丹蛭降糖胶囊调控β-catenin蛋白在高磷诱导的糖尿病大鼠下肢血管内皮细胞钙化中的作用

Effect of Danzhi Jiangtang Capsule on regulating β-catenin protein on high phosphorus-induced calcification of vascular endothelial cell in lower limb in diabetic mice

关键词:  糖尿病  丹蛭降糖胶囊  β-catenin  内皮细胞钙化

Key words:  diabetes  Danzhi Jiangtang Capsule  β-catenin  calcification of endothelial cell

摘要:

[目的] 探讨丹蛭降糖胶囊调控β-链蛋白（β-catenin蛋白）在高磷诱导的糖尿病大鼠下肢血管内皮细胞钙化中的作用。[方法] 分离糖尿病大鼠和健康大鼠下肢股动脉内皮细胞进行体外培养，分为空白组、高磷组、丹蛭组、氯化锂（LiCL）组；调整稳转内皮细胞（EC细胞）状态，除空白组外，其余各组加入高磷培养基（含有10 mmol/L β-甘油磷酸盐、50 mg/mL维生素C和1×10^-7 mol/L胰岛素）诱导细胞钙化，同时丹蛭组和LiCL组给予10%含药血清（丹蛭降糖胶囊）干预，48 h后LiCL组给予20 mmol/L LiCl继续干预48 h。采用DNA断裂的原位末端标记（TUNEL）法检测细胞凋亡，噻唑蓝（MTT）法检测细胞增殖，逆转录-聚合酶链反应（RT-PCR）法检测α平滑肌肌动蛋白（α-SMA）、β-catenin、碱性磷酸酶（ALP）、骨桥蛋白（OPN）、骨形态发生蛋白2（BMP2）、卷曲同源物1（FZD1）mRNA表达水平；激光共聚焦显微镜观察细胞β-catenin蛋白定位。[结果] 与空白组比较，高磷组细胞凋亡数、钙离子含量、α-SMA、β-catenin、ALP、OPN、BMP2及FZD1表达量增多，细胞增殖数量减少（P＜0.01）；激光共聚焦显微镜观察结果表明β-catenin多表达于细胞核内。与高磷组比较，丹蛭组细胞凋亡数、钙离子含量、α-SMA、β-catenin、ALP、OPN、BMP2及FZD1表达量减少，细胞增殖数量增多（P＜0.01），激光共聚焦显微镜下观察显示细胞核内β-catenin表达量减少。与丹蛭组比较，LiCL组细胞凋亡数、钙离子含量、α-SMA、β-catenin、ALP、OPN、BMP2及FZD1表达增加，细胞增殖数量减少（P＜0.01），激光共聚焦显微镜下观察显示细胞核内β-catenin表达量增加。[结论] 丹蛭降糖胶囊可以减轻糖尿病大鼠下肢内皮细胞钙化，机制是通过下调β-catenin在细胞核内的表达，减少β-catenin蛋白合成，抑制下游细胞表型基因的转录，达到防治内皮细胞血管钙化的目的。

Abstract:

[Objective] To investigate the regulating effect of Danzhi Jiangtang Capsule on high phosphorus-induced calcification of vascular endothelial cell in lower limb in diabetic mice.[Methods] Endothelial cells were isolated from femoral artery of diabetic mice and healthy mice and both cultured in vitro. They were divided into blank group,high phosphorus group,Danzhi group and LiCL group. After adjusting the stable state of EC cells,except for the blank group,the other groups were added with high phosphorus medium (containing 10 mmol/L β-glycerophosphate-50 mg/mL vitamin C and 1×10^-7m insulin) to induce cell calcification. Meanwhile,Danzhi group and LiCL group were given 10% drug-containing serum (Danzhi hypoglycemic capsule) for 48 h. LiCL group was given 20 mmol/L LiCL for further intervention for 48 h.Cell apoptosis was detected by TUNEL assay and cell proliferation was detected by MTT assay.The mRNA expression levels of α-SMA,β-catenin,alkaline phosphatase (ALP),osteopontin (OPN),bone morphogenetic protein 2(BMP2) and curly homolog 1(FZD1) were detected by reverse transcription-polymerase chain reaction (RT-PCR). The localization of β-catenin protein was observed by confocal laser microscopy.[Results] Compared with blank group,apoptosis number,Ca²⁺ content,expression levels of α-SMA,β-catenin,ALP,OPN,BMP2 and FZD1 were significantly increased in high phosphorus group,and cell proliferation number was significantly decreased (P<0.01). Confocal laser microscopy showed that β-catenin was mostly expressed in the nucleus. Compared with the high phosphorus group,the apoptosis number,Ca²⁺ content,α-SMA,β-catenin,ALP,OPN,BMP2 and FZD1 expression of Danzhi group were significantly decreased,and the number of cell proliferation was significantly increased (P<0.01). Microscopic observation showed that the expression of β-catenin in the nucleus was decreased. Compared with DanZhi group,the apoptosis number,Ca²⁺ content,α-SMA,β-catenin,ALP,OPN,BMP2 and FZD1 expression were increased in LiCL group,and the number of cell proliferation was significantly decreased (P<0.01). Microscopic observation showed that the expression of β-catenin in the nucleus was increased.[Conclusion] Danzhi Jiangtang Capsule can reduce the calcification of lower limb endothelial cells in diabetic mice by down-regulating the expression of β-catenin in the nucleus,reducing the synthesis of β-catenin protein,inhibiting the transcription of phenotypic genes in downstream cells,and preventing and controlling vascular calcification of endothelial cells.