藏红花素通过调控PI 3K/Akt信号通路抑制成骨细胞凋亡引起的骨质疏松

Crocetin Inhibits Osteoblast Apoptosis by Regulating PI 3K/Akt Signaling Pathway Caused by Ovariectomy

目的探究藏红花素通过调控PI 3K/Akt信号通路抑制成骨细胞凋亡引起的骨质疏松。方法建立去卵巢骨质疏松小鼠模型,再每天给予雌激素(25μg/kg)和藏红花素(20mg/kg)灌胃治疗,另设假手术组,每天给予等量蒸馏水灌胃。术后4周开始连续灌胃12周,称量动物体质量,双能X线测量骨密度,苏木精-伊红(hematoxylin-eosin,HE)染色观察骨组织病理形态,全自动生化分析仪检测小鼠血清钙(S-Ca)、血清磷(S-P)、尿钙(U-Ca)、尿磷(U-P)、尿肌酐(Cr)含量,ELISA检测小鼠血清骨钙素(osteocalcin,OC)和碱性磷酸酶(alkaline phosphatase,ALP)水平。体外原代培养小鼠成骨细胞,利用ALP染色鉴定成骨细胞。加入藏红花素和LY249002,Western blot法检测磷脂酰肌醇3激酶(PI 3K)、蛋白激酶(Akt)及其磷酸化水平的蛋白表达,流式细胞术检测细胞凋亡。结果与假手术组比较,模型组小鼠体质量迅速增加,骨密度降低,骨小梁分布稀疏,尿液中U-Ca、U-P、Cr含量升高,血清中OC、ALP水平也升高,骨组织中p-PI 3K、PI 3K、p-Akt、Akt蛋白表达下调;与模型组比较,雌激素组和藏红花素组小鼠体质量增加缓慢,骨密度增加,改善骨小梁分布和排列,降低尿液中U-Ca、U-P、Cr含量和血清中OC、ALP水平,上调了骨组织中p-PI 3K、PI 3K、p-Akt、Akt蛋白表达。在成骨细胞中,与假手术组比较,模型组成骨细胞凋亡增多,下调p-PI 3K、PI 3K、p-Akt、Akt蛋白表达;与模型组比较,藏红花素组抑制了成骨细胞凋亡,上调了p-PI 3K、PI 3K、p-Akt、Akt蛋白表达;与藏红花素组比较,藏红花素+LY249002组抵消了藏红花素对成骨细胞凋亡的抑制作用。结论藏红花素抑制了骨质疏松症发生和进展,可能是通过调控PI 3K/Akt信号通路抑制成骨细胞凋亡来介导的。

Objective To explore the mechanism of crocin inhibiting osteoporosis caused by ovariectomy by regulating PI 3K/Akt signaling pathway to inhibit osteoblast apoptosis.Methods Couse model of ovariectomized osteoporosis was established,and then estrogen(25μg/kg)and crocin(20mg/kg)intragastric treatment were given every day.Another sham operation group was given the same amount of distilled water every day.Four weeks after the operation,the stomach was continuously gavaged for 12 weeks.The weight of the animals was weighed.The bone density was measured by dual-energy x-ray.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of bone tissue.Calcium(S-Ca),serum phosphorus(SP),urinary calcium(U-Ca),urinary phosphorus(U-P),urine creatinine(Cr)content,ELISA detection of mouse serum osteocalcin(OC)and alkaline phosphatase(ALP)level.Mouse osteoblasts were cultured in vitro,and ALP staining was used to identify osteoblasts.Adding crocin and LY249002,Western blot was used to detect the protein expression of phosphatidylinositol 3-kinase(PI 3K),protein kinase(Akt)and their phosphorylation levels.Flow cytometry was used to detect apoptosis.Results Compared with the sham-operated group,the model group′s body weight increased rapidly,the bone density decreased,the trabecular bones were sparsely distributed,the U-Ca,U-P,Cr contents in urine increased,and the serum OC and ALP levels also increased,the protein expression of p-PI 3K,PI 3K,p-Akt,Akt in bone tissue was down-regulated.Compared with the model group,the weight of mice in the estrogen group and crocin group increased slowly,the bone density increased,and the trabecular bone improved distribution and arrangement reduce U-Ca,U-P,Cr content in urine and OC and ALP levels in serum,and up-regulate the protein expression of p-PI 3K,PI 3K,p-Akt and Akt in bone tissue.In osteoblasts,compared with the sham-operated group,the model composed of osteoblasts increased apoptosis and down-regulated p-PI 3K,PI 3K,p-Akt,and Akt protein expression.Compared with the model group,the crocin group inhibited osteoblasts apoptosis up-regulated the expression of p-PI 3K,PI 3K,p-Akt and Akt protein.Compared with the crocin group,the crocin+LY249002 group offset the inhibitory effect of crocin on apoptosis of osteoblasts.Conclusion Crocin inhibits the progression of osteoporosis,which may be mediated by regulating PI 3K/Akt signaling pathway and inhibiting osteoblast apoptosis.