| Objective: To clarify the therapeutic effect of Jiaweidan Xuankoukang on oral submucosal fibrosis;It was determined that Jiawendan Xuankoukang mediated Wnt/ β-catenin signaling pathway by regulating Axin.Methods:(1)Animal experiments: OSF model was established by arecoline,and high-dose,medium-dose and low-dose intervention therapy was given to Jiawei Dan Xuankou Kang.The pathological changes of buccal mucosa were observed by HE staining,and the expression and localization characteristics of β-catenin protein were observed by immunohistochemistry.The expression of Wnt,Axin,β-catenin and APC in Wnt/ β-catenin signaling pathway was detected by Western blot.The m RNA expression levels of downstream genes cylin D1 and C-myc were determined by q RT-PCR;(2)Cell experiment:OSF cell model of HUVECs was simulated by arecoline,and supplemented with Weidan Xuankukang medicated serum,Axin blocker and Axin agonist were given for intervention.Morphological changes of cells in each group were detected by cell imaging analysis.The cell survival rate of each group was detected by CCK8.The cell migration of each group was observed by Giemsa staining.The cell proliferation of each group was observed by crystal violet staining.Tunel was used to detect cell apoptosis in each group.The expression of endothelial mesenchymal transmutation related proteins E-cadherin,N-cadherin,Vimentin and Snai1 in each group by immunofluorescence;Expression patterns of TGF-β,Axin2,Wnt1,β-catenin,cylin D1 and C-myc related proteins and genes in Wnt/ β-catenin signaling pathway were respectively determined by Western blotting and PCR.Results:(1)Animal experiments: The oral mucosa in the modeling area of rats was pale in color,tough in texture,and had a sense of fibrous cord.Compared with the normal group,part of the mucosal epithelium in the model group was atrophied and thinned,and the epithelial spike process was atrophied and disappeared.Compared with the model group,part of the oral mucosal epithelium in the Jiaweidan Xuankou kang high-dose and medium-dose groups was intact,and the shape of the epithelial spike process returned to normal.β-catenin protein expression was increased in the model group,and immunohistochemical mapping showed that β-catenin protein was mainly distributed in the basal layer of epithelium.Compared with the normal group,Wnt andβ-catenin protein expression were increased,while APC and Axin protein expression were decreased.Compared with model group,the protein expressions of Wnt and β-catenin decreased and the protein expressions of APC and Axin increased in high-dose and medium-dose groups of Jiaweidan Xuankou Kang.In the cylin D1,cylin D1 and C-myc fields,respectively,compared with the normal group,the model group had a cylin D1 and cylind1 respectively,whereas the Jiaweidan Xuankoukang high and medium dose groups had a cylind1 and lower C-myc expressions compared with the model group.Cell experiment: Cell imaging analysis results showed that HUVECs cells in both the model group and the blank serum group were significantly damaged,with decreased cell number,cell lysis and intracellular lysosome increase.The cell damage was further aggravated after Axin blocker WIKI4 was given,while Axin agonist HLY78 could alleviate the cell damage of HUVECs.The cell damage model was repaired after the intervention of Jiaweidan Xuankoukang medicated serum and its combination with Axin stimulant HLY78,and the intervention of Weidan Xuankoukang medicated serum and Axin blockerwiki4 could reverse the damage of HUVECs cells to a certain extent.The results of CCK-8 showed that the survival rate of human umbilical vein endothelial cells decreased significantly in model group and blank serum group.Compared with the model group,the survival rate of HUVECs cells in Axin blocker WIKI4 group further decreased,while Axin agonist HLY78 could effectively improve the survival rate of HUVECs cells.After the intervention of Jiaweidan Xuankoukang medicated serum and Jiaweidan Xuankoukang combined with Axin agonist HLY78,The survival rate of HUVECs cells increased significantly,and Weidan Xuankoukang medicated serum combined with Axin blocker WIKI4 could improve the survival rate of HUVECs cells to a certain extent.Giemsa staining showed that the number of HUVECs cells in the model group and the blank serum group was significantly reduced,and the cell body was enlarged,showing irregular polygon shape.Compared with the model group,the cell damage was further aggravated in the Axin blocker group,and the Axin agonist HLY78 could improve these abnormal changes in the above cells.After the intervention of Jiaweidan Xuankou Kang medicated serum and its combination with Axin agonist HLY78,the cell morphology was normalized.Moreover,after the intervention of Weidan Xuankoukang medicated serum and Axin blocker WIKI4,the abnormal changes of cell morphology and number could be reversed to a certain extent.Giemsa staining showed that the number of HUVECs cells in the model group and the blank serum group was significantly reduced,and the cell body was enlarged,showing irregular polygon shape.Compared