| Part ⅠUpregulated expression of ASIC3 is involved in RTX-induced postherpetic neuralgia of ratsObjectiveTo investigate the role of acid-sensitive ion channel 3(ASIC3)in resiniferatoxin(RTX)-induced postherpetic neuralgia of rats and its possible mechanism.Methods1)Effects of intraperitoneal injection of RTX on the behaviors of mechanical pain and thermal pain in ratsUsing a random number table method,healthy adult male SD rats were divided into 2 groups:vehicle group(Vehicle group)and model group(RTX-PHN group).The rats in the model group received a single intraperitoneal injection of RTX(200μg/kg)under isoflurane anesthesia,and the vehicle group was injected with the same volume of vehicle.The RTX vehicle was a mixture of 10%Tween 80,10%ethanol and normal saline.Von Frey Fiber Filament and thermal pain stimulator were used to measure the mechanical pain threshold and thermal pain threshold.2)Effects of intraperitoneal injection of RTX on ASIC3 mRNA and protein in DRG of ratsHealthy adult male SD rats were randomly divided into 2 groups:vehicle group(Vehicle group)and model group(RTX-PHN group).All rats were sacrificed on the 14th day after RTX injection and the dorsal root ganglia of L4～6 were taken.qPCR and Western blot were used to detect the expression of ASIC3 mRNA and protein.Immunofluorescence technology was used to detect the cellular distribution of ASIC3 in DRG neurons of rats.3)Effect of Intrathecal Injection of ASIC3 Inhibitor Amiloride on Pain Behavior in RatsHealthy adult male SD rats were randomly divided into 7 groups:normal control group(Control group),vehicle group(Vehicle group),model group(RTX-PHN group),PBS group(RTX+PBS group),Amiloride 50μg group(RTX+Ami50 group),Amiloride 100μg group(RTX+Ami100 group)and Amiloride 200μg group(RTX+Ami200 group).In the RTX+Ami group,different concentrations of amiloride were injected intrathecally on the 7th day after the injection of RTX,once a day for 7 consecutive days.Von Frey Fiber Filament was used to measure the mechanical pain threshold of rats.4)The effect of intrathecal injection of amiloride on the expression of TRAF6,nNOS and C-fos proteinHealthy adult male SD rats were randomly divided into 3 groups:vehicle group(Vehicle group),model group(RTX-PHN group),and Amiloride group(RTX+Ami group).In the RTX+Ami group,intrathecal injection of amiloride was started on the 7th day after RTX injection,once a day,for 7 consecutive days.On the 14th day after surgery,all rats were sacrificed and L4～6 dorsal root ganglia were taken.Western blot was used to detect the expression of TRAF6,nNOS and C-fos protein.5)The effect of intrathecal injection of amiloride on the excitability of DRG neurons in ratsHealthy adult male SD rats were randomly divided into 3 groups:vehicle group(Vehicle group),model group(RTX-PHN group),and Amiloride group(RTX+Ami group).In the RTX+Ami group,intrathecal injection of amiloride was started on the 7th day after RTX injection,once a day,for 7 consecutive days.On the 14th day after surgery,all rats were sacrificed and L4～6 dorsal root ganglia were taken.Whole-cell patch clamp electrophysiological technique was used to detect the excitability of DRGs neurons in rats.Result1)The behavioral results of rats showed that the mechanical pain threshold was decreased and the thermal pain threshold was increased after RTX treatment.indicating that the postherpetic neuralgia(PHN)model was successfully established.In contrast,RTX vehicle injection did not affect the mechanical pain and thermal pain thresholds of rats.2)qPCR and Western Blot results showed that RTX injection increased the expression of ASIC3 mRNA and protein in DRG;immunofluorescence results showed that ASIC3 and NeuN were coexpressed in rat DRG neurons,and the protein fluorescence of ASIC3 and NeuN co-labeled in the model group was significantly enhanced.3)A single intrathecal injection of different doses of amiloride could relieve mechanical pain in PHN rats in a dose-dependent manner.Among them,50 μg amiloride was less effective,and 200 μg amiloride had an effect equivalent to 100 μg.At the same time,100 μg amiloride had no significant effect on the mechanism threshold of healthy control rats.Continuous intrathecal injection of 100μg amiloride effectively reduced the pain induced by RTX.4)Western Blot results suggested that RTX injection increased the levels of TRAF6,nNOS and Cfos proteins in DRG,and intrathecal injection of amiloride could inhibit the expression of TRAF6,nNOS and C-fos in DRG of rats.5)Electrophysiological results showed that intraperitoneal injection of RTX could increase the excitability of DRG neurons in rats,and amiloride could reverse the RTX-induced increase in the excitability of rat DRG neurons.ConclusionThe up-regulation of ASIC3 expression is involved in the formation of postherpetic neuralgia induced by RTX in rats,Intrathecal injection of amiloride can reduce the pain response in