2022, Vol. 26 ›› Issue (20): 3121-3126

Lipopolysaccharide stimulates the expression of interleukin-6 and nuclear factor kappa B receptor activator ligand in mouse MLO-Y4 osteoblasts

Liu Qiqi1, Liu Min2, Yang Jian1, Yu Ke1   

Abstract: BACKGROUND: Previous studies have confirmed that lipopolysaccharide could up-regulated the expression of interleukin-6, an inflammation-related cytokine, in mouse MLO-Y4 osteocytes, and interleukin-6 could further up-regulate the expression of receptor activator of nuclear factor κB ligand (RANKL), a factor related to osteoclastogenesis, in osteoblasts. PI3K/AKT signaling pathway is an important regulatory pathway in bone metabolism. Verifying whether this signaling pathway is involved in the production of inflammatory factors induced by lipopolysaccharides is greatly beneficial to studying the mechanism of bone resorption that is caused by bacterial inflammation.
OBJECTIVE: To verify whether PI3K/AKT signaling pathway is involved in the expression of interleukin-6 and RANKL in mouse MLO-Y4 osteocytes stimulated by lipopolysaccharides.
METHODS: MLO-Y4 osteoblasts were stimulated with 0 or 100 µg/L lipopolysaccharides for 1, 2, 4, 6, and 8 hours. Then the mRNA expressions of interleukin-6 and RANKL were detected by real-time quantitative polymerase chain reaction, and the protein expression of RANKL and p-AKT were detected by western blot. MLO-Y4 osteocytes in logarithmic growth was divided into seven groups: no treatment group (control group), 100 µg/L lipopolysaccharide group, 1 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 2.5 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 5 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 10 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group, 20 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of interleukin 6 and RANKL. MLO-Y4 osteocytes were divided into four groups: no treatment group (control group), 100 µg/L lipopolysaccharide group, dimethyl sulfoxide+100 µg/L lipopolysaccharide group, and 10 µmol/L PI3K inhibitor+100 µg/L lipopolysaccharide group. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of RANKL, and western blot was used to detect the protein expression of RANKL and p-AKT.
RESULTS AND CONCLUSION: After lipopolysaccharide stimulation, the mRNA expression of interleukin-6 and RANKL in osteocytes was increased first and then decreased; however, the mRNA expression levels were higher than those in the control group at each time point (P < 0.05). After lipopolysaccharide stimulation, the protein expression of RANKL and p-AKT in osteocytes was significantly higher than that in the control group (P < 0.05) at each time point, although the protein expression in lipopolysaccharide groups was increased first and then decreased. PI3K inhibitor of 1-10 µmol/L could decrease the mRNA expression of interleukin 6 and RANKL in lipopolysaccharide-induced osteocytes in a concentration-dependent manner, while 20 and 10 µmol/L PI3K inhibitor showed similar effects. Both dimethyl sulfoxide and PI3K inhibitors could inhibit the increase in the protein and mRNA expression of RANKL and the protein expression of p-AKT in osteocytes induced by lipopolysaccharide. Furthermore, PI3K inhibitor showed the better inhibitory effect. These results indicate that lipopolysaccharide stimulates the expression of interleukin-6 and RANKL in mouse MLO-Y4 osteocytes through the PI3K/AKT signaling pathway.
Key words: osteocyte, interleukin-6, lipopolysaccharide, PI3K/AKT, receptor activator of nuclear factor-κB ligand, bone resorption, phosphorylation, PI3K inhibitor