| [Objective]:The purpose of this study was to observe the expression of the stress gene NDRG2 in astrocytes in neuropathic pain(NP)by in vivo and in vitro experiments,and to investigate whether NDRG2 can regulate the activation of astrocytes and participate in the regulation of EAAT2 during the process of NP occurrence and development[Method]:In the first part,42 male SD rats weighing 180-230 g were randomly divided into Sham group(n=6)and SNL group(n=36).The Sham group only exposed spinal nerves without ligation,and spinal nerve ligation was performed in the SNL group.Rats' paw mechanical withdrawal threshold(MWT)and paw thermal withdrawal latency(TWL)were measured on the 1st day before surgery and on post-operative day(POD)1,3,7,10,14 and 21.Rats in the SNL group were sacrificed at each time point after the behavior test.Lumbar enlargement of the spinal cord was performed to detect the protein content and mRNA level of NDRG2 by Western blotting(WB)and Quantitative real-time polymerase chain reaction(qPCR).Immunofluorescence(IF)was used to detect the expression of NDRG2 and GFAP in dorsal horn of spinal cord on the 10th day after operation.In the second part,36 male SD rats were randomly divided into 3 groups(n=12):Sham+saline group,Sham+fluorocitrate(FC)group and Sham+NDRG2 adenovirus(AD-NDRG2-RNAi)group.An open field test(OFT)was performed 1 day before the administration and POD 3,7,10 after the administration to evaluate the effect of the intrathecal injection on the motor function of the rats.In the third part,144 male SD rats were divided into the following six groups randomly and equally(n=24):Sham+saline(Sham+Veh),Sham+AD-NDRG2-RNAi(Sham+AD),Sham+fluorocitrate(Sham+FC),SNL+saline(SNL+Veh),SNL+AD-NDRG2-RNAi(SNL+AD)and SNL+fluorocitrate(SNL+FC).The first cohort(n=12)were used to measure the MWT and TWL of the plantar 1 day before surgery and the 1st,3rd,7th and 10th days after the operation.The second cohort(n=12)were sacrificed at POD 10 to detect the expression of NDRG2,GFAP and EAAT2 proteins,levels of NDRG2 and EAAT2 mRNA,immunoreactivity of NDRG2 and GFAP and the levels of inflammatory cytokines(TNF-?,IL-1?,IL-6,IL-4 and IL-10)in spinal dorsal horn.In the fourth part,the primary rat astrocytes were extracted and cultured and divided into 4 groups:normal astrocyte(Con+Veh),normal astrocyte+AD-NDRG2-RNAi(Con+AD),normal astrocytes+lipopolysaccharide(LPS+Veh)and astrocytes+lipopolysaccharide+AD-NDRG2-RNAi(LPS+AD)groups.WB was used to detect the expression of NDRG2,GFAP and EAAT2 in four groups,IF was used to detect the expression of NDRG2,GFAP and EAAT2 and the levels change of inflammatory cytokines(TNF-?,IL-1?,IL-6,IL-4 and IL-10)were detected by ELISA.In the fifth part,the primary rat astrocytes were extracted and cultured and divided into 4 groups:normal astrocyte(Con+Veh),normal astrocyte+AG490(Con+AG490),normal astrocytes+lipopolysaccharide(LPS+Veh)and astrocytes+lipopolysaccharide+AG490(LPS+AG490)groups.WB was used to detect the protein contents of NDRG2,GFAP,EAAT2,p-STAT3 and t-STAT3 in four groups.[Results]:The MWL and TWL of the rats in the SNL group were significantly decreased.The protein contents and mRNA levels of NDRG2 were significantly increased and reached peak at POD 10.Immunofluorescence data showed colocalization of NDRG2 and GFAP in the dorsal horn of spinal cord.Compared with the SNL+Veh group,the MWT and TWL in SNL+Ad and SNL+FC groups significantly increased from days 7-10.Compared with the Sham+Veh group,GFAP and NDRG2 protein content and NDRG2 mRNA expression increased significantly,with EAAT2 protein content and mRNA expression decreased significantly in SNL+Veh group at POD 10.Compared with the SNL+Veh group,the GFAP and NDRG2 protein content and NDRG2 mRNA expression decreased significantly,with EAAT2 protein content and mRNA expression increased significantly in SNL+Ad and SNL+FC groups at POD 10.Immunofluorescence data showed colocalization of NDRG2 and GFAP in the dorsal horn of spinal cord.The increased expression of NDRG2 and GFAP in the dorsal horn of spinal cord was associated with the activation of astrocytes at POD 10.Compared with the Sham+Veh group,the expression of TNF-?,IL-1?,IL-6 and IL-6/IL-10 increased while IL-4 expression decreased significantly in SNL+Veh group.Compared with the SNL+Veh group,the expression of TNF-?,IL-1?,IL-6 and IL-6/IL-10 decreased while IL-4 and IL-10 expression increased significantly in SNL+Ad group.Compared with Con+Veh group,the expression of GFAP protein was increased while NDRG2 and EAAT2 protein significantly decreased in Con+AD group.LPS treated astrocytes showed significantly increased GFAP and NDRG2 protein,and decreased EAAT2 protein expression.Besides,the expression of TNF-?,IL-1?,IL-6,IL-4 and IL-10 was significantly increased in LPS+Veh group compard with Con+Veh group.Compared with LPS+Veh group,the expression of GFAP and NDRG2 protein were significantly decreased while the EAAT2 protein expression was significantly increased and the expression of TNF-?,IL-1?,IL-6,IL-4 and IL-10 were significantly decreased in LPS+AD group.The immunofluorescence assay results of the four groups showed that GFAP co-localized with NDRG2 and EAAT2.Compared with Con+Veh group,the expression of NDRG2,GFAP and p-STAT3 proteins in Con+AG490 group were significantly decreased.LPS induced in significantly increased expression of GFAP,NDRG2 and EAAT2 proteins while EAAT2 expression was decreased.Compared with LPS+Veh group,the expression of NDRG2,GFAP and p-STAT3 proteins were significantly decreased,while EAAT2 was increased in LPS+AG490 group[Conclusion]:The expression of NDRG2 in NP may play a role in regulating the activation of astrocytes and the expression of inflammatory factors in NP.NDRG2 may regulate the expression of EAAT2 on astrocyte membrane through JAK/STAT signaling pathway,thus affecting the regulation of glutamate metabolism in synaptic gap. |
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