with the model group,the cell damage was further aggravated in the Axin blocker group,and the Axin agonist HLY78 could improve these abnormal changes in the above cells.After the intervention of Jiaweidan Xuankou Kang medicated serum and its combination with Axin agonist HLY78,the cell morphology was normalized.Moreover,after the intervention of Weidan Xuankoukang medicated serum and Axin blocker WIKI4,the abnormal changes of cell morphology and number could be reversed to a certain extent.Crystal violet staining showed that the migration of HUVECs cells increased significantly in model group and blank serum group.Compared with model group,Axin blocker group further increased HUVECs cell migration,while Axin agonist group significantly decreased HUVECs cell migration.The cell migration of HUVECs was significantly reduced when Jiaweidan Xuankoukang medicated serum and Jiaweidan Xuankoukang combined with Axin agonist HLY78 were administered,and the abnormal cell migration of HUVECs was also significantly improved in Jiaweidan Xuankoukang combined with Axin blocker group.Tunel staining showed that apoptosis of HUVECs cells increased significantly in model group and blank serum group.Compared with model group,apoptosis of HUVECs cells in Axin blocker group was further aggravated,while HLY78 in Axin agonist group could effectively inhibit apoptosis of HUVECs cells.The number of apoptotic cells decreased significantly when Jiaweidan Xuankoukang medicated serum and Jiaweidan Xuankoukang combined with Axin agonist HLY78 were administered,and the combination of Weidan Xuankoukang and Axin blocker could also inhibit the aggravated apoptosis of HUVECs cells caused by WIKI4.Immunofluorescence assay results showed that the expression of mesenchymal transmutation related protein E-cadherin in HUVECs cells was significantly decreased in model group and blank serum group,while the expression of N-cadherin,Vimentin and Snai1 was significantly increased.Compared with model group,E-cadherin in HUVECs cells of Axin blocker group was further down-regulated,and N-cadherin,Vimentin and Snai1 proteins were also down-regulated to varying degrees.When treated with Axin agonist HLY78,supplemented with Weidan Xuankoukang medicated serum and supplemented with Weidan Xuankoukang combined with Axin agonist HLY78,E-cadherin protein expression in HUVECs cells was significantly increased,while N-cadherin,Vimentin and SNAI1 protein expression was significantly decreased.Among them,Jiaweidan Xuankoukang combined with Axin agonist has better effect;Western blotting results showed that the expression levels of TGF-β/Axin path-related proteins,including TGF-β,Wnt1,β-catenin,cylin D1 and C-myc,respectively,in HUVECs model group and blank serum group were significantly increased,whereas Axin2 protein expression was significantly decreased.Compared with model group,Axin blocker group showed further down-regulated Axin2 protein expression in HUVECs cells and further up-regulated β-catenin,cylin D1 and C-myc protein expression in cylind1 respectively.Group compared with model group,Axin agonist HUVECs Axin2 protein expression in cells increased significantly,while beta catenin,cylin D1,C-myc protein expression significantly lower),when given to add jan.1994-mar.1996:as dept.manager xuan mouth drug-containing serum and joint Axin blockers and exciting agent,HUVECs TGF-beta cells,Wnt1,beta-catenin,cylin D1,C-myc expression were significantly lower,and Axin2 protein expression significantly lower,with added jan.1994-mar.1996: as dept.manager xuan mouth kang joint Axin agonist effect is better,and can reverse the protein expression of Axin blockers caused by abnormal;The results of PCR detection showed that compared with normal group,the expression levels of TGF-β,Wnt1,β-catenin,cylin D1 and C-myc were significantly increased in HUVECs model group and blank serum group,whereas the expression levels of Axin2 gene were significantly decreased.Compared with model group,Axin2 gene expression in HUVECs cells in Axin blocker group was further down-regulated,β-catenin,cylin D1 and C-myc gene expressions in cylind1,respectively,and Axin2 gene expression in Axin agonist group was significantly increased.β-catenin,cylin D1 and C-myc gene expressions were significantly down-regulated;Compared with model group,TGF-β,Wnt1,β-catenin,cylin D1 and C-myc gene expressions in HUVECs were significantly reduced and Axin2 gene expression was significantly reduced after treatment with Jiaweidan Xiangukang drug containing serum combined with Axin blockers and agonists respectively.Conclusion: 1 The rat OSF lesion model can be established by local injection of arecoline + local stimulation.2 Added Wei Dan Xuan Kou Kang can improve the symptoms of OSF rat model and inhibit the Wnt/ β-catenin signaling pathway.3 Added Wei Dan Xuan Kou Kang can improve arecoline induced human umbilical vein endothelial cell injury.4.Jiawei Dan Xuankoukang inhibits the expression of Wnt/ β-catenin signaling pathway related proteins and downstream genes by regulating Axin. |
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