| Objective: to study the effects of chrono-acupuncture on nerve electrical activity of acupoint area and P2X3 receptor in acpoint area,dorsal root ganglion and spinal cord of rats in different conditions and to reveal the peripheral mechanism of chrono-acupuncture analgesia based on dynamic acupoint reaction.Methods: the experiment consisted of three parts.First,clean SD male rats were domesticated in animal rhythm room for 7 days and were randomly divided into control group and acupuncture group.Each group had 6 sub-groups,which were ZT0,ZT4,ZT8,ZT12,ZT16 and ZT20 sub-groups respectively.At each time point,the rats in the acupuncture group were treated with acupuncture at the Zusanli acupoint(ST36)on the right side(twisted 5 min per 5 min,lasted for 30 min)and the electrical activity of the peroneal nerve was recorded by Power Lab.While the electrical activity of the peroneal nerve of rats in the control group was recorded by Power Lab without acupuncture treatment.Second,the domesticated rats were randomly divided into control group which was injected intradermally with 0.1 ml saline on the right foot pad of the rats and model replication group which was injected intradermally with 0.1 ml complete Freund’s adjuvant on the right foot pad of the rats.After the model was successfully established,the model replication group was randomly divided into model group and model-acupuncture group.On the second day of successful model establishment,at each time point,the rats in the model-acupuncture group were treated with acupuncture at the Zusanli acupoint(ST36)on the affected side(twisted 5 min per 5 min,lasted for 30 min)and the electrical activity of the peroneal nerve was recorded by Power Lab.While the electrical activity of the peroneal nerve of rats in control group and model group was recorded by Power Lab without acupuncture treatment.Third,the grouping method of part three was the same as part two.Based on the results of part two and previous experiments,the best time point and the worst time point were chosen respectively.At each time point,the rats in the model-acupuncture group were bound and treated with acupuncture at the Zusanli acupoint(ST36)on the affected side(twisted 1 min per 5 min,lasted for 30 min).The rats in control group and model group were bound without acupuncture treatment.After the end of treatment on the seventh day,the expression of P2X3 receptor in local skin tissue,dorsal root ganglia(L4-L6),and spinal cord(L4-L6)was detected by immunohistochemistry.Results:1.Acupuncture at different time points could promote the number of neural discharge under physiological conditions.Compared with control group,the difference in the acupuncture group at ZT0,ZT8,ZT12 and ZT20 respectively was statistically significant and corresponding P value was 0.002,0.000,0.044 and 0.000 respectively.While the difference at ZT4 and ZT16 was no statistical significance and corresponding P value was 0.470,0.237 respectively.There was an interaction between the time factor and the number of neural discharge in each group.Compared with the control group,the number of neural discharge in the acupuncture group was statistically significant at different time points,P=0.000,P<0.05.2.Acupuncture at different time points could promote the number of neural discharge and reduce the average amplitude of neural discharge under pathological conditions.The statistical analysis of the number of neural discharge in local acupoint was as follows: compare with control group,the difference in the model group at ZT12 and ZT16 respectively was statistically significant and corresponding P value was 0.002,0.000 respectively.While the difference at ZT0,ZT4 ZT8 and ZT20 was no statistical significance.Compare with control group,the difference in the model-acupuncture group at ZT8 and ZT20 respectively was statistically significant and corresponding P value was 0.000,0.013 respectively.Compare with model group,the difference in the model-acupuncture group at ZT4,ZT8,ZT12,ZT16 and ZT20 respectively was statistically significant and corresponding P value was 0.001,0.002,0.000,0.000 and 0.004 respectively.The statistical analysis of the average amplitude of neural discharge in local acupoint was as follows: compare with control group,the difference in the model group at ZT4,ZT8,ZT12,ZT16 and ZT20 respectively was statistically significant and corresponding P value was 0.000,0.000,0.007,0.009 and 0.002 respectively.Compare with control group,the difference in the model-acupuncture group at ZT0,ZT4,ZT12,ZT16 and ZT20 respectively was statistically significant and corresponding P value was 0.001,0.000,0.003,0.016 and 0.003 respectively.Compare with model group,the difference in the model-acupuncture group at ZT4,ZT8,ZT12 and ZT20 respectively was statistically significant and corresponding P value was 0.000,0.000,0.037 and 0.009 respectively.There was an interaction between the time factor and the number of neural discharge in each group.The difference of the number of neural discharge among control group,model group and model-acupuncture group was statistically significant at different time points,P=0.000,P<0.05.There was an interaction between the time factor and the average amplitude of neural discharge in each group.The difference of the average amplitude of neural discharge among control group,model group and model-acupuncture group was statistically significant at different time points,F=17.433,P=0.000,P<0.05.3.In skin of Zusanli(ST36): compared with control group,the expression of P2X3 receptor in the model group was significantly increased at ZT8,P=0.000,P<0.01.compared with model group,the expression of P2X3 receptor in the model-acupuncture group was significantly decreased at ZT8,P=0.000,P<0.01.Compared with control group,the expression of P2X3 receptor in the model group was significantly increased at ZT16,P=0.003,P<0.01.Compared with model group,the difference of P2X3 receptor in the model-acupuncture group was no significant at ZT16.4.In dorsal root ganglion(L4-L6): compared with control group,the expression of P2X3 receptor in the model group was significantly increased at ZT8,P=0.000,P<0.01.compared with model group,the expression of P2X3 receptor in the model-acupuncture group was significantly decreased at ZT8,P=0.009,P<0.01.Compared with control group,the expression of P2X3 receptor in the model group was significantly increased at ZT16,P=0.000,P<0.01.Compared with model group,the difference of P2X3 receptor in the model-acupuncture group was no significant at ZT16.5.In spinal cord(L4-L6): compared with control group,the expression of P2X3 receptor in the model group was significantly increased at ZT8,P=0.000,P<0.01.compared with model group,the expression of P2X3 receptor in the model-acupuncture group was significantly decreased at ZT8,P=0.000,P<0.01.Compared with control group,the expression of P2X3 receptor in the model group was significantly increased at ZT16,P=0.000,P<0.01.Compared with model group,the difference of P2X3 receptor in the model-acupuncture group was no significant at ZT16.Conclusions:1.The nerve electrical activity in local acupoint is time-dependent.Not only acupuncture at different time,but the functional state of the body has different effects on nerve electrical activity in local acupoint.2.The mechanisms of chrono-acupuncture analgesia may be related to the effects of acupuncture at different time points on the expression of P2X3 receptor in the local acupoint,dorsal root ganglion and spinal cord. |
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