Study On The Mechanism Of JiaKangNing Capsule In The Treatment Of Cell Proliferation In Graves Disease Based On Autophagy

BackgroundGraves disease,also known as toxic diffuse goiter,is the most common cause of hyperthyroidism.At present,with the aggravation of environmental pollution,quickening pace of life and increasing pressure,the incidence of GD is on the rise.The annual prevalence rate of GD is about 0.02%-0.03%.In the lifetime,about 3%of women and 0.5%of men will suffer from GD.All age groups can suffer from GD,and the peak age of GD is 30-60 years old.The study has shown that the pathogenesis of GD is an autoimmune disorders which is mainly induced by factors such as genetic factors,environmental factors,infection,mental trauma,and so on.It is due to the production of thyroid stimulating hormone(TSH)receptor antibody(Thyrotropin receptor antibody,TRAb)bind TSH receptors on thyrocytes,resulting in the excessive cell proliferation and thyroid hormone secretion,presenting goiter and hyperthyroidism.Therefore,TSAb induced cell proliferation is the basic pathological changes in the pathogenesis of GD,so it is of great theoretical and practical value to study its mechanism and intervention measures to prevent the development of GD.Autophagy is the main pathway for the degradation of macromolecules in eukaryotic cells.It is involved in the regulation of cell growth,proliferation,differentiation,senescence,apoptosis and other important cellular physiological and pathological processes,which plays an important role in the maintenance of cell homeostasis.Aoutophagy is regulated by a series of autophagy related genes and signal transduction pathways.Mammalian target of rapamycin is an important serine-threonine protein kinase,which is a downstream kinase of PI3K/Akt pathway,and participates in regulation of proliferation and autophagy activity of thyroid cells by activating downstream p70 ribosomal protein S6 kinase.In the process of autophagy,beclinl is the main regulator of autophagy at the starting stage.Microtubule related protein light chain 3 has two forms,LC3Ⅰ and LC3Ⅱ.When autophagy occurs,the LC3Ⅰ is modified by a ubiquitin like process and is combined with the phosphatidyl ethanolamine on the surface of the autophagic membrane to form LC3Ⅱ.In addition,the combination of ATG12 and ATG5 can form Atgl 2-Atg5-Atgl 6L complex under the action of ATG16,which is helpful to the formation of autophagy by combining with LC3 system.In recent years,with the in-depth study of autophagy mechanism,people try to prevent and treat diseases by regulating autophagy.Autophagy related proteins have become a new candidate target for disease intervention,and have attracted close attention from medical researchers.To study the role of autophagy in TSAb stimulating the proliferation of thyroid epithelial cells by,which is of great guiding significance in regulating the autophagy of thyroid cells in the prevention and treatment of GD.Studying the autophagy mechanism of TSAb stimulating the proliferation of thyroid epithelial cells is of great significance for the prevention and treatment of GD by regulating the autophagy of thyroid cells.Jia Kang Ning capsule is a traditional Chinese medicine developed by Professor Lin Lan,which has the functions of nourishing yin and suppressing yang and resolving phlegm and resolving nodus.Clinical studies have shown that JKN has significant advantages in alleviating thyroid enlargement,reducing clinical symptoms and adverse reactions,It confirmed the clinical effectiveness of JKN in the treatment of GD under the guidance of TCM theory.In order to further clarify the mechanism of JKN in the treatment of GD,we take the cell proliferation as the breakthrough point to study the biological behavior and mechanism of JKN interfering with GD.In our previous study,we found that JKN may inhibit the proliferation of thyroid cells by downregulating Akt/mTOR and ERK1/2 signaling pathways.In order to further study whether JKN can regulate autophagy activity by regulating mTOR signaling pathway and how JKN inhibits cell proliferation through autophagy,this study will introduce the role of autophagy in the proliferation of GD thyroid cells and the mechanism of the therapeutic effect-of JKN.While enriching the theory of traditional Chinese medicine,it also provides experimental basis for the mechanism of Chinese medicine multi-target and multi link in the control of GD.The study is supported by the National Natural Science Foundation of China(81573961).Objective1.To clarify the pathological process of autophagy involved in GD cell proliferation and the intervention effect of Chinese medicine Jia Kang Ning capsule.2.To further explore the mechanism of JKN inhibiting GD cell proliferation from the perspective of regulating mTOR/p70S6K signaling pathway and affecting the expression of autophagy related genes.Methods1.In vivo part:using TSHR-A subunit recombinant adenovirus intramuscular injection of female Balb/c mice to establish a GD animal model.The validity of the model was identified by observing the expression of TRAb and T4 in serum and the proliferation of GD thyroid cells was confirmed by histopathological observation,immunohistochemistry and Western blot detecting the expression of Ki67 and Cyclin D1 protein respectively.The morphology of autophagic body was observed by transmission electron microscopy,the distribution of autophagy specific protein LC3 and the expression of LC3B-Ⅱ and Beclin 1 were detected by Western blot,to clarify the expression of