Study Of Yisui Tongjing Prescription On Regulatory T Cells Of Experimental Autoimmune Neuritis (ean) Rats

Purpose:To study Yisui Yongjing prescription on the Guillain-Barre Syndrome (GBS) classic animal model - experimental autoimmune neuritis (EAN) laboratory rats pathomorphology ultrastructure of peripheral nerve, nerve electrophysiological function, proliferation functions of spleen lymphocytes, serum IL-10 and TGF-βlevel, the spleen, thymus, lymph cells CD4+CD25+T level, and probe into the cellula immune mechanism of GBS, the mechanism of Yisui Yongjing prescription in treating GBS and its therapeutic effects, and thus provide the experimental basis for Yisui Yongjing prescription, enrich the Encephala Theory in traditional Chinese medicine (TCM) and promote the theory and practice in treating the Flaccidity disease by TCM.Methods:The immune Lewis rat models of EAN were made by P0180-199 Polypeptide and IFA. One hundred successful models from EAN Lewis rats were randomly divided into 5 groups: the model group, immunoglobulin group, high dose Yisui Yongjing prescription group, medium dose group and low dose group, 20 in each group. Twenty normal and healthy rats were taken as the control group.The immune globulin group was treated with 480mg / (kg ? d) immunoglobulin per rat by intraperitoneal injection from the 11th day, and 5 times in all;the high dose group, medium dose group and low dose group were treated by stomach feeding from the 11th day to the day they were killed, and the doses were 2.0ml/d, 1.5ml /d, and 1.0ml/d respectively. On the 10th day, 5 in the control group and 5 in the model group were killed, and on the 18th, 25th, and 39th day respectively, 5 were killed randomly to test the sciatic nerve motor conduction velocity in each group.The changes in ultrastructure of sciatic nerve pathology were observed by electron microscopy; the proliferation functions of spleen lymphocytes were tested by CCK-8 reagent; the serum IL-10 and TGF-βexpression level was tested by ELISA method; the ratios of the spleen, thymus, groin lymph cells CD4+CD25+T were tested by the flow cytometry, and their expression levels of modulation T cells, Foxp3mRNA were tested by RT-PCR method.Results:1.Pathological changes of the sciatic nerve of rats in each group were shown by the electron microscopy. On the 18th day, the model group has changes in the encephala nerve fiber structure, myelination overlapping and corrugating, and axonal was separated from the layers of the myelinated nerve fiber sheath. On the 25th day, myelination was in the process of disappearing or dissolving. And on the 39th day, the trend was more conspicuous. After treatment, the demyelinated nerve fiber sheath was improved and with a more orderly structure.More collagen fibers can be seen and the growth of the myelinated nerve fiber as well.2. The sciatic nerve motor conduction velocity was tested by BL-420. The result shows the velocity in the model group decreased after the 10th day, and the tendency continued on the 18th and 25th day, but began to increase on the 39th day. The velocity of the Yisui Yongjing prescription group and immunoglobulin group is higher than that in the model group on the 10th , 18th, and 25th day, and no significant difference(P>0.05)was shown between the high dose Yisui Yongjing prescription group and the immunoglobulin group on the 39th day. The velocity was higher than that in the medium dose group and low dose group(P<0.01), but lower than that in the control group(P<0.01).3. The experiment of the proliferation functions of lymphocytes showed that the proliferation intensity of the reaction to the stimulation of P0 Polypeptide and ConA increased continuously, and climaxed at the 25th day, then began to decrease. But the intensity of the model group was higher than that in the Yisui Yongjing groups. The intensity of the immunoglobulin group began to decrease on the 18th day. the proliferation functions of lymphocytes was back to normal generally in the immunoglobulin group and Yisui Yongjing prescription groups on the 39th day, and was better than that in the model group.4. The serum IL-10 level of the model group increased on the 10th day, and significantly on the 18th day, but decreased on the 25th day. The level related positively with the seriousness of EAN rats. And TGF-βlevel was lower than that in the control group on the 10th and 18th day, and nearly the same as the control group on the 25th day, and higher than the model group. The level was positively related to the improvement of the EAN rats. The IL-10 level of the immunoglobulin group, high dose Yisui Yongjing group was lower than that of the model group on the four given dates, but the level of TGF-βwas higher than the model group.5. The ratio of CD4+CD25+/ CD4+ in the spleen, thymus, lymph cells of the model group decreased on the 10th day, and so did the Foxp3 mRNA expression level. On the 18th day, the peak of the clinical symptoms, the tendency was more conspicuous, and the CD4+CD25+T rate and the Foxp3 mRNA expression level were the lowest in the model group. On the 25th day, the model rats were in the process of recovery. The number of Treg cells and Foxp3 mRNA expression level increased accordingly.The treatment for the Yisui Yongjing prescription group and immunoglobulin group had positive therapeutic effect on CD4+CD25+T cells. The Foxp3 expression level of the thymus and lymph cells was even better than that the immunoglobulin group. On the 39th day, the therapeutic effects were almost the same as the immunoglobulin group and high dose Yisui Yongjing group, and were better than those of the medium dose and low dose groupsConclusions:1. The Yisui Yongjing prescription significantly improved the sciatic nerve fiber structure of the EAN rats, reduced the myelination loss, caused the myelination layers to align orderly and become clearer. Yisui Yongjing prescription contributes to the recovery and the reproduction of the damaged nerves.2. The Yisui Yongjing prescription can improve EAN rats sciatic nerve motor conduction velocity3. The Yisui Yongjing prescription can restrain the abnormal proliferation reaction of the EAN model rats spleen to the specific antigen or nonspecific antigen, and help to alleviate the immune damage of the CD4+T lymphocyte.4. The Yisui Yongjing prescription can decrease the serum IL-10 expression level in the EAN model rats, the enhance the TGF-βexpression level, thus help to restore the balance of the model rats cells5. The Yisui Yongjing prescription helps to restore the normal number and function of the Treg cells in the thymus, spleen and groin lymph of the EAN rats. With the recovery of the Treg function, the Yisui Yongjing prescription exerts stronger retraining effects on the CD4+T, and contributes positively to the restoration of the immune balance and the termination of the inflammation damage to the peripheral nerve.