High concentration of IL-17A inhibits autophagy of osteoclast precursor cells and inhibits osteoclast differentiation through PI3K/Akt pathway
10.3760/cma.j.cn121113-20240125-00057
- VernacularTitle: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机制研究
- Author:
Shujie YUAN
1
;
Hao TANG
;
Shida ZHU
;
Kai CHEN
;
Chuntao LIANG
;
Yuanxin LI
;
Hongkai WANG
Author Information
1. 桂林医学院第二附属医院骨科，桂林 541199
- Keywords:
Osteoclasts;
Autophagy;
Interleukin-17;
PI3K/Akt pathway
- From:
Chinese Journal of Orthopaedics
2024;44(15):1025-1033
- CountryChina
- Language:Chinese
-
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 differentiation 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 tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclast differentiation. The number of autolysosomes was observed under transmission electron microscope. RAW264.7 was treated with IL-17A. Western blot was used to detect the relative expression levels of p-Akt/Akt, p-mtor/mTOR, p-PI3K/PI3K, p-ULK1/ULK1, Cleaved-caspase3/caspase3, Beclin1/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%, respectively, 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 showed 0.69±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 differentiation by inhibiting autophagy of osteoclast precursor cells through PI3K/Akt pathway.
