A Study On The Mechanism Of Proton Sensing Receptor Regulates The Apoptosis Of Human Nucleus Pulposus Cells

Objective:1.To study the effect of acid environment on the apoptosis of nucleus pulposus cells of human intervertebral disc.2.To explore whether there are OGR1 subfamily receptors in the nucleus pulposus cells of human intervertebral disc,and whether acid environment could activate OGR1 subfamily receptors in the nucleus cells of human intervertebral disc.3.To investigate the molecular mechanism of acidic environment regulates the apoptosis of nucleus pulposus cells of human intervertebral disc and provide theoretical basis for prevention and treatment of intervertebral disc degeneration.Methods:The excised human intervertebral discs were obtained,and the nucleus pulposus were carefully separated and cultured in vitro.The morphology of the nucleus pulposus cells of human intervertebral disc was observed under the inverted phase contrast microscope.The cultured cells were observed and identified by rayleigh giemsa,toluidine blue,safranin O and type II collagen immunocytochemical staining.Next,The P3 cells were randomly divided into three groups:cells in the control group were cultured with common medium（PH 7.4）,cells in the acidic group were cultured with acidic medium（PH 6.8）,cells in the inhibited group were cultured with acidic medium（PH 6.8）+Cucl₂（acidic proton receptor inhibitor）.After 24 hours,the morphological changes of cells nuclei after DAPI staining in the control group and the acid group were observed by laser confocal microscope.The apoptosis rates of nucleus pulposus cells in the control group and the acidic group were detected by flow cytometry.The gene expression of OGR1 subfamily receptors（OGR1、G2A、TDAG8 and GPR4）in each group were detected by real-time quantitative PCR（qRT-PCR）.The effect of acid stimulation on Ca²⁺level in nucleus pulposus cells were observed by laser confocal microscopy.The protein expression of Bcl-2,Bax and Caspase-3 in each group were detected by enzyme-linked immunosorbent assay（Elisa）.Results:Under inverted phase contrast microscope,the obtained cells after the digestion of nucleus pulposus were small spherical.The nuclei were clear and bright.The cytoplasm was rich.The cells floated in the cell culture medium,and sometimes a few cells that had not been completely digested and held together were also seen.The primary nucleus pulposus cells attached to the wall for a long time.After 24hours,a few cells began to attach to the wall slowly.After 7 days,most cells attached to the wall.The attached cells gradually changed to short fusiform or polygon.The nuclei were round and large.The cytoplasm was rich and contained the secretory corpuscle.About 7 days or so,the cells began to connect with each other,and the cells were monolayer.After 2 to 3 weeks,the cells growth was getting faster and faster,and appeared active cells proliferation and fusion.Under inverted phase contrast microscope,cells can be seen to spread over the bottom of the culture bottle,and multiple layer cells could be seen at the active site.Compared with the primary nucleus pulposus cells,the attachment time of the submedullary nucleus pulposus cells was shortened obviously,about 2 to 3 hours.The speeds of cells proliferation and fusion were also accelerated,and the cell bottom was filled with cells about 7days.The results of rayleigh giemsa showed that the cultured cells in vitro were short fusiform or polygon.The nuclei in the center were rounded or elliptic and dyed red and purple.The cytoplasm was rich and stained slighted.The results of toluidine blue,safranin O and type II collagen immunocytochemical staining were positive,and it was proved that the cells cultured in the experiment were nucleus pulposus cells.The results of DAPI staining showed that the nuclei of nucleus pulposus cells were blue fluorescence under laser confocal microscope.The nuclei in the control group were round or elliptic and intact.The chromatin was uniform.The nuclei in the acidic group were irregular and appeared nuclear pyknosis and granular substance.The cells apoptosis rates detected by flow cytometry in the control group and the acid group were 1.03±0.30 and 37.14±2.14 respectively.There were statistical significant（P<0.05）.The results of qRT-PCR showed that OGR1,G2A and GPR4mRNA were expressed in human nucleus pulposus cells,but no TDAG8mRNA expression was observed.The expression of OGR1mRNA was significantly higher in the acidic group（P<0.05）.In the inhibited group,the expression of OGR1mRNA was significantly decreased（P<0.05）,while G2A and GPR4mRNA did not change significantly（P>0.05）.The results of laser confocal microscopy showed that the Ca²⁺levels increased both in the control group and the inhibited group after stimulation with acidic conditions.The highest fluorescence intensity of nucleus pulposus cells in the control group was 4095,and the highest fluorescence intensity was 1536 in the inhibited group.The intracellular Ca²⁺level was significantly increased by acid stimulation,and the increase of intracellular Ca²⁺level was inhibited by the inhibitor.The results of Western Blot showed that there were expressions of OGR1,Calpain and Calcineurin in nucleus pulposus cells of human intervertebral disc under acid environment.The expressions of OGR1,Calpain and Calcineurin were inhibited after the inhibitor was added.The results of Elisa showed that the acidic environment could inhibit the expression of Bcl-2 protein and promote the expression of Bax and Caspase-3 protein in nucleus pulposus cells（P<0.05）.The inhibitor could promote the expression of Bcl-2 protein and inhibit the expression of Bax and Caspase-3 protein（P<0.05）.Conclusion:1.Acidic environment could promote the apoptosis of nucleus pulposus cells of human intervertebral disc.2.OGR1 subfamily receptors are present in the nucleus pulposus cells of human intervertebral disc.The acidic environment could activate OGR1 receptor in the nucleus pulposus cells of human intervertebral disc.3.In acidic environment,the activation of OGR1 receptor in the nucleus pulposus cells of human intervertebral disc could trigger the increase of intracellular Ca²⁺level.Ca²⁺could activate the Ca²⁺sensitive proteins-Calpain and Calcineurin.Activated Ca²⁺sensitive proteins-Calpain and Calcineurin could further inhibit the Bcl-2 protein and activate Bax and Caspase-3 protein,which ultimately leads to cell apoptosis.