2023, Vol. 27 ›› Issue (8): 1205-1210

Naringin regulates the function of RAW264.7 macrophages to affect the osteogenic differentiation of MC-3T3-E1 cells

Tang Liang1, Li Xiheng1, Niu Ruijuan1, Li Xinyue1, Zou Xinying1, Mao Tianjiao2, Li Jiang1, 2   

Abstract: BACKGROUND: Naringin, as a monomer of traditional Chinese medicine, has the anti-inflammatory and anti-cancer effects and can promote osteogenesis. RAW264.7 cells play an important role in bone destruction. Some studies have found that reducing inflammation can promote the osteogenic differentiation of MC-3T3-E1 cells. So, it may promote osteogenic differentiation by inhibiting inflammation-related pathways.
OBJECTIVE: To observe whether naringin can regulate the function of RAW264.7 cells to affect the osteogenic differentiation of MC-3T3-E1 cells.
METHODS: The cytotoxicity of different concentrations of naringin to RAW264.7 cells induced by lipopolysaccharide was tested by cell counting kit-8 method. RAW264.7 cells were divided into seven groups: control (DMEM culture), model group (1 mg/L lipopolysaccharide), and naringin groups (1 mg/L lipopolysaccharide+50, 100, 150, 200, 250 μmol/L naringin). In different concentration naringin groups, the mRNA levels of inflammatory factors were detected by RT-PCR. Data of three naringin groups with better anti-inflammatory concentrations (150, 200, 250 μmol/L) were selected for follow-up experiments. RAW264.7 cells were divided into four groups: inflammation model, 150, 200, 250 μmol/L naringin groups. The above four groups were used as inflammatory supernatant. The inflammatory supernatant prepared above was mixed with osteogenic induction medium in a ratio of 1:1 to co-culture MC-3T3-E1 cells. Culture cells were divided into five groups: control group, inflammation model group, 150, 200, 250 μmol/L naringin groups. Osteogenesis-related gene detection, alkaline phosphatase staining, and alkaline phosphatase activity experiment were carried out on 7 and 14 days of culture.
RESULTS AND CONCLUSION: 1 mg/L lipopolysaccharide has no toxic effect on macrophages, but 200 μmol/L naringin showed a cytotoxic effect on macrophages induced by lipopolysaccharide (P < 0.05). In the process of screening the anti-inflammatory concentration of naringin, the concentration of naringin at 150, 200, and 250 μmol/L compared with the control group had good anti-inflammatory effects (P < 0.05). Therefore, 150, 200, and 250 μmol/L naringin were used for subsequent experiments. Alkaline phosphatase staining results showed that compared with the control group, 200 μmol/L naringin had the best dyeing result and the dyeing effect was better on 14 days than 7 days. The mRNA expression level of osteogenesis-related genes in the 200 μmol/L naringin was the closest to that in the control group and the inflammation model group had the worst osteogenic effect (P < 0.05). In the alkaline phosphatase activity experiment, compared with the control group and inflammation model group, 200 μmol/L naringin showed the strongest alkaline phosphatase activity on 7 days of culture (P < 0.05). Compared with the control group and inflammation model group, 200 μmol/L naringin showed no statistical significance in the alkaline phosphatase activity on 14 days of culture. Whilst compared with the inflammation group, the strongest alkaline phosphatase activity was found in the 200 μmol/L naringin group. The above results show that naringin can improve the osteogenic differentiation of MC-3T3-E1 cells in the inflammatory state by regulating the function of RAW264.7 macrophages.
Key words: naringin, anti-inflammation, osteogenesis, MC-3T3-E1 cell, RAW264.7 cell