| Objective:The purpose of this study is to observe the changing law of acid-sensitive ion channels(ASICs)during CSD and the pretreatment effect of acupuncture,and to study the mechanism of acupuncture preventive treatment of migraine from the perspective of inhibiting the activation of acid-sensitive ion channels.Method:The SD rats were randomly divided into blank group,sham operation group,model group,acupuncture pretreatment group,and western medicine pretreatment group.Among them,the acupuncture pretreatment group was treated to Yanglingquan and Taichong points 14 days before CSD modeling Electrical acupuncture stimulation,intensity 1m A,density wave,2/15 Hz,20 minutes,1 time/day;the western medicine pretreatment group was given flunarizine hydrochloride 20 mg/kg intraperitoneal injection,1 time 14 days before CSD modeling Day;blank group,sham operation group and model group do not do any intervention except daily grabbing.Except for the blank group,all groups were anesthetized after 14 days.The sham operation group was stimulated with 0.9% Nacl to stimulate the dura,and the remaining groups were used to stimulate the dura with 1mol/L KCL to replicate the migraine CSD model,which was detected by Western Blot and immunofluorescence.The content of ASIC1 in the cortex of the migraine side and ASIC3 in the nucleus of the trigeminal spinal tract in each group of rats;immunohistochemical method was used to observe the protein distribution of CGRP in the trigeminal ganglion and the nucleus of the spinal trigeminal nerve in each group of rats Situation:ELISA method was used to detect the content of 5-HT and CGRP in the serum of rats in each group.Result:(1)The effect of acupuncture pretreatment on the content of ASIC1 in the cortex and nucleus of the trigeminal nerve spinal tract of migraine CSD model mice:(1)Western Blot detected the content of ASIC1 in the rat cortex,the results showed: blank group,sham operation group and model group There is a statistical difference in content(P<0.05);there is a statistical difference between the western medicine pretreatment group and the model group(P<0.05),although there is no statistical difference between the acupuncture pretreatment group and the model group(P>0.05),But the content of ASIC1 in the cortex of the acupuncture pretreatment group is less than that of the model group,and there is a downward trend.Although there is no statistical difference between the acupuncture pretreatment group and the western medicine pretreatment group,the content of ASIC1 in the acupuncture pretreatment group and the western medicine pretreatment group has a further downward trend than that of the western medicine pretreatment group.(2)The results of immunofluorescence detection of ASIC1 in rat cortex showed that the target antibody ASIC1 was green in rat brain cortex.The expression of the target antibody ASIC1 in the cortex of each group of rats can be clearly observed.The image shows that it is consistent with the results of the protein content determination.ASIC1 is also expressed in the cortex of the blank group of normal rats and sham-operated rats,and the migraine CSD model In mice,the expression of ASIC1 in the mouse cortex is more dense,and the number is significantly increased.The figure shows that the distribution of the two pretreatment groups is significantly less than that of the model group,and the range is also more scattered than the model group.(3)Western Blot detects the content of ASIC3 in the nucleus of the spinal tract of trigeminal nerve in rats.The results showed that: the blank group,the sham operation group and the model group were statistically different(P<0.05).Although there was no statistical difference between the western medicine pretreatment group and the acupuncture pretreatment group compared with the model group(P>0.05),the content of ASIC3 in the trigeminal tract nucleus of the two intervention groups was further decreased compared with the model group trend.There was no statistical difference between the western medicine pretreatment group and the acupuncture pretreatment group(P>0.05),but the content of ASIC3 in the acupuncture pretreatment group had a downward trend compared with the western medicine pretreatment group.(4)The results of immunofluorescence detection of ASIC3 in the nucleus of the spinal trigeminal nerve in rats showed that the expression of the target antibody ASIC3 in each group of rats could be clearly observed.The image showed that the distribution results were consistent with the protein content determination results,and it was normal in the blank group.ASIC3 is also expressed in mice and sham-operated rats,while in rats in the migraine CSD model group,ASIC3 is more intensively expressed in the nucleus of the trigeminal nerve spinal tract,and the number is significantly increased.The acupuncture pretreatment group and the western medicine pretreatment group have two Group,the expression of ASIC3 appears scattered,and even has a shrinking trend.(2)The effect of acupuncture preconditioning on the expression of CGRP in the trigeminal ganglion and trigeminal spinal nucleus of migraine CSD model mice:(1)The immunohistochemical results showed that CGRP was in the blank group of normal rats and sham-operated rats There is expression in the trigeminal ganglion.The immunohistochemical staining score results show that the expression in the model group,acupuncture group,and western medicine pretreatment group is higher than that of the blank group and the sham operation group(P<0.05),and the staining intensity of the model group is significantly higher Acupuncture pretreatment group and western medicine pretreatment group.The staining intensity in the two intervention groups showed a decreasing trend in the acupuncture pretreatment group and western medicine pretreatment group.(2)The immunohistochemical results showed that CGRP was in the trigeminal spinal nucleus of the control group and the sham operation group.The immunohistochemical staining score results showed that the expression of the model group,the acupuncture pretreatment group,and the western medicine pretreatment group was higher than that of the blank group and the sham operation group(P<0.05).The staining intensity of the model group was compared with the acupuncture pretreatment group.The treatment group and the western medicine pretreatment group increased significantly,and the staining intensity in the two intervention groups showed a decreasing trend in the western medicine pretreatment group and the acupuncture pretreatment group.(3)The effect of acupuncture pretreatment on the levels of 5-HT and CGRP in the serum of migraine CSD model mice:(1)Enzyme-linked immunosorbent assay(ELISA)detects the level of 5-HT in the serum,the results show that: the blank group and There was a statistical difference between the sham operation group and the model group(P<0.05),and the 5-HT content was significantly higher than that of the model group(P<0.05).Compared with the model group,the western medicine pretreatment group and the acupuncture pretreatment group were statistically different(P<0.05),and the 5-HT content was higher than that of the model group(P<0.05);the acupuncture pretreatment group and the western medicine pretreatment group There was no statistical difference between the two intervention groups in the two groups(P>0.05),but the 5-HT content of the acupuncture pretreatment group was further increased compared with the western medicine pretreatment group.And compared with the blank group,the acupuncture pretreatment group and the western medicine pretreatment group were statistically different(P<0.05).(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of CGRP in the serum.The results showed that there was no statistical difference between the blank group and the sham operation group(P>0.05),and the content of CGRP was lower than that of the model group(P<0.05).Although there is no statistical difference between the intervention groups in the acupuncture pretreatment group and the western medicine pretreatment group,the content of CGRP in the acupuncture pretreatment group and the western medicine pretreatment group has a further decrease and decline trend compared with the blank group.There was no statistical difference(P>0.05).The CGRP content of the acupuncture pretreatment group and the western medicine pretreatment group was lower than that of the model group(P<0.05).Conclusion:1.Acupuncture at Taichong and Yanglingquan points and flunarizine hydrochloride can prevent CSD.2.Acupuncture at Taichong and Yanglingquan points with flunarizine hydrochloride may prevent CSD by inhibiting the activation of acid-sensitive ion channels,thereby reducing cortical ASIC1 and trigeminal spinal nucleus ASIC3 protein content,and inhibiting trigeminal ganglia and trigeminal spinal tracts The expression of CGRP protein in the nucleus,downregulates the content of calcitonin gene-related peptide in the blood,and up-regulates the content of serotonin in the blood. |
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