| Purpose:Subacute thyroiditis(SAT)is a common disease of thyroiditis,according to the manifestations of the disease,it can be divided into acute stage,remission stage and recovery stage.At present,it has been confirmed that the treatment of SAT in acute stage is the key to the disease,and it is also the advantage stage of TCM treatment.Firstly,through literature research,this paper systematically traces the origin of the theory of “dissipating excessive depression of fire”,and then discusses the theoretical basis of composition of traditional Chinese medicine(TCM)compound Yingtongyin which based on the theory of “dissipating excessive depression of fire”.Secondly,through clinical observation,we objectively evaluates the efficacy and safety of TCM compound Yingtongyin in the treatment of SAT in the acute stage with the syndrome of excessive heat and toxin.Then,through Meta analysis,to verify the efficacy and safety of ascending Yang and clearing away heat method in treating SAT and provide evidence-based medical evidence.Finally,through in vitro experimental study,to observe the Chinese medicine compound Yingtongyin intervention of adenovirus-induced thyroid follicular epithelial Nthy-roi3-1 cell infected pyroptosis process,explain the possible treatment mechanism of SAT.Material and method:1.Theoretical study on the application of the theory “dissipating excessive depression of fire”in the treatment of SAT in the acute stage with the syndrome of excessive heat and toxin.1.1Tracing back to the origin of the theory “dissipating excessive depression of fire”: The literature of “dissipating excessive depression of fire” is deeply excavated,and the theoretical connotation of “dissipating excessive depression of fire” is combed by using the methods of philology and logic.The content includes the respective connotation of “fire”,“depression”and “dissipate” respectively.The cognition of “dissipating excessive depression of fire” and its classic representative prescriptions are described respectively.1.2 The thoughts inheritance: From the definition,etiology,pathogenesis,and prescription of SAT,explained Professor Gao Tianshu's concept in SAT treatment.Based on the theory of“dissipating excessive depression of fire”,this paper discusses the theoretical basis and the thought of composition of Yingtongyin,in the treatment of acute SAT with the symdrome ofexcessive heat and toxin.2.The Clinical observation of “ascending Yang and clearing away heat” method in the treatment of SAT in acute stage with the syndrome of excessive heat and toxin In this study,randomized and controlled clinical trials were carried out.The observed cases were all the first untreated patients with SAT in the Department of Endocrinology,Affiliated Hospital of Liaoning University of traditional Chinese medicine from January 2017 to December 2019.A total of 60 qualified patients were included in this study.According to the diagnostic criteria,the patients were included in the study,which was also in accordance with the syndrome of excessive heat and toxin according to the guiding principles for clinical research of new Chinese medicine in 2002.The patients were randomly divided into the western medicine group and the traditional Chinese medicine group.In western medicine group,prednisone acetate tablet was taken and in the Chinese medicine group,Yingtongyin was prescribed.The specific composition of Yingtongyin is as follows: cohosh,bupleurum,Bombyx mori,mint,Coptis,Scutellaria,burdock,licorice root,Platycodon,isatis root,forsythia,radix,Cicada exuviate,Curcuma.The experimental observation period is 4 weeks,the follow-up time is 12 weeks.The observation indexes include pain improvement degree,pain relief time,antipyretic time,goiter shrink degree and regression time,TCM symptom improvement rate,TCM syndrome improvement rate,thyroid function,ESR,recurrence rate,hypothyroidism incidence,safety index,compliance and adverse events.3.Meta analysis of the efficacy and safety of “ascending Yang and clearing away heat”method in the treatment of SAT Based on seven databases including China biomedical literature database(CBM),CNKI China Journal Full-text Database,Wanfang Data knowledge service platform,VIP Chinese science and technology journal database,Pub Med database,EMBASE database,Cochrane control experiment center database,a total of 243 SAT documents were collected.After eliminating duplication,review,system evaluation articles and screening out the papers such as inconsistent research content and lax design,Meta analysis was used to verify the effectiveness and safety of “ascending Yang and clearing away heat” method in the treatment of SAT,including the overall effective rate,improvement rate of TCM symptom score,improvement rate of TCM syndrome,pain relief degree and time,antipyretic time,goitershrink degree and regression time,palpitation,irritable,thyroid function,ESR,adverse reactions,recurrence rate of SAT,incidence of hypothyroidism.4.In vitro experimental study of “ascending Yang and clearing away heat” on the pyroptosis process of thyroid follicular epithelial Nthy-roi3-1 cell infection induced by adenovirus4.1 Preparation of serum containing medicine: 50 healthy,male,clean,200 ± 20 g weight Wistar rats(purchased in Liaoning Changsheng Biotechnology Co.Ltd.,animal license No.:scxk(Liao)2010-0001)were