To Investigate The Role Of The Chinese Herbal Medicine Xianhuayin On The Reversal Of Premalignant Mucosal Lesions In The Golden Hamster Buccal Pouch With Ultramicrostructure And Immunohistochemical Method

Objective:There was a correlation between oral mucosal premalignant lesion and carcinoma of oral mucosa. These years, people had been paid more and more attention on the carcinoma of oral mucosa preservation and treatment to cut down the disease incidence of carcinoma of oral mucosa. Now, there were lots of therapeutic tool for oral mucosal premalignant lesion. And mainly divide into drug treatment and un-drug treatment. The un-drug treatment usually included operation treatment,laser therapy,freezing therapy and so on. But these ways had indication confine and higher recurrence of post-treatment, especially could not prevent the new lesion appearance in other part of oral cavity. So these defects greatly cut down the un-drug treatment possibility. Medical doctor treat such as ratinoic acid would had adverse reaction if long-term using. So, the emphasis of oral mucosal premalignant lesion drug treatment was to prevent the new lesion appearance in other part of oral cavity and deeply developed. And Chinese herbal medicine properly had these ideal condition. These years research showed that Chinese herbal medicine had played markedly therapeutic effect during the oral mucosal premalignant lesion treatment process. And it had become to oral mucosal premalignant lesion research hot spot. The topic investigated the role of Xianhuayin on the reversal of the premalignant lesion in the mucosa of cheek pouch in golden hamster by using immunohistochemical methodand electron microscope by premalignant lesion animal model generation.Methods:A total of 55 SPF LVG Syrian golden hamsters (6 weeks old, weight: 80 g) were randomly divided into untreated control group (n=5) and experimental group (n=50). Animals in the experimental group were used to generate the oral mucosal premalignant lesion models. Of these 50 animals with oral mucosal premalignant lesions, 10 hamsters were treated with normal saline (NS-treated group); 10 hamsters were untreated (premalignant lesion group); 30 hamsters were treated with Xianhuayin (Xianhuayin-treated group). The buccal mucosa of 50 hamsters in the experimental group was painted with DMBA (0.5% in acetone) as described previously. Application with DMBA was performed three times a week for a continuous of 6 weeks. Then intragastic administration was performed once a day for a continuous 8 weeks in Xianhuayin-treated group and NS-treated group. All the animals were sacrificed and the tissue samples were collected at the same time. To investigate the role of Xianhuayin on the reversal of the premalignant lesion in the mucosa of cheek pouch in golden hamster by using immunohistochemical method,electron microscope.Results:1 Premalignant lesion animal model generationSix weeks after treatment with DMBA acetone solution, in the experimental group, both sides of the cheek pouch mucosa of hamsters became adhered, lacking of elasticity, congested, anabrotic and streak-like ruffle. Histological analysis was performed by randomly collecting ten part of lesion tissues, which had different of visual manifestation. Variable degree of epithelial dysplasia was observed under light microscopic . So six weeks after treatment with DMBA acetone solution, it had successfully generated the premalignant lesion animal model.2 Role of Xianhuayin on the reversal of premalignant lesion in the oral buccal mucosa of golden hamsters2.1 Visual observationsThe cheek pouch mucosa of hamsters in the untreated-group exhibited smooth surface, light pink color, transparency, elasticity and clear blood vessels. Six weeks after treatment with DMBA acetone solution, the cheek pouch mucosa of hamsters in the experimental group became adhered, rough, lacking of elasticity, congestion, anabrosis and streak-like ruffle was evident . Eight weeks after intragastric administration with Xianhuayin, the adherence of cheek pouch mucosa became mitigated, smooth surface appeared gradually, and congestion as well as anabrosis disappeared, but the elasticity was still relatively poorer than the normal tissues. No significant changes were observed after the hamsters in the premalignant lesion group were intragastrically administered with normal saline .2.2 Light microscopic observationsBuccal mucosa of the normal hamsters in the untreated group was keratinized and stratified squamous epithelium under light microscope. Lamellar connective tissues, small amount of sporadic fibrocytes and blood vessels were present between epithelium and muscular layer . Variable degree of epithelial dysplasia were observed in the hamster of premalignant group 8 weeks after painting with DMBA was stopped. Eight weeks after treatment with Xianhuayin, the irregular epithelial mucosa gradually became distinct, the layer of epithelium was recovered to normality, basilar membrane cells were regularly organized and the number of atypical cells was reduced . No significant changes were observed after hamsters were intragastrically administered with normal saline .2.3 SEM analysisScanning Electronic Microscopic (SEM) analysis showed that in the hamster of untreated-group, the buccal mucosa contained polygonal cells and clear cell bridge. The surface of the individual cells exhibited honeycomb structures . Eight weeks after painting with DMBA was stopped, the epithelial cells were morphologically irregular, overlapped and loosened. Swelling and widening micro-crests were irregularly arranged and appeared as gravel shape. Eight weeks after intragastric administration with Xianhuayin, most of the cell surface exhibited honeycomb structures and continuous cellular border was observed in the hamsters of Xianhuayin-treated group . No significant changes were observed for the hamsters in NS-treated group compared with those in the premalignant group.2.4 TEM analysis Transmission Electronic Microscopic (TEM) analysis showed that in the hamsters of untreated group, buccal mucosal epithelial cells were morphologically regular, closely connected and contained abundant desmosomes. The tonofibrils were developed normally. Eight weeks after painting with DMBA was stopped, desmosomes and tonofibrils were significantly reduced, intercellular gap was increased, the shape of epithelial cells were irregular, the morphologically irregular nucleolus was marginally gathered and the caryotheca was invaginated. Eight weeks after intragastric administration with Xianhuayin, the widening intercellular gap became reduced gradually, desmosomes and tonofibrils were gradually becoming abundant and the cells were becoming morphologically regular. No significant difference was observed between the hamsters in NS-treated group and those in the premalignant group.3 Expression of EZH2,PCNA in different group after Xianhuayin treatmentEZH2,PCNA were rarely expressed or lower expressed in the untreated group and Xianhuayin-treated group, and they were higher expressed in the premalignant group and NS-treated group.The rate of EZH2,PCNA high expression of the premalignant lesion group compared with the untreated control group's, it had statistical difference (P<0.05); The rate of EZH2,PCNA high expression of the Xianhuayin-treated group compared with the premalignant lesion group's, it had statistical difference (P<0.05); The rate of EZH2,PCNA high expression of the Xianhuayin-treated group compared with the untreated control group's, it had no statistical difference (P>0.05); The rate of EZH2,PCNA high expression of the NS-treated group compared with the Xianhuayin-treated group's, it had statistical difference (P<0.05)Statistical analysis, there was a correlation between EZH2 and PCNA, and the expression goodness of fit had statistical difference.Conclusion:1 Six weeks after treatment with DMBA acetone solution, it had successfully generated the premalignant lesion animal model.2 Eight weeks after intragastric administration with Xianhuayin, congestion as well as anabrosis disappeared; the cells were becoming morphologically regular; the number of atypical cells was reduced; the rate of EZH2,PCNA high expression obviously decreased. It's concluded that Xianhuayin had the obvious role on the reversal of premalignant mucosal lesions in the golden hamster buccal pouch.