目的:探讨靶向抑制3型脱碘酶(Dio3)对脓毒症骨骼肌线粒体的保护作用及其机制。
方法:(1)体内实验:采用腺相关病毒(AAV)特异性靶向Dio3在大鼠胫骨前肌的表达,并使用盲肠结扎和穿孔(CLP)制作脓毒症大鼠模型。雄性SD大鼠分为shNC+Sham组,shD3+Sham组,shNC+CLP组,shD3+CLP组采用随机数字表法,每组8只大鼠。CLP建模后,收集胫骨样本并进行蛋白质印迹分析以评估Dio3,过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC1α)的蛋白质水平,和沉默调节蛋白1(SIRT1)。实时荧光定量聚合酶链反应(RT-qPCR)用于检测包括甲状腺激素受体(THRα,THRβ),单羧酸转运蛋白10(MCT10),线粒体DNA(mtDNA),和PGC1α。采用透射电子显微镜研究线粒体形态。(2)体外实验:涉及培养C2C12成肌细胞,使用慢病毒干扰Dio3表达,用脂多糖(LPS)处理细胞,构建内毒素细胞模型。C2C12细胞分为shNC组,shD3组,shNC+LPS组,和shD3+LPS组。进行免疫荧光共定位分析以确定PGC1α的细胞内分布。进行联合免疫共沉淀测定和Western印迹以评估PGC1α的乙酰化水平。
结果:(1)体内实验:与shNC+Sham组相比,shNC+CLP组骨骼肌中Dio3蛋白的表达明显增加(Dio3/β-微管蛋白:3.32±0.70vs.1.00±0.49,P<0.05),然而,shD3+Sham组差别无统计学意义。相对于shNC+CLP组,shD3+CLP组中的Dio3表达显着降低(Dio3/β-微管蛋白:1.42±0.54vs.3.32±0.70,P<0.05)。与shNC+CLP组相比,shD3+CLP组T3调节基因表达恢复[THRαmRNA(2-ΔΔCt):0.67±0.05vs.0.33±0.01,THRβmRNA(2-ΔΔCt):0.94±0.05vs.0.67±0.02,MCT10mRNA(2-ΔΔCt):0.65±0.03vs.0.57±0.02,均P<0.05]。电镜形态学分析提示shNC+CLP组骨骼肌线粒体损伤明显,而shD3+CLP组表现出明显的改善。与shNC+Sham组相比,shNC+CLP组显着减少线粒体数量(细胞/HP:10.375±1.375vs.13.750±2.063,P<0.05),而shD3+CLP组与shNC+CLP组相比,线粒体数量显著增加(细胞/HP:11.250±2.063vs.10.375±1.375,P<0.05)。与shNC+Sham组相比,shNC+CLP组mtDNA的表达明显减少(拷贝:0.842±0.035vs.1.002±0.064,P<0.05)。尽管shD3+CLP组和shNC+CLP组之间的mtDNA表达没有检测到差异,但与shD3+Sham组相比,发现显着增加(拷贝:0.758±0.035vs.0.474±0.050,P<0.05)。在shD3+CLP组中,相对于shNCCLP组,PGC1α的表达在转录和蛋白质水平上均显着提高[PGC1αmRNA(2-ΔΔCt):1.49±0.13vs.0.68±0.06,PGC1α/β-微管蛋白:0.76±0.02vs.0.62±0.04,均P<0.05]。(2)体外实验:LPS处理C2C12细胞24小时后,PGC1α的细胞定位变得弥散;干扰Dio3表达促进了PGC1α向核周区域和细胞核的易位。此外,shD3+LPS组的乙酰化PGC1α水平明显低于shNC+LPS组(乙酰化PGC1α/β-微管蛋白:0.59±0.01vs.1.24±0.01,P<0.05),而去乙酰化剂SIRT1的表达在Dio3抑制后显著升高(SIRT1/β-微管蛋白:1.04±0.04vs.0.58±0.03,P<0.05)。当使用EX527抑制SIRT1活性时,与shD3LPS组相比,PGC1α蛋白表达显着降低(PGC1α/β-微管蛋白:0.92±0.03vs.1.58±0.03,P<0.05)。
结论:抑制骨骼肌中的Dio3通过激活SIRT1减少PGC1α的乙酰化,促进PGC1α的核转位,从而提供针对脓毒症诱导的骨骼肌线粒体损伤的保护。
OBJECTIVE: To investigate the protective effects and mechanisms of targeted inhibition of type 3 deiodinase (Dio3) on skeletal muscle mitochondria in sepsis.
