Bioinformatics Analysis And Expression Verification For The New Pathogenic Genes Related To Chronic Pain

Objective:Using bioinformatics technology to mine the new pathogenic genes and analyze the expression profile of differential expressed genes. Besides, to observe the time-dependent variation tendency of pain behavior and investigate the mRNA and protein expression level as well as the distribution in different DRG neuron subtypes, and to verify the possibility of the gene mining method based on bioinformatics technology and the potential effect of mined new pathogenic gene in the neuropathic pain.Methods:1. The microarray data was downloaded from online database GEO. The data were normalized, filtered and compared by BRB ArrayTools. In order to make sure that the expression of obtained new pathogenic genes was not reported in the L4and L5DRG of SNL model, the differential genes were detected in data mining platforms and compared with the existing Meta analysis research. Meanwhile, DAVID6.7database was applied for the analysis of differential expressed genes and related physiopathologic change in uninjured DRG.2.65adult male SD rats were randomly divided into3groups: Normal group (n=15), Sham group (n=25) and SNL group (n=25). For the rats of SNL group, left L5spinal nerve was ligated, while L5spinal nerve was only exposed without ligation for the rats of sham group, and the normal group was not treated by any surgery. The mechanical threshold values were measured before the surgery and1,4,7,10,13,16,19,22,25and28days after SNL surgery. The rats were scarified respectively at3,7,14,21and28days after SNL surgery, and the L4and L5DRG at both sides were obtained to verify the expression level of new pathogenic genes by RT-PCR and Western Blot experiments.3.36adult male SD rats were randomly divided into3groups: Normal group (n=9), Sham group (n=9) and SNL group (n=18). For the rats of SNL group, the left L5spinal nerve was ligated, while L5spinal nerve was only exposed without ligation for the rats of sham group and the normal group was not treated by any surgeries. The mechanical threshold values were measured1day before the surgery and1,3,5,7,9,11and14days after SNL surgery. The rats were then perfused and scarified respectively at3,7and14days after SNL surgery, and the L4and L5DRG at the operative side were obtained to verify the distribution of new pathogenic gene in different DRG neuron subtypes by immunofluorescence technology.Results:1.123genes which expressed differentially in L4and L5DRG were obtained by bioinformatics technology. After further literature mining,34genes were related with the neuropathic pain, and expressions of20genes in them were not reported. NSF has the strong and correlational evidence with neuropathic pain based on the exsited Meta analysis. Besides, the differentially expressed genes induced the change of ion channel, ion transportation, neuron damage, immune response and vesicle transportation in the uninjured L4DRG.2. The50%mechanical paw withdrawal threshold (50%MWT) values were not significantly different among3groups before SNL surgery (P>0.05). After the SNL surgery,50%MWT values of SNL group rats decreased significantly compared with Normal and Sham group (P<0.05). Meanwhile, some behavior changes such as everted foot and closed toes were observed on the rats of SNL group.3. Through the analysis of RT-PCR and Western Blot, the mRNA and protein expression level of NSF decreased significantly in ipsilateral L4and L5DRG of SNL rats while compared with the DRGs in contralateral sides, Normal and Sham group.4. In the normal rats, the NSF was distributed widely in NF200-, IB4-and CGRP-positive DRG neurons. And most of the NSF positive neurons were small and medium size neurons. In the SNL rats, the NSF positive rates in IB4and CGRP positive neurons decreased significantly (P<0.05), and the area distribution of NSF positive neurons moved to the large and medium size. Conclusions:1. The bioimformatics technology can effectively mine the potential pathogenic genes of neuropathic pain, which can be verified by further molecular biology and histology experiments.2. The significant decreases of NSF mRNA and protein levels in L4and L5DRG of SNL rats as well as the persistent pain-related behaviors were observed, which can indicate that the expression change of NSF plays a role in the neuropathic pain.3. The wide distribution of NSF in normal DRG neurons implies that NSF can regulate the transmission of both innocuous and noxious sensory information. After SNL surgery, the area of NSF positive neurons were changed, and the positive rates in IB4and CGRP positive neurons were deceased, which implies a more important role of the NSF expression in IB4and CGRP positive neurons.