| Diabetic neuropathic pain,a common,difficult to treat complication in diabetic: cause serious harmful effects on patients' mental health and living standard,and make great harm to human beings.Activated microglia cells produce several neurotoxic factors,such as pro-inflammatory cytokines and nitric oxide(NO),which contribute to the occurrence of DNP.Recent study shows that activation of spinal microglia P2Y13 receptor is involved in diabetic neuropathic pain rats.In addition,parallel relationship between microglial activation and pro-inflammatory cytokines is observed in diabetic rats,which suggest that abnormal activation of microglia in the spinal dorsal horn is closely associated with the occurrence of inflammatory reaction.The upregulation of JAK2/STAT3 and the phosphorylation of NMDA receptor subtype NR2 B were involved in the hyperalgesia of DNP rats.However,it is unclear whether spinal P2Y13 receptor plays a crucial role in inflammatory responses in spinal cord of diabetic rats,and whether spinal P2Y13 receptor plays a crucial role in the IL-1?,IL-6,JAK2,STAT3 expression and NR2 B phosphorylation in dorsal spinal cord of diabetic rats.In this work,we will use some methods include morphology,ethology,molecular biology to investigate the effects of MRS2211 on the expression of IL-1?,IL-6,JAK2,STAT3 and NR2 B phosphorylation in spinal dorsal horn in a rat model of diabetic neuropathic pain.We also speculate the role of P2Y13 receptor in ADP?S-induced the increased expression of IL-6,JAK2 and STAT3 in cultured dorsal spinal cord microglia cells.Objective: 1.The aim of this study is to explore the possible role of dorsal spinal cord P2Y13 receptor in STZ-induced diabetic neuropathic pain.2.The effects of MRS2211 on the expression of IL-6 and JAK2,STAT3 of dorsal horn microglia in vitro.Methods:1.All rats were randomized into five groups(n=8):?1 normal group(intrathecal saline),?2 Diabetic neuropathic pain(DNP,intrathecal saline),?3 ~?5 DNP+MRS2211 group(intrathecal injection of MRS2211,10,50,100pmol/L).Diabetic neuropathic pain model was induced by a single intraperitoneal injection of STZ at a dose of 50mg/kg.After two weeks,diabetic neuropathic pain model was confirmed when thermal pain threshold decreased significantly.Then,MRS2211 or saline were administered twice per day for 4 weeks.All rats were tested for thermal withdrawal latency(TWL)at the plantar surface of the hindpaw 1 day before and 2th,4th and 6th week after STZ injection.The expression of P2Y13,Iba-1,IL-1? and IL-6 in dorsal horn was examined by RTFQ-PCR on the 2th,4th and 6th weekend after STZ injection,respectively.The expression of IL-1? and IL-6 in dorsal horn was examined by ELISA on the 2th,4th and 6th weekend after STZ injection,respectively.The expression of JAK2/STAT3 and NR2B(p Tyr1336,p Tyr1472)were examined by western blot on the 2th,4th and 6th weekend after STZ injection,respectively.2.Primary cultures of microglia were prepared from dorsal spinal cord of new born SD rats(<3d).The cells were seeded on 12-well plates at a cell density of 5×105/well for 48 h.These cells were randomized into four groups(n=3): normal group(high glucose medium),ADP?S(10?mol/L,3h)group,MRS2211(10?mol/L,20min)+ ADP?S.The effects of ADP?S(ADP analogue)on the expression of Iba-1,IL-6 and JAK2,STST3 by using western blot.Result: 1.The results of thermal pain threshold show that there was no significant difference in TWL before STZ injection,and 2 weeks after STZ injection,rats showed significant decrease in TWL as compared to control group.TWL in the vehicle-treated DNP rats decreased at every time point(P<0.05).Subsequently,MRS2211 was given from day 15 after STZ injection for 28 consecutive days.The result show that compared with DNP group,the TWL were significantly increased after MRS2211(10,50 and 100pmol/L)administration on 4th weekend(10pmol/L: P<0.05;50pmol /L,100pmol/L: P<0.01)but not on 6th weekend.It appears that MRS2211 exerts hardly any anti-hyperalgesic effect of DNP rats.2.The PTFQ-PCR results indicated that compared with control group,the expression of P2Y13,Iba-1,IL-1? and IL-6 was significantly increased in dorsal horn on 2th,4th and 6th weekend after STZ injection(P<0.01).Intrathecal administration of MRS2211(100pmol/L)suppressed the increased content of P2Y13,Iba-1,IL-1? and IL-6 in spinal dorsal horn mainly at the end of 4th week(P<0.05)but not at the end of 6th week of diabetic rats.3.The ELISA results indicated that compared with control group,the expression of IL-1? and IL-6 was significantly increased in dorsal horn on 2th,4th and 6th weekend after STZ injection(P<0.01).Intrathecal administration of MRS2211(100pmol/L)suppressed the increased content of IL-1? and IL-6 in spinal dorsal horn mainly at the end of 4th week(P<0.05)but not at the end of 6th week of diabetic rats.4.Western blot results showed that compared with control group,the expression of JAK2 in the dorsal spinal cord was significantly increased at 2,4 and 6 weeks after STZ injection(P<0.01).The expression of STAT3 in the dorsal spinal cord was significantly increased at 4 and 6 weeks after STZ injection(P<0.01).Intrathecal administration of MRS2211(100pmol/L)suppressed the increased expression of JAK2,STAT3 in spinal dorsal horn mainly at the end of 4th week(P <0.05)but not at the end of 6th week of diabetic rats.5.The expression of NR2B(p Tyr1336,p Tyr1472)in the dorsal spinal cord was significantly increased at 2,4 and 6 weeks after STZ injection(P<0.05).Intrathecal administration of MRS2211(100pmol/L)suppressed the increased expression of p Tyr1336 NR2 B in spinal dorsal horn mainly at the end of 4th week(P<0.05)but not at the end of 6th week of diabetic rats.However,the expression of p Tyr1472 NR2 B was not impaired by MRS2211 in diabetic rats.6.In cultured dorsal spinal cord microglia cells,stimulation of microglia cells with 10 ?M ADP?S for 3h led to a robust increase of Iba-1,IL-6,JAK2,STAT3 protein(JAK2:P<0.05;Iba-1,IL-6,STAT3 : P<0.01).MRS2211 show a moderate degree of inhibition with ADP?S stimulatory effect on these increased protein expression(P<0.05).Conclusion: 1.Intrathecal MRS2211 produces antinociceptive effect in the early stage of DNP.2.MRS2211 inhibits the levels of IL-6,which subsequently inhibits the activation of the JAK2/STAT3 signalling pathway.In addition,intrathecal MRS2211 can also decrease p Tyr1336NR2 B in dorsal horn,which may be one of the mechanisms of MRS2211 analgesia at the Level of the spinal cord in diabetic rats.3.The activation of P2Y13 receptor may increase the expression of JAK2/STAT3 in protein level by promoting the expression of IL-6 in cultured microglia. |
PDF Full Text Request