Study On Digital Gene Expression Profiles And Molecular Networks Of Effective Treatment Of Lumbar Intervertebral Disc Protrusion By Electroacupuncture At

Objectives:1. To find the pathogenesis of lumbar disc herniation (LDH) at the transcriptomics level.2. To explore the mechanism of the efficacy of the electro-acupuncture (EA) deeply inserting the "Five Points for LDH" at the transcriptomics level.Methods:1. Clinical screening of ribonucleic acid sequencing (RNA-seq) samples: ① Groups and treatments:According to the diagnostic criteria, inclusion and exclusion criteria, thirty clinical LDH patients were involved into LDH group. There were six treatment sessions of EA deeply acupuncture treatment of the "Five Points for LDH" which represents BL25, BL26 and Jiaji points of L4, L5, S1 on the affected side:deeply inserting the "Five Points for LDH" by sterile needles (Φ= 0.35mm, length= 75mm), using electric acupuncture apparatus for 25 minutes. The Japanese orthopaedic association assessment (JOA) scores of lumbar were obtained before and after treatment to calculate the improvement rate of the of treatment. The top eight of thirty patients, whose levels were beyond "good", were chosen for sequencing. Also, we formulated the inclusion and the exclusion criteria, and brought in a control group of healthy non-patients of the same ages and genders. ②Sampling:the blood of the peripheral veins were acquired from the LDH group before and after the six treatments, as well as the blood of the peripheral veins from the healthy control group after they were involved. We chose the blood samples of the top eight patients and the healthy control group for sequencing. For each sample of the individuals, the total RNA of the white blood cells were extracted separately, marked as:Group 1 (LDH group before treatments), Group 2 (LDH group after treatments), Group 3 (healthy control group).2. RNA-seq:①Sequencing method:First, we prepared and identified the total RNA of white blood cells. Second, we separated and quantified the mRNA, building a library of the mRNA. Lastly, we operated on the computer. Based on the computer results, we analyzed the digital gene expression profiling (DGE) of all the groups. ② Data analysis:we compared the DGE all the groups, and screened the significant differential genes that reversed the direction in the pathogenesis and in the treatment process; we analyzed those genes using the gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), and ingenuity pathway analysis (IPA) to find the pathogenesis of LDH and the mechanism of the efficacy of the EA deeply inserting the "Five Points for LDH" at the transcriptomics level.3. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR):we verified the results of RNA-seq by qRT-PCR to guarantee the accuracy and reliability of this study. Firstly, the reverse transcription of total RNA was designed, the qPCR primers were designed and tested, and then the qPCR was carried out on the machine, and the data were generated and analyzed.Results:1. Results of the clinical screening:we got the peripheral venous blood samples of the top eight patients and the healthy control group for sequencing. For each sample of the individuals, the total RNA of the white blood cells were extracted separately, marked as: Group 1 (LDH group before treatments), Group 2 (LDH group after treatments), Group 3 (healthy control group).2. Results of RNA-seq:① Differential genes:we compared the data from Group 1 with those from Group 3, obtaining 120 differential genes related to the LDH pathogenesis. Groups 1 and 2 have shown 76 differential genes, seventeen of which overlapped with the 120 genes mentioned before. The seventeen genes have reversed the expression directions, from pathogenesis to healing process. They coincide with the pathogenesis of the LDH and the mechanism of EA deeply inserting the "Five Points for LDH". ② The analytical results of the seventeen genes are as follows:(1) the GO enrichment results mentioned water balance; (2) the KEGG enrichment results did not show relation to any pathways; (3) the IPA enrichment results:A) the pathway enrichment results came up with the IL-17 signal transduction pathways, with CCL2 gene resides in the cell nucleus through it. We suggest that upregulation of CCL2 will cause and aggravate the pain from the LDH, and it can also affect IL-17 signal conductive pathway to aggravate the immune and inflammatory reaction. B) the network enrichment results pointed out a molecular network of inflammatory reactions and immune diseases, in which differential genes CCL2, IFI27 and non-differential genes TNF, IFNG who located in the center interwork with each other. We suggest that upregulation of CCL2 will cause and aggravate the degeneration of the disc, as well as the inflammatory reaction through the network. The express direction of all the differential genes in the pathogenesis of the LDH reversed in the mechanism of the efficacy of EA deeply inserting the "Five Points for LDH".3. Results of qRT-PCR:we used qRT-PCR to test four significant genes carried out by RNA-seq, CCL2, IFI27, TNF and IFNG. The results showed that the expression of CCL2 and IFI27 in three samples was more obvious, and the expression of TNF and IFNG was not obvious, which was the same as that of RNA-seq. This confirms the accuracy and reliability of the study.Conclusions:1. Part of the pathogenesis of the LDH are as follows: ① The water imbalance of lumbar intervertebral disc cells can cause degeneration, which leads to LDH. ② Upregulation of CCL2 gene can cause and aggravate the pain of LDH. ③ Upregulation of CCL2 gene can affect IL-17 signal transduction pathway and exacerbate the inflammatory reaction of the LDH. ④ Upregulation of CCL2 and IFI27 genes can cause and increase the pain of LDH and degeneration and inflammatory reaction through the network related to TNF and IFNG.2. The mechanism of the efficacy of EA deeply inserting the "Five Points for LDH" are as follows: ①EA deeply inserting the "Five Points for LDH" can play a positive role in the process of water transport in the lumbar intervertebral disc, and delay the degeneration. ② EA deeply inserting the "Five Points for LDH" down-regulates the expression of CCL2 gene to relieve the pain of LDH. ③ EA deeply inserting the "Five Points for LDH" down-regulates the expression of CCL2 gene to alter the IL-17 signaling transduction pathway to reduce the immune and inflammatory reaction of the LDH.④EA deeply inserting the "Five Points for LDH" down-regulates the expression of CCL2 and IFI27 genes, which affect the interaction network related to TNF and IFNG gene, to reduce the immune and inflammatory reaction of the LDH and relieve the pain, as well as delay the degeneration.