Effects Of Rho Kinase Inhibitor Fasudil On Glial Cell Activation Induced By Tooth Movement Pain

Background:The pain caused by orthodontic treatment seriously affects the physical and mental health of patients.The pain caused by tooth movement is mainly mediated by the medullary dorsal horn(MDH).Non-steroidal anti-inflammatory drugs(NSAIDs),as the most widely used orthodontic analgesic drugs,have been proven to have side effects of interfering with bone remodeling and reducing the rate of tooth movement,which makes their clinical application controversial.Therefore,it is imminent to find a better way of orthodontic pain relief.In recent years,studies have shown that glial cells play an important role in the acute attack and maintenance stages of maxillofacial pain,and the activation of glial cells can be mediated by Rho-related protein kinase(ROCK).Fasudil,as a ROCK inhibitor,has gradually become a new hotspot in the research of analgesia.Objective:This study intended to establish a model of experimental tooth movement pain in rats,and to observe the effect of experimental tooth movement pain on the state of microglia and astrocytes in rat MDH,exploring the relationship between orthodontic pain and glial cells.At the same time,by applying the ROCK inhibitor fasudil to the experimental tooth movement pain model in rats,observing its effect on glial cells,and exploring the potential therapeutic effect of fasudil on orthodontic pain,for clinical new ideas for managing orthodontic pain.Methods:1.A helical tension spring was placed between the incisor and the first molar of SD rats to establish a model of experimental tooth movement in rats,through the Von Frey mechanical pain sensitivity test,the pain degree of experimental tooth movement in rats was evaluated.2.By immunofluorescence staining,we observed the expression of nociceptive neuron reactive protein c-Fos in the MDH,we analyzed the activation degree and type of microglia,and the activation degree and morphological changes of astrocytes.3.Through q RT PCR,the expression levels of inflammation-related cytokines and pain-related genes in MDH were detected.4.By intraperitoneal injection of fasudil,we evaluated the changes in the pain degree of experimental tooth movement in rats,observed the expression changes of the pain response protein c-Fos in MDH,analyzed the activation degree of microglia and the activation degree,morphological changes of astrocytes,the expression changes of the above-mentioned inflammatory factors and pain genes were evaluated.5.At the same time,we measured the mesial movement distance of the first molars of rats with a vernier caliper to evaluated the effect of fasudil on the experimental tooth movement distance of rats.Results:1.After the establishment of the experimental tooth movement pain model,the cFos protein in rat MDH was strongly expressed on the first day of tooth movement,and then gradually decreased.2.The specific marker OX 42 related to the activation of microglia was significantly express from the first day after tooth movement,the expression level reached the peak on the third day after tooth movement,and then gradually decreased.Among them,the activated microglia cells were mainly of M1 type.3.The expression of GFAP,a specific marker of astrocyte activation,began to express significantly on the third day of tooth movement,and reached its peak on the fifth day,and the morphology of astrocytes also became complex,showing the number of branches increases,the cell body became larger,and then its shape gradually recovered.4.After the treatment of ROCK inhibitor fasudil,the expression of the above related proteins showed a downward trend.Fasudil significantly reduced the expression of protein c-Fos,inhibited the activation of microglia,increased the proportion of M2 glial cells in activated microglia,and inhibited the activation of astrocytes,and restored its form to its resting state.5.After the experimental tooth movement pain model established,the m RNA levels of pro-inflammatory factors IL-1β and TNF-α peaked at three days after tooth movement,and then gradually decreased,the m RNA levels of anti-inflammatory factors TGF-β was the lowest on the third day of tooth movement,and then gradually increased.After fasudil treatment,the expression of pro-inflammatory factors showed a downward trend,while the expression of anti-inflammatory factors showed an upward trend.After the experimental tooth movement pain model was established,the expression of pain-related genes CSF-1,t-PA,CTSS and BDNF at the m RNA level increased significantly,and could be inhibited by fasudil.6.The application of fasudil had no significant effect on the distance of tooth movement in rats.Conclusion:Experimental tooth movement pain can cause asynchronous activation of microglia and astrocytes in the MDH area of rats,and cause a local inflammatory microenvironment in the medulla oblongata,The ROCK inhibitor fasudil can relieve the pain of experimental tooth movement in rats by inhibiting the activation of glial cells and reduce the level of local inflammation in the medulla oblongata with no effect on the distance of experimental tooth movement.