| Graves’ disease(GD),also known as toxic diffuse goiter,is an autoimmune thyroid disease that is stimulated by a patient’s anti-thyroid hormone receptor antibody(TRAb)on thyroid cells.The TSH receptor,which causes an increase in thyroid hormone production and release,is also the most common cause of hyperthyroidism.A cross-sectional study in 10 cities in China showed that the prevalence of GD was 0.8%in areas with excessive iodine intake;in areas with sufficient iodine intake,the prevalence of GD was 0.5%.GD can easily cause damage to the nervous system,circulatory system,eye lesions,blood system,digestive system,etc.,and cause harm to human health.Genetic,environmental,infection,social stress and other factors lead to the combination of TRAb and TSHR site of thyroid follicular epithelial membrane.TSAb in TRAb has TSH-like effect,persistent stimulation of TSHR,resulting in increased cAMP content and excessive proliferation of thyroid cells.The amount of T3 and T4 released increased.Compared with normal population,the level of Bcl-2 in thyroid cells of GD patients is higher than that of normal people.The thyroid tissue of GD patients proliferates excessively and apoptosis decreases.In the process of intervening in the process of GD,Chinese medicine has opened a lot of exploration and achieved fruitful results.Chinese medicine Jiakangning Capsule has been used in clinical practice for more than 30 years,which have the effect of nourishing yin and yang,and dissipating phlegm.In a randomized,controlled,blinded trial,332 patients with GD were enrolled,including 270 in the treatment group and 62 in the control group.The treatment group was treated with hyperthyroidism combined with standard dose 1/2 antithyroid drug,and the control group was treated with placebo.Add standard dose anti-thyroid drugs.A total of 24 weeks were observed,followed up every 4 weeks,and followed up for 2 years after the observation.The results showed that Jiakangning Capsule can significantly improve the clinical symptoms and thyroid function of patients;it can reduce the amount of western medicine,reduce liver damage,improve hematopoietic function;and has anti-recurrence effect,and the clinical curative effect is exact.Previous studies have found that Jiakangning Capsule can promote the apoptosis and autophagy of GD cells.However,the specific signaling pathway deserve further study.Objective:(1)To clarify the regulation of Jiakangning Capsule on autophagy and apoptosis of thyroid cells.(2)To explore the relationship between the effect of Jiakangning Capsule on autophagy and apoptosis and the AMPK-mTOR signaling pathway.Methods:(1)In vivo experiments:In this study,BALB/c female mice were transfected with Ad-TSHRa-289 to establish a GD animal model,and the mold formation rate of T4 and TRAb was detected.Jiakangning Capsule and the positive control drug methimazole were observed for 5 weeks,and the pathological state of thyroid tissue was observed.Electron microscopic observation of autophagosomes in mouse thyroid tissue;qRT-PCR was used to detect the expression levels of LC3,Beclinl,Atg5,bcl-2,Bax and Caspace-3 genes in mouse thyroid tissue.Western blot was used to detect the expression levels of LC3BI/II,Beclinl,Atg5,bcl-2,Bax,Caspace 3,AMPK,p-AMPK,mTOR and p-mTOR in mouse thyroid tissue.(2)In vitro experiments:M22 induced human thyroid follicular epithelial cell line(Nthy-ori-3-1)to establish a GD cell model.CCK8 was used to detect the inhibition rate of Nthy-ori-3-1 proliferation in high,medium and low concentrations of hyperthyroidism drug.After treatment with M22 to induce Nthy-ori-3-1 cells,the amount of cAMP released from the supernatant of each group was detected by ELISA.The apoptosis of Nthy-ori-3-1 cells was detected by flow cytometry.The autophagic flow was detected by laser confocal fluorescence microscopy.qRT-PCR was used to detect the effect of serum containing Jiakangning on the expression of LC3,Beclinl,Atg5,bcl-2,Bax and Caspace 3 mRNA in human thyroid follicular epithelial cells induced by M22;Western blot was used to detect M22 induced by serum Effects of LC3BⅠ/Ⅱ,Beclinl,Atg5,bcl-2,Bax,Caspace 3,AMPK,mTOR,p-AMPK,and p-mTOR proteins in human thyroid follicular epithelial cells.Results:(1)Establishment of GD animal model and protective effect of Jiakangning Capsule on thyroid tissue of mice:In this animal experiment,the normal group had a T4 level of 43.73±6.98 ng/mL and a TRAb of 2.99±0.34 ng/mL.The T4 level of the model group was 156.57±23.29 ng/mL,and the TRAb was 20.00±3.51 ng/mL.The T4 and TRAb levels in the model group were significantly higher than those in the normal group,the difference was statistically significant(p<0.01),and the GD model rate was as high as 70%.The thyroid gland of the normal group was attached to the side of the trachea,the volume was normal,the color was reddish,and the thyroid weight was 0.0033±0.0005 g.The thyroid volume of the model group was significantly increased,the color was dark red,and the thyroid weight was 0.0162±0.0013 g.In the model group,the thyroid volume of the mice was significantly