Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts

To evaluate the effects of high glucose on oxidative stress, autophagy, and apoptosis in MC3T3-E1 cells co-cultured with Raw264.7 cells.

①Raw264.7 cells were separated into 3 groups based on the glucose levels in the medium (25, 30, and 35 mmol/L). Flow cytometry was used to detect CD86 expression. RT-qPCR was employed to detect the mRNA expression levels of IL-6, TNF-α, iNOS, NOX2, and p47phox in Raw264.7 cells. Western blotting was used to detect the protein expression level of iNOS. The DCFH-DA probe was used to measure the level of reactive oxygen species (ROS) in Raw264.7 cells. ②MC3T3-E1 cells were divided into 3 groups: normal glucose group (5.5 mmol/L glucose, 5.5-Mono), high glucose mono-culture group (35 mmol/L glucose, 35-Mono) and high glucose co-culture group (co-cultured with Raw264.7 for 24 h, 35-Co). The ROS level of MC3T3-E1 cells were detected by DCFH-DA probe. The autophagic level was assessed by transmission electron microscopy (TEM) and the expression of related proteins was detected with Western blotting. Flow cytometry was applied to determine the rate of apoptosis.

Compared with the normal glucose group, the expression level of CD86 in Raw 264.7 cells was increased in other high glucose groups. Compared with the 25 mmol/L group, the mRNA levels of IL-6, TNF-α, iNOS, NOX2, and p47phox were increased (P<0.05), the protein level of iNOS was elevated (P<0.05) and the ROS level in the Raw264.7 cells was raised in the 35mmol/L group as well. Compared with the normal glucose group, the ROS level of MC3T3-E1 cells in the high glucose groups was increased, especially in high glucose co-culture group. The number of autophagolysosomes in the MC3T3-E1 cells were increased sharply in the high glucose co-culture group. The protein levels of LC3-II and SQSTM1/p62 in MC3T3-E1 cells were increased significantly in the high glucose co-culture group (P<0.05). The apoptotic rate of MC3T3-E1 cells was significantly higher in the high glucose co-culture group than the high glucose mono-culture group (P<0.05).

Co-culture with M1-polarized Raw264.7 cells under high glucose induces apoptosis in MC3T3-E1 cells, which may be related to the production of ROS and over-enhancement of autophagy in MC3T3-E1 cells.