The Role Of Spinal Tetrahydrobiopterin In The Development Of Hyperalgesia Induced By Chronic Restraint Stress In Rats

ObjectiveTo investigate the mechanisms of tetrahydrobiopterin in the development of hyperalgesia(SIH)induced by chronic restraint stress.Methods1.Twenty-eight male SD rats,weighing 190-220 g,were randomly divided into two groups: Con group and RS group.Restraint stress was performed at 9:00 AM to 15:00 PM for seven consecutive days and food and water were removed during the period of the time that the RS rats were kept in the plastic tube.The Mechanical Paw Withdrawal Threshold(PWMT),Thermal Paw Withdrawal Latency(PWTL)and Tail Flick Latency(TFL)were performed on the first day before establishing RS models,and on 3rd,5th,7th day after the end of RS models.After measuring the mechanical and thermal threshold on 7th day of RS models,rats were sacrificed to remove the lumbar enlargements,which were used to measure the protein and m RNA expression of spinal GCH1,i NOS,n NOS and e NOS by Western Blot and q RT–PCR.The concentration of spinal NO was detected by the nitrate reductase method,and the content of plasma BH4 and spinal BH4 were measured by high performance liquid chromatography(HPLC).Mass Array system was used to detect the methylation level of gene GCH1.2.Fifty-one male SD rats,weighing 190-220 g,both performed the operation of intrathecal catheter.Rats after seven days recovery of successfully intrathecal catheter implantation were randomly divided into five groups: Vehicle + RS group,BH4 + RS group,DAHP + RS group,L-Name + RS group and L-Name + BH4 + RS group.Intrathecal injection for seven consecutive days was performed 15 min prior to RS and L-Name in the L-Name + BH4 + RS group was injected 30 min prior to BH4.We detected the PWMT,PWTL and TFL on the first day before establishing RS models,and on 3rd,5th,7th day after the end of RS models.Lumbar enlargements in the Vehicle + RS group,BH4 + RS group and DAHP + RS group obtained on the 7th of RS after detecting the pain threshold were used to detect the protein and m RNA expression of i NOS,n NOS and e NOS by Western Blot and q RT–PCR.The concentrations of spinal NO in the Vehicle + RS group,DAHP + RS group,BH4 + RS group,L-Name + RS group and L-Name + BH4 + RS group were detected by the nitrate reductase method.3.Thirty-two male SD rats,weighing 190-220 g,both performed the operation of intrathecal catheter.Rats after seven days recovery of successfully intrathecal catheter implantation were randomly divided into three groups: Vehicle + RS group,7-NI + RS group and 1400 W + RS group.Intrathecal injection was performed 15 min prior to RS and last for seven consecutive days.We detected the PWMT,PWTL and TFL on the first day before RS models,and on 3rd,5th,7th day after the end of RS models.Lumbar enlargements in the Vehicle + RS group,7-NI + RS group and 1400 W + RS group obtained on the 7th of RS after detecting the pain threshold were used to measuring the concentration of spinal NO by the nitrate reductase method.Results1.Compared with the Con group,the PWMT,PWTL and TFL in the RS group both decreased robustly(P < 0.05);Compared in group,the PWMT,PWTL and TFL in the RS group presented in a time-dependent decrease(P < 0.05),while no significant difference in the Con group rats.The results of Western Blot and q RT–PCR indicted that the m RNA and protein expression of spinal i NOS in the RS group were significantly up-regulated on the 7th day(P < 0.05),while the expression of spinal n NOS and e NOS were slightly up-regulated(P > 0.05).The content of spinal NO was robustly increased in the RS group(P < 0.05).Moreover,the content of plasma BH4 and spinal BH4 also significantly increased(P < 0.05).GCH1 gene was hypomethylated in the location of CPG 14,15(27909945,2790993)and CPG39(27909657)in the RS rats by the Mass Array system(P = 0.001,0.016,respectively).2.Compared with the Vehicle + RS group,the PWMT,PWTL and TFL were significantly decreased in the BH4 + RS group(P < 0.05),and increased in the DAHP + RS,L-Name + RS and L-Name + BH4 + RS group(P < 0.05);compared in group,the pain threshold was in a time-dependent manner in the BH4 + RS group,DAHP + RS,L-Name + RS and L-Name + BH4 + RS group and reached the highest or lowest level on the 7th day.The results of Western Blot and q RT–PCR suggested that spinal i NOS expression significantly up-regulated in the BH4 + RS group(P < 0.05)and down-regulated in the DAHP + RS group(P > 0.05),while the expression of spinal n NOS and e NOS were not significant altered in the three group(P > 0.05).The concentration of spinal NO was slightly increased in the BH4 +RS group and significantly decreased in the DAHP + RS,L-Name + RS and L-Name + BH4 + RS group(P < 0.05)compared with the Vehicle + RS group by the nitrate reductase method.3.Compared with the Vehicle + RS group,the PWMT,PWTL and TFL were significantly increased in a time-dependent manner both in the 7-NI + RS and 1400 W + RS group.The concentration of spinal NO was robustly decreased in the 1400 W + RS group(P < 0.05),while no significant difference was observed in the 7-NI + RS group(P > 0.05).Conclusion1.The expression of spinal GCH1 was significantly up-regulated and the level of GCH1 methylation was robustly decreased in the chronic RS models,suggesting that the de novo synthesis pathway of BH4 was involved in the regulation of SIH.2.i NOS-NO cascade system modulate the role of BH4 in the SIH in the spinal cord.In general,spinal BH4 mediates the occurrence and development of hyperalgesia induced by the chronic RS;the decrease of GCH1 methylation induced by chronic stress can increase the expression of GCH1 and induce the synthesis of BH4,and the i NOS-NO cascade system may be an important downstream mechanism of BH4.