The Role Of P2x₇ Receptor In Dental Pulp Tissue In A Rat Model Of Acute Pulpitis Pain

Objective:To investigate the interrelationship among the expression of P2X₇receptor,pulpitis and pulpitis pain,and to explore the role of P2X₇receptor activation in the formation and maintenance of acute pulpitis pain in an lipopolysaccharide-induced acute pulpitis rat model.Methods:A total of 60 adult Sprague Dawley rats（clean grade,male,210±10 g）were randomly divided into normal control group,normal saline（normal saline,NS）group and lipopolysaccharide（LPS）group（n=12）.The rat model of acute pulpitis was established in accordence with the standard protocol in rats of NS group and LPS group.The head-withdrawal reflex threshold values of normal control group,NS group and LPS group rats were measured 15 min before modeling and 24 hours after modeling,and the pain responses of facial trigeminal nerve distribution area to mechanical stimulation was evaluated before and after modeling.After the detection of facial pain threshold,the pathological changes of dental pulp tissue of rats in normal control group,NS group and LPS group were observed by Hematoxylin-Eosin stain（HE）staining,and the expression areas of P2X₇receptor in dental pulp tissue of normal control group,NS group and LPS group were tested by immunohistochemistry.The expression levels of P2X₇receptor protein in dental pulp of rats in each group were detected by Western Blot.Another 24 rats were selected to establish an acute pulpitis model（induced by LPS）.They were randomly divided into LPS+DMSO group,LPS+0.1 mg A-740003 group,LPS+1 mg A-740003group and LPS+10 mg A-740003 group（n=6）.24 hours after the establishment of the model,25μL DMSO and different doses of selective P2X₇receptor antagonist A-740003（0.1 mg/kg,1 mg/kg,10 mg/kg,n=6）were injected into the periodontal ligament space of the experimental rats in each group.The head-withdrawal reflex thresholds of 15 min before drug administration,and 15,30,45 and 60 min after drug administration were measured to explore the role of P2X₇receptor activation in acute pulpitis pain.Results:1.Rat head-withdrawal reflex thresholds before and after modeling.Fifteen minutes before modeling,the head-withdrawal reflex thresholds of normal control group,NS group and LPS group were 31.37±1.84 g,31.98±1.43 g and 31.05±2.86 g,respectively.Twenty-four hours after modeling,the head-withdrawal reflex thresholds of normal control group,NS group and LPS group were 30.83±2.21 g,20.20±1.36 g and 15.11±0.93 g,respectively,and the threshold values of head-withdrawal reflex in NS group and LPS group were significantly lower than normal control group（P<0.001）,and the pain threshold in LPS group was far lower than NS group（P<0.001）.2.HE staining.In the normal control group,evenly distributed fibroblasts were found in the crown pulp and root pulp tissues without an obvious inflammatory cell aggregation.In NS group,only fibroblasts gathered in the coronal pulp and only sparse neutrophils and lymphocytes gathered in the center of the mass.In LPS group,a large number of fibroblasts,neutrophils and lymphocytes were gathered densely and focally.The necrosis and exfoliation of the dental pulp tissue appeared in the center of inflammation,and with small abscess formation in some areas,along with telangiectasia and loss of continuity of odontoblast layer.3.Immunohistochemical staining.In normal control group,the positive expression of P2X₇receptor mainly existed in the odontoblast layer,and there was unobvious positive expression of P2X₇receptor in the dental pulp tissue of all segments of the root.In the NS group,the positive expression of P2X₇receptor in the odontoblast layer of the crown pulp,root neck and middle root was light brown granule,its expression was weakly positive,and the P2X₇receptor was sparsely distributed in the apical segment.In LPS group,the expression of P2X₇receptor was dark brown granules with strongly positive representations in all segments of crown pulp and root.4.Western Blot detection.Twenty-four hours after modeling,the dental pulp tissues of normal control group,NS group and LPS group were taken and positive bands of P2X₇receptor protein expression were found by WB.Statistical analysis showed that the relative expression of P2X₇receptor protein in NS group was significantly higher than that in normal control group（P<0.001）.The expression of P2X₇receptor protein in LPS group was markedly higher than that in NS group（P<0.001）.5.Effect of A-740003 preconditioning on head-withdrawal reflex threshold in rats of acute pulpitis pain.There were interactive effects between administration time and dose in each group（P<0.05）.Before administration,there was no significant difference in head-withdrawal reflex threshold between LPS+DMSO,LPS+0.1 mg A-740003 group,LPS+1 mg A-740003 group and LPS+10 mg A-740003 group（P>0.05）.There was no significant difference in the threshold values of head-withdrawal reflex in LPS+DMSO group at 15 min pre-administration and at each observation time point post-administration（P>0.05）.There was no significant difference in the threshold of head-withdrawal reflex between LPS+0.1 mg A-740003 group and LPS+DMSO group pre-and post-administration（P>0.05）.The threshold values of head-withdrawal reflex of LPS+1 mg A-740003 group and LPS+10 mg A-740003 group rats were significantly higher than that in LPS+DMSO group at 30 min post-administration,and reached the peak value at 60 min after administration（P<0.001）.60 min post-administration,the threshold of head-withdrawal reflex in LPS+10 mg A-740003 group was significantly higher than that in LPS+1 mg A-740003 group（P<0.05）.Conclusion:1.Acute pulpitis induced by LPS can up-regulate the expression level of P2X₇receptor in odontoblast layer of dental pulp and activate its function.Within 24 hours after modeling,the expression level of P2X₇receptor protein was related to the inflammation severity and the pain intensity of pulpitis.2.Local injection of P2X₇receptor antagonist A-740003 into the periodontal ligament can significantly and dose dependently relieve the pain of acute pulpitis in rats,indicating that the P2X₇R activation in dental pulp tissue plays a driving role in acute pulpitis pain.