| Background and Objective: Diabetic neuropathic pain(DNP)is a common complication in type 2 Diabetes mellitus(DM),which is prone to co-morbidity with neuropsychiatric diseases,such as depression(Major depressive disorder,MDD).When patients with DNP combined with depressive symptoms,the condition is often more serious than single DNP or MDD,and it is also more difficult to treat.Even if the combination of analgesic and antidepressant drugs is used,it is difficult to completely relieve the symptoms.Effective therapeutic drugs for DNP combined with MDD is still lack in clinic.P2X4 receptor,a kind of purinergic receptor,is widely present in the central nervous system(CNS),which plays an important role in the pathological development of DNP and MDD.Long non-coding RNA(lnc RNA)is a transcription product that does not have the function of coding protein.It is highly expressed in CNS,and involved in the maintenance of normal physiological function of the brain and the occurrence and development of neuropsychiatric diseases.This study aims to investigate whether the treatment of MSTRG.81401 with short hairpin RNA(shRNA)could regulate P2X4 receptor in the hippocampus and spinal cord,affect the comorbidity of DNP and MDD in type 2 diabetic rats,and provide experimental basis for finding clinical treatment of DNP combined with MDD.Methods: Adult male SD rats were randomly divided into four groups: Control group,Model group,Model+ MSTRG.81401 shRNA group,and Model+ scramble shRNA group.DNP combined with MDD animal models were established.The rats were fed with high glucose and high fat for 4 weeks,and then streptozocin(STZ)was injected intritoneally after 12 hours of fasting and water abstinence.Fasting blood glucose>7.8mmol/L was measured as type 2 diabetic rats.The change of mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)was observed.Rats with pain sensitivity were screened out,and received chronic unpredictable mild stimulation for 4 weeks.Then sucrose preference test(SPT),forced swimming test(FST)and open-field test(OFT)were used to select the rats with MDD.Quantitative real time PCR(Q-PCR)was used to screen the effective shRNA interference sequence of MSTRG.81401.The rats in Model+ MSTRG.81401 shRNA group and Model+scramble shRNA group were injected with the same amount of MSTRG.81401 shRNA and scramble shRNA by stereotaxer,respectively.The expression of P2X4 receptor m RNA and MSTRG.81401 in the hippocampus and spinal cord of rats in each group was detected by Q-PCR.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of inflammatory cytokines interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum of rats in each group.Western blotting(WB)was used to detect the protein levels of P2X4 receptor,IL-1β and TNF-α in the hippocampus and spinal cord of rats.The phosphorylation level of ERK1/2 was also detected by WB.The co-expression of P2X4 receptor and astrocyte marker GFAP in the hippocampus and spinal cord of rats was detected by double immunofluores-cence method.Results: 1.Behavioral test: Compared with the Control group,the MWT,TWL,OFT and SPT of the Model group were significantly decreased(p<0.01),and the immobility time of FST was significantly increased(p<0.01).Compared to the Model group,the MWT,TWL,OFT and SPT in the Model + MSTRG.81401 shRNA group were significantly increased(p<0.01),and the FST immotility time was significantly decreased(p<0.01)after the injection of MSTRG.81401 shRNA.There were no significant differences in the MWT,TWL,OFT,SPT and FST in the Model+scramble shRNA group compared with the Model group(p>0.05).2.Q-PCR results: P2X4 receptor m RNA and MSTRG.81401 in the hippocampus and spinal cord of rats in the Model group and Model +scramble shRNA group were significantly higher than those in the Control group(p<0.01),P2X4 receptor m RNA and MSTRG.81401 in the Model +MSTRG.81401 shRNA group were significantly lower than those in the Model group(p<0.01),while there was no statistically significant difference between the Model group and Model+scramble shRNA group(p>0.05).3.ELISA results: IL-1β and TNF-α levels of serum in the Model group and Model +scramble shRNA group were significantly higher than those in the Control group(p<0.01),and IL-1β and TNF-α levels of serum in the Model +MSTRG.81401 shRNA group were significantly lower than those in the Model group(p<0.01),but there was no statistical significance between the Model group and Model +scramble shRNA group(p>0.05).4.Western blot results: The protein levels of P2X4 receptor,IL-1β and TNF-α in the hippocampus and spinal cord of rats in the Model group and Model +scramble shRNA group were significantly increased compared with the Control group(p<0.01),and the protein levels of P2X4 receptor,IL-1β and TNF-α in the hippocampus and spinal cord of rats in the Model + MSTRG.81401 shRNA group were significantly decreased compared with the Model group(p<0.01).There were no significant differences in the protein levels of P2X4 receptor,IL-1β and TNF-α between the Model group and Model +scramble shRNA group(p>0.05).The level of p-ERK1/2 in the hippocampus and spinal cord of the Model group and Model + scramble shRNA group were significantly higher than the Control group(p<0.05),while the level of p-ERK1/2 of the hippocampus and spinal cord in the Model + MSTRG.81401 shRNA group was significantly lower than the Model group(p<0.05).5.Double-label immunofluorescence results: P2X4R-GFAP was co-expressed in the hippocampus and spinal dorsal horn of rats.The co-expression of P2X4 receptor and GFAP in the hippocampus and spinal dorsal horn of rats in the Model group and Model +scramble shRNA group was significantly higher than that in the Control group(p<0.01),and the co-expression of P2X4 receptor and GFAP in the hippocampus and spinal dorsal horn of rats in the Model + MSTRG.81401 shRNA group was significantly lower than that in the Model group(p<0.01),but there was no significant difference between the Model group and Model +scramble shRNA group(p>0.05).Conclusion: Lnc RNA MSTRG.81401 shRNA can alleviate diabetic neuropathic pain and depressive behavior by down-regulating the expression of P2X4 receptor in the hippocampus and spinal cord of rats.The mechanism may be that down-regulated P2X4 R inhibits the release of inflammatory cytokines IL-1β and TNF-α and decreases the phosphorylation level of ERK1/2. |
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