| Objective: Neuropathic pain(NP)refers to pain caused by damage or disease affecting the somatosensory nervous system,with a prevalence of about 6.9% to 10%.Different from acute pain caused by tissue damage,NP has the characteristics of severe pain,long course,complex pathogenesis and pathological changes,and no effective treatment.Long-term pain will not only affect the patient’s ability to sleep,work and live,but also increase the incidence of depression,anxiety,and other affective disorders.Despite the continuous progress in basic research on pain medicine and clinical diagnosis and treatment techniques,the occurrence and development mechanisms of neuropathic pain have not been elucidated.Therefore,it is important and significant to detect the pathogenesis of neuropathic pain,find the key points for early diagnosis and therapy,and prevent and treat neuropathic pain.Bromodomain-containing Protein 4(BRD4)is the most important functional protein in the bromodomain and extra-terminal domain(BET)family.It recruits transcriptional complexes by binding to histone acetylated lysine residues to regulate gene expression and plays an important role in processes such as cell proliferation and inflammation.Recent studies have confirmed that BET protein is involved in the process of neuropathic pain caused by spinal cord injury.Targeted inhibition of BET protein can reduce the reactivity of microglia after spinal cord injury,improve hyperalgesia caused by spinal cord injury.In this study,we aimed to explore the role of BRD4 in rat neuropathic pain.Through an indepth study of its possible mechanism of action,we aimed to provide new targets for the early diagnosis and intervention of patients with neuropathic pain.Methods: Part 1: To clarify the changes of BRD4 expression in the spinal cord of rats with neuropathic pain and its effect on mechanical allodynia.1.To construct a neuropathic pain model in rats to observe the abnormal of pain behavior and the content of BRD4 in the spinal cord of rats over time.The experimental rats were randomly divided into sham group and SNI group.The SNI group underwent SNI surgery to construct a neuropathic pain model in rats.The sham group only exposed the nerves but did not damage them.Models at different time points were constructed on the 1st,3rd,7th,10 th,and 14 th days after SNI,respectively,to detect the paw withdrawal threshold(PWT)in each group.We used immunofluorescence and Western Blotting to detect the BRD4 content in spinal cord,and we analyzed the relationship between the different expressions and the pain behavior of rats.2.To observe the effect of inhibiting BRD4 on mechanical allodynia in SNI rats.The experimental rats were randomly divided into BRD4 inhibitor group(I-BET762 group)and inhibitor control group(Vehicle group).The rats in each group received SNI surgery,and intrathecal injection was performed on the 10 th day after surgery.The paw withdrawal threshold of rats in each group was measured to reflect mechanical allodynia.3.To observe the effect of reduced BRD4 expression on mechanical allodynia in SNI rats.The experimental rats were randomly divided into specific si RNA-1 group,si RNA-2 group,si RNA-3 group and control group(si RNA-NC group).SNI surgery was performed on all groups,and specific si RNA was given to each group.The PWT in SNI rats was measured,and the expression level of BRD4 in spinal cord of affected side was detected by Western Blotting.Part 2: To explore the regulatory mechanism of differential expression of BRD4 in neuropathic pain1.We used real-time PCR to detect the relative expression of mi R-200a-3p/miR-141-3p in the spinal cord of SNI rats.The experimental rats were randomly divided into SNI group and sham group,and the contents of mi R-200a-3p/mi R-141-3p in the spinal cord of rats was analyzed on at the 10 th day after the SNI operation2.Constructed dual-luciferase reporter gene vectors and used the dual-luciferase reporter gene experiment to detect the direct binding between mi R-200a-3p/mi R-141-3p and BRD4 3’UTR.3.To detect the effect of mi R-200a-3p and mi R-141-3p on BRD4 expression and mechanical allodynia.By injecting agomir,antagomir to overexpress and silence mi R-200a-3p/mi R-141-3p,we used Western Blotting to analyze the expression of BRD4 in SNI rat spinal cord tissue samples,and the changes of paw withdrawal threshold were analyzed.The experimental rats were randomly divided into mi R-200a-3p agomir group,mi R-141-3p agomir group,mi RNA agomir NC group,mi R-200a-3p antagomir group,mi R-141-3p antagomir group,mi RNA antagomir NC group,SNI group and sham group.To observe the effect of mi R-200a-3p/mi R-141-3p levels on pain behavior and BRD4 in SNI rats,and to clarify the upstream regulatory mechanism of BRD4 in neuropathic pain.4.To observe the effect of mi R-200a-3p/mir-141-3p on mechanical allodynia after inhibiting BRD4 function.Based on inhibition of BRD4 function with BRD4 inhibitor IBET762,mir-200a-3p/Mir-141-3p were overexpressed or silenced,respectively,to detect the changes of pain behavior in rats.Based