Li Fuxing, Sheng Zhiyong, Zhou Yihua, Li Jingying, Xu Jianning
Department of Intensive Care Unit, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China. Corresponding author: Xu Jianning, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 Dec;33(12):1428-1433. doi: 10.3760/cma.j.cn121430-20210108-00043.
OBJECTIVE: To explore the potential mechanism of chitinase-3-like protein 1 (CHI3L1) involved in skeletal muscle stem cell injury induced by sepsis. METHODS: Six different concentrations of lipopolysaccharide (LPS) were used to stimulate mouse skeletal muscle satellite cells cultured in vitro. Enzyme linked immunosorbent assay (ELISA) and cell counting kit-8 (CCK-8) were used to determine the optimal concentration. The overexpression and interference vectors of CHI3L1 were constructed to transfect skeletal muscle satellite cells, and the transfection efficiency was verified by polymerase chain reaction (PCR) and Western blotting. The cells were randomly divided into blank control group (cells without any intervention), model group (LPS-stimulated untransfected cells), overexpressing CHI3L1 group (LPS-stimulated cells transfected with CHI3L1 plasmid), overexpressing CHI3L1 control group [LPS-stimulated cells transfected with negative control (NC) plasmid], CHI3L1 interference group [LPS-stimulated cells transfected with CHI3L1 small interfering RNA (siRNA)], CHI3L1 interference control group (LPS-stimulated cells transfected with CHI3L1-siRNA NC). The levels of extracellular caspase-1 and interleukin-1β (IL-1β) were detected by ELISA. The protein expressions of intracellular IL-1β, signal transducters and activator of transcription 3 (STAT3), protein kinase B (Akt) and phosphorylated Akt (p-Akt) were detected by Western blotting. RESULTS: According to the results of CCK-8 and ELISA, the best concentration of 5 mg/L LPS was selected for the subsequent experiment. The transfection was validated by PCR and Western blotting. Compared with the blank control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular Akt, p-Akt, and IL-1β were significantly increased in the model group [IL-1β (ng/L): 11.22±0.55 vs. 8.63±0.63, caspase-1 (pmol/L): 9.47±0.22 vs. 8.65±0.15, Akt/GAPDH: 1.36±0.12 vs. 1.06±0.15, p-Akt/GAPDH: 0.78±0.07 vs. 0.09±0.01, IL-1β/GAPDH: 1.38±0.12 vs. 0.18±0.03, all P < 0.05]. Compared with the model group and the overexpressing CHI3L1 control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly increased in the overexpressing CHI3L1 group [IL-1β (ng/L): 14.93±0.97 vs. 11.22±0.55, 9.38±0.40, caspase-1 (pmol/L): 10.35±0.03 vs. 9.47±0.22, 8.46±0.24, p-Akt/GAPDH: 1.21±0.04 vs. 0.78±0.07, 0.63±0.04, IL-1β/GAPDH: 1.87±0.08 vs. 1.38±0.12, 1.51±0.17, all P < 0.05]. Compared with the model group and the CHI3L1 interference control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly decreased in the CHI3L1 interference group [IL-1β (ng/L): 8.98±0.73 vs. 11.22±0.55, 10.44±0.65, caspase-1 (pmol/L): 7.61±0.63 vs. 9.47±0.22, 8.37±0.38, p-Akt/GAPDH: 0.50±0.04 vs. 0.78±0.07, 0.94±0.06, IL-1β/GAPDH: 0.77±0.02 vs. 1.38±0.12, 1.13±0.07, all P < 0.05]. CONCLUSIONS: CHI3L1 may mediate the damage of skeletal muscle stem cells in sepsis by increasing the expression of caspase-1 and IL-1β. CHI3L1 may be involved in the regulation of Akt signaling pathway in skeletal muscle stem cells, but has no significant effect on STAT3 signaling pathway.
目的：探讨几丁质酶-3样蛋白1（CHI3L1）参与脓毒症诱导的骨骼肌干细胞损伤的潜在机制。 方法：采用六种不同浓度的脂多糖（LPS）刺激体外培养的小鼠骨骼肌卫星细胞。使用酶联免疫吸附测定（ELISA）和细胞计数试剂盒-8（CCK-8）确定最佳浓度。构建CHI3L1的过表达和干扰载体转染骨骼肌卫星细胞，并通过聚合酶链反应（PCR）和蛋白质印迹法验证转染效率。将细胞随机分为空白对照组（未进行任何干预的细胞）、模型组（LPS刺激的未转染细胞）、CHI3L1过表达组（用CHI3L1质粒转染的LPS刺激细胞）、CHI3L1过表达对照组[用阴性对照（NC）质粒转染的LPS刺激细胞]、CHI3L1干扰组[用CHI3L1小干扰RNA（siRNA）转染的LPS刺激细胞]、CHI3L1干扰对照组（用CHI3L1-siRNA NC转染的LPS刺激细胞）。通过ELISA检测细胞外半胱天冬酶-1和白细胞介素-1β（IL-1β）的水平。通过蛋白质印迹法检测细胞内IL-1β、信号转导子和转录激活因子3（STAT3）、蛋白激酶B（Akt）和磷酸化Akt（p-Akt）的蛋白表达。 结果：根据CCK-8和ELISA结果选择5 mg/L LPS作为后续实验的最佳浓度。通过PCR和蛋白质印迹法验证转染。与空白对照组相比，模型组细胞外IL-1β、半胱天冬酶-1水平以及细胞内Akt、p-Akt和IL-1β的蛋白表达显著升高[IL-1β（ng/L）：11.22±0.55 vs. 8.63±0.63，半胱天冬酶-1（pmol/L）：9.47±0.22 vs. 8.65±0.15，Akt/GAPDH：1.36±0.12 vs. 1.06±0.15，p-Akt/GAPDH：0.78±0.07 vs. 0.09±0.01，IL-1β/GAPDH：1.38±0.12 vs. 0.18±0.03，均P<0.05]。与模型组和CHI3L1过表达对照组相比，CHI3L1过表达组细胞外IL-1β、半胱天冬酶-1水平以及细胞内p-Akt和IL-1β的蛋白表达显著升高[IL-1β（ng/L）：14.93±0.97 vs. 11.22±0.55，9.38±0.40，半胱天冬酶-1（pmol/L）：10.35±0.03 vs. 9.47±0.22，8.46±0.24，p-Akt/GAPDH：1.21±0.04 vs. 0.78±0.07，0.63±(0.04)，IL-1β/GAPDH：1.87±0.08 vs. 1.38±0.12，1.51±0.17，均P<0.05]。与模型组和CHI3L1干扰对照组相比，CHI3L1干扰组细胞外IL-1β、半胱天冬酶-1水平以及细胞内p-Akt和IL-1β的蛋白表达显著降低[IL-1β（ng/L）：8.98±0.73 vs. 11.22±0.55，10.4(4)±0.65，半胱天冬酶-1（pmol/L）：7.61±0.63 vs. 9.47±0.22，8.37±0.38，p-Akt/GAPDH：0.50±0.04 vs. 0.78±0.07，0.94±0.06，IL-1β/GAPDH：0.77±0.02 vs. 1.38±0.12，1.13±0.07，均P<0.05]。 结论：CHI3L1可能通过增加半胱天冬酶-1和IL-1β的表达介导脓毒症中骨骼肌干细胞的损伤。CHI3L1可能参与骨骼肌干细胞中Akt信号通路的调节，但对STAT3信号通路无显著影响。
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