Decoction mediates spinal cord repair in vitro and in vivo through the PI3K-AKT signaling pathway

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to find the potential molecular mechanism of traditional Chinese medicine in the treatment of spinal cord injury Methods: Total RNA was isolated and purified using TRIzol reagent following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 . The RNA integrity was assessed by Bioanalyzer 2100 with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module under 94℃ 5-7min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase , which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP Solution . An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 following the vendor's recommended protocol. Results:In this study, we obtained: chromosome: 23, Genes (G): 32623, Transcripts (T): 40808, GO Annoated: 20795, KEGG Annoated: 8299; in further pathway analysis, it was shown that PI3K-AKT signaling pathway may be the underlying molecular mechanism. Conclusions: Our study represents the first detailed analysis of the spinal cord transcriptome generated by RNA-seq technology with biological replicates. Our results suggest that Modified Erxian decoction may mediate spinal cord repair through the PI3K-AKT pathway. Spinal Cord Injury in Wistar Rats and Intervention Treatment with Modified Erxian Decoction, RNA-Seq Analysis of Spinal Cord Tissue, The experiment was divided into treatment group (T_Spinal group, MED treatment after injuring the spinal cord), control group (C_Spinal group, distilled water after injuring the spinal cord) and sham group (sham group, only the lamina was opened without injuring the spinal cord), and all experiments were repeated three times.