization of M1-type microglia and promoting the polarization of M2-type microglia. |
PDF Full Text Request