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Osmolarity fluctuations are inevitable within the nucleus pulposus (NP). However, the effects of osmolarity on NP cell apoptosis within the organ-cultured disc remain unclear. The objective of this study was to investigate effects of different osmolarity levels (hypo-, iso-, and hyper-) and osmolarity modes (constant and cyclic) on NP cell apoptosis in a disc perfusion culture and to study the role of the ERK1/2 pathway in this regulatory process. Porcine discs were cultured for 7 days in different osmotic medium, including constant hypo-, iso-, and hyper-osmolarity (330, 430, and 550 mOsm/L, respectively) and cyclic-osmolarity (430 mOsm/L for 8 h, followed by 550 mOsm/L for 16 h). The role of the ERK1/2 pathway was investigated by using the pharmacological inhibitor U0126. NP cell apoptosis was analyzed by TUNEL staining, caspase-3 activity, gene expression of Bcl-2, Bax and caspase-3 and protein expression of cleaved caspase-3, and cleaved PARP. Our results showed that NP cell apoptosis was increased in hypo- and hyper-osmolarity cultures compared to iso- or cyclic-osmolarity culture, whereas the level of apoptosis in the iso-osmolarity culture was lower than that in the cyclic-osmolarity culture. When the ERK1/2 pathway was inhibited in the iso- and cyclic-osmolarity cultures, the level of NP cell apoptosis was significantly increased. In conclusion, the effects of osmolarity on NP cell apoptosis depend on the osmolarity level (hypo-, iso-, or hyper-) and osmolarity mode (constant or cyclic). Futhermore, inhibition of the ERK1/2 pathway promotes NP cell apoptosis in this process. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:86-92, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Objective To investigate the role of aquaporin-3 (AQP-3) in hyperosmolarity-induced apoptosis of cultured rat nucleus pulposus (NP) cells. Methods Rat NP cells were cultured under different osmotic pressures (330 and 550 mOsm/kg), and the proliferation and apoptotic rate of the cells were determined using CCK-8 assay and flow cytometry, respectively. Western blotting and real-time quantitative PCR were used to detect the expression levels of apoptosis-related molecules (Bcl-2, Bax, cleaved caspase 3/caspase 3), and immunocytochemistry, Western blotting and real-time quantitative PCR were used to detect the expression of AQP-3 in the cells. The effect of lentivirus-mediated AQP-3 overexpression on apoptosis of NP cells was investigated after hyperosmolar exposure. Results An osmotic pressure of 550 mOsm/kg, as compared with 330 mOsm/kg, significantly decreased NP cell proliferation, increased the cell apoptotic rate, upregulated the expression of the pro-apoptotic molecules Bax and cleaved caspase 3/caspase 3 (P < 0.05), and down-regulated the expression of the anti-apoptotic molecule Bcl-2 (P < 0.05). The cells with lentivirus-mediated AQP-3 overexpression showed an obviously lowered apoptotic rate following the hyperosmolar exposure and significantly reduced expression of Bax and cleaved caspase 3/caspase 3 (P < 0.05) with an enhanced expression of Bcl-2 (P < 0.05). Conclusion Hyperosmolar exposure significantly promotes apoptosis of NP cells and reduce the expression of AQP-3. AQP-3 overexpression can partly attenuate apoptosis of NP cells following a hyperosmolar exposure.

Background and Purpose: There is an urgent but unmet need for mitigating radiation-induced intestinal toxicity while radio sensitising tumours for abdominal radiotherapy. We aimed to investigate the effects of metformin on radiation-induced intestinal toxicity and radiosensitivity of colorectal tumours.Experimental Approach: Acute and chronic histological injuries of the intestine from mice were used to assess radioprotection and IEC-6 cell line was used to investigate the mechanisms in vitro. The fractionated abdominal radiation model of HCT116 and HT29 tumour grafts was used to determine the effects on colorectal cancer.Key Results: Metformin alleviated radiation-induced acute and chronic intestinal toxicity by optimising mitophagy which was AMPK-dependent. In addition, our data indicated that metformin increased the radiosensitivity of colorectal tumours with P53 mutation both in vitro and in vivo.Conclusion and Implications: Metformin may be a radiotherapy adjuvant agent for colorectal cancers especially those carrying P53 mutation. Our findings provide a new strategy for further precise clinical trials for metformin on radiotherapy. [ABSTRACT FROM AUTHOR]

Background/Aims: Mechanical overloading-induced nucleus pulposus (NP) apoptosis plays an important role in the pathogenesis of intervertebral disc degeneration. N-cadherin (N-CDH)-mediated signaling preserves normal NP cell phenotype. This study aims to investigate the effects of N-CDH on NP cell apoptosis under high-magnitude compression and the underlying mechanism behind this process. Methods: Rat NP cells seeded on scaffold were perfusion-cultured using a self-developed perfusion bioreactor for 5 days and experienced different magnitudes (2% and 20% compressive deformation, respectively) of compression at a frequency of 1.0 Hz for 4 hours once per day. The un-loaded NP cells were used as controls. Lentivirus-mediated N-CDH overexpression and inhibitor LY294002 were used to further investigate the role of N-CDH and PI3K/Akt pathway under highmagnitude compression, respectively. NP cell apoptosis was evaluated by caspase-3 activity measured using a commercial kit, flow cytometry, and expression of apoptosis-related molecules analyzed by real-time PCR and western blotting assays. Results: High-magnitude compression significantly increased apoptotic NP cells, caspase-3 activity and expression of pro-apoptotic molecules (Bax and caspase-3/cleaved caspase-3), but decreased expression of anti-apoptotic molecule (Bcl-2). High-magnitude compression decreased expression of N-CDH, p-Akt and p-GSK-3β. However, N-CDH overexpression attenuated NP cell apoptosis and increased expression of p-Akt and p-GSK-3β under high-magnitude compression. Further decreased expression of p-GSK-3β, but had no significant effects on N-CDH expression under high-magnitude compression. Conclusion: N-CDH can attenuate NP cell apoptosis through activating the PI3K/Akt-GSK-3β signaling under high-magnitude compression. [ABSTRACT FROM AUTHOR]