| ObjectiveTo provide basis for the clinical and laboratory study of Bushen Huoxue Decoction treating patient with chronic non-myelopathic cervical spinal cord compression(CNMCSCC)and to find the prognostic factors for this disease,we observed the characteristics of the non-myelopathic symptoms of these patients,and analysed the symptoms based on the Traditional Chinese Medicine theory.After establishing the CNMCSCC model in rats and verificating it prelimitively,we detected the inflammation in the compressed spinal cord,and then we intended find whether using Bushen Huoxue Decoction or inhibiting the NF-kappa B signal pathway could downregulate the inflammation.So that the further research targeting the pathogenesis and treatment of cervical spinal cord compression can be benefited.Methods:85 patients with the CNMCSCC were enrolled in the outpatient department of Guangdong Province Traditional Chinese Medical Hospital from May 2013 to October 2014.The last follow-up was completed in February 2017.The patients were divided into low group(less than 2)and high group(greater than or equal to 2)according to the difference of cervical Japanese Orthopaedic Association(JOA)scores assessing at the beginning and the last follow-up.Sagittal cervial spinal cord compression ratio on MRI,the detailed localization of neck or upper arm pain(occipital,posterior neck,shoulder,elbow,part between the scapulas,upper arm),different types of pain(tingling,soreness,distending pain),muscle tension,range of cervical motion and the cervical instablity were observed between two groups.In the CNMCSCC rat model study,Thirty 300g-350g Sprague Dawley(SD)male rats were randomly assigned into control(sham operation)group and model group equally.Basso Beattie Bresnahan(BBB)scores were used to assess the neurological function of rats.The rats with damaged neurological function(BBB scores less than 19)were removed.Morphological changes of spinal cord were observed by hematoxylin-eosin(HE)staining.After 1 week and 1 month of modeling,we used magnetic resonance to determine whether there might be inflammatory lesions around or in the compression part.Drawing materials was carried out after 1 month after modeling.In the inflammatory mechanism study,SD rats were randomly divided into control(sham operation)group and model group equally with each group of 20 rats.Rats in both group were sacrificed for research at 1 month after modeling,respectively.In the study about the effect of Bushen Huoxue Decoction and the NF-kappa B signaling pathway on inflammation in CNMCSCC rats,the rats were divided into BSHX group(Bushen Huoxue Decoction gavage,BSHX),Inhibitor group(intraperitoneal injection of Ammonium pyrrolidinedithiocarbamate,PDTC),BSHX combing Inhibitor group(BSHX gavage and intraperitoneal injection of PDTC)and model group with 20 rats in each group.All rats were obtained after 1 month of modeling.The mRNA of p65,TNF-alpha,IL-1 beta,IL-6 in the spinal cord were tested by polymerase chain reaction(PCR).The protein expression of phosphorylated-p65(pp65)and TNF-alpha were detected by Western Blot(WB).Enzyme-linked immunosorbent assay was used to determine IL-6,IL-1 beta content in spinal cord.Neural cells(Neun marked),activated microglia(CD68 marked),activated NF-kappa B cells(p65 marked)were labeled by Immunohistochemistry(IHC),and the number of positive cells were counted.Continuous assessment(BBB scores)of rats with neurological function and neurological impairment was carried out in rats.Fingerprint and chromatographic analysis were applied to find out main components of Bushen Huoxue prescription.Results:The clinical observation results showed that more patients in high group were presented with tingling pain.(P<0.05)Cervical instability might be related to the decrease of cervical JOA score(-0.26).There was no significant difference of BBB score in rats with cervical spinal cord compression and control rats.MRI showed that the cervical spinal cord was compressed mildly in the CNMCSCC rats and no edema around compressing site at 1 week and 1 month after modeling.HE staining showed that the neurons and glial cells in the two groups were not different in distribution and morphology at 1 week and 1 month after modeling.In the inflammatory mechanism study,the mRNA expression of p65,IL-1 beta,IL-6 and TNF-alpha in 1 month rised significantly in the model group compared to control group.Western blot showed that the pp65 and TNF-alpha in the model group were higher than that in the control group.IHC found that the number of positive cells marked by p65 or CD68 increased significantly,but no difference in the number of neurons.The IL-6 and IL-1 beta in the CNMCSCC rats enhanced significantly at each time point by using ELISA method.The BBB score was no significant difference between the two groups in the 1 months after modeling.In the study about the effect of Bushen Huoxue Decoction and the NF-kappa B signaling pathway on inflammation in the CNMCSCC rats,mRNA expression of p65 increased in model group compared with BSHX group,Inhibitor group and BSHX combining Inhibitor group.In addition to the mRNA expression of IL-6 group having no significant difference between the inhibitor group and model group,mRNA expression of IL-6,TNF-alpha and IL-1 beta was lower than that in the model group significantly.The mRNA of TNF-alpha in BSHX combining Inhibitor group lowered significantly than that in the BSHX group and inhibitor group.Western Blot showed that the pp65 was reduced in BSHX combing Inhibitor group compared to other groups.In contrast with the model group,pp65 was not significantly decreased in the BSHX group or the Inhibitor group.The expression of TNF-alpha in model group was higher than that in other groups.Besides that,the TNF-alpha of BSHX group and Inhibitor group was higher than that of BSHX combing Inhibitor group.(P<0.05)Immunohistochemistry(IHC)found that the number of CD68 positive cells was higher in the model group than that in other groups(P<0.05),and the number of CD68 positive cells in Inhibitor group was higher in contrast with BSHX combing Inhibitor group.The number of p65 positive cells was significantly higher in the model group than that in other groups.No difference in the number of neurons had been observed.ELISA showed that the IL-6 and IL-1 beta in the model group increased significantly compared to other groups.Further more,IL-6 and IL-1 beta in the Inhibitor group and BSHX group were significantly advanced than that in BSHX combing Inhibitor group.Main content of Bushen Huoxue Decoction included Curculigo glycosides,epimedium glycoside,tanshinone ?A,monoester aconite alkaloids.Conclusion:1 In the CNMCSCC patients presented with tingling pain,or cervical instability on MRI,the long-term cervical JOA score decreased rate may be greater.Besides that cervical instability might be related to the decrease of cervical JOA score.(r=-0.264)2 There is an inflammatory response in the compressed spinal cord of the CNMCSCC rat model,and being regulated by activation of the NF-kappa B pathway.3 Bu Shen Huo Xue Decoction has inhibitory effect on spinal cord inflammation,and same as inhibiting the NF-kappa B signal pathway or combination of them.But it is not comfirmed Bu Shen Huo Xue Decoction downregulated the inflammatory raction by inhibiting NF-kappa B signal pathway.4 Bu Shen Huo Xue Decoction is benefical for treating patient with CNMCSCC. |
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