ABSTRACT: purpose:The goals of this study is to investigate rat quadriceps muscle responses treated with different catgut embedding acupoint in various molecular pahtways by RNA-seq and real-time quantitative PCR methods and evaluaate protocols for optimal high-throughput data analysis. Methods:mRNA profiles of rat quadriceps muscle responses treated with catgut embedding acupoint 1 and 2 group,treadmill group and control were generated by sequencing, in sextuplicate, utilizing Illumina HiSeq 4000 platform. The raw RNA-seq data was filtered to remove adaptor contamination, low quality bases, and undetermined beses utilizing Cutadapt software. The filtered reads were aligned and mapped to the reference genome using Hisat 2.0. RT-qPCR validation was performed using SYBR green assays. Results:The transcription profiling results showed that there were 346 differentially expressed genes (DEGs) between the A and B groups. These included 221 up-regulated DEGs and 125 down-regulated DEGs. There were 363 DEGs between the B group and the C, which included 144 up-regulated DEGs and 219 down-regulated DEGs. There were 458 DEGs between the B group and D which included 248 up-regulated DEGs and 210 down-regulated DEGs. Through GO functional annotation, the DEGs were divided into three categories: cellular components, biological processes, and molecular functions. The above three functions were further divided into positive regulation of transcription by RNA polymerase II, and RNA polymerase II on transcription. Negative regulation, participation in nuclear synthesis, synthesis with cytoplasm, participation in extracellular exosome synthesis, participation in protein binding, and participation in metal ion binding, etc. After enrichment analysis, 16 metabolic pathways were found to be significantly enriched (p<0.05), which included chemical carcinogenesis, metabolism of cytochrome P450 to exogenous substances, circadian entrainment, circadian rhythm, protein digestion and absorption, PPAR Signaling pathways, drug metabolism - cytochrome P450, parathyroid hormone synthesis, secretion and action, steroid hormone biosynthesis, leishmaniasis, arachidonic acid metabolism, bile secretion, measles, linoleic acid metabolism, fat digestion and absorption, and fluid shear stress and atherosclerosis. By inspecting the bubble enrichment plot, we decided that the following would be important targets of our future studies: protein digestion and absorption; PPAR signaling pathway; synthesis, secretion and action of parathyroid hormone and other metabolic pathways.DEGs that enriche into pathways were verified by RT-qPCR. Conclusions: Our study provides new insights on the interaction between catgut embedding acupoint and rat quadriceps muscle responses.
