| Chronic pain,also known as "immortal cancer",has a long course and is hard to cure.The long-term suffering of chronic pain has brought serious harm to the patients' physical and mental health and quality of life,and the patients have paid a heavy economic cost for the treatment of chronic pain.In recent years,with the continuous improvement of cell and molecular research technology,the research on the pathogenesis of chronic pain has made great progress.Peripheral and central hyperalgesia has been proved to be the most important neurological mechanism leading to chronic pain.However,due to the lack of clear molecular mechanism of the disease,the lack of effective diagnosis standards and treatment programs,the current treatment effect is still not optimistic.The most commonly used painkillers are opioids and NSAIDs.Because of the limited therapeutic effect of NSAIDs on pain,they can not be widely used as analgesics.Opioids are more likely to cause serious side effects such as mental addiction,respiratory depression,nausea and vomiting,resulting in their use being greatly limited.Therefore,it is of great medical and social significance to study the pathogenesis of chronic pain for researchers to find new targets,explore new methods and formulate new strategies in pain treatment.It is pointed out that the c AMP-PKA signaling pathway plays an important role in the formation of neuropathic pain.It not only mediates peripheral hyperalgesia caused by inflammatory,but also participates in many regulation pathways of neuropathic pain.At the same time,it has also been pointed out that hyperpolarization activated cyclic nucleotide gated channels(HCN)are closely related to the occurrence of neuropathic pain.According to previous literature reports,we found that c AMP-PKA and HCN are involved in the formation of inflammatory hyperalgesia;other reports pointed out that in a variety of physiological activities,the activation of HCN channel is regulated by c AMP PKA signal pathway.Therefore,we further designed experiments to explore how c AMPPKA signaling pathway and HCN channel participate in and mediate the formation of inflammatory hyperalgesia through the application of animal behavioral detection and molecular biological detection technology.Part 1 To explore the effect of c AMP-PKA signal pathway on basic pain threshold in normal physiological state and the role of hyperalgesia in inflammatory pathological state Objective: To verify the effect of c AMP-PKA pathway on basic pain threshold in physiological state and hyperalgesia in inflammatory pathological state.Methods: 16 mice in WT group,PKA?KO group and forsklin group were randomly divided into two groups according to different groups,six groups in total;three groups of mice were used for physiological state research,and three groups of mice were used for inflammatory pathological state research;by measuring the mechanical and thermal pain thresholds of WT mice,PKA?KO mice and forsklin mice in physiological state,we can judge whether the three groups of mice have different responses to mechanical and thermal stimulation;the other three groups of mice were injected with Freund's complete adjuvant on one side of the hind foot to establish the inflammatory hyperalgesia model,and the mechanical pain threshold and thermal pain threshold of the three groups of mice were observed before and after the establishment of the model(6h,12 h,24h,48 h,96h),To determine whether there are differences in mechanical and thermal hyperalgesia among the three groups of mice in inflammatory hyperalgesia pathological state.Results: In normal physiological state,compared with WT group,PKA?KO group showed a significant decrease in response to mechanical and thermal stimulation,with a significant increase in pwtl and pwmt values(P<0.001),while forsklin group showed a significant increase in response to mechanical and thermal stimulation,with a significant decrease in pwtl and pwmt values(P<0.001);the results of repeated measurement analysis of variance showed that: After CFA injection,mechanical and thermal hyperalgesia were found in all the three groups,and pwtl and pwmt decreased significantly(P<0.001);The results of intergroup comparison showed that the pwtl and pwmt values of PKA?KO group were significantly higher than those of WT group at different time points(P<0.001),while the pwtl and pwmt values of forsklin group were lower than those of WT group after CFA injection(P<0.001).Conclusion: In physiological state,c AMP-PKA signaling pathway may play an important role in CO-sensing external mechanical stimulation and thermal stimulation,while in inflammatory hyperalgesia pathological state,c AMP-PKA