骨碎补总黄酮对口腔种植骨整合中牙槽骨成骨细胞增殖和凋亡的影响研究

Effects of total flavonoids of rhizomadrynariae on proliferation and apoptosis of alveolar bone osteoblasts during osseointegra⁃tion of oral implants

目的 探讨骨碎补总黄酮(total flavonoids of rhizomadrynariae, TFRD)对牙槽骨成骨细胞的增殖和凋亡的影响及其机制。方法 分离和培养大鼠牙槽骨成骨细胞。将细胞分为对照组、低剂量TFRD组(50 mg/L)、高剂量TFRD组(100 mg/L);NC mimic和miR-93-5p mimic转染至成骨细胞后,与高剂量的TFRD孵育,分别记为TFRD+NC mimic组和TFRD+miR-93-5p mimic组;CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡;采用RT-qPCR和Western blotting分别检测miR-93-5p、成骨分化相关基因(Osteriex和RUNX2)和WNT通路相关基因(Dishevelled和β-catenin)的表达水平;ALP染色和活性测试检测细胞成骨分化。结果 TFRD处理后的牙槽骨成骨细胞增殖能力增加(P<0.05),细胞凋亡率降低(P<0.05),ALP活性、Osteriex和RUNX2的表达增加(P<0.05),miR-93-5p的表达降低(P<0.05),FZD6、Dishevelled和β-catenin的表达增加(P<0.05),激活WNT通路。过表达miR-93-5p逆转TFRD对成骨细胞的保护作用,导致细胞增殖能力降低(P<0.05),细胞凋亡率增加(P<0.05),ALP活性和Osteriex和RUNX2的表达降低(P<0.05),miR-93-5p的表达增加(P<0.05),FZD6、Dishevelled和β-catenin表达减少(P<0.05),抑制WNT通路。结论 TFRD通过miR-93-5p/FZD6/WNT通路促进牙槽骨成骨细胞增殖,抑制凋亡。

Objective To investigate the effects of total flavonoids of rhizomadrynariae(TFRD)on the proliferation and apoptosis of alveolar bone osteoblasts and its mechanism.Methods Osteoblasts from rat alveolar bone were isolated and cultured.The cells were divided into control group,low⁃dose TFRD group(50 mg/L)and high⁃dose TFRD group(100 mg/L).After transfection with NC mimic and miR⁃93⁃5p mimic,osteoblasts were incubated with high⁃dose TFRD,which were classified as TFRD+NC mimic group and TFRD+miR⁃93⁃5p mimic group,respectively.Cell proliferation was detected by CCK⁃8 method.Cell apoptosis was detected by flow cytometry.The expression of miR⁃93⁃5p,osteogenic differentiation⁃related genes(Osteriex and RUNX2),and WNT pathway⁃related genes(Di⁃shevelled andβ⁃catenin)were detected by RT⁃qPCR and Western blotting,respectively.Osteogenic differentiation was detected by ALP staining and activity test.Results After TFRD treatment,the proliferation ability of alveolar osteoblasts was increased(P<0.05);ap⁃optosis rate was decreased(P<0.05);ALP activity and the expression of Osteriex and RUNX2 was increased(P<0.05);the expression of miR⁃93⁃5p was decreased(P<0.05);the expression of FZD6,Dishevelled andβ⁃catenin was increased(P<0.05),activating the WNT pathway.Overexpression of miR⁃93⁃5p reversed the protective effect of TFRD on osteoblasts,leading to the reduction of cell pro⁃liferation(P<0.05),the increase of apoptosis rate(P<0.05),the decrease of ALP activity and Osteriex and RUNX2 expression(P<0.05),the increase of miR⁃93⁃5p expression(P<0.05),the reduction of FZD6,Disevelled andβ⁃catenin expression(P<0.05),and the inhibition of WNT pathway.Conclusion TFRD promotes proliferation and inhibits apoptosis of alveolar bone osteoblasts through miR⁃93⁃5p/FZD6/WNT pathway.