Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Noto, Ishikawa 927-0553, Japan.
Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Noto, Ishikawa 927-0553, Japan.
Comp Biochem Physiol A Mol Integr Physiol. 2016 May;195:26-31. doi: 10.1016/j.cbpa.2016.01.022. Epub 2016 Feb 2.
Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3h of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal-less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18h of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in osteoclasts using the TUNEL (TdT-mediated dUTP nick end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3h of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure.
利用同时存在于钙化骨基质中的破骨细胞和成骨细胞的鱼鳞片，我们研究了低强度脉冲超声（LIPUS）对破骨细胞和成骨细胞的影响。在 LIPUS 处理后 3 小时孵育时，破骨细胞标志物如抗酒石酸酸性磷酸酶（TRAP）和组织蛋白酶 K 的 mRNA 表达显著降低，而成骨细胞标志物骨钙素、远系同源盒 5、runt 相关转录因子 2a 和 runt 相关转录因子 2b 的 mRNA 表达显著增加。然而，在孵育 6 和 18 小时时，至少在当前条件下，破骨细胞和成骨细胞标志物的 mRNA 表达都没有改变。使用 LIPUS 处理的斑马鱼鳞片的 GeneChip 分析，我们发现细胞死亡相关基因在 LIPUS 处理后上调。实时 PCR 分析表明，凋亡相关基因的表达也显著增加。为了确认凋亡在 LIPUS 作用下破骨细胞中的参与，通过自体移植鳞片在金鱼中诱导破骨细胞。此后，使用 TUNEL（末端转移酶介导的 dUTP 缺口末端标记）法在破骨细胞中检测与凋亡相关的 DNA 片段化。TRAP 染色的破骨细胞的多核被 TUNEL 标记。TUNEL 染色表明，在孵育 3 小时时，LIPUS 处理显著增加了金鱼鳞片中凋亡破骨细胞的数量。因此，我们首次证明 LIPUS 直接作用于破骨细胞，并得出结论，LIPUS 直接导致暴露后不久破骨细胞凋亡。
