UNASSIGNED: MV modeling was performed on C57 mice and DB mice, and the control group was set up in each group. After 12 h of mechanical ventilation, the muscle strength of the diaphragm was measured, and the muscle fiber immunofluorescence staining was used to verify the successful establishment of the MV model. RNA sequencing (RNA-seq) method was used to detect mRNA expression levels of the diaphragms of each group, and then differential expressed gene analysis, Heatmap analysis, WGCNA analysis, Venn analysis, GO and KEGG enrichment analysis were performed. qRT-PCR was used to verify the expression of the selected mRNAs.
UNASSIGNED: Our results showed that, compared with C57 control mice, the muscle strength and muscle fiber cross-sectional area of mice after mechanical ventilation decreased, and DB mice showed more obvious in this respect. RNA-seq showed that these differential expressed (DE) mRNAs were mainly related to genes such as extracellular matrix, collagen, elastic fiber and Fbxo32. GO and KEGG enrichment analysis showed that the signaling pathways associated with diabetes were mainly as follows: extracellular matrix (ECM), protein digestion and absorption, PI3K-Akt signaling pathway, calcium signaling pathway, MAPK signaling pathway and AGE-RAGE signaling pathway in diabetic complications, etc. ECM has the closest relationship with VIDD in diabetic mice. The key genes determined by WGCNA and Venn analysis were validated by quantitative real-time polymerase chain reaction (qRT-PCR), which exhibited trends similar to those observed by RNA-seq.
UNASSIGNED: VIDD can be aggravated in diabetic environment. This study provides new evidence for mRNA changes after mechanical ventilation in diabetic mice, suggesting that ECM and collagen may play an important role in the pathophysiological mechanism and progression of VIDD in diabetic mice, and provides some clues for the research, diagnosis, and treatment of VIDD in diabetic context.
■对C57小鼠和DB小鼠进行MV建模,每组设对照组。机械通气12小时后,测量了隔膜的肌肉力量,肌纤维免疫荧光染色验证MV模型的成功建立。RNA测序(RNA-seq)法检测各组膈肌mRNA表达水平,然后进行差异表达基因分析,热图分析,WGCNA分析,维恩分析,进行GO和KEGG富集分析。使用qRT-PCR来验证所选择的mRNA的表达。
■我们的结果表明,与C57对照小鼠相比,机械通气后小鼠肌力和肌纤维横截面积下降,DB小鼠在这方面表现得更明显。RNA-seq表明这些差异表达(DE)mRNA主要与细胞外基质等基因相关,胶原蛋白,弹性纤维和Fbxo32。GO和KEGG富集分析表明,与糖尿病相关的信号通路主要如下:细胞外基质(ECM)、蛋白质消化吸收,PI3K-Akt信号通路,钙信号通路,MAPK信号通路和AGE-RAGE信号通路在糖尿病并发症中的作用,等。ECM与糖尿病小鼠的VIDD关系最密切。WGCNA和Venn分析确定的关键基因通过定量实时聚合酶链反应(qRT-PCR)进行验证,其表现出与RNA-seq观察到的趋势相似。
■VIDD可在糖尿病环境中加重。本研究为糖尿病小鼠机械通气后mRNA的变化提供了新的证据,提示ECM和胶原可能在糖尿病小鼠VIDD的病理生理机制和进展中起重要作用,并为研究提供了一些线索,诊断,和糖尿病背景下VIDD的治疗。