The Study On The Mechanism Of Acid-Sensing Ion Channels 1a Involved In The Repair Of Degenerated Intervertebral Disc By Transplantation Of Bone Marrow Mesenchymal Stem Cells

Low back pain (LBP) is one of the most debilitating conditions, resulting in substantial socioeconomic and health-care consequences. The most frequent cause of LBP is lumbar intervertebral disc degeneration (IVDD). Current treatment options that address the pain and disability resulting from lumber disc degeneration include non-operative approaches and surgery such as discectomy, spinal fusion and disc arthroplasty. However, these treatments do not preserve the function of the intervertebral disc (IVD). Various biological therapies have attempted to prevent or to reverse the disc degenerative process, and they do this by utilizing growth factors, cell transplantation, and gene therapy. However the biochemical and biophysical changes in a degenerated IVD will present a harsh microenvironment for both the endogenous and transplanted cells in cell-based therapy. The disc microenvironment is characterized by low pH, reduced oxygen supply, and reduced nutrition as well as high osmolarity. Studies demonstrated that the most challenging chemical condition in this microenvironment is its matrix acidity, which has a potentially negative effect on cell viability and function. Activation or sensitization of Ca2+-permeable ASICla is responsible for acidosis-mediated cell injury caused by Ca2+ influx in neurons and chondrocytes.Acid-sensing ion channels (ASICs) are H+-gated voltage-insensitive ion channels, and belong to the degenerin/epithelial Na+channel superfamily. The ASIC family has six subunit proteins (ASICla, ASIClb, ASIC2a, ASIC2b, ASIC3 and ASIC4) that are encoded by four genes (ASIC1, ASIC2, ASIC3 and ASIC4). Homomeric ASIC1a channels are Na+ and Ca2+ permeable, whereas other combinations are only permeable to Na+. Activation or sensitization of Ca2+-permeable ASIC la is responsible for acidosis-mediated ischemic brain injury and rheumatoid arthritis caused by Ca2+influx in neurons and chondrocytes, respectively. Intracellular Ca2+([Ca2+]i) represents a prevalent signaling mechanism and is involved in processes such as cell division and injury. However, it is uncertain whether ASIC la mediates directly Ca2+ influx and increased [Ca2+]i in transplanted BMSC in disc. It is also uncertain whether ASIC 1a contributes to BMSC injury through an acidosis-evoked increase in [Ca2+]i. Therefore, this study is divided into the following four parts: Part 1:Investigate the curative effects of transplantation of BMSC on intervertebral disc regeneration Part 2:Investigate the expression of ASIC 1a in rat nucleus pulposus tissue and BMSC Part 3:Investigate the acidosis-mediated ASIC 1a activation and BMSC injury Part 4:Investigate the mechanism of ASIC 1a-mediated BMSC injuryPart 1:Investigate the curative effects of transplantation of BMSC on intervertebral disc regenerationPurpose:Investigate the curative effects of transplantation of BMSC on intervertebral disc regeneration.Method:Forty-eight rabbits were used to establish disc degeneration model by stabbing the annulus fibrosus and then divided into four groups randomly, i.e. two weeks after wards, BMSC or phosphate-buffered saline (PBS) were transplanted into degenerated discs (BMSC group and PBS group), while the operated rabbits without implantation of BMSC or PBS served as the sham group and the rabbits without any operation were used as the control group. At weeks two, six and ten after implantation, the T2 values and disc height indices (DHI) were calculated by magnetic resonance imaging (MRI 3.0 T), and the type â…¡ collagen (COL2) and aggrecan (ACAN) gene expression in degenerated discs were detected by real-time reverse tran-scription polymerase chain reaction (RT-PCR). T2 relaxation time for the nucleus pulposus were correlated with ACAN or COL2 expression by regression analysis.Result:Disorganised fibres, cell clusters and interlamellar glycosaminoglycan (GAG) matrix were observed in degenerated discs. The gene expression of COL2 and ACAN increased during ten weeks after transplantation as well as the T2 signal intensity and T2 relaxation time. The DHI in BMSC group decreased more slowly than that in PBS group. T2 relaxation time correlated significantly with the gene expression of ACAN and COL2 in nucleus pulposus. Conclusion:Transplantation of BMSC is able to promote the regeneration of degenerated discs to some extent. During the follow-up of 10 weeks, gene expression of ACAN and COL2 increases, while the DHI still decreases progressively. Thus, the repair efficiency of BMSC on disc degeneration needs to be improved.Part 2:Investigate the expression of ASIC la in rat nucleus pulposus tissue and BMSCPurpose:Investigate the expression of ASIC 1a in rat nucleus pulposus tissue and BMSC.Method:RT-PCR, Western blot and Immunohistochemistry were used to dectect the expression of ASIC 1a. Compared the expression of ASIC la in normal and degenerated rat nucleus pulposus tissue. Compared the expression of ASIC 1a in rat BMSC before and after co-culture with degenerated rat nucleus pulposus cells.Result:The results showed that ASIC 1a was expressed in rat nucleus pulposus tissue and BMSC. The expression of ASIC 1a was much high in degenerated rat nucleus pulposus tissue than normal. Co-culture with degenerated rat nucleus pulposus increased the expression of ASIC1a in BMSC.Conclusion:ASIC 1a is expressed in rat nucleus pulposus tissue and BMSC. Disc degeneration induces the expression of ASIC 1a.Part 3:Investigate the acidosis-mediated ASICla activation and BMSC injuryPurpose:Investigate the acidosis-mediated ASIC1a activation and BMSC injure.Method:Intracellular calcium ([Ca2+]i) was determined by Ca2+-imaging using Fura-2-AM. Cell vitality, apoptosis and cell cycle following acid exposure were determined using MTT assay, annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual-staining and cell cycle analysis. Effects of treatment with psalmotoxin-1 (PcTX1, blocker of ASIC1a) on [Ca2+]i and cell survival were investigated.Result:Acid exposure increased in [Ca2+]i, cell apoptosis and cell cycle arrest, decreased cell vitality. When BMSC were treated with PcTX1, acid induced increases in [Ca24]i were significantly inhibited. PcTX1 treatment also resulted in increased cell vitality, decreased cell apoptosis and cell cycle arrest in acid condition.Conclusion:The ASICla on BMSC can be activated in acid condition. The activation of ASIC1a induced BMSC injury.Part 4:Investigate the mechanism of ASICla-mediated BMSC injuryPurpose:Investigate the mechanism of ASIC1a-mediated BMSC injury.Method:Effects of treatment with PcTX1 on acid-induced activation of calcium-dependent proteases was investigated. BAD phosphorylation and the activation of caspase-9, caspased-8 and caspase-3 in BMSC was also investigated using western blot after acid exposure. The morphology of mitochondria was observed by electron microscope after acid exposure.Result:We demonstrated that the acid-induced [Ca2+]i increase via ASICla is involved in BMSC apoptosis. Moreover, blocking of ASIC1a-mediated [Ca2+]i elevation inhibited activation of acid-induced calcium-dependent proteases and decreased BAD phosphorylation in BMSC. Decreased BAD phosphorylation resulted the activation of caspase-9 and caspase-3, which was inhibited by PcTX1.Conclusion:ASIC1a activation in BMSCs may trigger Ca2+-dependent proteases activity and signaling, which leads to apoptosis of BMSCs in IVDs.