摘要
目的 探讨高浓度IL-17A通过PI3K/Akt通路抑制破骨细胞前体细胞自噬抑制破骨细胞分化的影响及分子机制.方法 采用50 ng/ml核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)诱导破骨细胞前体细胞(osteoclast precursor cells,OCPs),建立破骨细胞分化模型.将高浓度IL-17A(100 ng/ml)作用于破骨细胞分化模型,将RAW264.7细胞分为阴性对照组及加入RANKL的阳性对照组、IL-17A组、IL-17A+LY294002组;破骨细胞原代细胞(bone marrow derived macrophages,BMMs)分为加入巨噬细胞集落刺激因子(macrophagecolony stimulating factor,M-CSF)的阴性对照组及加入M-CSF与RANKL的阳性对照组、IL-17A组、IL-17A+LY294002组.通过IL-17A作用OCPs,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色观察破骨细胞分化的数量;透射电镜观察自噬小体的数量;通过IL-17A作用 RAW264.7,Western blot检测 p-Akt/Akt、p-mTOR/mTOR、p-PI3K/PI3K、p-ULK1/ULK1、Cleaved-caspase3/caspase3、Beclin1/β-Actin的相对表达量;通过IL-17A作用RAW264.7,流式细胞术检测RAW264.7细胞凋亡率;通过IL-17A与PI3K抑制剂LY294002作用OCPs,TRAP染色观察破骨细胞分化的数量.结果 TRAP染色示RAW264.7细胞阴性对照组、阳性对照组、IL-17A组阳性细胞比例分别为1.33％±0.58％、1 00％±3.01％、51.11％±4.02％,IL-17 A组低于阳性对照组,差异有统计学意(t=16.970,P＜0.05);BMMs细胞阴性对照组、阳性对照组、IL-17A组阳性细胞比例分别为1.67％±0.58％、100％±1.01％、50.33％±2.52％,IL-17A组低于阳性对照组,差异有统计学意义(t=31.770,P＜0.05).透射电镜示RAW264.7细胞阳性对照组自噬小体数量为3.67±1.53,高于IL-17A组的0.67±0.58,差异有统计学意义(t=3.182,P＜0.001);BMMs细胞阳性对照组自噬小体数量为3.00±1.00,高于IL-17A组的0.33±0.58,差异有统计学意义(t=4.000,P＜0.001).Western blot结果示 RAW264.7 细胞阳性对照组 p-Akt/Akt、p-mTOR/mTOR、p-PI3K/PI3K、p-ULK1/ULK1、Cleaved-caspase3/caspase3、Be-clin1/β-Actin 分别为 0.69±0.03、0.69±0.13、1.47±0.13、0.78±0.04、0.66±0.10、0.82±0.03,IL-17A 组分别为 0.89±0.04、1.14±0.18、1.87±0.04、0.53±0.09、0.93±0.02、0.54±0.03,差异均有统计学意义(P＜0.05).流式细胞术检测示RAW264.7细胞阳性对照组细胞凋亡率为6.92％±0.62％,低于IL-17A组的12.12％±0.69％,差异有统计学意义(t=9.747,P＜0.001).使用LY294002后的TRAP染色示RAW264.7细胞阴性对照组、阳性对照组、IL-17A组、IL-17A+LY294002组阳性细胞比例分别为 9.00％±2.00％、158.33％±3.51％、100％±2.65％、128.99％±4.01％,IL-17A+LY294002 组高于 IL-17A 组,差异有统计学意义(t=10.470,P＜0.001);BMMs细胞阴性对照组、阳性对照组、IL-17A组、IL-17A+LY294002组阳性比例分别为8.01％±0.99％、151.67％±4.51％、100％±3.61％,IL-17A+LY294002组高于IL-17A组,差异有统计学意义(t=6.535,P＜0.001).结论 高浓度IL-17A通过PI3K/Akt通路抑制破骨细胞前体细胞自噬抑制破骨细胞分化.
