| Chronic pulpitis is a chronic inflammatory pulp disease that mainly manifests severe pain. It badly affects the health and the quality of patient’s life, while it has more influence on children which might cause unilateral mastication affecting the maxillary and facial morphology. However, its central regulatory mechanism is not fully elucidated. When pulp is inflammated or injuried, cell morphology, gene expression and synaptic connections change occur in medullary dorsal horn, so as to participate in the process of central sensitization at medullary levels in inflammatory pain, formatting the characteristic symptoms of pulpitis pain. Now more and more evidences have showen that mi RNA is a new way of gene regulatation to participate in pain-related gene expression in dorsal root ganglia and spinal cord dorsal horn. But so far, the central regulation of mi RNA involved in pulpitis pain is still a new field, which has not been reported yet.Our previous study indicated that pulp exposure method can produce desired chronic pulpitis hyperalgesia mode. Compared with the control group, gene chip assay showed that some mi RNA expression changed in rat medullary dorsal horn after one dayof pulp exposure with significant mi R-34 a reduction. The literature supports that mi R-34 a regulates expression and activity of various kinds of pain related genes. These studies provide a new idea of central regulatory mechanism of chronic pain pulpitis: Will mi R-34 a serve as a way to participate in pain-related gene expression in the medullary dorsal horn under pain states? Based on the above hypothesis, we designed the following experiments in order to explore the central regulatory mechanism of chronic pulpitis pain, which might provide new targets and new ideas for the development of analgesic drugs.[Objective]This research intends to explore the central regulation of mi R-34 a in chronic pulpitis pain and screen its target gene(SIRT1). By detecting the expression changes of mi R-34 a and SIRT1 at different time points after tooth pulp exposure and cellular localization in medullary dorsal horn, the relation between mi R-34 a, SIRT1 and chronic pulpitis pain was explored; Overexpression of mi R-34 a and SIRT1 in medullary dorsal horn by virus infection and agonist injection was carried out to investigate whether mi R-34 a takes part in the central regulation of chronic pulpitis pain by inhibition of SIRT1, so as to provide novel targets and new ideas for the development of analgesic drugs.[Methods]1. SD rats were randomly divided into the control group and pulpitis group; after anesthesia, the left maxillary first molars of pulpitis group were drilled with a 1/2 tungsten round bur and exposed to the oral cavity. The control group was not treated after anesthesia. The total time of face grooming was tested to assess pain response in rats before or 1, 3, 7, 14 days after operation, and medulla oblongata and pulp were collected after the behavior test to prepare frozen sections and paraffin sections. Immunofluorescence staining was used to detect expression of TNF-α protein in dental pulp and c-Fos protein in medulla oblongata. HE staining was used to observe pulp inflammation. 2. After establishing chronic acute pulpitis model, gene chips assay was carried out in medulla oblongata with vast expression changes of varieties of mi RNAs includingdownregulation of mi R-34 a. Quantitative real-time PCR were further used to detect the expression of mi R-34 a at different time points; the localization of mi R-34 a in which cell types was visualized by FISF and immunofluorescence double-labeling technique. Through literature review and software, the potential target genes of mi R-34 a were predicted and further screened by real-time quantitative PCR. q PCR and immunofluorescence were further used to validate the target gene expression and cell type positioning. 3. After surgical exposure of medullary dorsal horn, lenti-virus infection was carried out to overexpress mi R-34 a. The dynamic changes of mi R-34 a and SIRT1 in medullary dorsal horn were detected, and chronic pulpitis pain behaviours in rats were observed. After intrathecal catheter in cistern, SIRT1 agonist(resveratrol, Res) was continuous administrated. SIRT1 expression in medullary dorsal horn was detected, and the chronic pulpitis pain symptoms in rats were observed.[Results]1. Pain behavior results showed that pulpitis pain symptom was time correlated and reached a peak at 1 day, and then gradually reduced; the results of HE staining showed that 1, 3, 7, 14 days after operation, inflammation progressed in the pulp horn, the cervial third, the middle and the apical third of radicular pulp; TNF-α was mainly expressed in the inflamed pulp tissue, and the highest expression was in acute stage(1d); immunofluorescent staining showed that 2h after dental operation, c-Fos positive cells in the medullary dorsal horn were significantly increased, suggesting that cells in the medullary dorsal horn were activated. 2. q PCR results showed that compared with the control group, mi R-34 a expression was significantly reduced except 14 days after operation(P<0.05). Immunofluorescence and FISH results showed that there were lots of mi R-34 a hybridization signal in medullary dorsal horn and mi R-34 a coexisted with Neu N, suggesting that mi R-34 a expresses in neurons; Compared to the control group, mi R-34 a was significantly decreased(P <0.05). Based on software prediction and literature, we selected Bcl-2,SIRT1, P53, HGF, Notch-1, USF-1 and Met as the target candidates. SIRT1 was finally proven to be the downstream target of mi R-34 a through q PCR and immunofluorescence in chronic pulp inflammatory pain. SIRT1 expression was significantly upregulated and reached the peak 3 days after operation, then gradually decreased(P<0.05). Immunofluorescence staining results showed that there were many SIRT1 active cells in medullary dorsal horn which coexisted with neurons, but not with DAPI, suggesting that SIRT1 mainly located in neuronal cytoplasm. 3. After virus infection, the degree of pain in pulpitis rats was significantly increased(P <0.05); compared with the control group, mi R-34 a expression was significantly increased by 3.25 times while SIRT1 was 0.45 times lower(P<0.05). After continuous administration of SIRT1 agonists(resveratrol) to cistern, SIRT1 expression was significantly increased compared with the control group(P<0.05); meanwhile, the degrees of pain was alleviated notably comparing with the control group(P<0.05).[Conclusions] 1. Pulp exposure method is simple and can effectively simulate the process of clinical pulpitis, and thus establish an ideal experimental model of hyperalgesia pulpitis. 2. In the medullary dorsal horn, mi R-34 a and SIRT1 mainly express in neurons; under chronic pulpitis state, mi R-34 a is significantly reduced, while SIRT1 is significantly increased with time, suggesting a close relationship between mi R-34 a, SIRT1 and chronic pulpitis pain. 3. SIRT1 has a neuroprotective effect, and overexpression of SIRT1 play obvious analgesic effect on chronic pulpitis pain. 4. Mi R-34 a can inhibit the expression of SIRT1, and thus participate in the central regulation of chronic pulpitis pain. |
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