| Objective:To observe the dynamic expression changes of NgR in injured neurons and the intervention effect of Wenshen Tongdu decoction(Jisuikang)on the spinal cord injury,and explore the mechanism of Wenshen Tongdu decoction to promote spinal cord repair through regulating NgR/RhoA/ROCK signaling pathway.Methods:Part Ⅰ:Soaking time,adding water,decocting time and decocting times were used as influencing factors.HPLC method was used to determine astragaloside Ⅳ and tanshinone ⅡA,and the effect of various factors on the water extraction process of Wenshen Tongdu decoction was analyzed in combination with the extraction rate.Part Ⅱ:Rat neurons were isolated in vitro,purified and cultured,and the nuclear antigen(NeuN)was identified by immunofluorescence,and the neuronal injury model was made by glutamate and identified by TUNEL method.The rats were divided into five groups:1d,3d,7d,14d and control group.NgR and NeuN double staining,Western blot and qPCR were used to observe the expression of NgR in injured neurons.The PC 12 cells model was used to replace neurons and intervene with rat medicated serum(blank control group,prednisone group,and Wenshen Tongdu decoction group)after injuried model prepared,and NgR immunofluorescence staining and qPCR were performed on 1d,3d,and 7d to observe the expression of NgR.Part Ⅲ:The SD rat spinal cord injury model was prepared by modified Allen’s method,and the experiment was divided into model group,prednisone group,Wenshen Tongdu decoction group,and sham operation group.The motor function of rats was evaluated by BBB score and inclined plate test,Golgi staining and transmission electron microscope were used to observe the microstructure of nerve tissue,TUNEL and NeuN fluorescence double labeling method was used to detect neuronal apoptosis.Part Ⅳ:The rat spinal cord injury model was prepared,and the experiment was divided into model group,prednisone group,Wenshen Tongdu decoction group,sham operation group.The expression and localization of local NgR were detected by the fluorescence double-labeling method of NeuN and NgR,and the expression of NgR,RhoA,and ROCK in the injured area were detected by immunohistochemistry,Western blot,and qPCR.Results:Part Ⅰ:The best water extraction technology of Wenshen Tongdu decoction was as follows:soaking time 1 hour,adding 12 times the amount of water,decocting 3 times,1 hour each time.Part Ⅱ:The results of immunofluorescence,Western blot and qPCR showed that the expression of NgR in injured neurons was the highest at 1d,which was significantly different from that in the control group(P<0.05),and decreased gradually at 3d,7d and 14d;The results of immunofluorescence and qPCR showed that the expression of NgR in the PC12 cells of the Wenshen Tongdu decoction group decreased on the 7th day,which was significantly different from that of the blank control group(P<0.05).Part Ⅲ:Compared with the model group at 28 days,both BBB score and inclined plate test showed that Wenshen Tongdu decoction could promote the recovery of motor function in rats with spinal cord injury;Golgi staining and transmission electron microscopy both indicate that Wenshen Tongdu decoction could improve the microstructure of neurons(mitochondria,endoplasmic reticulum,dendritic spines,etc.);TUNEL and NeuN fluorescent double labels indicate that Wenshen Tongdu decoction could reduce the apoptosis rate of neurons in rats with spinal cord injury(P<0.05).Part Ⅳ:NeuN and NgR fluorescence double labeling method showed that Wenshen Tongdu decoction could inhibit the expression of NgR in spinal cord injury rats;Immunohistochemistry,Western blot,and qPCR all show that Wenshen Tongdu decoction could inhibit the expression of NgR,RhoA,ROCK in the injured area of spinal cord injury rats(P<0.05).Conclusion:The best water extraction technology of Wenshen Tongdu decoction was as follows:soaking time 1 hour,adding 12 times the amount of water,decocting 3 times,1 hour each time;Wenshen Tongdu decoction can promote neuronal axon regeneration by inhibiting NgR/RhoA/ROCK pathway,thereby improving Motor function of rats with spinal cord injury. |
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