Based On TSHR And Related Signal Transduction Pathways To Verify The Pathogenesis Of Graves' Disease Goiter And The Research On The Intervention Effect Of Nourishing Yin And Reducing Fire

Objective:This study investigated the pathogenesis of GD goiter based on TSHR and signal transduction pathways of thyroid cell proliferation and apoptosis after receptors.Application of Ziyin Jianghuo Sanjie compound for intervention treatment,to verify its effect on the proliferation and apoptosis of FRTL cells induced by thyroid stimulating antibodies(TRAb),provide a new theoretical basis for the prevention and treatment of GD goiter.Research method:1 Drug-containing serum preparation:A total of 10 male and female Wis t a rats(200g±30/head)for 6-8 weeks,adaptive feeding for 2 weeks,divided into control group,ziyin Jianghuo Sanjie Formula Low Concentration Group,in the concentration group of Ziyin Jianghuo Sanjie Recipe,there are five groups of Ziyin Jianghuo Sanjie formula high concentration group and Saizhi group,blood was taken from the abdominal aorta 1 hour after the last gastric lavage,separation of serum,0.22um filter sterilization,after inactivation at 56? for 30 min,store at-20? until use.2 Culture and dosing of thyroid cells:Take FRTL-5 rat thyroid cell line,to recover,the recovered cells are centrifuged in a centrifuge tube containing complete culture solution,the cells are then cultured in the medium,observe the cell morphology.Generation cell density grows to about 90%,the cells were subcultured at a ratio of 1:2.The cells were resuspended after centrifugation.Take an appropriate amount of cell suspension and dilute it with PBS,drop the diluted cells into the counting plate,place the cells under a microscope for cell counting.Finally,the first three generations of cells in good logarithmic growth phase were cryopreserved.Cell dosing is to culture FRTL-5 cell line in complete medium,wait until the cells grow to 80%-85%,the cells were grouped experimentally and cultured in the incomplete medium taken out of gonadotropin and starved,several groups of cells are normal group:Add normal rat serum;Model group:Add M22 and normal rat serum;Low concentration square group:Add M22 and low-concentration Chinese medicine-containing serum;Medium concentration square group:Add M22 and Chinese medicine serum;High concentration square group:Add M22 and high-concentration Chinese medicine-containing serum;Saizhi group:M22 and methimidazole tablets(saizhi)drug-containing serum were added to observe the cell growth.3 Detection of cell proliferation in each group by MTT method;Flow cytometry to detect cell death;Western blot was used to detect the expression of PKA C-a,p-PKA C(Thr197),CREB,p-CREB(Ser133),ERK,and p-ERK(Thr202/Tyr204).Results:1 MTT method to detect cell proliferation in each group Detection of OD value of FRTL-5 cells in each group by MTT method,the results show that,compared with the normal group,the OD value of the model group was significantly increased;The concentration of the traditional Chinese medicine in the group of Ziyin Jianghuo Sanjie is lower than that in the model group,the difference was statistically significant(P<0.05);Ziyin Jianghuo Sanjie Chinese medicine high concentration group is lower than the model group,there was a statistically significant difference(P<0.05);Saiji group is lower than model group,there was a statistically significant difference(P<0.05);There was a statistically significant difference between the Ziyin Jianghuo Sanjie Chinese medicine group,the low-,medium-,and high-concentration group and the Saizhi group(P<0.05).2 Flow cytometry detection of apoptosis in each group Ziyin Jianghuo Sanjie Chinese medicine can induce apoptosis of FRTL-5 cell at low,medium and high concentrations,medium and high concentrations,and its apoptosis rate increased with increasing concentration.Flow cytometry was used to detect the apoptosis of each group,compared with the normal group,the apoptosis rate of the model group was significantly increased.The apoptosis rate of the Ziyin Jianghuo Sanjie Chinese medicine low concentration group was higher than that of the model group,the difference was statistically significant(P<0.05);The apoptosis rate of the concentration group of Ziyin Jianghuo Sanjie Chinese medicine is higher than that of the model group,there was a statistically significant difference(P<0.05);The apoptosis rate of the high-concentration Chinese medicine group of Ziyin Jianghuo Sanjie was higher than that of the model group,there was a statistically significant difference(P<0.05);The apoptosis rate of Saizhi group was higher than that of model group,the difference was statistically significant(P<0.05).There was a statistically significant difference between the low-,medium-,and high-dose groups of Ziyin Jianghuo Sanjie and the Saizhi group(P<0.05).3 Western blot detection of protein expressionWestern blot was used to detect protein expression after drug intervention,expression of p-PKA C and PKA C-? protein in rat thyroid cells in each group,type group was significantly higher than the normal group,the difference was statistically significant(P<0.05).The apoptosis rate of the concentration group of Ziyin Jianghuo Sanjie Chinese medicine is lower than that of the model group,the difference was statistically significant(P<0.05);The relative expression of Ziyin Jianghuo Sanjie Chinese medicine high concentration group was lower than that of the model group,The difference was statistically significant(P<0.05);The relative expression level in Saiji group was lower than that in model group,The difference was statistically significant(P<0.05).There was a statistically significant difference between the low-,medium-,and high-dose groups of Ziyin Jianghuo Sanjie and the Saizhi group(P<0.05).Western blot was used to detect protein expression after drug intervention,the relative expression of p-CREB/?-actin in the model group was significantly higher than that in the normal group,the difference was statistically significant(P<0.05).The relative expression of Ziyin Jianghuo Sanjie Chinese medicine in the concentration group was lower than that in the model group,the difference was statistically significant(P<0.05);The relative expression of Ziyin Jianghuo Sanjie Chinese medicine high-concentration group was lower than that of the model group,The difference was statistically significant(P<0.05);The relative expression in the control group was lower than that in the model group,the difference was statistically significant(P<0.05).There was a statistically significant difference between Ziyin Jianghuo Sanjie Chinese medicine group and Saizhi group(P<0.05).Western blot detection of p-ERK/?-actin protein expression,the model group was significantly higher than the normal group,the difference was statistically significant(P<0.05).The relative expression of the Ziyin Jianghuo Sanjie Chinese medicine low concentration group was lower than that of the model group,the difference was statistically significant(P<0.05);The apoptosis rate of the concentration group of Ziyin Jianghuo Sanjie Chinese medicine is lower than that of the model group,the difference was statistically significant(P<0.05);The relative expression of Ziyin Jianghuo Sanjie Chinese medicine high concentration group was lower than that of the model group,the difference was statistically significant(P<0.05);The relative expression of the Saizhi group was lower than that of the model group,the difference was statistically significant(P<0.05).There was a statistically significant difference between Ziyin Jianghuo Sanjie Chinese medicine group and Saizhi group(P<0.05).Conclusion:1 Ziyin Jianghuo Sanjie Chinese medicine-containing serum can effectively inhibit the excessive proliferation of FRTL-5 cells under the action of TRAb,ziy is also dose-dependent.2 Ziyin Jianghuo Sanjie Chinese medicine-containing serum can induce apoptosis of FRTL-5 cells under TRAb,results are dose-dependent.3 Ziyin Jianghuo Sanjie can effectively down-regulate the expression of PKA,CREB,ERK and other TRAb related receptor pathway-related proteins.This may be the key to the treatment of GD with Ziyin Jianghu Sanjie.