title: Lentiviral shRNA silencing of CHOP inhibits apoptosis induced by cyclic stretch in rat annular cells and attenuates disc degeneration in the rats.
abstract: The expression of CHOP (C/EBP homologous protein), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress induced by cyclic stretch and leads to rat AF cells apoptosis. However, whether the suppression of CHOP can inhibit apoptosis and attenuates disc degeneration by cyclic stretch remains unclear. The aim of this study was to evaluate the suppressive effects of lentiviral CHOP shRNA on apoptosis induced by cyclic stretch in rat annulus fibrosus (AF) cells in vitro and disc degeneration of rat lumber spine in vivo. Lentiviral CHOP shRNA was constructed and introduced into AF cells. After stretched by the Flexcell Tension Plus system with 20% elongation for 36 h, silencing of the CHOP gene was identified by RT-PCR and Western blot. Inhibition of apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining. The effect of CHOP shRNA on disc degeneration was determined in vivo by using a rat model. At 7 weeks after intradiscal injection of the control or CHOP shRNA in the L4/L5 and L5/L6 discs, disc degeneration was assessed by X-ray examination, magnetic resonance imaging (MRI) assessment, and HE and TUNEL staining. A significant decrease in CHOP mRNA and protein expression was detected in AF cells with CHOP shRNA transfection after 36 h stretch. There was a significant decrease in apoptotic incidence in cells treated with CHOP shRNA, which was parallel to the expression of CHOP. Injection of CHOP shRNA in vivo resulted in the improvement in MRI and histologic score, and decrease in the apoptosis in the disc. No significant change in disc height was observed. In conclusion, a novel lentiviral vector expressing CHOP shRNA efficiently inhibits apoptosis in rat AF cells by silencing CHOP expression. In a rat model, intradiscal injection of CHOP shRNA induces the suppression of disc degeneration. The therapeutic effects of lentiviral CHOP shRNA should be further explored.
authors.affiliation: Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200092, China.
authors.forename: Yue-Hui
authors.initials: YH
authors.lastname: Zhang
authors.forename: Chang-Qing
authors.initials: CQ
authors.lastname: Zhao
authors.forename: Lei-Sheng
authors.initials: LS
authors.lastname: Jiang
authors.forename: Li-Yang
authors.initials: LY
authors.lastname: Dai
mesh_terms: D000818:Animals; D017209:Apoptosis; D001696:Biomechanical Phenomena; D002478:Cells, Cultured; D015536:Down-Regulation; D005260:Female; D020868:Gene Silencing; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007403:Intervertebral Disc / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy*; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D051742:Transcription Factor CHOP / Q000235:genetics* / Q000378:metabolism
chemical_list: C497251:Ddit3 protein, rat; D034741:RNA, Small Interfering; D051742:Transcription Factor CHOP