| ObjectiveWnt/β-catenin signal is closely related to cell proliferation,differentiation and tissue development[1].In recent years,it has also received extensive attention in the field of neuropathic pain research.Trigeminal neuralgia（TN）is a common neuropathic pain（NP）in clinic,and its pathogenesis has not been fully elucidated.This project intends to establish a rat model of chronic compression of the trigeminal root entry zone,analyze the expression characteristics of Wnt/β-catenin signaling related molecules in the TREZ area of TN model rats,and clarify the activation type of astrocytes and its relationship with Wnt/β-catenin signaling pathway activation is related to the two,and further application of lentivirus-mediated Sh RNA targeting to silence the expression ofβ-catenin in the TREZ region,to explore the Wnt/β-catenin signaling and astrocytes involved in TN peripheral sensitization Possible mechanism.Methods:1.Adult male SD rats were randomly divided into 4 groups:sham operation group（Sham group）,TN group,TN+LV-scramble sh RNA group,TN+LV-β-catenin sh RNA group.The TN rat model was established by compressing TREZ through the intracranial cannula of the right infraorbital fissure thin guide wire.von Frey wire was used to detect the orofacial mechanical pain threshold of each group of rats.2.Western blot detection of Wnt signaling related proteins such as Wnt signaling pathway ligands Wnt-3a,Activeβ-catenin,Frizzled receptor Frizzled 8,complement C1q,and other Wnt signaling pathway ligands in the TREZ region of the Sham group and the TN group at 7,14,21,and 28 days after surgery.The expression of interleukin IL-18,tumor necrosis factor TNF-αand other inflammation-related mediators,as well as the changes in the expression of activeβ-catenin,C1q after the local targeted knockdown ofβ-catenin by TREZ.Immunofluorescence double-labeling method was used to detect the expression and localization of Activeβ-catenin in the TREZ.3.Using immunofluorescence methods,observe the co-localization of glial fibrillary acidic protein GFAP and complement C3,the markers of astrocytes in the TREZ area 14days and 21 days after surgery,and study the proportion of GFAP/C3 co-labeled immuno-double positive processes,The ratio of the total number of GFAP immunopositive processes.Results:1.The results of orofacial mechanical pain threshold showed that the mechanical pain threshold of the TN group decreased significantly on the 14 and 28 days after operation,and the difference was statistically significant compared with the sham group（p<0.05）.Compared with the TN+LV-scramble sh RNA group,the mechanical pain threshold of rats in the TN+LV-β-catenin sh RNA group did not change significantly at 21 days after surgery,and it increased significantly at 28 days after surgery.2.Western Blot results showed that the protein expression of Wnt-3a,Frizzled 8,Activeβ-catenin,C1q was significantly increased in the TN group,and C1q expression was significantly down-regulated after specific silencing ofβ-catenin.3.The immunofluorescence results showed that the expression of Activeβ-catenin in the astrocytes in the TREZ area of the TN group after chronic compression injury was significantly increased,and the co-localization of Iba1 positive cells and Activeβ-catenin could be seen.In addition,the number of GFAP/C3 double-positive processes in the TN group,TN+LV-scramble sh RNA group and the adjacent peripheral GFAP/C3 double-positive processes increased significantly at different time points after chronic compression injury,with the most significant increase at 14 and 21 days after surgery.After targeted knockdown ofβ-catenin,the fluorescence expression intensity of C3 in the TREZ area of the TN+LV-β-catenin sh RNA group and the number of GFAP/C3 double positive processes in the total GFAP immunopositive processes were significantly reduced,compared with the TN+LV-scramble sh RNA group,The difference is significant（p<0.05）.Conclusions:1.Chronic mechanical compression up-regulates the expression of Wnt-3a,Frizzled 8,Activeβ-catenin and A1 astrocyte activation in the trigeminal root entry zone;2.Wnt3a/β-catenin/C1q signal-mediated A1 astrocytes activation participated in TN peripheral sensitization. |
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