Effect Of Astragalus Polysaccharide On Biological Behavior Of Oral Fibroblasts And Immunological Condition Of Rats With RAU

Objective:1. To investigate the influences of astragalus polysaccharide (APS) on biological behavior of oral fibroblasts in primary culture.2. To establish an experimental RAU model in SD rats by immunization and to explore the effects of APS on T-lymphocyte subsets and the serum levels of TNF-a in rats with RAU.Methods:1. The primary oral fibroblasts were cultured by covering coverslip in vitro. Inverted phase contrast microscope and immunocytochemistry method were used to observe the efficiency and biological characteristics of oral fibroblasts and to identify the cell origin, respectively.2. The effects of different concentrations of APS on oral fibroblasts proliferation were determined by MTT assay. The change of the total content of protein synthesized by oral fibroblasts was measured using Coomassie brilliant blue.3. Immunization was used to establish RAU models. All rats were randomly divided into five groups:normal control group (A), negative control group (B), 100mg/kg APS treatment group (C), 200mg/kg APS treatment group (D),300mg/kg APS treatment group (E). After 20 days'treatment, rats'oral mucosa tissues were collected to observe the oral mucosa pathological changes. The values of T-lymphocyte subsets in peripheral blood and the serum expression levels of TNF-a in SD rats of each group were measured by flow cytometry (FCM) and ELISA assay, respectively.Results:1. The identification of oral fibroblastsAfter primary incubation for 3～14 days, some fibroblasts emigrated from tissue. Cultured primary cells were elongated fusiform with several different length of processes and plump body. The cells exhibited radical and whirlpool-shaped array under inverted microscope. The immunochemistry study showed the positive staining of vimentin and the negative staining of cytokeratin in cultured cells.2. The effect of APS on the proliferation of oral fibroblastsCompared with the control group, APS at the concentration of 2.5μg/ml,10μg/ml,40μg/ml,160μg/ml could increase oral fibroblasts proliferation significantly after treatment for 24 hours and 48 hours, respectively (P<0.05). After 72 hours, APS at the concentration of 10μg/ml,40μg/ml,160μg/ml also promote the proliferation of fibroblasts (P<0.05), and 160μg/ml APS showed the most significant effect on promoting cell proliferation at this time. The difference between 2.5μg/ml APS group and control group was not significant (P>0.05).3. The effect of APS on the total content of protein of oral fibroblastsCompared with the control group, the total content of protein synthesized by oral fibroblasts increased significantly after treatment with APS at the concentration of 10μg/ml,40μg/ml,160μg/ml for 72 hours (P<0.05). The increase in the total content of protein of cells was the most obvious in 160μg/ml APS group. Whereas, the difference between 2.5,μg/ml APS group and control group was not significant (P>0.05).4. The effect of APS on T-lymphocyte subsets of the peripheral blood in rats with RAUCompared with normal control group (A), the values of CD4+and CD4+/CD8+significantly decreased, while CD8+obviously increased in negative control group (P<0.05). After 20 days' APS administration, the values of CD4+and CD4+/CD8+rose again in rats of APS treatment groups (C～E). Furthermore, there is a significant difference between 300 mg/kg APS treatment group (E) and negative control group (B) (P<0.05).5. The effect of APS on the serum expression levels of TNF-a in rats with RAUThe serum expression levels of TNF-a in negative control group (B) were significantly higher than normal control group (A) (P<0.05).Compared with negative control group, the serum TNF-a levels in APS treatment groups decreased after APS administration. The difference was significant (P<0.05). In addition, the inhibiting effect of high-dose (300mg/kg) APS on TNF-a expression was greater than median-dose (200mg/kg) APS and low-dose (100mg/kg) APS.Conclusions:APS at the concentration of 10μg/ml～160μg/ml can significantly promote proliferation and protein synthesis of oral fibroblasts. Experimental RAU model can be successfully established in SD rats by immunization. The oral lesions caused by this method are similar to human RAU. Therefore, this RAU model can provide an effective means for the study of RAU treatment. In RAU model rats, both the CD4+and the ratio of CD4+/CD8+are obvious lower than the normal control group, while the CD8+increase significantly, and the serum expression levels of TNF-a are obvious higher as well. However, the ratio of T-lymphocyte subsets and the serum expression of TNF-a become normal after APS administration. These results suggest that APS can effectively promote oral ulcer healing, improve the abnormal immune state and suppress the expression of related cytokines in a dose-dependent manner, thus providing scientific and theoretical basis for the application of APS for RAU.