| objective:The transient receptor potential cation channel subfamily V member 1(TRPV1)is a non-selective cation voltage-gated channel,expressed in the peripheral and central nervous systems and involed in pain conduction and integration.Its agonist Capsaicin patch has been approved by the FDA for the treatment of postherpetic neuralgia.It is an effective target for the treatment of neuropathic pain.Pregabalin is the first-line drug for the treatment of PHN,acting on the voltage-gated calcium channel in the central nervous system,to inhibit the transmission of pain signals in the central nervous system.Therefore,we suspect that pregabalin may also relieve neuropathic pain through spinal cord TRPV1.In order to verify the above hypothesis,we investigated the effect of pregabalin and/or TRPV1 antagonist capsazepine(CPZ)on the expression of TRPV1 in the spinal cord based on chronic constriction injury model,and explored whether pregabalin can exert an analgesic effect through spinal cord TRPV1.Methods:1.Animal grouping and model replication:Sixty healthy male Sprague-Dawley rats were randomly divided into five groups.Sham group (n=12):control group,the operation was the same as CCI group,but no nerve was ligated.CCI group(CCI group,n=12):right sciatic nerve was exposed and ligated to induce chronic constriction injury of CCI animal models;CCI+PGB group(n=12):CCI animal models were replicated.After pain threshold was measured 7~thh days,pregabalin was administered intragastrically 30 mg/kg/d for7days;CCI+CPZ group(n=12):CCI animal models were replicated.After pain threshold was measured 7~thh days,TRPV1 antagonist capsazepine was administered intraperitoneally with 2 mg/kg/d for 7days;CCI+CPZ+PGB group(n=12):CCI animal models were replicated.After pain threshold was measured7~thh days,TRPV1 antagonist capsazepine was administered intraperitoneally with 2 mg/kg/d,and pregabalin was administered intragastrically 30 mg/kg/d half a hour later for 7days.2.Pain threshold detection:The pain threshold was measured in rats in each group by Von Frey Filament preoperative,3,5,7 and 14 days after surgery respectively.3.Detection of proteins TRPV1 in spinal cord:Six rats in each group were randomly selected and sacrificed on the 14~thh day after pain threshold detection for detecting TRPV1 by immunohistochemistry and another six rats were for Western blot.Results:1.Rat models:One rat died after anesthesia and three rats had no significant pain threshold change after surgery in sham group.Four?five and three rats had no significant pain threshold change respectively in CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group,thus those animals model were considered to be failed.Another sixteen rats were selected and divided into groups according to the random method.A total of sixty rats were analyzed in this study.2.Pain threshold change:There was no statistical difference in all groups preoperative(P?>?0.05);There was no statistical difference among all time points in sham group(P?>?0.05);There was significantly decreased after operation,compared to the preoperative in CCI group?CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05).Compared with sham group,it was significantly decreased at 14~stt day in CCI group?CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);Compared with CCI group,it was significantly increased at 14~stt day in CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);Compared with CCI+PGB group,it was significantly increased at 14~stt day in CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);Pain threshold in CCI+CPZ+PGB group was higher than CCI+CPZ group at 14~stt day,but without statistical difference(P?>0.05).3.3.Detection of TRPV1 in spinal cord:(1)Immunohistochemistry results(IOD/Area):Compared with sham group,it was significantly increased at 14~stt day in CCI group?CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);Compared with CCI group,it was significantly decreased at14~stt day in CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);Compared with CCI+PGB group,it was significantly decreased at 14~stt day in CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);TRPV1 in spinal cord in CCI+CPZ+PGB group was lower than CCI+CPZ group at 14~stt day,but without statistical difference(P?>0.05).(2)Western blot results:Compared with sham group,it was significantly increased at 14~stt day in CCI group?CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);Compared with CCI group,it was significantly decreased at14~stt day in CCI+PGB group?CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);Compared with CCI+PGB group,it was significantly decreased at 14~stt day in CCI+CPZ group?CCI+CPZ+PGB group(P?<?0.05);TRPV1 in spinal cord in CCI+CPZ+PGB group was lower than CCI+CPZ group at 14~stt day,but without statistical difference(P?>0.05).Conclusion:1.The mechanical pain threshold reduced and the expression of TRPV1increased in spinal cord in CCI rats.2.Both pregabalin and CPZ could increase the mechanical pain threshold and reduce the expression of TRPV1 in spinal cord in CCI rats.3.The effect increasing the mechanical pain threshold and reducing the expression of TRPV1 of Pregabalin associated with CPZ was stronger than pregabalin,but without difference compared with CPZ.Therefore,the analgesic effect of pregabalin on neuropathic pain could be through down-regulation of the expression of the TRPV1 in spinal cord.The interaction between pregabalin and CPZ is not clear. |
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