Preliminary Study Of Multifidus In Elderly Women Based On Rna-seq Molecular Mechanism Of Lipid Infiltration

Chapter 1 IL-6 is a vital factor of multifidus lipid infiltration in lumbar spinediseasesObjective : Multifidus lipid infiltration is an important influencing factor of ageing-related lumbar degenerative diseases.In this study,we explored the differential gene expression of multifidus lipid infiltration in elderly women by RNA-Seq technique and further screened the key influencing factors by combining with bioinformatics analysis.Methods:Twelve cases of multifidus tissues were collected from October to December2021 in patients who underwent surgical treatment for lumbar spine diseases at the Department of Orthopedics,Gongli Hospital,Pudong New Area,Shanghai,China,and preoperatively analyzed according to MRI imaging measurements,including 6 cases in the multifidus lipid infiltration group and 6 cases in the multifidus normal group,and analyzed by RNA-Seq technology to screen out target genes with significant differences in expression,combined with bioinformatics analysis to screen key genes and related signaling pathways.Finally,immunohistochemistry(IHC)staining and quantitative real-time fluorescence PCR(q PCR)were performed to verify the key genes in the two groups.Results : Based on the results of RNA-Seq analysis,a total of 495 differentially expressed genes were obtained,and the key gene(Interleukin-6)IL-6 was highly expressed in the lipid infiltrated tissues,while bioinformatics analysis predicted that the highly expressed IL-6 may co-regulate with mitogen-activated protein kinases(MAPK)signaling pathway.kinases(MAPK)signaling pathway to regulate lipid infiltration in multifid muscles.The results of immunohistochemical staining and quantitative PCR showed that the expression levels of IL-6 and MAPK were significantly increased(P < 0.05)in the group with lipid infiltration compared to the normal group.Conclusion:The key factors IL-6 and MAPK signaling pathways are closely related to the development of lipid infiltration in multifidus muscle in lumbar spine diseases and may be potential diagnostic and therapeutic targets.Chapter 2: Regulatory mechanisms of IL-6 and MAPK signaling pathways involvedin lipogenesis in human skeletal muscle cellsObjective:A lipogenesis model was constructed by inducing lipogenic differentiation of skeletal muscle cells to explore the regulatory role of IL-6/MAPK signaling pathway involved in skeletal muscle lipogenesis.Methods: Isolated normal multifidus tissue was selected for cell culture and differentiation,and an in vitro model was constructed by co-culture of IL-6 stimulating factor with human skeletal muscle cells,combined with protein blotting(Western bloting,WB)analysis and oil red staining to detect the expression levels of lipid metabolism indexes such as perilipin and adiponectin and MAPK.Subsequently,IL-6 inhibitor(Sarilumab)and MAPK inhibitor(SB203580)were added to verify the regulatory roles of IL-6 and MAPK in cellular lipogenesis.Finally,the effects of IL-6 knockdown on cell viability and apoptosis and the expression levels of lipid metabolism indexes were further assessed according to CCK-8,flow cytometry and PCR assays.Results:IL-6 induced increased lipogenesis in skeletal muscle cells,and the levels of perilipin and MAPK expression were considerably increased in the IL-6-treated group compared with the blank control group(P < 0.05),and the induction of lipogenesis in skeletal muscle cells was apparently attenuated by the addition of Sarilumab.Meanwhile,the levels of perilipin and MAPK expression in cells were clearly reduced after the addition of Sarilumab compared with the IL-6-treated group(P < 0.05).In addition,the expression of perilipin and adiponectin was greatly reduced in cells after the addition of SB203580 compared with the IL-6-treated group(P < 0.05).Finally,cell viability was notably enhanced and apoptosis was enormously reduced after knockdown of IL-6,and the expression of perilipin and adiponectin was hugely upregulated at the m RNA level(P < 0.05).Conclusion:IL-6 may activate the MAPK signaling pathway to promote the regulation of skeletal muscle lipogenesis,and provide a new theoretical basis for the early diagnosis and treatment of lipid infiltration in multifidus.