| Sjogren's syndrome (SS) is a chronic autoimmune disease that primarily affects the salivary and lacrimal glands. Characterized histologically by lymphocytic infiltration of the affected glands and clinically by the picture of oral dryness. The syndrome may manifest a systemic disease with widespread autoimmune manifestations and pronounced immunologic features. Various autoantibodies in serum are tested in many patients with hyperimmunoglobulinemia. By now, there has been no effective treatment for dry mouth except for symptomatic therapies such as taking frequent sips of water, the use of artificial saliva in modern medicine. Traditional Chinese Medicine (TCM) is playing an important role in treating SS in our country, which is effective in improving symptoms of patients who are administered with pleasure to grow enthusiasm in work and a joy of living with little toxic and side-effect. However, the clear target and mechanism of TCM against SS is still unknown.Jinxueyuan Granules as an important preparation in Jiangsu Province Hospital of TCM is a clinically effective Chinese medicine treating SS. In order to provide the experimental basis for the therapeutic action and mechanism of SS and clinical using of Jinxueyuan granules, we investigated the effects of Jinxueyuan granules on the immune function of SS model animals, the distribution of water channel protein (aquaporin,AQP5) and the changes of intracellular calcium concentration of rat submandibular gland cells, ranging from molecular level to in vivo.Objective:To develop a Lewis rat model by immunity similar to SS and observe the therapeutic effects of Jinxueyuan granules, examine the expression of AQP5 in rats salivary gland. The influence of Jinxueyuan granules on intracellular calcium concentration were investigated on cultured submandibular gland cells of rats.Methods:Establishing a Lewis rat model of SS by immunity according to the literatures, Lewis rats were divided randomly into 5 groups (control, SS model, adjuvant, low-dosage and high-dosage Jinxueyuan granules). The submandibular gland (SMG) antigen of Lewis rats was purified by differential centrifugation. Then Lewis rats of SS model and drug therapeutic groups were sensitized on multiple spots with SMG antigen in complete Freund adjuvant (CFA). These rats had received different treatment for 21 days. During the whole experiment, the volume of salivary secretion was determined every three days, body weight of rats and the consumption of food and water were measured every day. Finally, the organ indexes including those of SMG, spleen and thoracic gland were calculated, pathologic slices were made by HE stain, and the characteristic of the AQP5 expression was analyzed in submandibular gland by immunohistochemistry.In vitro experiment, primary submandibular gland cells were cultured from younger rats. and dyed with Fluo-3. The changes of light density of cells under confocal laser microscopy were investigated by adding Jinxueyuan granules of different concentration.Results:1. The HE stain showed that the model rats had moderate lymphocytes infiltration in substantial with the destruction of ducts. The SMGs index of the model rats were bigger than normal ones. Besides, the salivary secretion of SS model rats also was obviously decreased from modeling 10 days, the water uptake of them was obviously increased compared with the normal group. From these experimental results, it can be concluded that the rat model of SS were successfully established.2. Jinxueyuan granules could significantly reduce the lymphocytes infiltration, alleviate the SMG swell, decrease the immune organ indexes, and promote the salivary secretion of model animals. The changes of the weight, the consumption of food and the consumption of water of the SS model animals were observed dynamically and Jinxueyuan granules in mild concentration could improve the symptom of SS rats.3. There were much more AQP5 protein on the salivary glands cell membrane in the normal rats, and AQP5 protein was also expressed on the cell membrane in the adjuvant group, but significant decreased while comparing to the normal group. The acinus was analysed in the SS rats. The expression of APQ5 protein was significantly decreased in the apical membrane of surviving acinus, and there was a significant difference while comparing to the normal group. The APQ5 protein was expressed in most of cell membrane of the SS rats, and it was significantly increased comparing to the model group, distributed equally.4. The calcium-dependent secretion of the salivary glands is one of an important way of salivary glands secreting, the Jinxueyuan granules promoted the calcium-dependent secretion of the salivary gland by increasing the intracellular calcium concentration of the salivary gland cells, and these could partly explain the mechanism of action of Jinxueyuan granulesConclusion:These results show that Jinxueyuan granules might treat SS by inhibiting the enhancement of immune function of SS model animals and reducing the lymphocytes infiltration, increasing the expression of the AQP5. Salivary gland fluid secretion induced by Jinxueyuan Granules might be related with the increase of rat gland intracellular calcium concentration. |
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