基于PI3K/Akt/GSK3β/NFATc1通路分析沉默miR-664-3p对去卵巢骨质疏松大鼠的干预作用

Intervention effect of silencing miR-664-3p on ovariectomized osteoporotic rats based on PI3K/Akt/GSK3β/NFATc1 pathway analysis

目的探究基于磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/糖原合成酶(GSK)3β/活化T细胞核因子(NFATc1)通路分析沉默miR-664-3p对去卵巢骨质疏松大鼠的干预作用。方法选取30只SD雌性大鼠,将其中10只设为正常组,剩余20只建立去卵巢骨质疏松大鼠模型,并随机分为模型组、沉默组。检测对比骨密度变化,Micro-CT扫描重建股骨微结构,采用酶联免疫吸附试验、免疫透射比浊法分别对血清骨钙素(OC)水平、碱性磷酸酶(ALP)含量进行检测,通过Western印迹法检测各组骨髓基质细胞(BMSCs)的PIK3、Akt、GSK3β、NFATc1的磷酸化水平,miR-664-3p表达。结果模型组骨细胞指数、骨密度Tb.N、Tb.Th、BV/TV均低于正常组,Tb.Sp、miR-664-3p、PI3K、Akt、NFATc1均高于正常组,差异均有统计学意义(P<0.05);沉默组TB.Sp、miR-664-3p、PI3K、Akt、NFATc1均低于模型组,骨密度、骨细胞指数及Tb.N、Tb.Th、BV/TV、GSK3β高于模型组,差异均有统计学意义(P<0.05)。结论沉默miR-664-3p通过对PI3K/Akt/GSK3β/NFATc1通路调控促进BMASc增殖,对成骨细胞凋亡产生抑制,从而达到治疗骨质疏松目的。

Objective To explore the intervention effect of silencing miR-664-3p on ovariectomized osteoporotic rats based on PI3K/Akt/GSK3β/NFATc1 pathway analysis.Methods Thirty female SD rats were selected,10 of them were set as normal control group,and the remaining 20 were set up ovariectomized osteoporosis rat model,and randomly divided into model and silence groups.The changes of bone density were detected and compared,micro-CT scanning was used to reconstruct the femoral micro-structure,and the serum osteocalcin(OC)level and alkaline phosphatase(ALP)content were detected by enzyme-linked immunosorbent assay and immunotransmission turbidimetry respectively.The phosphorylation levels of phosphatidylinositol-3 kinase(PIK3),protein kinase B(Akt),glycogen synthase 3β(GSK3β)and activated T cell nuclear factor(NFATc1)in bone marrow stromal cells(BMSCs)and expression of miR-664-3p were determined by Western blot.Results The osteocyte index and bone density of model group were lower than those of normal group and silent group(P<0.05).The osteocyte index and bone density of silent group were lower than those of normal group and higher than those of model group(P<0.05).The Tb.N,Tb.Th,BV/TV of model group were lower than those of normal group and silent group,and Tb.Sp was higher than that normal group and silent group(P<0.05).Tb.N,Tb.Th,BV/TV of silent group were lower than those of normal group and higher than those of model group,Tb.Sp was higher than that of normal group and lower than that of model group(P<0.05).miR-664-3p,PI3K,Akt and NFATcl in ovariectomized osteoporosis model group were higher than those of normal group and silent group,and GSK3βwas lower than that of normal group and silent group(P<0.05).miR-664-3p,PI3K,Akt and NFATcl in silent group were higher than those of normal group and lower than those of silent group,and GSK3βwas lower than that of normal group and higher than that of silent group(P<0.05).Conclusions Silencing miR-664-3p could promote BMASc proliferation and inhibit osteoblast apoptosis by regulating PI3K/Akt/GSK3β/NFATc1 pathway,thus achieve the goal of inhibiting osteoporosis.