on the BRD4 inhibitor I-BET762 to inhibit BRD4 function,mi R-200a-3p/mi R-141-3p was overexpressed and silenced to detect the changes of pain behavior in rats.The experimental rats were randomly divided into 7 groups: mi R-200a-3p agomir group,mi R-141-3p agomir group,mi RNA agomir NC group,mi R-200a-3p antagomir group,mi R-141-3p antagomir group,mi RNA antagomir NC group and sham group.It was further confirmed that mi R-200a-3p/mi R-141-3p affected the occurrence and development of neuropathic pain by regulating the expression level of BRD4.Part 3: To confirm the downstream signaling pathways involved in the regulation of BRD4 in neuropathic pain1.To detect the expression of c-myc and ROS markers(8-OXO-d G,HO-1)in spinal cord of SNI rats.In this part of the experiment,the rats were divided into SNI group and sham group,and Western Blotting was used to detect the differential expression of c-myc,8-OXO-d G,and HO-1 in the spinal cord of rats was detected on the 10 th day after the operation.2.To clarify the regulation of BRD4 on c-myc.BRD4 was overexpressed by cell transfection technology,the expression of c-myc was detected by real-time PCR,and the expression of BRD4 and c-myc was detected by Western Blotting and cell immunofluorescence.The regulatory effect of BRD4 on c-myc was verified at the cellular level.3.To detect the changes of c-myc and ROS in spinal cord of SNI rats after inhibition of BRD4 function.The SNI rats were randomly divided into I-BET762 group and vehicle group,and Western Blotting was used to detect the expression changes of c-myc,8-OXOd G and HO-1 in the spinal cord of rats at 10 days after operation.To observe whether BRD4 may regulate neuropathic pain through c-myc during pain in SNI rats.4.To observe the effects of BRD4 on mechanical allodynia and ROS levels after inhibiting c-myc function.10058-F4 was applied to inhibit the function of c-myc,and the expression and function of BRD4 were changed by injection of mi R-200a-3p/mi R-141-3p antagomir and I-BET762.The experimental rats were randomly divided into 8 groups: 10058-F4 group and vehicle group,SNI group,mi R-200a-3p antagomir group,mi R-141-3p antagomir group,mi RNA antagomir NC group,I-BET762 group and DMSO group.The changes of pain behavior and the expression of 8-OXO-d G and HO-1 in rats were detected.It was verified that the regulation of neuropathic pain by BRD4 was dependent on c-myc-mediated ROS.Results: Part 1:1.The expression of BRD4 in the right spinal cord of the rats increased,and the expression of BRD4 reached a peak on the 10 th day after surgery;the immunofluorescence showed that the fluorescence intensity of BRD4 at the dorsal horn of the spinal cord on the affected side was significantly enhanced,indicating that the expression of BRD4 was increased and co-expressed with the dorsal horn neurons in the spinal dorsal horn.2.After intrathecal injection of BRD4 inhibitor I-BET762,the PWT of SNI rats was significantly increased,which could improve mechanical pain in rats’ sensitive situations.3.After intrathecal injection of specific siRNA,the PWT of SNI rats was significantly increased,which could improve the mechanical hyperalgesia of the rats.And the expression of BRD4 in the right spinal cord of SNI rats was significantly decreased.Part 2:1.The expression of mi R-200a-3p/mi R-141-3p in the spinal cord at the right side of SNI rats was significantly decreased.2.The direct binding of mi R-200a-3p/miR-141-3p to BRD4 3’UTR was verified by a dual-luciferase reporter gene assay.3.The injection of mi R-200a/141-3p agomir can overexpress mi RNAs and decrease the expression of BRD4 in the spinal cord of SNI rats,and the PWT of rats was also significantly higher.After injection of mi R-200a/141-3 antagomir silenced mi RNAs,the PWT of the antagomir group was significantly lower and the expression of BRD4 in the spinal cord of SNI rats was significantly increased.4.After inhibiting the function of BRD4 and then injecting mi R-200a/141-3p agomir/antagomir,the PWT had no significant difference PWT between the two groups.Part 3: 1.The expressions of c-myc and ROS in the spinal cord of the rats were significantly increased.2.BRD4 was overexpressed in 293 T cells,the expression of c-myc in the cells after overexpression of BRD4 was significantly increased.3.After the injection of I-BET762 to inhibit the function of BRD4,the expressions of c-myc and ROS in the spinal cord were significantly decreased.4.After inhibiting the function of c-myc,and then changing the expression of BRD4,the PWT and ROS in the spinal cord showed that there was no significant difference.Conclusions:Part 1: The expression of BRD4 increased in SNI rat’ spinal cord and increased with the degree of pain sensitivity.BRD4 was involved in the pathogenesis of neuropathic pain in rats.Part 2: mi R-200a-3p/mi R-141-3p affected neuropathic pain by regulating BRD4 expression.Part 3: BRD4 affected the level of ROS by targeting c-myc and ultimately participates in the regulation of neuropathic pain. |
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