increased and the color was deepened.HE staining of thyroid gland in mice under light microscope showed that the thyroid follicular epithelial cells in the normal group were round and arranged neatly.The wall of the follicular cavity was thin and smooth,and it was filled with colloid.In the model group,the thyroid follicular epithelial cells were of different sizes,arranged disorderly,irregularly shaped,and the wall of the follicular cavity was thickened.The proliferating cells in the luminal wall formed papillary bulges into the cavity,and the intraluminal colloid was significantly reduced.The mice in the normal group had no obvious mental and abnormal activities.The mice in the model group showed irritability,weight loss,dampness in the cage,frequent activities,and increased excretion.This animal experiment showed that the serum T4 and TRAb of the MMI group and the JKN group decreased compared with the GD group after 5 weeks of intragastric administration,and the difference was statistically significant.The serum T4 level in the MMI group was 92.76±9.65 ng/mL,and the TRAb level was 12.16±3.83 U/mL.The serum T4 level in the JKN group was 108.36±20.89 ng/mL,and the TRAb level was 12.64±1.78 U/mL.MMI and JKN mice were more susceptible to irritation and activity than the GD group.In histopathological changes,compared with the GD group,the thyroid enlargement of the MMI group was slightly relieved and the color was lighter;in the JKN group,the thyroid gland was less enlarged than the GD group,and the color was reddish.In the thyroid weight comparison,the weight of the thyroid gland in the MMI group was 0.0097±0.0010 g;the weight of the thyroid gland in the JKN group was 0.0100±0.0035 g.MMI and JKN can effectively reduce the thyroid volume of mice,and the difference is statistically significant.In HE staining,the structure of thyroid follicular epithelial cells in the JKN group was restored,the follicles were flat,and the gelatin color in the follicles was light.Both MMI group and JKN group can reduce the papillary bulge in the thyroid follicular cavity,improve the excessive proliferation of thyroid follicular epithelial cells,and increase the gelatinity in the thyroid follicular cavity.(2)Effects of Jiakangning Capsule on autophagy,apoptosis and AMPK-mTOR signaling pathway in vivo:The protein of anti-apoptotic factor Bcl-2 in mouse thyroid tissue was compared between JKN group and MMI group compared with GD group.Gene expression decreased,and protein and gene expression levels of Bax and Caspace-3 increased.The expression levels of autophagy-associated LC3BI/II,Beclinl and Atg5 proteins and genes in mouse thyroid tissue were increased in JKN group and MMI group compared with GD group.Transmission electron microscopy showed that a large number of autophagosomes with double-layer membrane structure appeared in the thyroid tissue of JKN group.Western medicine MMI showed a small amount of autophagosomes.After the intervention of Jiakangning Capsule and western medicine methimazole,the expression of p-AMPK protein in the thyroid tissue of mice increased,while the expression of p-mTOR protein decreased.(3)Effects of Jiakangning on autophagy,apoptosis and AMPK-mTOR signaling pathway in vitro:The release of cAMP in the model group was significantly increased compared with the normal group.Jiakangning can reduce cAMP release compared with the model group.Jiakangning can significantly increase the early apoptotic rate,late apoptotic rate and total apoptotic rate of Nthy-ori-3-1 cells.The autophagic flow was observed by laser confocal fluorescence microscopy.The results showed that Jiakangning could significantly increase autophagy.The expression levels of p-AMPK protein in the high,medium and low dose groups of Jiakangning increased significantly,and the expression level of p-mTOR protein decreased significantly.The genes and proteins of apoptosis-related Bcl-2,Bax and Caspace-3 were detected by qRT-PCR and Western blot.The results showed that Jiakangning could down-regulate the expression of Bcl-2 and up-regulate Bax and Caspace-3.The genes and proteins of autophagy-related LC3,Beclinl and Atg5 were detected by qRT-PCR and Western blot,respectively.The results showed that the drug-containing serum of Jiakangning could up-regulate the expression of LC3,Beclinl and Atg5 proteins and genes,and promote autophagy.Silencing of AMPK by siRNA technology showed that JKN capsule still promoted apoptosis and enhanced autophagy level,but the level of autophagy and apoptosis was significantly lower than that of siRNA-Control group.Conclusion:(1)Jiakangning Capsule can effectively reduce the serum T4 and TRAb levels of GD model mice,improve the thyroid enlargement state of GD mice,reduce the thyroid weight of GD mice,and alleviate the irritability of GD mice.(2)In vitro and in vivo studies showed that Jiakangning promoted apoptosis and increased autophagy.The regulation of Jiakangning on apoptosis and autophagy of GD cells may be through activation of AMPK-mTOR signaling pathway. |
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