Spine Orthopedics Department, The First Hospital of Hunan University of Chinese Medicine, China.
Wangjing Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing, China.
J Orthop Surg (Hong Kong). 2022 Sep-Dec;30(3):10225536221135474. doi: 10.1177/10225536221135474.
This study aims to explore the potential mechanism of Achyranthoside D (AD) in improving intervertebral disc (IVD) degeneration (IDD).
The IDD model of SD rats and nucleus pulposus cells (NPCs) was established by lumbar cone annulus puncture and tert-butyl peroxide, respectively. Cell proliferation was detected by CCK8 assay. Apoptosis was detected by flow cytometry and TUNEL staining. IVD tissue injury was observed by HE staining. Alcian blue staining observed the glycoprotein secretion in IVD. Monodansylcadaverin (MDC) staining was used to detect the formation of autophagosomes. The LC3 expression was tested by immunofluorescence. The type II collagen, aggrecan and MMP3 expression were detected by ELISA. RT-qPCR was used to detect the Casp 3, Bax, Bcl2, Acan, Col2a1 and Mmp3 expression. The LC3, P62, type II collagen, aggrecan, Beclin1, Akt, MMP3, p-mTOR, PI3K, mTOR, p-PI3K and p-Akt expression were analyzed by western blot.
The IVD tissue damage and apoptosis occurred in the Model group, and the glycoprotein secretion decreased. Compared with Model group, AD-H group alleviated the injury of IVD tissue, inhibited the apoptosis of cells, and increased the secretion of glycoprotein. 40 μg/mL AD restored the proliferation activity of NPCs. Compared to the Normal group, the NPCs apoptosis increased, the Collagen II, aggrecan and Bcl2 expressions were significantly decreased, the MMP3, Bax and Casp 3 expression were significantly increased, and the LC-3 II/I expression in IVD tissues were increased significantly in Model group, all of which was reversed in AD group. AD promoted the p-Akt, p-PI3K, p-mTOR, LC-3 II/I and Beclin1 expression, inhibited the P62 expression to alleviate the damage of nucleus pulporeus cells and the degeneration of IVD.
AD improved IDD by affecting the PI3K/Akt/mTOR pathway and autophagy.
本研究旨在探讨牛膝多糖（AD）改善椎间盘退变（IDD）的潜在机制。
通过腰椎锥间盘环穿刺和叔丁基过氧化物分别建立 SD 大鼠 IDD 模型和髓核细胞（NPC）模型。通过 CCK8 检测细胞增殖。通过流式细胞术和 TUNEL 染色检测细胞凋亡。通过 HE 染色观察椎间盘组织损伤。通过阿尔辛蓝染色观察椎间盘内糖蛋白分泌。通过单丹磺酰尸胺（MDC）染色检测自噬体的形成。通过免疫荧光检测 LC3 表达。通过 ELISA 检测 II 型胶原、聚集蛋白聚糖和 MMP3 的表达。通过 RT-qPCR 检测 Casp 3、Bax、Bcl2、Acan、Col2a1 和 Mmp3 的表达。通过 Western blot 分析 LC3、P62、II 型胶原、聚集蛋白聚糖、Beclin1、Akt、MMP3、p-mTOR、PI3K、mTOR、p-PI3K 和 p-Akt 的表达。
模型组椎间盘组织损伤和细胞凋亡，糖蛋白分泌减少。与模型组相比，AD-H 组减轻了椎间盘组织损伤，抑制了细胞凋亡，增加了糖蛋白分泌。40μg/ml AD 恢复了 NPCs 的增殖活性。与正常组相比，模型组 NPCs 凋亡增加，Collagen II、aggrecan 和 Bcl2 表达显著降低，MMP3、Bax 和 Casp 3 表达显著增加，椎间盘组织 LC-3 II/I 表达显著增加，AD 组均得到逆转。AD 促进了 p-Akt、p-PI3K、p-mTOR、LC-3 II/I 和 Beclin1 的表达，抑制了 P62 的表达，从而减轻了 NPCs 的损伤和椎间盘的退变。
AD 通过影响 PI3K/Akt/mTOR 通路和自噬来改善 IDD。
