| Objective: To observe the therapeutic effect of Atractylenolide Ⅲ（ATL-Ⅲ）on spinal cord injury（SCI）in rats,and to confirm the neuroprotective effect of ATL-Ⅲ on SCI.To explore the protective mechanism of ATL-Ⅲ on SCI,so as to provide theoretical and experimental basis for the application o f ATL-Ⅲ in the treatment of central nervous system diseases such as SCI in the future.Methods:（1）SD rats were randomly divided into sham,control and ATL-Ⅲ group.The SCI model was constructed by PSI-IH impactor.（2）At different time points post SCI,the motor function was evaluated by Basso,Beattie and Bresnahan（BBB）motor rating scale,grid walking test and footprint analysis experiment.（3）The rats were perfused on the 42 days after SCI.The spinal cord lesion area and myelin retention area were measured by specific staining,such as hematoxylin eosin（HE）staining and Luxol fast blue（LBF）staining.The survival of neuron was detected by N issl toluidine blue staining.（4）Immunohistofluorescence（IF）and flow cytometry（FCM）were used to detect the activation and polarization of microglia/macrophages on the 7 days post injury.（5）The BV2 microglial cell line stimulated by lipopolysaccharide（LPS）was used to construct the neuroinflammatory model in vitro.It was divided into control,LPS,ATL-Ⅲ（1μM）,ATL-Ⅲ（10μM）and ATL-Ⅲ（100μM）group.（6）CCK-8 kit was used to detect the effects of ATL-Ⅲ on the viability of BV2 microglia.（7）On the 7days after SCI and 24 h after LPS stimulation,Western blot was used to detect the expressions of M1 polarization-related factors i NOS,TNF-α,IL-1β,and IL-6,and M2polarization-related factors Arg-1 and IL-10,and the phosphorylation of NF-κB,MAPK,PI3K/Akt signaling pathways both in vivo and vitro.Results:（1）BBB score showed that ATL-Ⅲ could significantly improve motor function score at 14-42 days post injury on rats（P<0.05）;At 28,35 and 42 days after injury,the grid walking test error rate in ATL-Ⅲ treatment group was significantly lower than that in the control group（P<0.01）;Footprint analysis showed that the score of ATL-Ⅲ treatment group on the 42 day was higher than that of the control group（P<0.05）.（2）The results of HE staining and LFB staining showed that compared with the control rats,the rats in ATL-Ⅲ administration group had significantly smaller lesion areas and larger LFB positive areas at the epicenter（0）and the lateral（+）and tail（-）sides 1mm away from the epicenter（P<0.05）;Nissl staining showed that the number of residual motoneurons at the lateral（+）and tail（-）sides 3mm and 4mm away from the epicenter in ATL-Ⅲ treatment group was more than that in the control group（P<0.05）.（3）IF and FCM showed that microglia/macrophages were activated and the number of cells increased significantly after SCI,while ATL-Ⅲ administration could inhibit the number and proportion of activated microglia/macrophages（P<0.05）.ATL-Ⅲ administration can inhibit the transformation of microglia into M1 phenotype and enhance the transformation of microglia into M2 phenotype,so as to regulate the polarization of microglia/macrophages M1/M2（P<0.05）.（4）The results of WB showed that the protein expression of M1 microglia/macrophages representative factors i NOS,TNF-α,IL-1β,and IL-6 in ATL-Ⅲ treatment group was significantly lower than that in the control group（P<0.05）;The protein expressions of M2 microglia Arg-1 and IL-10 was significantly higher than those in the control group（P<0.05）.It was also found that ATL-Ⅲ could reduce the phosphorylation of p65,IκBα in NF-κB signaling pathway（P<0.05）,the phosphorylation of JNK and p38 in MAPK signaling pathway（P<0.05）and significantly increased the phosphorylation of Akt signaling pathway.Conclusions: ATL-Ⅲ promotes the histological repair and motor function recovery of SCI rats,promotes the survival of neuron and has neuroprotective effect.ATL-Ⅲ inhibits the polarization of microglia/macrophages to M1 and promotes the polarization to M2,so as to play an anti-inflammatory role,which may be one of the key mechanisms for ATL-Ⅲ to play a neuroprotective role. |
PDF Full Text Request