randomly divided into 5 groups(blank group,low-dose group,middle dose group,high-dose group and prednisone group),10 rats in each group.Yingtongyin,the experimental compound,was gavaged in low dose group,middle dose group and high dose group according to 0.5,1.0 and 2.0 times of normal adult's dose respectively.After conversion,the drug concentration in the low dose group = 0.33g/ml,the middle dose group = 0.67g/ml,the high dose group = 1.32g/ml,the dose of each gavage is 1ml / 100 g,three times a day,for 7 days.Prednisone which normal adults dose as 30 mg / day,dissolved in 0.9% sodium chloride to 0.156 mg / ml,and administered by gavage at a dose of 1 ml /100 g in prednisone group.The rats in the blank group were given 1 ml / 100 g normal saline.The rats were killed after 7 days of continuous gavage.Blood was collected from abdominal aorta.After centrifugation,inactivation of complement and filtration of bacteria,they were placed in EP tube and sealed in refrigerator at-80 ?.4.2 Cultured cells: The normal human thyroid follicular epithelial Nthy-roi3-1 cells were purchased from Shanghai Hengsheng Biotechnology Co.Ltd.and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin streptomycin in cell incubator(temperature 37 ?,5% CO2 and relative saturation humidity),1:2 subcultured.4.3 Adenovirus infection: Nthy-roi3-1 cells were randomly divided into 2 groups,blank group and model group.After the model group infected by adenovirus,then divided into 5 groups which were prednisone group,low dose group,middle dose group and high dose group and model group.Each group of cells were added with the serum,in which the blank group was normal rat serum + normal non adenovirus infected cells;the model group was normal rat serum + adenovirus infected cells;the prednisone group was prednisone drug-containing serum with adenovirus infected cells;the low-dose group of traditional Chinese medicine serum with adenovirus infected cells;the middle-dose group of traditional Chinese serumwith adenovirus infected cells;the high-dose group of traditional Chinese medicine with adenovirus infected cells.The contents of Caspase-1,T3,T4,LDH,IL-1 ?,IL-18,GSDMD-N,Caspase-1,NLRP-3 and ELAVL-1 were detected by immunofluorescence,immunochemiluminescence,colorimetry,ELISA and Western blot respectively.The expression of Tg,TPO,Gaspase-1,NLRP-3 and ELVAVL-1 m RNA was detected by PCR,and the level of cell pyroptosis was detected by annexin V-FITC / PI staining.Results:1.Theoretical study on the application of the theory of “dissipating excessive depression of fire” in the treatment of SAT in the acute stage with the syndrome of excessive heat and toxin This paper expounds the concept and meaning of “fire”,“depression” and “dissipating”respectively,and explains the causes and types of “fire depression”,the indication and specific methods of “dissipating”.By comparing the theoretical cognition of “fire depression"in different dynasties,we concludes that the theoretical meaning of “fire depression" has changed from the meaning of “five movements and six qi depression" to the publication of sweating in the Jin,Yuan and Tang Dynasties,and finally to the treatment of fiery diseases by“adroitly guiding actions according to circumstanses”.This paper systematically expounds the classic and effective representative prescriptions of “dissipating excessive depression of fire”.Based on the theory of “dissipating excessive depression of fire”,this paper deeply inherits Professor Gao Tianshu's theory on the treatment of SAT in the acute stage with the syndrome of excessive heat and toxin,and explains the Yingtongyin's basis.2.Clinical observation on the efficacy and safety of “ascending Yang and clearing away heat”method in the treatment of SAT in the acute stage with the syndrome of excessive heat and toxin2.1 VAS pain score: At the second week of treatment,the effect of TCM group on improving VAS pain score was better than that of prednisone group(P = 0.020 < 0.05);at the fourth week of treatment,the effect of TCM group and prednisone group was similar,there was no statistical difference.(P=0.307>0.05).2.2 Measurement of goitre degree: Palpate the goitre degree of patients in each group before treatment,2 weeks,4 weeks and 16 weeks respectively.The goitre degree of patients in thetwo groups gradually decreases with the time flows,and there is no statistical significance between the TCM group and prednisone group at 2 weeks and 4 weeks(2 weeks: P = 0.281 >0.05,4 weeks: P = 0.875 > 0.05).Follow up observation at the 16 th week,the goitre degree in TCM group was significantly lower than that in the prednisone group(P = 0.039 < 0.05).2.3 Antipyretic time and pain disappearance time: There was no significant difference in the antipyretic time between TCM group(5.90 ± 2.81 days)and prednisone group(6.60 ± 4.06days)(P = 0.375 > 0.05).The pain disappearance time(6.87 ± 2.35 days)in the TCM group was significantly shorter than prednisone group(10.00 ± 5.78 days),and the difference was statistically significant(P = 0.025 < 0.05).2.4 TCM symptom quantification score and TCM symptom efficacy: After 4 weeks' treatment,compared with before treatment,TCM symptom quantification score in TCM group(3.57 ± 4.20)and prednisone group(7.37 ± 6.75)was significantly lower(P = 0.000 <0.01,P = 0.000 < 0.01),and TCM