Liu Xi, Qian Fan, Fan Qiwei, Lin Li, He Meiyao, Li Peizhi, Cai Hongmei, Ma Lisha, Cheng Xin, Yang Xuesong
Division of Histology and Embryology, International Joint Laboratory for Embryonic Development & Prenatal Medicine, Medical College, Jinan University, Guangzhou, 510632, China.
Key Laboratory for Regenerative Medicine of the Ministry of Education, Jinan University, Guangzhou, 510632, China.
J Orthop Translat. 2021 Dec 3;31:52-61. doi: 10.1016/j.jot.2021.10.009. eCollection 2021 Nov.
Diabetes mellitus could cause numerous complications and health problems including abnormality of endochondral bone formation during embryogenesis. However, the underlying mechanisms still remain obscure.
Streptozotoci (STZ) was injected to induce pregestational diabetes mellitus (PGDM) mouse model. The femurs of E18.5 mouse embryos from control and PGDM groups were harvested. Morphological staining was implemented to determine the abnormality of the bone development. The expressions of the key genes participating in osteogenesis (e.g., Sox9, Runx2, and Osterix), the NF-κB signaling molecules (e.g., P50, P65, IκBα), and the corresponding regulatory factors (e.g., Bmp2, phospho-p38) were evaluated by immunofluorescence, quantitative PCR and western blot. Finally, in vitro chondrocyte differentiation model was employed to verify the role of NF-κB on the expressions of chondro-osteogenic markers.
Alcian blue/alizarin red double staining and H&E staining demonstrated the restriction of skeletal development and relatively extended hypertrophic zone at growth plate in E18.5 STZ-induced diabetic mouse embryos compared to the control. Immunofluorescent staining and qPCR showed that Sox9 expression increased, while Runx2 and Osterix expressions decreased in the growth plate of the offspring of PGDM mice. Immunofluorescence of P65 manifested the activation of NF-κB signaling in growth plate in PGDM mouse embryos. Furthermore, the relatively extended hypertrophic zone was also observed in the growth plate of the NF-κB-activated transgenic mice, as well as the activated p65 up-regulated the expression of Bmp2 and p-p38. In ATDC5 cells, we could observe the high glucose up-regulated the P50 and P65 expressions and down-regulated IκBα expression, but the high glucose-activated NF-κB signaling could be reversed by addition of Bay (inhibitor of NF-κB signaling). The expression changes of Bmp2, Sox9 and Runx2 in presence of high glucose were resumed too.
Our data revealed that NF-κB signaling was involved in mediation effects of dysfunctional trans-differentiation of hypertrophic chondrocytes in the embryonic growth plate induced by maternal diabetic mellitus.
糖尿病可引发多种并发症及健康问题，包括胚胎发育过程中软骨内骨形成异常。然而，其潜在机制仍不明晰。
注射链脲佐菌素（STZ）诱导孕前糖尿病（PGDM）小鼠模型。采集对照组和PGDM组E18.5小鼠胚胎的股骨。进行形态学染色以确定骨骼发育异常。通过免疫荧光、定量PCR和蛋白质印迹法评估参与成骨的关键基因（如Sox9、Runx2和Osterix）、NF-κB信号分子（如P50、P65、IκBα）以及相应调节因子（如Bmp2、磷酸化p38）的表达。最后，采用体外软骨细胞分化模型验证NF-κB对软骨成骨标志物表达的作用。
阿尔新蓝/茜素红双重染色和苏木精-伊红染色显示，与对照组相比，E18.5 STZ诱导的糖尿病小鼠胚胎生长板处骨骼发育受限且肥大区相对延长。免疫荧光染色和qPCR表明，PGDM小鼠后代生长板中Sox9表达增加，而Runx2和Osterix表达降低。P65的免疫荧光显示PGDM小鼠胚胎生长板中NF-κB信号激活。此外，在NF-κB激活的转基因小鼠生长板中也观察到肥大区相对延长，并且激活的p65上调了Bmp2和磷酸化p38的表达。在ATDC5细胞中，我们观察到高糖上调了P50和P65的表达并下调了IκBα的表达，但添加Bay（NF-κB信号抑制剂）可逆转高糖激活的NF-κB信号。高糖存在时Bmp2、Sox9和Runx2的表达变化也得以恢复。
我们的数据表明，NF-κB信号参与了母体糖尿病诱导的胚胎生长板中肥大软骨细胞功能失调转分化的介导作用。
