| Pain is a complex feeling,an unpleasant feeling and emotional experience,often accompanied by actual or potential tissue damage,which is one of the most common clinical conditions,and has significant age differences.Chronic pain originates from nociceptors in the peripheral nervous system.Currently,the knowledge of thermal nociception is mainly confined to temperature sensors,including TRPV1 channel,a transient receptor potential cation channel expressed in nociceptive neurons of dorsal root ganglion?DRG?.Nociceptor neuronal excitability increase or improve the response to stimuli is an important mechanism for pain produced.The action potential generated by the activation of the voltage-gated sodium channel on the neuronal cell membrane is also one of the main factors for pain production.The loss of thermal sensors only partially impairs the sense of thermal injury,and the transduction of noxious signals also needs the assistance of sodium channel?Nav?,but the regulatory mechanism has not been found.Therefore,this paper focuses on two knockout mice of TRP channel and Na channel,based on the development of knockout mice,based on the age change of knockout mice,The effects of TRP channel subtypes and Na+channel subtypes on mechanical pain and thermal hyperalgesia during development were investigated by using behavioral,molecular biology?fluorescence quantitative PCR?and electrophysiology techniques.It further reveals the cellular and molecular regulatory mechanism of the interaction between the two?mechanical pain and heat pain?during development.Experimental method:1.The mechanical and thermal pain thresholds of TRPV1 genotype mice(i.e.TRPV1+/+,TRPV1+/-,TRPV1-/-)and Nav1.6 genotype mice(i.e.Nav1.6+/+,Nav1.6+/-,Nav1.6-/-)were measured by von Frey hair pain test and hot plate method.Since Nav1.6-/-mice died naturally on about 21 days,no follow-up experiments could be carried out.2.The expression of TRP channel subtypes and Na+channel subtypes in DRG neurons was detected by qRT-PCR in 1W-8W TRPV1 knockout mice and Nav1.6knockout mice under normal physiological conditions.3.The whole-cell patch clamp method was used to detect the early developmental current changes of DRG neurons in Nav1.6 and TRPV1 channels.The results are as follows:1.Behavioral results:Continuous 43?thermal stimulation,compared with wild-type mice,TRPV1 gene knockout had no effect on the growth of mice,but reduced the weight of mice;Compared with wild-type mice,the mechanical pain threshold of TRPV1+/-,TRPV1-/-mice increased at 1W,and there was significant difference at 1W?P<0.05?.There was no significant difference at 3W-8W.After 5W,the mechanical pain threshold of three types of mice increased.For thermal stimulation,the latency of left and right foot contraction of TRPV1+/-,TRPV1-/-mice increased,with significant difference at 1W and 2W?P<0.05?,there was no difference in mechanical pain threshold between 3W-8W mice of the above three types.It can be seen that the growth of mice's body length did not change after 43?thermal stimulation and TRPV1 gene knockout,but the weight loss,the early mechanical pain threshold increased,and the latency of thermal pain response increased,which affected the early development of mice.The mice were born 1W-8W,and the body length and body weight of TRPV1 wild type mice were higher than 43?heat-stimulated TRPV1 wild type mice,and there was significant difference?P<0.05?.It is suggested that continuous 43?thermal stimulation affects the growth and development of mice;In the mechanical stimulation experiment,mechanical pain was more sensitive to discontinuous 43?thermal stimulation;The threshold of contraction latency was gradually increased when the change of contraction latency induced by thermal stimulation was detected;Compared with the wild type TRPV1 mice stimulated by 43?at birth,the threshold of foot contraction latency of the wild type TRPV1 mice stimulated by 43?at discontinuous temperature increased significantly?P<0.05?.It can be seen that discontinuous 43?thermal stimulation affected the growth status and pain perception of mice.2W may be a turning point in the development of mice.The mechanical thresholds of Nav1.6+/+and Nav1.6+/-genotypes increased