Gang Zheng1, Zongyou Pan2, Yu Zhan3, Qian Tang1, Fanghong Zheng4, Yifei Zhou5, Yaosen Wu5, Yulong Zhou1, Deheng Chen1, Jiaoxiang Chen1, Xiangyang Wang5, Weiyang Gao5, Huazi Xu6, Naifeng Tian7, Xiaolei Zhang8. 1. Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China; Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou 325000, Zhejiang Province, China. 2. Department of Orthopaedics, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China. 3. Department of Chemoradiation Oncology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China. 4. The Second School of Medicine, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China. 5. Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China; Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou 325000, Zhejiang Province, China; The Second School of Medicine, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China. 6. Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China; Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou 325000, Zhejiang Province, China; The Second School of Medicine, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China. Electronic address: spinexu@163.com. 7. Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China; Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou 325000, Zhejiang Province, China; The Second School of Medicine, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China. Electronic address: fengnq@163.com. 8. Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China; Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou 325000, Zhejiang Province, China; The Second School of Medicine, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China; Chinese Orthopaedic Regenerative Medicine Society, China. Electronic address: zhangxiaolei@wmu.edu.cn.
Abstract
OBJECTIVE: Excessive apoptosis and senescence of nucleus pulposus (NP) cells are major pathological changes in intervertebral disc degeneration (IVDD) development; previous studies demonstrated pharmacologically or genetically stimulation of autophagy may inhibit apoptosis and senescence in NP cells. Transcription factor EB (TFEB) is a master regulator of autophagic flux via initiating autophagy-related genes and lysosomal biogenesis. This study was performed to confirm whether TFEB was involved in IVDD development and its mechanism. METHODS: TFEB activity was detected in NP tissues in puncture-induced rat IVDD model by immunofluorescence as well as in tert-Butyl hydroperoxide (TBHP), the reactive oxygen species (ROS) donor to induce oxidative stress, treated NP cells by western blot. After TFEB overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. In in vivo study, the lentivirus-normal control (LV-NC) or lentivirus-TFEB (LV-TFEB) were injected into the center space of the NP tissue, after 4 or 8 weeks, Magnetic resonance imaging (MRI), X ray, Hematoxylin-Eosin (HE) and Safranin O staining were used to evaluate IVDD grades. RESULTS: The nuclear localization of TFEB declined in degenerated rat NP tissue as well as in TBHP treated NP cells. Applying lentivirus to transfect NP cells, TFEB overexpression restored the TBHP-induced autophagic flux blockage and protected NP cells against apoptosis and senescence; these protections of TFEB are diminished by chloroquine-medicated autophagy inhibition. Furthermore, TFEB overexpression ameliorates the puncture-induced IVDD development in rats. CONCLUSIONS: Experimental IVDD inhibited the TFEB activity. TFEB overexpression suppressed TBHP-induced apoptosis and senescence via autophagic flux stimulation in NP cell and alleviates puncture-induced IVDD development in vivo.
OBJECTIVE: Excessive apoptosis and senescence of nucleus pulposus (NP) cells are major pathological changes in intervertebral disc degeneration (IVDD) development; previous studies demonstrated pharmacologically or genetically stimulation of autophagy may inhibit apoptosis and senescence in NP cells. Transcription factor EB (TFEB) is a master regulator of autophagic flux via initiating autophagy-related genes and lysosomal biogenesis. This study was performed to confirm whether TFEB was involved in IVDD development and its mechanism. METHODS:TFEB activity was detected in NP tissues in puncture-induced rat IVDD model by immunofluorescence as well as in tert-Butyl hydroperoxide (TBHP), the reactive oxygen species (ROS) donor to induce oxidative stress, treated NP cells by western blot. After TFEB overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. In in vivo study, the lentivirus-normal control (LV-NC) or lentivirus-TFEB (LV-TFEB) were injected into the center space of the NP tissue, after 4 or 8 weeks, Magnetic resonance imaging (MRI), X ray, Hematoxylin-Eosin (HE) and Safranin O staining were used to evaluate IVDD grades. RESULTS: The nuclear localization of TFEB declined in degenerated rat NP tissue as well as in TBHP treated NP cells. Applying lentivirus to transfect NP cells, TFEB overexpression restored the TBHP-induced autophagic flux blockage and protected NP cells against apoptosis and senescence; these protections of TFEB are diminished by chloroquine-medicated autophagy inhibition. Furthermore, TFEB overexpression ameliorates the puncture-induced IVDD development in rats. CONCLUSIONS: Experimental IVDD inhibited the TFEB activity. TFEB overexpression suppressed TBHP-induced apoptosis and senescence via autophagic flux stimulation in NP cell and alleviates puncture-induced IVDD development in vivo.