Abstract
Objective To investigate the effect and molecular mechanism of high concentration of IL-17A on osteoclast differentiation by inhibiting autophagy of osteoclast precursor cells through PI3K/Akt pathway.Methods With RANKL(50 ng/ml)inducing osteoclast precursor cells(osteoclast we cells,OCPs),osteoclast differentiation model is set up.In osteoclast differen-tiation model of high levels of IL-17A(100 ng/ml),RAW264.7 cells were divided into negative control CTR-N group,CTR-R group with RANKL,IL-17A group,IL-17A+LY294002 group.BMMs were divided into negative control CTR-N group with M-CSF,CTR-R group,IL-17A group and IL-17A+LY294002 group with M-CSF and RANKL.IL-17A was applied to OCPs,and tar-trate-resistant acid phosphatase(TRAP)staining was used to observe the number of osteoclast differentiation.The number of autol-ysosomes was observed under transmission electron microscope.RAW264.7 was treated with IL-17A.Western blot was used to de-tect the relative expression levels of p-Akt/Akt,p-mtor/mTOR,p-PI3K/PI3K,p-ULK1/ULK1,Cleaved-caspase3/caspase3,Be-clin1/β-actin.The apoptosis rate of RAW264.7 cells treated with IL-17A was detected by flow cytometry.OCPs were treated with IL-17A and PI3K inhibitor LY294002,and TRAP staining was used to observe the number of osteoclast differentiation.Results The TRAP staining showed that the positive ratio for RAW264.7 cells CTR-N group,CTR-R group,IL-17A group was 1.33％±0.58％,100％±3.01％,51.11％±4.02％with that of IL-17A significantly lower than CTR-R group(t=16.970,P＜0.05).The positive rates of BMMs in the CTR-N group,CTR-R group and IL-17A group were 1.67％±0.58％,100％±1.01％and 50.33％±2.52％,re-spectively,with that of IL-17A group significantly lower than CTR-R group(t=31.770,P＜0.05).Transmission electron microscopy showed that the number of autophagosomes in RAW264.7 cells in CTR-R group and IL-17A group were 3.67±1.53 and 0.67±0.58,respectively,with significant difference between the groups(t=3.182,P＜0.05).While in BMMs cells CTR-R group and IL-17 the numbers of autophagosome were 3.00±1.00 and 0.33±0.58 with significant difference(t=4.000,P＜0.05);Western blot results showed0.69±0.03、0.69±0.13、1.47±0.13、0.78±0.04、0.66±0.10、0.82±0.03 for RAW264.7 cells CTR-R group Akt/Akt,p-mTOR/mTOR,p-PI3K/PI3K,p-ULK1/ULK1,Cleaved caspase3/caspase3,Beclin1/β-Actin and 0.89±0.04、1.14±0.18、1.87±0.04、0.53±0.09、0.93±0.02、0.54±0.03 for RAW264.7 cells IL-17A group p-Akt/Akt,p-mTOR/mTOR,p-PI3K/PI3K,p-ULK1/ULK1,Cleaved caspase3/caspase3,Beclin1/β-Actin with significant difference(t=6.708;t=3.497;t=5.424;t=4.542;t=4.638;t=11.220,all P＜0.05);Flow cytometry detection showed that in CTR-R group,IL-17A RAW264.7 cells apoptosis rates of group A were 6.92％±0.62％,12.12％±0.69％,with significant difference between the two groups(t=9.747,P＜0.05);After using LY294002 TRAP staining,it showed a positive result of 9.00％±2.00％,158.33％±3.51％,100％±2.65％and 128.99％±4.01％for CTR-N,CTR-R,IL-17A and IL-17A+LY294002 in RAW264.7 cells respectively with significant difference between IL-17A+LY294002 group and the IL-17A in group A(t=10.470,P＜0.05).For BMMs cells CTR-N,CTR-R group,IL-17A in group,IL-17A+LY294002 group,the positive rate was 8.01％±0.99％,151.67％±4.51％,100％±3.61％,with significant difference between IL-17A+LY294002 group and IL-17A group(t=6.535,P＜0.05).Conclusion High concentration of IL-17A inhibits osteoclast differ-entiation by inhibiting autophagy of osteoclast precursor cells through PI3K/Akt pathway.
基金项目
国家自然科学基金地区项目(81960172)
广西自然科学基金面上项目(2020GXNSFAA238014)
广西医疗卫生重点培育学科建设项目(桂卫科教发[2022]4号)
Guangxi Medical and health key cultivation discipline construction project(gwkjf[2022]4)
维普
万方数据