| Objective:Firstly,we characterized a novel animal model of acute itch and touch-evoked itch induced by Methglyoxal(MGO),an intermediate product of glucose metabolism,in ICR mice.The pharmacological,behavioral methods,live cell imaging and molecular cell biology were used to investigate the molecular mechanism underlying MGO-induced itch.Secondly,we established streptozotocin(STZ)-induced diabetic mouse model to detect the behavioral changes of spontaneous itching,chemical stimulation-induced itch and touch-evoked itch in diabetic mice.We explored the molecular mechanism of MGO signaling mediating changes of itch and pain threshold in diabetic mice by using pharmacological,behavior and molecular biological methods.Methods:(1)Male adult ICR mice were intradermally injected different doses of MGO on the cheek and the nape of the neck and the itch or pain behavior of the mice was observed and quantitatively analyzed.(2)Application of pharmacological methods by blocking C fibers,depletion of mast cells,morphine,naloxone,chlorpheniramine,U0126,MGO scavenger,T-type calcium channel blockers,TRPA1 blocker,TRPV1 blockers,and TRPV1 and TRPA1 knockout mice to study the molecular mechanism underlying MGO-induced acute itch.Intrathecal injection of MGO scavenger,TRPA1,TRPV1 blockade and so on to study the molecular mechanism underlying changes of MGO-induced pain threshold(3)Western blotting technique was used to determine the expression of p-ERK after MGO incubation with ND7-23 DRG cell line.Western blotting was used to determine the spinal p-ERK expression in mouse spinal cord and DRG tissue induced by intradermal injection of MGO alone,intradermal injection of TRPA1,TRPV1 blocker or 30 minutes before the injection of antioxidant NAC,PBN and then intradermal injection of MGO.(4)The effects of MGO on the accumulation of intracellualr reactive oxygen species(ROS)in ND7-23 cell line were investigated by using flow cytometry.(5)Spontaneous itch,chemicals-induced itch and touch-evoked itch were quantified in a diabetes mouse model induced by a single intraperitoneal injection of STZ(100 mg/kg).The effects of application of MGO scavenger,TRPA1,TRPV1 blockers on itch behaviors were also examined.(6)The expression of TRPA1 and TRPV1 in diabetic mice were determined by fluorescence quantitative PCR and Western blotting analysis.(7)TRPV1 and TRPA1 plasmids were transfected respectively in HEK-293 cell line cultured in vitro.Immunofluorescence technique was used to determine the expression of TRPV1 and TRPA1.The effects of MGO on the activities of TRPV1 and TRPA1 were determined by using calcium imaging on live cell imaging workstation.Results:(1)Intradermal injection of MGO induced dose-dependent scratching behavior and touch-evoked itch behavior in the cheek model and the back of the neck model in mice.(2)Behavioral pharmacological results showed that blocking of C fiber,depletion of mast cells,intradermal injection of naloxone,chlorpheniramine,MGO scavenger could significantly inhibited MGO-inlduced itch in mice(P<0.05).Intadermal injection of MGO scavenger could also significantly inhibit MGO-induced touch-evoked itch(P<0.05).(3)Live cell calcium imaging analysis showed that MGO could increase intracellular calcium concentration in HEK293 cells which transfected with TRPA1(P<0.05),but not TRPV1(P>0.05),suggesting that MGO selectively activated TRPA1 channel.(4)Behavioral results showed that the application of TRPA1 blocker HC030031 or A967079 significantly inhibited MGO-induced acute itch(P<0.05)and touch-evoked itch(P<0.05),whereas the administration of TRPV1 blocker capsazepine(CPZ)was ineffective(P>0.05).In addition,MGO-induced acute itch was significantly reduced in TRPA1 knockout mice(P<0.05),but was not significantly affected in TRPV1 knockout mice(P>0.05).(5)Intradermal injection of MGO induced increased expression of p-ERK in spinal cord and DRG tissues(P<0.05),and the expression of p-ERK in spinal cord was significantly decreased by application of a TRPA1 blocker HC030031(P<0.05),rather than a TRPV1 blocker CPZ(P>0.05).(6)Incubation of MGO induced the increase of ROS in DRG cell line ND7-23 cells(P<0.05),while the antioxidant PBN can inhibit the accumulation of ROS induced by MGO(P<0.05).Behavioral results demonstrated that intraperitoneal injection of antioxidant PBN or NAC significantly reduced MGO-induced acute itch(P<0.05)and the expression of p-ERK in the spinal cord of mice(P<0.05).(7)STZ-induced diabetic mice did not show any signs of spontaneous itch.Chemicals-induced itch behavior significantly decreased in diabetic mice(P<0.05).Strikingly,touch-evoked itch significantly increased in diabetic mice(P<0.05).MGO scavenger and TRPA1 blocker could effectively relieve touch-evoked itch in diabetic mice(P<0.05),but not TRPV1 blockers(P>0.05).(8)The mRNA expression of TRPV1 in DRG of diabetic mice was significantly decreased(P<0.05),while the mRNA and protein expression of TRPA1 in DRG were significantly increased in diabetic mice(P<0.05).(9)Diabetic mice showed increased thermal pain threshold(P<0.05).Intrathecal injection of MGO scavenger and TRPA1 blocker HC030031 could inhibit diabetes-induced increase of thermal pain threshold(P<0.05),but not TRPV1 blocker CPZ(P>0.05).Systemic or intrathecal injection of MGO can cause elevated thermal pain threshold in normal mice(P<0.05).Intrathecal injection of MGO scavengers,TRPA1 blocker HC030031 can inhibit MGO-induced increase in thermal pain threshold in mice(P<0.05),but not TRPV1 blocker CPZ.Conclusions:(1)MGO induced acute itch and touch-evoked itch in mice.(2)TRPA1,rather than TRPV1,played a key role in MGO-induced increase in intracellular calcium concentration in DRG neurons and in acute itch behavior in mice.(3)MGO induced p-ERK activation in spinal cord and DRG,which play an important role in MGO-induced acute itch behavior in mice.(4)Oxidative stress plays an important role in MGO-induced acute itch and p-ERK expression.(5)MGO-TRPA1-pERK signaling pathway plays an important role in the itching of diabetic mice.(6)Diabetic mice increased the area of hot pain,which may be related to MGO activation of spinal cord TRPA1 channel. |
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