| Objective:To explore the establishment of the experimental model of nodular goiter on rats. To investigate the pathogeny of nodular goiter in rats. To observe the effect of "Huo-Xue-Xiao-Ying" on nodular goiter model rats. To observed the effect of "Huo-Xue-Xiao-Ying" on form of thyroid in nodular goiter model rats; to approach the effect of "Huo-Xue-Xiao-Ying" on apoptosis of thyrocyte in nodular goiter model rats; to approach the effect of "Huo-Xue-Xiao-Ying" on cell proliferation of thyrocyte in nodular goiter model rats; to approach the effect of "Huo-Xue-Xiao-Ying" on vascularization of thyroid in nodular goiter model rats; to approached the effect of "Huo-Xue-Xiao-Ying" on chemotaxis of thyrocyte in nodular goiter model rats.Methods:1To prepare the model of nodular goiter in rats and grouping:60SD male rats,weighing120-140g, were separated into six groups at random, scilicet, normal control group.model control group,L-T4 group, low dose " Huo-Xue-Xiao-Ying " group,medium dose " Huo-Xue-Xiao-Ying" group,high dose " Huo-Xue-Xiao-Ying " group. The subsequence five groups had been drenched0.1%PTU solution and normal the control group drenched equivalent physiological saline until the end of experiment. After8weeks, the L-T4group was drenched0.1mg/l L-T4solution;low、medium、 high dose "Huo-Xue-Xiao-Ying" groups each were drenched44g/l、88g/l、176g/1"Huo-Xue-Xiao-Ying" solution; the model control group was drenched equivalent distilled water. Which continue after8weeks, the rats were sacrificed under pentobarbital anaesthesia.2ml blood were collected. The thyroid glands were devided inio three parts:one was fixed with4%paraf ormaidehyde; another was fixed with3%glutar aldehyde for electron mieroscopy;the third was preserved in-20℃for Western blot analysis or flow cytometer detecting.2Morphological observation:section preparations were stained and electron microscope observed.3Western blotting:To detect the expression of PCNA in thyrocyte of rats.4Flow cytometer detecting:apoptosis of thyrocyte in rats.5ELISA method:to detect density of VEGF in serous of rats.6Immunohistochemical method:To detect the expression of FGF-2and CXCR-4in thyrocyte of rats. The results were analyzed quantified with the softwire Image-Pro Plus6.0.Result:1To observe ultrathin tissue slice on electron microscope indicated that.follicular cavity,the smaller expansion of endoplasmic reticulum.and the small particles of the common free ribosomes in the cytoplasm can be seen within the thyroid tissue of "Huo-Xue-Xiao-Ying" group rats compared with model group; there were more lysosomes compared with normal group.2The result of the expression of PCNA detected by Western blotting showed that,compared with model group,the expression of PCNA significantly decrease in normal group (P<0.01);L-T4and low,medium and high dose "Huo-Xue-Xiao-Ying" can decrease the expression of PCNA in thyroid cell of nodular goiter rats and it was reduced more significantly with the increased concentration of "Huo-Xue-Xiao-Ying", so the expression of PCNA in thyroid cell was the most significantly inhibited by high dose of "Huo-Xue-Xiao-Ying"(P<0.01).3Flow cytometer detecting apoptosis showed that,compared with model group, apoptosis rate significantly increased in thyroid cell of the normal group rat (P<0.01). L-T4and low, medium and high dose "Huo-Xue-Xiao-Ying" can promote apoptosis of thyroid cells in the model rat,and apoptosis is more obvious with the increase of the concentration of of " Huo-Xue-Xiao-Ying ", so high dose "Huo-Xue-Xiao-Ying" played the most significant role in promoting apoptosis in thyroid cells of rats (P<0.01).4The results of concentration of VEGF in serum detected by ELISA showed that, compared with the model group, the concentration of VEGF reduced in the serum of normal control rats (P<0.05); L-T4and low, medium and high dose can reduce the concentration of VEGF in the serum of rats, concentration of VEGF was reduced more significantly with the increase of the concentration of "Huo-Xue-Xiao-Ying", so the expression of VEGF in thyroid cell was the most significantly inhibited by high dose "Huo-Xue-Xiao-Ying"(P<0.01). 5The result of the expression of FGF-2detected by the immunohistochemistry assay show that, compared with normal control group, expression of FGF-2was strongly positive in thyroid tissue of model group rat; compared with the model group,The expression of FGF-2decreased in thyroid tissue of L-T4, low,medium and high dose "Huo-Xue-Xiao-Ying" group rats. Quantitative morphological analysis showed that the area and integrated optical density of positive expression of FGF-2in the model group was significantly larger than the normal group, L-T4group and group (P<0.01); the area and integrated optical density of positive expression of FGF-2in the low dose "Huo-Xue-Xiao-Ying" group was significantly larger than high dose "Huo-Xue-Xiao-Ying" group (P<0.01).6The result of the expression of CXCR-4detected by the immunohistochemistry assay show that, compared with normal control group, expression of CXCR-4was strongly positive in thyroid tissue of model group rat; compared with the model group,The expression of CXCR-4decreased in thyroid tissue of L-T4, low,medium and high dose "Huo-Xue-Xiao-Ying" group rats. Quantitative morphological analysis showed that the area and integrated optical density of positive expression of CXCR-4in the model group was significantly larger than the normal group, L-T4group and group (P<0.01); the area and integrated optical density of positive expression of CXCR-4in the low dose "Huo-Xue-Xiao-Ying" group was significantly larger than high dose "Huo-Xue-Xiao-Ying" group(P<0.01).Conclusion:1"Huo-Xue-Xiao-Ying" can improve the morphology of thyreoid in nodular goiter model rats. 2"Huo-Xue-Xiao-Ying" can inhibit cell proliferation of thyrocyte in nodular goiter model rats.3"Huo-Xue-Xiao-Ying" can promote apoptosis of thyrocyte in nodular goiter model rats,which takes on dose-effect relationship.4"Huo-Xue-Xiao-Ying" can inhibit vascularization of thyroid in nodular goiter model rats. Which takes on dose-effect relationship.5"Huo-Xue-Xiao-Ying" can inhibit chemotaxis of thyrocyte in nodular goiter model rats. |
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