Effects of chitinase-3-like protein 1 on the expression of inflammatory damage-related molecules in mouse skeletal muscle satellite cells induced by lipopolysaccharide / 中华危重病急救医学

ABSTRACT

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 transducer 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.