METHODS: (1) In vivo experiments: adeno-associated virus (AAV) was employed to specifically target Dio3 expression in the anterior tibial muscle of rats, and a septic rat model was generated using cecal ligation and puncture (CLP). The male Sprague-Dawley (SD) rats were divided into shNC+Sham group, shD3+Sham group, shNC+CLP group, and shD3+CLP group by random number table method, with 8 rats in each group. After CLP modeling, tibial samples were collected and Western blotting analysis was conducted to assess the protein levels of Dio3, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), and silence-regulatory protein 1 (SIRT1). Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was utilized to examine mRNA expression of genes including thyroid hormone receptors (THRα, THRβ), monocarboxylate transporter 10 (MCT10), mitochondrial DNA (mtDNA), and PGC1α. Transmission electron microscopy was employed to investigate mitochondrial morphology. (2) In vitro experiments: involved culturing C2C12 myoblasts, interfering with Dio3 expression using lentivirus, and constructing an endotoxin cell model by treating cells with lipopolysaccharide (LPS). C2C12 cells were divided into shNC group, shD3 group, shNC+LPS group, and shD3+LPS group. Immunofluorescence colocalization analysis was performed to determine the intracellular distribution of PGC1α. Co-immunoprecipitation assay coupled with Western blotting was carried out to evaluate the acetylation level of PGC1α.
RESULTS: (1) In vivo experiments: compared with the shNC+Sham group, the expression of Dio3 protein in skeletal muscle of the shNC+CLP group was significantly increased (Dio3/β-Tubulin: 3.32±0.70 vs. 1.00±0.49, P < 0.05), however, there was no significant difference in the shD3+Sham group. Dio3 expression in the shD3+CLP group was markedly reduced relative to the shNC+CLP group (Dio3/β-Tubulin: 1.42±0.54 vs. 3.32±0.70, P < 0.05). Compared with the shNC+CLP group, the expression of T3-regulated genes in the shD3+CLP group were restored [THRα mRNA (2-ΔΔCt): 0.67±0.05 vs. 0.33±0.01, THRβ mRNA (2-ΔΔCt): 0.94±0.05 vs. 0.67±0.02, MCT10 mRNA (2-ΔΔCt): 0.65±0.03 vs. 0.57±0.02, all P < 0.05]. Morphology analysis by electron microscopy suggested prominent mitochondrial damage in the skeletal muscle of the shNC+CLP group, while the shD3+CLP group exhibited a marked improvement. Compared with the shNC+Sham group, the shNC+CLP group significantly reduced the number of mitochondria (cells/HP: 10.375±1.375 vs. 13.750±2.063, P < 0.05), while the shD3+CLP group significantly increased the number of mitochondria compared to the shNC+CLP group (cells/HP: 11.250±2.063 vs. 10.375±1.375, P < 0.05). The expression of mtDNA in shNC+CLP group was markedly reduced compared with shNC+Sham group (copies: 0.842±0.035 vs. 1.002±0.064, P < 0.05). Although no difference was detected in the mtDNA expression between shD3+CLP group and shNC+CLP group, but significant increase was found when compared with the shD3+Sham group (copies: 0.758±0.035 vs. 0.474±0.050, P < 0.05). In the shD3+CLP group, PGC1α expression was significantly improved at both transcriptional and protein levels relative to the shNC+CLP group [PGC1α mRNA (2-ΔΔCt): 1.49±0.13 vs. 0.68±0.06, PGC1α/β-Tubulin: 0.76±0.02 vs. 0.62±0.04, both P < 0.05]. (2) In vitro experiments: post-24-hour LPS treatment of C2C12 cells, the cellular localization of PGC1α became diffuse; interference with Dio3 expression promoted PGC1α translocation to the perinuclear region and nucleus. Moreover, the acetylated PGC1α level in the shD3+LPS group was significantly lower than that in the shNC+LPS group (acetylated PGC1α/β-Tubulin: 0.59±0.01 vs. 1.24±0.01, P < 0.05), while the expression of the deacetylating agent SIRT1 was substantially elevated following Dio3 inhibition (SIRT1/β-Tubulin: 1.04±0.04 vs. 0.58±0.03, P < 0.05). When SIRT1 activity was inhibited by using EX527, PGC1α protein expression was notably decreased compared to the shD3+LPS group (PGC1α/β-Tubulin: 0.92±0.03 vs. 1.58±0.03, P < 0.05).
CONCLUSIONS: Inhibition of Dio3 in skeletal muscle reduced the acetylation of PGC1α through activating SIRT1, facilitating nuclear translocation of PGC1α, thereby offering protection against sepsis-induced skeletal muscle mitochondrial damage.