Hydrogen peroxide induces programmed necrosis in rat nucleus pulposus cells through the RIP1/RIP3‐PARP‐AIF pathway

This study aimed to systematically investigate whether programmed necrosis contributes to H₂O₂‐induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H₂O₂ for various time periods. The cell viability was measured using a cell counting kit‐8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis‐related molecules receptor‐interacting protein 1 (RIP1), receptor‐interacting protein 3 (RIP3), poly (ADP‐ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real‐time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin‐1 (Nec‐1), the PARP inhibitor diphenyl‐benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H₂O₂ induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec‐1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3‐mediated programmed necrosis, executed through the PARP‐AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1269–1282, 2018.

     

Combined treatment with a β3‐adrenergic receptor agonist and a muscarinic receptor antagonist inhibits detrusor overactivity induced by cold stress in spontaneously hypertensive rats

Aims

This study determined if combined treatment with the muscarinic receptor (MR) antagonist solifenacin and the β₃‐adrenergic receptor (AR) agonist mirabegron could inhibit detrusor overactivity induced by cold stress in spontaneously hypertensive rats (SHRs).

Methods

Thirty‐two female 10‐week‐old SHRs were fed an 8% NaCl‐supplemented diet for 4 weeks. Cystometric measurements of the unanesthetized, unrestricted rats were performed at room temperature (RT, 27 ± 2°C) for 20 min. The rats were then intravenously administered vehicle, 0.1 mg/kg solifenacin alone, 0.1 mg/kg mirabegron alone, or the combination of 0.1 mg/kg mirabegron and 0.1 mg/kg solifenacin (n = 8 each group). Five minutes later, the treated rats were exposed to low temperature (LT, 4 ± 2°C) for 40 min. Finally, the rats were returned to RT. After the cystometric investigations, the β₃‐ARs and M₃‐MRs expressed within the urinary bladders were analyzed.

Results

Just after transfer from RT to LT, vehicle‐, solifenacin‐, and mirabegron‐treated SHRs exhibited detrusor overactivity that significantly decreased voiding interval and bladder capacity. However, treatment with the combination of solifenacin and mirabegron partially inhibited the cold stress‐induced detrusor overactivity patterns. The decreases of voiding interval and bladder capacity in the combination‐treated rats were significantly inhibited compared to other groups. Within the urinary bladders, there were no differences between expression levels of M₃‐MR and β₃‐AR mRNA. The tissue distribution of M₃‐MRs was similar to that of the β₃‐ARs.

Conclusions

This study suggested that the combination of solifenacin and mirabegron act synergistically to inhibit the cold stress‐induced detrusor overactivity in SHRs. Neurourol. Urodynam. 36:1026–1033, 2017. © 2016 The Authors. Neurourology and Urodynamics Published by Wiley Periodicals, Inc.