rats.Part ⅡExogenous BDNF regulates the expression of ASIC3 signal through TrkB receptor in PC12 neural cellsObjectiveTo investigate the role of exogenous brain-derived neurotrophic factor(BDNF)in regulating ASIC3 signal through tyrosine kinase receptor B(TrkB)receptor in PC 12 neural cells.Method1)The effect of exogenous BDNF on TrkB and ASIC3 proteins in PC 12 cellsThe cultured PC 12 cells were divided into 4 groups using a random number table:normal control group(Control group)and different concentrations of BDNF treatment group(2ng/ml group),(20ng/ml group)and(200ng/ml group).The normal control group used RPMI 1640 medium,and the BDNF group was treated with 2ng/ml,20ng/ml and 200ng/ml exogenous BDNF in the medium for 20 minutes.The immunofluorescence method was used to detect the distribution of TrkB and ASIC3 in normal PC 12 cells,and the Western Blot method was used to detect the changes in the expression of TrkB and ASIC3 protein levels after BDNF treatment.2)The effect of TrkB antagonist ANA-12 on the expression level of ASIC3,TRAF6 and nNOS protein in PC 12 cells treated with exogenous BDNFThe cultured PC 12 cells were randomly divided into 3 groups:normal control group(Control group),BDNF treatment group(BDNF group)and ANA-12 group(BDNF+ANA-12 group).ANA-12 was added for pretreatment for 2h before BDNF treatment in ANA-12 group,At the end of treatment,three groups of cells were collected,and the Western Blot method was used to detect the changes in the expression levels of ASIC3,TRAF6 and nNOS in PC12 cells.3)The effect of ASIC3 shRNAs on the expression levels of ASIC3,TRAF6 and nNOS protein in PC 12 cells treated with exogenous BDNFa.The cultured PC 12 cells were randomly divided into 6 groups:normal control group(Control group),transfection reagent group(Vehicle group),negative control group(NC group),shRNA-59 group,shRNA-60 group,and shRNA-61 group.Except for the normal control group,the Vehicle group was treated with transfection reagent,and the other groups were transfected with the corresponding shRNA or negative sequence into PC 12 cells for 24 hours,respectively.At the end of transfection,6 groups of cells were collected to detect the changes of ASIC3 protein expression levels in PC 12 cells using the Western Blot method.b.The cultured PC 12 cells were randomly divided into 4 groups:normal control group(control group),BDNF-treated group(BDNF group),shRNA-treated group(BDNF+shRNA-61 group)and negative control group(BDNF+NC group).Except for the normal control group,the other three groups were treated with 20 ng/ml BDNF for 20 min,and the other three groups were transfected with shRNA or negative sequence for 24 h before BDNF treatment.At the end of treatment in each group,the cells were collected,Western blot was used to detect the changes in the expression levels of ASIC3,TRAF6 and nNOS proteins in PC 12 cells.4)The effect of ASIC3 shRNA on the expression level of inflammatory factors in PC 12 cells treated with exogenous BDNFThe cultured PC 12 cells were randomly divided into 4 groups:normal control group(Control group),BDNF treatment group(BDNF group),shRNA treatment group(BDNF+shRNA-61 group)and negative control group(BDNF+NC group).After the treatment of each group,the cells were collected,and the expression levels of inflammatory factors IL-1β,IL-6 and TNF-α in PC 12 cells were detected by qPCR method.5)The effect of ASIC3 shRNA on the expression level of Nitric oxide(NO)in PC 12 cells treated with exogenous BDNFThe cultured PC 12 cells were divided into 4 groups by random number table method:normal control group(Control group),BDNF treatment group(BDNF group),shRNA treatment group(BDNF+shRNA61 group)and negative control group(BDNF+NC group).At the end of treatment in each group,the cells were collected and the nitric oxide kit was used to detect the changes in the expression of NO in PC 12 cells.Result1)Immunofluorescence results showed that there was co-expression of TrkB and ASIC3 in PC 12 cells,Western Blot results showed that exogenous BDNF treatment of PC 12 could promote the expression of TrkB and ASIC3 in PC12 cells.2)Western Blot results showed that ANA-12 reverses the effect of BDNF and significantly reduces the protein expression levels of ASIC3,TRAF6 and nNOS.3)Western Blot results showed that ASIC3 shRNA significantly reduced the expression of ASIC3,and was able to reverse the effect of BDNF and reduce the protein expression levels of TRAF6 and nNOS.4)The qPCR results indicated that ASIC3 shRNA reduced the mRNA levels of inflammatory factors IL-1β,IL-6 and TNF-α.5)Nitric oxide results showed that ASIC3 shRNA significantly reduced the level of NO in PC 12 cells.ConclusionTrkB-ASIC3 signal was activated in PC 12 cells treated with exogenous BDNF,indicating that BDNF can regulate the expression of ASIC3 signal by binding to its high affinity receptor TrkB in PC 12 cells.Part ⅢBDNF/TrkB pathway regulates ASIC3 signal