益髓通经方对实验性自身免疫性神经炎大鼠调节性T细胞的影响
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摘要
目的:通过观察益髓通经方对格林-巴利综合征(GBS)的经典动物模型—实验性自身免疫性神经炎(EAN)大鼠周围神经病理形态学超微结构、神经电生理功能、脾淋巴细胞增殖功能及对血清IL-10、TGF-β水平和胸腺、脾、淋巴结CD4+CD25+T细胞mRNA表达水平的影响,探讨GBS的细胞免疫机制以及益髓通经方对本病的治疗机理,验证益髓通经方对GBS的临床疗效,进一步丰富中医“脑髓”理论,完善中医治疗痿病的理论和实践。
方法:采用P0180-199多肽及IFA免疫Lewis大鼠制备EAN动物模型。将造模成功的大鼠100只随机分为模型组、免疫球蛋白治疗组,益髓通经方高、中、低剂量组5组,每组20只,另取20只正常鼠作为空白对照组,共分为6组。免疫球蛋白治疗组于免疫后第11天起给予人免疫球蛋白注射液480mg/只·天腹腔注射治疗,共5天。益髓通经方高、中、低各组于免疫后第11天分别给予益髓通经方溶液2.0ml/d、1.5ml /d、1.0ml/d灌胃治疗直至处死;在免疫后第10天,空白组及模型组各取5只大鼠处死、在免疫后第18、25、39天各组分别随机抽取5只大鼠处死检测各组大鼠坐骨神经运动传导速度;应用光镜、电镜观察坐骨神经病理形态学及超微结构的变化;采用CCK-8试剂检测大鼠脾脏淋巴细胞增殖能力;ELISA法检测血清IL-10、TGF-β表达水平;采用流式细胞技术检测各组大鼠胸腺、脾、腹股沟淋巴结CD4+CD25+T/ CD4+T比率,并以RT-PCR法检测上述淋巴器官调节性T细胞Foxp3 mRNA表达水平。
结果:
1.通过电镜观察各组大鼠坐骨神经病理变化:模型组在免疫后第18天出现有髓神经纤维结构病变,髓鞘折叠起皱、轴索与髓鞘板层分离等改变,在第25天时出现较明显髓鞘脱失或髓鞘溶解,在39天时仍有广泛脱髓鞘及轴索崩解改变。经治疗,益髓通经方治疗组与免疫球蛋白组的髓鞘脱失情况得到明显改善,有髓纤维髓鞘板层结构整齐,同时还可见到胶原纤维聚集及有髓神经纤维新生等改变。
2.应用BL-420生物机能实验系统检测EAN大鼠坐骨神经运动传导速度。模型组在免疫后第10天起运动神经传导速度下降,在第18天、25天进一步下降,在第39天时神经传导速度略有改善。益髓通经方各治疗组及球蛋白治疗组在上述时间点均优于模型组,在第39天治疗4周时,益髓通经方高剂量组神经传导速度同球蛋白治疗组比较无显著性差异(P>0.05),优于中、低剂量组(P<0.01),但均不及空白组(P<0.01)。
3.淋巴细胞增殖实验显示,模型组及益髓通经方各治疗组对P0多肽及ConA的刺激增殖反应在第10天、18天、25天进行性增强,在第25天达到顶峰,随后下降,但模型组的增殖强度均高于同期中药治疗组。球蛋白治疗组淋巴细胞增殖强度在第18天起下降。在第39天时益髓通经方各治疗组同球蛋白组淋巴细胞增殖反应基本恢复正常,均优于模型组。
4.在免疫后第10天,模型组血清IL-10水平增高,在第18天显著增高,在25天起下降,与EAN大鼠疾病严重程度呈正相关;模型组血清TGF-β水平在免疫后第10天、第18天低于空白组,在免疫后第25天与空白组持平,在39天高于空白组,与EAN大鼠病情改善情况呈正相关。益髓通经方高剂量组及球蛋白治疗组IL-10水平在以上各时间点均低于模型组,而其TGF-β水平则高于模型组。
5.模型组在免疫后第10天,胸腺、脾、腹股沟淋巴结CD4+CD25+/ CD4+比率降低,相应的,其Foxp3 mRNA表达水平也降低。在免疫后第18天,即临床症状高峰期,这种趋势更加明显,模型鼠CD4+CD25+T比率及Foxp3 mRNA表达水平均处于谷底。在免疫后第25天,模型鼠进入恢复期,此时Treg细胞数量及Foxp3 mRNA表达水平也随之回升。经过药物治疗,益髓通经方高剂量组与免疫球蛋白治疗组在免疫后第18天均表现出对CD4+CD25+T细胞的改善作用,在胸腺、淋巴结Foxp3的表达方面,高剂量组甚至优于球蛋白组,在第39天时,高剂量组与球蛋白组作用持平,均优于中低剂量组。
结论:
1.益髓通经方可以使EAN模型鼠病变坐骨神经有髓神经纤维结构改善,髓鞘脱失减少,髓鞘板层排列整齐、清晰度增强,有助于受损神经修复和再生。
2.益髓通经方组能提高EAN大鼠坐骨神经运动传导速度。
3.益髓通经方能抑制EAN模型鼠脾淋巴细胞对特异性或非特异性抗原刺激的异常增殖反应,有助于减轻CD4+T淋巴细胞介导的免疫损伤。
4.益髓通经方能降低EAN模型鼠血清IL-10的表达水平,提高保护性因子TGF-β的表达水平,有助于恢复模型鼠细胞因子平衡状态。
5.益髓通经方有助于EAN大鼠胸腺、脾、腹股沟淋巴结调节性T细胞数量及功能恢复正常,随着调节性T细胞功能恢复,对CD4+T细胞的抑制作用增强,对于恢复免疫平衡状态,终止周围神经的炎性损伤起到积极作用。
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 groups
Conclusions:
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 velocity
3. 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 cells
5. 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.
