黄芪多糖对激素性股骨头坏死模型骨细胞凋亡和SP1/MEK/ERK轴的影响

Effect of astragalus polysaccharides on osteocyte apoptosis and SP1/MEK/ERK axis in cell models of steroid-induced osteonecrosis of femoral head

摘要:

目的 探究黄芪多糖对激素性股骨头坏死（SONFH）模型骨细胞凋亡和SP1/MEK/ERK轴的影响。方法 通过地塞米松诱导小鼠骨细胞（MLO-Y4）建立SONFH细胞模型，加入300 μg/mL黄芪多糖处理后通过MTT和流式细胞术检测骨细胞活性和细胞凋亡。进一步在细胞中过表达或者敲降SP1，通过Western blotting验证SP1表达对MEK/ERK通路活性的影响。结果 SONFH模型组48 h后光密度值较对照组降低（P <0.05），SONFH+黄芪多糖组较SONFH+PBS组升高（P <0.05）。SONFH模型组细胞凋亡率较对照组高（P <0.05），SONFH+黄芪多糖组较SONFH模型组低（P <0.05），SONFH模型组Bax、Cleaved-caspase-3蛋白相对表达量较对照组高（P <0.05），Bcl-2蛋白相对表达量较对照组低（P <0.05），SONFH+黄芪多糖组Bax、Cleaved-caspase-3蛋白相对表达量较SONFH模型组低（P <0.05），Bcl-2蛋白相对表达量较SONFH模型组高（P <0.05）。SONFH模型组SP1 mRNA、蛋白相对表达量较对照组高（P <0.05），SONFH+黄芪多糖组较SONFH模型组低（P <0.05）。SONFH+黄芪多糖组SP1 mRNA相对表达量较SONFH模型组低，SONFH+黄芪多糖+OE-SP1组较SONFH+黄芪多糖+OE-NC组高。SONFH模型组细胞活性较对照组降低（P <0.05），SONFH+黄芪多糖组较SONFH模型组升高（P <0.05），SONFH+黄芪多糖组与SONFH+黄芪多糖+OE-NC组比较，差异无统计学意义（P >0.05），SONFH+黄芪多糖+OE-SP1组较SONFH+黄芪多糖组降低（P <0.05）。SONFH模型组细胞凋亡率和Bax、Cleaved-caspase-3蛋白相对表达量较对照组高（P <0.05），SONFH+黄芪多糖组较SONFH模型组低（P <0.05），SONFH+黄芪多糖+OE-SP1组较SONFH+黄芪多糖+OE-NC组高（P <0.05）；SONFH模型组Bcl-2蛋白相对表达量较对照组低（P <0.05），SONFH+黄芪多糖组较SONFH模型组低（P <0.05），SONFH+黄芪多糖+OE-SP1组较SONFH+黄芪多糖+OE-NC组低（P <0.05）。OE-SP1组SP1 mRNA相对表达量较OE-NC组高，sh-SP1组较sh-NC组低。OE-SP1组SP1蛋白相对表达量较对照组、OE-NC组高（P <0.05），sh-SP1组较sh-NC低（P <0.05）；OE-SP1组MEK、p-ERK蛋白相对表达量较对照组、OE-NC组低（P <0.05），sh-SP1组较sh-NC高（P <0.05）。结论 黄芪多糖抑制了SONFH骨细胞活性，并促进了骨细胞凋亡，其治疗作用可能与SP1和MEK/ERK通路相关。

Abstract:

Objective To explore the effect of astragalus polysaccharides on osteocyte apoptosis and SP1/MEK/ERK axis in cell models of steroid-induced osteonecrosis of femoral head (SONFH).Methods The cell model of SONFH was established by treating mouse osteocytes (MLO-Y4) with dexamethasone. After modeling, the SONFH cells were treated with 300 μg/mL of astragalus polysaccharides, followed by the MTT assay and flow cytometry to detect the cell viability and apoptosis. Furthermore, SP1 was overexpressed or knocked down in SONFH cells, and the effect of SP1 expression on the activity of the MEK/ERK pathway was verified by Western blotting.Results The 48-hour optical density of the SONFH model group was lower than that of the control group (P < 0.05), while that in the SONFH + astragalus polysaccharides group was higher compared with the SONFH + PBS group (P < 0.05). The cell apoptosis rate in the SONFH model group was higher than that in the control group (P < 0.05), while that in the SONFH + astragalus polysaccharides group was lower compared with the SONFH model group (P < 0.05). Compared with the control group, the protein expressions of Bax and cleaved caspase-3 were higher, but the protein expression of Bcl-2 was lower in the SONFH model group (P < 0.05). Compared with the SONFH model group, the protein expressions of Bax and cleaved caspase-3 were lower, but the protein expression of Bcl-2 was higher in the SONFH + astragalus polysaccharides group (P < 0.05). The mRNA and protein expressions of SP1 in the SONFH model group were higher than those in the control group (P < 0.05), whereas those in the SONFH + astragalus polysaccharides group were lower compared with the SONFH model group (P < 0.05). The mRNA expression of SP1 in the SONFH + astragalus polysaccharides group was lower than that in the SONFH model group, while that in the SONFH + astragalus polysaccharides + OE-SP1 group was higher compared with the SONFH + astragalus polysaccharides + OE-NC group (P < 0.05). The cell viability in the SONFH model group was lower than that in the control group (P < 0.05), while that in the SONFH + astragalus polysaccharides group was higher compared with the SONFH model group (P < 0.05). There was no difference in the cell viability between the SONFH + astragalus polysaccharides group and the SONFH + astragalus polysaccharides + OE-NC group (P > 0.05), while the cell viability in the SONFH + astragalus polysaccharides + OE-SP1 group was lower compared with the SONFH + astragalus polysaccharides + OE-NC group (P < 0.05). The cell apoptosis rate and the protein expressions of Bax and cleaved caspase-3 in the SONFH group were higher than those in the control group (P < 0.05), while they were lower in the SONFH + astragalus polysaccharides group than in the SONFH model group, and were higher in the SONFH + astragalus polysaccharides + OE-SP1 group than in the SONFH + astragalus polysaccharides + OE-NC group (P < 0.05). The protein expression of Bcl-2 in the SONFH model group was lower than that in the control group (P < 0.05), while that in the SONFH + astragalus polysaccharides group was lower than that in the SONFH model group (P < 0.05), and that in the SONFH + astragalus polysaccharides + OE-SP1 group was even lower than that in the SONFH + astragalus polysaccharides + OE-NC group (P < 0.05). The mRNA expression of SP1 in the OE-SP1 group was higher than that in the OE-NC group, while that in the sh-SP1 group was lower than that in the sh-NC group. The protein expression of SP1 in the OE-SP1 group was higher than that in the control group and the OE-NC group (P < 0.05), and that in the sh-SP1 group was lower than that in the sh-NC group (P < 0.05). The protein expressions of MEK and p-MEK in the OE-SP1 group were lower than those in the control group and the OE-NC group (P < 0.05), whereas those in the sh-SP1 group were higher than those in the sh-NC group (P < 0.05).Conclusions Astragalus polysaccharide inhibits the viability of osteocytes in SONFH and promotes the apoptosis of osteocytes. The therapeutic effect of astragalus polysaccharide might be related to SP1 and the MEK/ERK pathway.