in RTX-induced postherpetic neuralgia of ratsObjectiveTo investigate the role of BDNF/TrkB pathway in regulating ASIC3 signal in RTX-induced postherpetic neuralgia of rats.Method1)The effect of intraperitoneal injection of RTX on the mRNA and protein of BDNF and TrkB in rat DRGHealthy adult male SD rats were randomly divided into 2 groups:vehicle group(Vehicle group)and model group(RTX-PHN group).All rats were sacrificed on the 14th day after RTX injection and the dorsal root ganglia of L4～6 were taken.qPCR and Western blot were used to detect the expression of BDNF and TrkB mRNA and protein.Immunofluorescence technology was used to detect the cellular distribution of BDNF in DRGs of rats and the co-localization of TrkB and ASIC3 in DRGs.2)The effect of intraperitoneal injection of ANA-12 on pain behavior in ratsHealthy adult male SD rats were randomly divided into 4 groups:vehicle group(Vehicle group),model group(RTX-PHN group),PBS group(RTX+PBS group),ANA-12 group(RTX+ANA-12 group).In addition to the vehicle group injected with RTX vehicle,the other three groups were intraperitoneally injected with RTX.The RTX+ANA-12 group and the RTX+PBS group were injected intraperitoneally with ANA-12 or PBS on the 7th day after RTX injection,once a day for 7 consecutive day.Von Frey Fiber Filament was used to measure the mechanical pain threshold of rats.3)The effect of intraperitoneal injection of ANA-12 on the expression of ASIC3,TRAF6,nNOS and C-fos proteinHealthy adult male SD rats were randomly divided into 3 groups:vehicle group(Vehicle group),model group(RTX-PHN group),and ANA-12 group(RTX+ANA-12 group).On the 14th day after surgery,all rats were sacrificed and L4～6 dorsal root ganglia were taken.Western blot was used to detect the expression of ASIC3,TRAF6,nNOS and C-fos protein.4)The effect of intraperitoneal injection of ANA-12 on the excitability of rat neuronsHealthy adult male SD rats were randomly divided into 3 groups:vehicle group(Vehicle group),model group(RTX-PHN group),and ANA-12 group(RTX+ANA-12 group).On the 14th day after surgery,all rats were sacrificed and L4～6 dorsal root ganglia were taken.Whole-cell patch clamp electrophysiological technique was used to detect the excitability of DRGs neurons in rats.5)The effect of intrathecal injection of TrkB agonist 7,8-dihydroxyflavone(7,8-DHF)and amiloride on pain behavior in ratsa.Divide healthy adult male SD rats into 4 groups randomly:normal control group(Control group),7,8-DHF 1mg/kg group(lmg DHF group),7,8-DHF 3mg/kg group(3mg DHF group),7,8-DHF 6mg/kg group(6mg DHF group).In the 7,8-DHF group,normal rats were injected intrathecally with different concentrations of 7,8-DHF.After administration,Von Frey Fiber Filament was used to measure the mechanical pain threshold of rats.b.Divide healthy adult male SD rats into 2 groups randomly:model group(RTX-PHN group),7,8DHF+amiloride group(RTX-PHN+DHF+Ami group).Rats in the two groups were intraperitoneally injected with RTX.Inthe RTX-PHN+DHF+Ami group,amiloride(100μg)and 7,8-DHF(3mg/kg)were injected intrathecally on the 7th day after RTX injection,once a day for 7 consecutive days.Von Frey Fiber Filament was used to measure the mechanical pain threshold of rats.6)The effect of intrathecal injection of 7,8-DHF and amiloride on the expression of TRAF6,nNOS and C-fos proteinHealthy adult male SD rats were randomly divided into 2 groups:model group(RTX-PHN group),7,8-DHF+amiloride group(RTX-PHN+DHF+Ami group).On the 14th day after surgery,all rats were sacrificed and L4～6 dorsal root ganglia were taken.Western blot was used to detect the expression of TRAF6,nNOS and C-fos protein.Result1)The results of qPCR and Western blot showed that RTX increased the expression of BDNF and TrkB in the dorsal root ganglia;immunofluorescence results showed that the increased BDNF may be derived from neuronal cells and satellite glial cells in DRG,and TrkB and ASIC3 co-localized on neuronal cells.2)Behavioral results showed that intraperitoneal injection of ANA-12 could reduce the pain response of PHN model rats,but did not affect the pain threshold of normal rats.3)Western blot results showed that intraperitoneal injection of ANA-12 could reduce the expression of ASIC3,TRAF6,nNOS and C-fos in DRG.4)Electrophysiological results showed that intraperitoneal injection of ANA-12 could reduce the excitability of DRG neurons in rats.5)Behavioral results showed that a single intrathecal injection of 7,8-DHF could cause pain response in rats,while the simultaneous use of 7,8-DHF and amiloride can alleviate the pain response in PHN model rats.6)Western blot results showed that the simultaneous use of 7,8-DHF and amiloride could reduce the expression of TRAF6,nNOS and C-fos proteins in DRG.ConclusionBDNF/TrkB pathway can play an important role in RTX-induced postherpetic neuralgia of rats by regulating ASIC3 signal. |
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