方法:采用P0180-199多肽及IFA免疫Lewis大鼠制备EAN动物模型。将造模成功的大鼠100只随机分为模型组、免疫球蛋白治疗组,益髓通经方高、中、低剂量组5组,每组20只,另取20只正常鼠作为空白对照组,共分为6组。免疫球蛋白治疗组于免疫后第11天起给予人免疫球蛋白注射液480mg/只·天腹腔注射治疗,共5天。益髓通经方高、中、低各组于免疫后第11天分别给予益髓通经方溶液2.0ml/d、1.5ml /d、1.0ml/d灌胃治疗直至处死;在免疫后第10天,空白组及模型组各取5只大鼠处死、在免疫后第18、25、39天各组分别随机抽取5只大鼠处死检测各组大鼠坐骨神经运动传导速度;应用光镜、电镜观察坐骨神经病理形态学及超微结构的变化;采用CCK-8试剂检测大鼠脾脏淋巴细胞增殖能力;ELISA法检测血清IL-10、TGF-β表达水平;采用流式细胞技术检测各组大鼠胸腺、脾、腹股沟淋巴结CD4+CD25+T/ CD4+T比率,并以RT-PCR法检测上述淋巴器官调节性T细胞Foxp3 mRNA表达水平。
结果:
1.通过电镜观察各组大鼠坐骨神经病理变化:模型组在免疫后第18天出现有髓神经纤维结构病变,髓鞘折叠起皱、轴索与髓鞘板层分离等改变,在第25天时出现较明显髓鞘脱失或髓鞘溶解,在39天时仍有广泛脱髓鞘及轴索崩解改变。经治疗,益髓通经方治疗组与免疫球蛋白组的髓鞘脱失情况得到明显改善,有髓纤维髓鞘板层结构整齐,同时还可见到胶原纤维聚集及有髓神经纤维新生等改变。
2.应用BL-420生物机能实验系统检测EAN大鼠坐骨神经运动传导速度。模型组在免疫后第10天起运动神经传导速度下降,在第18天、25天进一步下降,在第39天时神经传导速度略有改善。益髓通经方各治疗组及球蛋白治疗组在上述时间点均优于模型组,在第39天治疗4周时,益髓通经方高剂量组神经传导速度同球蛋白治疗组比较无显著性差异(P>0.05),优于中、低剂量组(P<0.01),但均不及空白组(P<0.01)。
3.淋巴细胞增殖实验显示,模型组及益髓通经方各治疗组对P0多肽及ConA的刺激增殖反应在第10天、18天、25天进行性增强,在第25天达到顶峰,随后下降,但模型组的增殖强度均高于同期中药治疗组。球蛋白治疗组淋巴细胞增殖强度在第18天起下降。在第39天时益髓通经方各治疗组同球蛋白组淋巴细胞增殖反应基本恢复正常,均优于模型组。
4.在免疫后第10天,模型组血清IL-10水平增高,在第18天显著增高,在25天起下降,与EAN大鼠疾病严重程度呈正相关;模型组血清TGF-β水平在免疫后第10天、第18天低于空白组,在免疫后第25天与空白组持平,在39天高于空白组,与EAN大鼠病情改善情况呈正相关。益髓通经方高剂量组及球蛋白治疗组IL-10水平在以上各时间点均低于模型组,而其TGF-β水平则高于模型组。
5.模型组在免疫后第10天,胸腺、脾、腹股沟淋巴结CD4+CD25+/ CD4+比率降低,相应的,其Foxp3 mRNA表达水平也降低。在免疫后第18天,即临床症状高峰期,这种趋势更加明显,模型鼠CD4+CD25+T比率及Foxp3 mRNA表达水平均处于谷底。在免疫后第25天,模型鼠进入恢复期,此时Treg细胞数量及Foxp3 mRNA表达水平也随之回升。经过药物治疗,益髓通经方高剂量组与免疫球蛋白治疗组在免疫后第18天均表现出对CD4+CD25+T细胞的改善作用,在胸腺、淋巴结Foxp3的表达方面,高剂量组甚至优于球蛋白组,在第39天时,高剂量组与球蛋白组作用持平,均优于中低剂量组。
结论:
1.益髓通经方可以使EAN模型鼠病变坐骨神经有髓神经纤维结构改善,髓鞘脱失减少,髓鞘板层排列整齐、清晰度增强,有助于受损神经修复和再生。
2.益髓通经方组能提高EAN大鼠坐骨神经运动传导速度。
3.益髓通经方能抑制EAN模型鼠脾淋巴细胞对特异性或非特异性抗原刺激的异常增殖反应,有助于减轻CD4+T淋巴细胞介导的免疫损伤。
4.益髓通经方能降低EAN模型鼠血清IL-10的表达水平,提高保护性因子TGF-β的表达水平,有助于恢复模型鼠细胞因子平衡状态。
5.益髓通经方有助于EAN大鼠胸腺、脾、腹股沟淋巴结调节性T细胞数量及功能恢复正常,随着调节性T细胞功能恢复,对CD4+T细胞的抑制作用增强,对于恢复免疫平衡状态,终止周围神经的炎性损伤起到积极作用。
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 groups
Conclusions:
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 velocity
3. 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 cells
5. 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.
引文
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