| Objectives:Inflammatory pain is pain triggered by the activation of inflammatory mediators associated with pain enhancement following peripheral tissue injury,and its mechanisms mainly involve sensitization at the peripheral and central levels.Inflammatory pain,a common clinical condition affecting 25-35% of adults worldwide,is currently treated mainly with NSAIDs but still suffers from inadequate pain relief and side effects such as gastrointestinal bleeding,acute myocardial infarction,heart failure and acute kidney injury,and therefore new therapeutic approaches are urgently needed.IRF7(Interferon regulatory factor IRF7 is a member of the interferon regulatory factor transcription factor family,which is involved in pathogen defense,autoimmunity,lymphocyte development,cell growth,etc.Its pro-inflammatory effects in lung inflammation,viral diseases,vascular regeneration,and cardiac hypertrophy have also been reported.The results of our group’s previous study showed that spinal IRF7 expression levels were significantly downregulated in a complete Freund’s adjuvant(CFA)-induced inflammatory pain model,suggesting that spinal IRF7 is involved in the process of inflammatory pain.However,it is not clear the role and mechanism of IRF7 in CFA-induced inflammatory pain.Method:1.A rat model of complete Freund’s adjuvant(CFA)-induced inflammatory pain was constructed to clarify the changes of IRF7 expression and cellular expression localization in the rat spinal cord.Fortyeight healthy adult male SD(Sprague Dawley)rats,weighing 180-220 g,were randomly divided into a sham-operated group(NS group)and a complete Freund’s adjuvant-induced inflammatory pain group(CFA group).The rats were injected with 100 ul of complete Freund’s adjuvant intradermally into the right hind toe to construct a complete Freund’s adjuvant-induced inflammatory pain rat model,and the sham-operated group was injected with an equal amount of 0.9% saline(NS)at the same location.The pain behaviors(mechanical pain,thermal pain)of the right hind limb of the rats were examined 1 day before and 1,3,7,14 and 21 days after the modeling of inflammatory pain,and the expression level and fluorescence intensity of IRF7 protein in the lumbar expansion(L4-L6 segment of spinal cord)were detected by protein immunoblotting(Western blotting)and immunofluorescence staining(IF).The expression level and fluorescence intensity of IRF7 protein in the spinal cord of the lumbar expansion(L4-L6 segments)were measured by western blotting and immunofluorescence(IF).Immunofluorescence staining(IF)was used to detect the localization of IRF7 expression in the rat spinal2.Intrathecal injection of adeno-associated virus AAV-IRF7 and verification of its overexpression spinal IRF7 protein expression effect and the effect on basal pain threshold in rats.Thirty-two healthy adult male SD rats,weighing 180-220 g,were randomly divided into four groups: shamoperated group(NS group),complete Fusarium adjuvant-induced inflammatory pain group(CFA group),CFA + AAV-IRF7 adenoassociated virus group(CFA + AAV-IRF7 group),CFA + negative control virus group(CFA + AAV-IRF7 group),inflammatory pain AAV-IRF7 and AAV-NC 10 ul were injected intrathecally into the CFA + AAV-IRF7 and CFA + AAV-IRF7 groups respectively on day 21 before modeling,and the changes in pain behavior(mechanical and thermal pain)in the right hind limb of mice were detected on days 1,3,7,10 and 14 after modeling,and inflammatory pain was modeled.On the 14 th day after pain modeling,the lumbar expansion(L4-L6 segment of spinal cord)was taken and the expression level of spinal IRF7 protein was detected by protein immunoblotting(Western blotting)and immunofluorescence staining(Immunofluorescence,IF).3.To verify the changes in the expression of NF-κB and related inflammatory factors downstream of IRF7 in the spinal cord.The spinal cord of rats with lumbar expansion(L4-L6 segments)in each of the above subgroups was examined by protein immunoblotting(Western blotting)and quantitative real-time polymerase chain reaction(qRT-PCR)to determine the expression levels of spinal NF-κB protein and TNF-α(Tumor necrosis factor-α),IL-6(Interleukin-6),IL-1β(Interleukin-1-beta)mRNA expression levels.Result:1.Compared with the NS group,mechanical pain thresholds in rats with fully Feverfew adjuvant-induced inflammatory pain were significantly lower on day 7 after modeling and persisted until day 21 after modeling;thermal pain thresholds were significantly lower on day 1 after modeling,and the differences were still statistically significant on day 21 after modeling.The expression level and fluorescence intensity of IRF7 protein in spinal cord lumbar expansion were significantly and gradually decreased from day 7 to day 14 after modeling compared with the NS group.IRF7 was mainly expressed in spinal cord dorsal horn neurons and to a lesser extent in spinal cord microglia.2.Rats in the CFA + AAV-IRF7 group had significantly higher mechanical pain thresholds from day 10 to day 14 after modeling compared with rats in the CFA + AAV-NC group,and rats in the CFA + AAV-NC group had significantly higher thermal pain thresholds from day 7 to day14 after modeling,when injected intrathecally with AAV-IRF7 and negative control virus AAV-NC,respectively.The spinal lumbar expansion IRF7 protein expression level and fluorescence intensity were significantly higher in the CFA + AAV-NC group rats compared with the CFA + AAV-NC group rats.3.Compared with the NS group,the protein and TNF-α and IL-6mRNA expression levels of NF-κB were significantly increased in the spinal cord of rats in the CFA group.Compared with the CFA + AAV-NC group and the CFA + AAV-IRF7 group,the protein of NF-κB and the expression levels of TNF-α、IL-1β and IL-6 mRNA in the spinal cord of rats were significantly decreased.Conclusion:In summary,our study shows that spinal IRF7 expression is downregulated in a rat model of inflammatory pain induced by complete Freund’s adjuvant.Overexpression of spinal IRF7 may negatively regulate the neuroinflammatory response in the spinal cord and alleviate inflammatory pain by inhibiting the expression of NF-κB and downstream proinflammatory factors. |
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