Neuroprotective Potential Of Gentongping In Rat Model Of Cervical Spondylotic Radiculopathy Targeting Ppar-γ Pathway

Objective:The rat model of cervical spondylotic radiculopathy(CSR)was induced by spinal cord injury(SCI)and the effect of peroxisome proliferator-activated receptor(PPAR-γ)as the active target protein of GTP was evaluated.The behavior,nerve electrophysiology,histopathology,inflammatory response,apoptosis and the cytoskeletal integrity of the spinal cord were studied.The aim of this study was to explore the pharmacological mechanisms of GTP on CSR,so that it could provide basis for clinical treatment.Methods:1.Model establishment and interventions: The non-toxic and stable 0.5mm diameter nylon fishline was inserted into the SD rat at C4~T1 position and established the animal model of cervical spondylosis radiculopothy.After the model was completed successfully,the intervention on each group of animals was implemented.The normal group,the sham operation group and the model group were given saline,the positive group was given fenbid at 4mg/mL,and the GTP group was given GTP granule at 0.32g/mL.The treatment was given according to the weight of 1mL/100 g dose,once a day,for 5 weeks.2.The spontaneous pain and gait scores of all the rats were evaluated once a week for 5 weeks.The detection of median nerve somatosensory evoked potential(SEP)was implemented before the surgery,after the surgery,and after 5 weeks of drug administration.And the amplitude(Amp)was detected for electrophysiological detection of all the rats in the group.Hematoxylin-eosin(HE)staining was used for the observation of the pathological changes of the motoneurons on the paraffin sections of the spinal cord and peripheral nerve tissues.Western blot ananysis was used to detect the protein expression levels of PPAR-γ,TNF-α,Bcl-2,Bax and NF200 in spinal cord neurons.Immunofluorescence assay was used to detect the expression level of TNF-α protein in spinal cord neurons.TUNEL assay was used to detect the apoptosis of neurons in the spinal cord.The expression of TNF-α cytokine in serum was detected by ELISA,which was as a supplementary experiment.3.GraphPad Prism 7 was used for statistical processing of the results.Results:After 5 weeks of administration,there was a significant difference between the model group and the fenbid group as well as the GTP group(P<0.05).The spontaneous pain and the gait score in the GTP were significantly improved in the CSR rats.There was significant difference in the Amplitude value of the operation rats and the results of the model group was obviously lower than the GTP group(P<0.05).HE staining showed motoneurons nuclei and cytoplasm dyeing lightly compared with normal group,the number of neurons significantly less than the normal group,presented as a kind of irregular form.In the GTP group,motoneurons nuclei and cytoplasm staining were significantly deeper than the model group,the number of neurons increased obviously,there were significant differences(P<0.05),the cell morphology also tended to the normal group.Western blot analysis showed that after the SCI operation,the protein level of PPAR-γ of the spinal cord in model group was decreased compared to normal group,in the GTP group the expression was increased compared to model group.The levels of Bcl-2 and NF200 in GTP group were significantly higher than that of model group,the expressions of TNF-α and Bax were significantly lower than the model group.Immunofluorescence experiment exhibited that the level of TNF-α in GTP group was significantly lower than that in the model group,and the GFAP expression was significantly higher than model group.TUNEL assay showed that the number of apoptosis in the GTP group was significantly lower than that in the model group.ELISA showed that the expression of TNF-α was significantly lower than that of the model group,this was consistent with the Western blot analysis and Immunofluorescence experiment of TNF-α.All the differences were statistically significant(P<0.05).Conclusion:These findings demonstrated that GTP could promote the expressions of PPAR-γ and some other related cytokines in the spinal cord,probably by regulating PPAR-γ related signaling pathways,by suppressing inflammation response,apoptosis,as well as by protecting the cytoskeletal integrity of the spinal cord,all of which ultimately play a neuroprotective role in CSR.