title: RIPK1/RIPK3/MLKL-mediated necroptosis contributes to compression-induced rat nucleus pulposus cells death.
abstract: The aim of this study was to systematically investigate the role of necroptosis in compression-induced rat nucleus pulposus (NP) cells death, as well as explore the underlying mechanisms involved. Rat NP cells underwent various periods of exposure to 1.0 MPa pressure. Cell viability and cell death were quantified by using cell counting kit-8 (CCK-8), and Calcein-AM/propidium iodine (PI) staining respectively. Necroptosis-associated target molecules receptor-interacing protein kinase 1 (RIPK1), phosphorylated RIPK1 (pRIPK1), receptor-interacing protein kinase 3 (RIPK3), phosphorylated RIPK3 (pRIPK3) and mixed lineage kinase domain-like (MLKL) were analyzed by Western-blot and RT-PCR. NP cells were also examined for morphological and ultrastructural changes, which can indicate necroptosis. To indirectly establish the presence of necroptosis, the RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 inhibitor GSK'872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) were utilized. The results established necroptosis was taking place in NP cells. The level of necroptosis increased in a time-dependent manner, and this effect was reduced by Nec-1 in vitro. Additionally, NP cells death were significantly attenuated following treatment with Nec-1, GSK'872 or NSA. SiRNA-induced knockdown of RIPK3 or MLKL increased cell survival rate, while knockdown of RIPK1 resulted in a decreased cell survival rate. In summary, RIPK1/RIPK3/MLKL-mediated necroptosis may play an important role in NP cells death induced by continuous mechanical stress. Treatment strategies which aim to regulate necroptosis may prove beneficial, by both reducing NP cells death and slowing IVD degeneration.
authors.affiliation: Department of Orthopaedic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou City, 450052, China. chensongfeng123@126.com.
authors.forename: Songfeng
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Lv
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Binwu
authors.initials: B
authors.lastname: Hu
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China. szwpro@163.com.
authors.forename: Zengwu
authors.initials: Z
authors.lastname: Shao
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Baichuan
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Kaige
authors.initials: K
authors.lastname: Ma
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Lin
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Min
authors.initials: M
authors.lastname: Cui
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D009336:Necrosis / Q000235:genetics / Q000473:pathology; D000070614:Nucleus Pulposus / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000187:drug effects / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*
chemical_list: D007093:Imidazoles; D007211:Indoles; D034741:RNA, Small Interfering; C507699:necrostatin-1; C000610381:MLKL protein, rat; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C586536:RIPK1 protein, rat; D053422:Receptor-Interacting Protein Serine-Threonine Kinases
keywords: Compression; Intervertebral disc degeneration; Necroptosis; Necrostatin-1 (Nec-1); Nucleus pulposus cells