signaling pathway may play a more important role in the formation of mechanical and thermal hyperalgesia.Part 2 To explore the effect of c AMP-PKA pathway on DRG neurons in normal physiological state and inflammatory hyperalgesia pathological state Objective: To verify the effect of c AMP-PKA pathway on the excitability of DRG neurons in physiological and inflammatory hyperalgesia pathological conditions.Methods: The source and grouping of the experimental mice were the same as the first part.After the behavioral test,the mice were killed and the DRG tissues were extracted.The whole-cell patch clamp technique was used for recording and analysis the active membrane characteristic(the basic intensity,frequency,amplitude,threshold and half width of action potential(AP))and passive membrane characteristics(resting potential(RMP),membrane resistance(RM),membrane capacitance(CM)).We can determine whether the excitability of DRG neurons in different groups of mice in different conditions is different,by observing the active and passive membrane characteristics of DRG neurons in three groups of mice between physiological conditions and after CFA injection for 24 h.Results: In physiological state,RMP,RM and CM of DRG neurons in the three groups were not different(P>0.05),while the base intensity and frequency of AP were significantly different: compared with WT group,the base intensity in PKA?KO group was increased significantly,and the frequency was decreased significantly(P<0.01),while the base intensity in forsklin group was decreased significantly,and the frequency was increased significantly(P<0.01);there was no difference in the amplitude,threshold and half width of AP among the three groups(P>0.05).24 hours after CFA injection,compared with the physiological state,the excitability of DRG neurons in the three groups increased significantly,which showed that the base intensity of AP was decreased significantly,and the frequency was increased significantly,and the difference was statistically significant(P<0.01);The results of intergroup comparison showed that compared with WT mice,the base intensity in PKA?KO group was increased significantly,and the frequency was decreased significantly(P<0.01),while the base intensity in forsklin group was decreased significantly,and the frequency was increased significantly(P<0.01);There was no significant difference in amplitude,threshold,half width,and RMP,RM,CM between the three groups(P>0.05).Conclusion: c AMP-PKA signaling pathway may play an important role in the excitability of DRG neurons in physiological state and hyperactivity of DRG neurons in inflammatory hyperalgesia pathological state Part 3 To study the effect of c AMP-PKA signaling pathway on HCN channel of DRG neurons in inflammatory hyperalgesia Objective: To verify whether c AMP-PKA signal pathway is involved in the activation of HCN channel of DRG neurons in inflammatory hyperalgesia.Methods: The source,grouping and tissue extracting methods of the experimental mice were the same as the second part,which will not be described here;Western blotting and real-time fluorescent quantitative PCR(q PCR)were used to detect the HCN channel protein and gene in different groups of mice in physiological and inflammatory pain pathological conditions.The expression level of HCN 2 channel protein and gene in different groups of mice in the two conditions was observed to verify whether c AMP-PKA signaling pathway is involved in the activation of HCN channel in DRG neurons during the process of inflammatory hyperalgesia.Results: In physiological state,compared with WT group,the expression of HCN2 protein and HCN2 m RNA in PKA?KO group decreased significantly(P<0.05,P<0.01),while in forsklin group,the expression of HCN2 protein and HCN2 m RNA increased significantly(P<0.05,P<0.01).In the inflammatory hyperalgesia pathological state,The expression levels of HCN2 protein and HCN2 m RNA in DRG of three groups of mice were significantly higher than those in physiological state(P<0.05,P<0.01).The results of intergroup comparison showed that compared with WT group,the expression of HCN2 protein and HCN2 m RNA in PKA?KO group decreased significantly(P<0.05,P<0.01),while in forsklin group,the expression of HCN2 protein and HCN2 m RNA increased significantly(P<0.05,P<0.01).Conclusion: 1.Compared with the physiological state,the opening of HCN2 channel in DRG neurons of mice in inflammatory pain pathological state increased significantly and was closely related to the level of c AMP-PKA.2.The activation of HCN pathway may be involved in the signal transmission of c AMPPKA in inflammatory hyperalgesia. |
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