Abstract: BACKGROUND: Leonurine has many biological activities such as improving microcirculation, anti-oxidation, anti-apoptosis, scavenging free radicals, anti-inflammation, and anti-fibrosis, and can promote osteogenic differentiation of bone marrow mesenchymal stem cells, which has the potential to be applied in the treatment of periodontitis.
OBJECTIVE: To explore the anti-inflammatory and osteogenic effects of leonurine loading into chitosan/sodium glycerophosphate/sodium alginate hydrogel.
METHODS: (1) Chitosan/sodium glycerophosphate/sodium alginate hydrogel (blank hydrogel) and chitosan/sodium glycerophosphate/sodium alginate/leonurus alkali hydrogel were prepared respectively. RAW 264.7 and MC3T3-E1 cells were inoculated with the two kinds of hydrogel. The cytotoxicity of hydrogels was detected by CCK-8 assay and live/dead cell staining. (2) RAW 264.7 cells were cultured in five groups. The blank group was cultured for 24 hours routinely. The lipopolysaccharide group was treated with lipopolysaccharide. The simple hydrogel group was treated with lipopolysaccharide and blank hydrogel. The drug-loaded hydrogel group was treated with lipopolysaccharide and drug-loaded hydrogel. The inhibitor group was treated with lippolysaccharide, drug-loaded hydrogel, and PI3K inhibitor LY294002. 24 hours later, mRNA expression of inflammation-related factors was detected by qRT-PCR. Western blot assay was utilized to detect the protein expression of inflammation-related factors and PI3K/AKT signaling pathway. (3) MC3T3-E1 cells were inoculated in four groups. The blank group was cultured without any material. The simple hydrogel group was treated with blank hydrogel. The drug-loaded hydrogel group was treated with drug-loaded hydrogel. The inhibitor group was treated with drug-loaded hydrogel and PI3K inhibitor LY294002 for 7 days. Alkaline phosphatase staining was performed. mRNA expression levels of osteogenic factors were detected by qRT-PCR. The protein expression levels of the PI3K/AKT signaling pathway were detected by western blot assay.
RESULTS AND CONCLUSION: (1) The results of CCK-8 assay and live/dead cell staining showed that the two kinds of hydrogels had no cytotoxic effect and had good cytocompatibility. (2) Compared with the blank group, the mRNA and protein expression levels of interleukin 6, tumor necrosis factor α, and interleukin 1β were significantly increased (P < 0.05), and the protein expression levels of p-AKT, p-PI3K, p-p65, and p-IκBα were significantly increased in the lipopolysaccharide group (P < 0.05). Compared with lipopolysaccharide group, mRNA and protein expression levels of the above indexes were decreased in drug-loaded hydrogel group (P < 0.05). Compared with the drug-loaded hydrogel group, the mRNA and protein expression levels of the above indexes were decreased in the inhibitor group (P < 0.05). (3) The activity of alkaline phosphatase in drug-loaded hydrogel group was higher than that in the blank group, simple hydrogel group, and inhibitor group (P < 0.05). Compared with blank group, the mRNA expression levels of alkaline phosphatase, Runx2, osteocalcin, and type I collagen were increased (P < 0.05), and the protein expression levels of p-AKT and p-PI3K were increased in the simple hydrogel group (P < 0.05). Compared with the simple hydrogel group, the mRNA and protein expression levels of the above indexes were increased in the drug-loaded hydrogel group (P < 0.05). Compared with the drug-loaded hydrogel group, the mRNA and protein expression levels of the above indexes were decreased in the inhibitor group (P < 0.05). (4) These findings conclude that chitosan/sodium glycerophosphate/sodium alginate/leonurine hydrogel has anti-inflammatory and osteogenic effects, which may be related to the regulation of PI3K/AKT signaling pathway. 

Key words: periodontitis, hydrogel, chitosan, sodium alginate, leonurine, inflammatory factor, osteogenesis