autophagy in the proliferation of thyroid cells in GD model mice and the intervention effect of JKN.In order to further study the molecular mechanism of autophagy in the process of JKN interference in GD cell proliferation,RT-PCR and Western-blot were used to detect the expression level of ATG12,ATG5,ATG16L,p-mTOR and p-p70S6K at the gene and protein level.2.In vitro part:the Nthy-ori-3-1 cells of normal thyroid epithelial cells were induced by TSAb monoclonal antibody(M22),and the cell proliferation model of GD in vitro was established.The aging and dose effect relationship of M22 induced proliferation of Nthy-ori-3-1 cells was detected by CCK8.Then the cell proliferation inhibition rate,cell cycle distribution,the expression level of cyclin Cyclin D1 and the changes of cAMP release in the supernatant were detected by CCK8,flow cytometry,Western blot and ELISA.The ultrastructural changes of autophagic bodies,the accumulation of green fluorescent spots in autophagic vesicles and the expression of LC3B-Ⅱ/LC3B-Ⅰand p62 were separately detected by transmission electron microscopy,immunofluorescent labeling and Western blot,by which the mechanism of autophagy and the therapeutic effect of JKN was determined.To determine whether JKN could interfere with the excessive proliferation of thyroid cells by regulating autophagy,the autophagy inhibitor 3-MA and the inducer,rapamycin,were used to verify its mechanism.The mRNA and protein expression of autophagy related proteins LC3B-II,ATG12,ATG16L,p-mTOR and p-p70S6K were observed,and the results of animal experiments were verified at the cellular level.Results1.JKN intervention could significantly reduce the level of serum TRAb and T4 in the GD model mice,improve the morphology and pathology of thyroid enlargement,and reduce the expression level of the proliferation marker protein Ki67 and the cyclin Cyclin D1 in the thyroid cells of the GD model mice.2.Transmission electron microscopy showed that there were more heterochromatin in the thyroid follicle and the appearance of autophagic in the non double layer membrane of GD mice.After JKN intervention,nuclear condensation and chromatin accumulation were observed in cells,and the structure of autophagosome and vacuoles increased significantly;Western blot results suggested that JKN could increase the expression level of autophagy protein LC3BII and Beclin 1 in GD mice.3.The results of Western blot and RT-PCR showed that JKN could increase ATG5,ATG12,ATG16L protein and gene expression levels in thyroid tissues of GD mice,and reduced the expression levels of p-mTOR,p-P70S6K protein and mTOR and p70S6K genes in GD mice.4.The results of CCK8 and ELISA showed that the proliferation activity of Nthy-ori-3-1 cells induced by M22 increased significantly,accompanied by an increase in cAMP release in the supernatant.After treatment with different concentrations of JKN,both medium and high doses significantly inhibited the proliferation activity of the model cells and significantly reduced the amount of cAMP released;Flow cytometry and Western blot showed that JKN inhibited the cell cycle and the expression level of cyclin Cyclin D1 in the model cells.5.Transmission electron microscopy and fluorescence labeling showed that JKN increased the number of autophagy in GD model cells;And Westen blot showed that JKN increased the level of LC3BII/LC3BI protein while decrease the level of p62 protein expression.6.The effect of JKN intervention on autophagy flow was detected by Western blot.The results showed that compared with the model group,the expression level of LC3Ⅱ/LC3Ⅰprotein in the model +CQ group increased significantly,and the expression level of LC3Ⅱ/LC3Ⅰ protein in the JKN group and the JKN+CQ group both increased significantly;Compared with model +CQ group,the expression level of LC3Ⅱ/LC3Ⅰprotein in JKN +CQ group increased significantly(P<0.05).7.The results of Western blot and RT-PCR showed that JKN treatment could increase the protein and gene expression levels of LC3b,ATG12 and ATG16L in GD model cells;And reduced the expression levels of p-mTOR,p-P70S6K protein and mTOR and p70S6K genes in GD model cells.Conclusion1.JKN can reduce thyroid hormone abnormalities and thyroid autoantibody level in GD model mice,improve thyroid enlargement and pathological morphology,significantly inhibit the proliferation protein Ki67 of model cells,and reduce the expression level of related proliferation marker protein and cyclin D1protein.2.It is feasible to establish GD thyroid cell proliferation model by stimulating Nthy-ori-3-1 thyroid cells with 1ug/ml M22 at 24h.High dose drug-containing serum can significantly inhibit the proliferation of GD thyroid cells and the release of cAMP from the supernatant,inhibit the cell cycle to G0G1 phase,and reduce the level of CyclinD1 protein expression.3.The autophagic ultrastructure,immunofluorescence staining and autophagic marker protein were used to demonstrate that JKN could promote the formation of GD cells autophagy.4.JKN can significantly Autophagy flow test showed that the mechanism of JKN promoting LC3B-Ⅱ expression may be related to the formation of autophagy and the inhibition of its degradation process,and intervention the formation of autophagy conjugated gene ATG12-ATG5-ATG16L complex may be one of its mechanisms.5.Further analysis from the gene and protein level showed that JKN may interfere with the formation of autophagy by regulating the mTOR/p70S6K signaling pathway and play a role in the inhibition of the proliferation of thyroid cells.