with age.Compared with wild type mice,the mechanical pain threshold of Nav1.6+/-mice aged1W-7W decreased,and there were significant differences between 6W and 7W?P<0.01?,but at 8W,there is no difference in pain threshold between the two genotype mice;The length and weight of Nav1.6+/+,Nav1.6+/-mice were the same under continuous 43?heat stimulation;the length and weight of Nav1.6-/-mice decreased,and died about 21 days;With the increase of week age,the pain threshold of Nav1.6+/-mice was lower than that of wild type mice.The latency of foot contraction in two genotypes of mice showed a decreasing trend.Compared with wild type mice,the latency of heat pain response in Nav1.6 gene deletion(Nav1.6+/-)mice increased significantly?P<0.05?under continuous 43?heat induction.The growth of body length was slowed down and the sensitivity of mechanical pain and thermal pain was increased when the Nav1.6 gene was deleted(Nav1.6+/-)or knocked out(Nav1.6-/-).2.Hyperalgesia related changes in gene expression and development of the DRG mRNAThe mRNA expression of the pain-sensitive gene in TRPV1+/+,TRPV1+/-,TRPV1-/-,Nav1.6+/+,Nav1.6+/-mouse DRG neurons was detected by qRT-PCR.The result shows:Na+channel subtypes and TRP channel subtypes were expressed in DRG neurons of three genotypes of TRPV1 mice.With the increase of age,the expression of TRP channel subtypes and Na+channel subtypes showed an upward trend.After TRPV1 gene knockout,TRPA1 gene and TRPV1 gene expression were down-regulated compared with wild type mice.On the contrary,the expression of Na+channel subtypes is up-regulated,which was most significant at the age of 2 weeks.These results suggest that TRPV1 gene knockout differentially regulates the expression of Na+and TRP channels.Na+channel subtypes and TRP channel subtypes were expressed in DRG neurons of Nav1.6 knockout mice.With the increase of age,the expression of wild-type mice showed a downward trend,while that of heterozygous mice showed an upward trend,especially at the age of 3 weeks after birth.Na+channel subtypes,TRPA1,TRPV1,TRPV2 and TRPV4 were up-regulated after Nav1.6 gene knockout,suggesting that Nav1.6 gene knockout affected the expression of Na channel and TRP channel.Therefore,the knockout of TRPV1 and Nav1.6 genes affects the expression of Na+and TRP channels,suggesting that there is an interactive compensatory mechanism between the two channel subtypes.3.Developmental changes of TRPV1 and Na+channel currentsThe current expression and development in DRG neurons of TRPV1+/+,TRPV1+/-,TRPV1-/-mice were detected by whole cell patch clamp technique.The results showed that the peak current of 2W of TRPV1+/-mice was about 2300 pA,the inversion potential was 30 mV,and the activation potential was 50 mV.The peak current of 4W of TRPV1+/+mice is about 4000 pA,the reversal potential is 18 mV,and the activation potential is 60mV.The peak current of 5W in TRPV1+/+mice is about 2400 pA,the inversion potential is 25 mV,and the activation potential is 60 mV.Compared with 4W and 5W in TRPV1+/+mice,the peak current of 5W mice became smaller and the inactivation was slowed down.The 2W currents of TRPV1 genotype mice were compared.After TRPV1 gene knockout,the peak current decreased,Na+channel inhibited activation,and delayed inactivation.Conclusion:1.It was revealed that the basal metabolism of mice was decreased and the perception of harmful stimuli was decreased after 43?heat stimulation and TRPV1gene knockout.Among them,1W-2W is the key period.2.It was revealed that discontinuous 43?thermal stimulation affected the growth of body length and body weight of mice,and decreased the perception of harmful stimuli.Among them,1W-2W is the key period.3.It was revealed that the body length of mice was not affected by 43?heat stimulation and Nav1.6 gene knockout,while the weight loss was more sensitive to pain.4.It is revealed that the knockout of TRPV1 and Nav1.6 gene affects the expression of Na+channel and TRP channel.It is speculated that there is an interactive compensatory mechanism between the two channels.Na+channel can not only mediate the transmission of action potential,but also the corresponding heat. |
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