symptom score in TCM group was significantly lower than that in prednisone group(P = 0.009 < 0.01),and TCM symptom efficacy was significantly better than that in prednisone group(P = 0 026<0.05).2.5 Thyroid function: After 4 weeks of treatment,compared with before treatment,FT3 and FT4 in TCM group and prednisone group decreased significantly,TSH increased significantly,but there was no significant difference between the two groups(FT3: P = 0.774 < 0.05,FT4:P = 0.264 > 0.05,TSH: P = 0.186 > 0.05).2.6 ESR: At 2 and 4 weeks of treatment,the ESR level of TCM group and prednisone group decreased,but there was no significant difference between the two groups(2 weeks: P =0.744 > 0.05,4 weeks: P = 0.526 > 0.05).2.7 Recurrence rate and incidence of hypothyroidism: At the 16 th week,there was no case recurrence in the TCM group and 1 case recurrence in the prednisone group.There were 2cases of hypothyroidism in the TCM group(incidence of hypothyroidism is 6.7%),and 3cases in the prednisone group(incidence of hypothyroidism is 10%).There was no significant difference between the two groups in terms of recurrence rate and incidence of hypothyroidism(P = 0.313 > 0.05,P = 064 > 0.05).2.8 Safety index: The results showed that except the total number of leukocytes(8.03 ± 1.47 *109 / L)in prednisone group increased significantly after treatment(within the normal range),the other indexes,such as blood,urinary regular test and ECG in the two groups were normal.2.9 Adverse events and compliance: 1 case in prednisone group and 0 case in TCM group occur adverse events and there was no significant difference between the two groups(P =0.313 > 0.05).In terms of compliance,95.87 ± 5.70% in the TCM group and 94.10 ± 6.17%in the prednisone group,all of the two groups were more than 80%.There was no statistical significance(0.227 > 0.05)?3.Meta analysis of the efficacy and safety of “ascending Yang and clearing away heat”method in the treatment of SAT Through strict screening,223 unqualified literatures were excluded.20 literatures were included in this study,with a total of 1445 cases.3.1 “Ascending Yang and clearing away heat” method can improve the overall efficiency of SAT treatment and significantly improve the efficiency of syndrome.In the aspect of improving SAT symptoms,“ascending Yang and clearing away heat” method is superior to western medicine in relieving anterior neck pain,shortening the pain time,reducing goiter shrink,reducing palpitation,irritable,etc.It is equivalent to western medicine in antipyretic time and eliminating goiter time.3.2 In terms of test indicators,the “ascending Yang and clearing away heat” method can reduce FT3 and FT4 levels,but the results need more clinical observations to prove it.The method of “ascending Yang and clearing away heat” is similar to western medicine in increasing TSH and decreasing ESR,and combination of Chinese and Western treatment shows better efficiency than western medicine alone.3.3 In terms of adverse reactions,recurrence rate of SAT and hypothyroidism,the “ascending Yang and clearing away heat” method is superior to the conventional western medicine.4.In vitro experimental study of “ascending Yang and clearing away heat” method on the process of pyroptosis death of Nthy-roi3-1 cells infected by adenovirus4.1 Detection of Caspase-1 by immunofluorescence: The fluorescence intensity of the model group was significantly enhanced,while the fluorescence of each TCM group and prednisone group was weakened in varying degrees.Immunofluorescence observation showed that Caspase-1 was largely expressed in the cytoplasm after modeling,and the experimental drugs in each group could descend the expression level of caspase-1 in varying degrees.4.2 Detection of T3 and T4 in cell supernatant by immunochemiluminescence : T3 and T4 in model group were significantly higher than those in blank group,suggesting that there was destruction of thyroid follicular epithelial cells and release of thyroid hormone after model establishment.Compared with the model group,the T3 and T4 levels of each treatment group were descended in different levels,among which the T3 levels of middle dose,high dose group and prednisone group were descending significantly compared with the model group,and there was no significant difference among the those three groups.The T4 level,including middle dose(9.483 ± 0.194 ng / ml),high dose group(10.267 ± 0.121 ng / ml),prednisone group(10.233 ± 0.258 ng / ml)and the model group compared with prednisone group(26.033± 0.258 ng / ml),the T4 level in the middle dose group of traditional Chinese medicine decreased significantly(P = 0.000 < 0.01).4.3 Determination of LDH content in cell supernatant by colorimetry: Compared with the blank group,the cell death rate in the model group was significantly higher,compared with the model group.The cell death rate in the other drug groups was decreased to some extent,among which the cell death rate in the middle dose group(14.267 ± 0.476%,P = 0.000<0.01)and the high dose group(14.700 ± 0.623%,P=0.000<0.01)was significantly lower than that in the prednisone group(18.983 ± 0.691%),and the difference was statistically significant.4.4 The levels of IL-1 ? and IL-18 in the supernatant of the model group were detected by ELISA: Compared with the blank group,the levels of IL-1 ? and IL-18 in the supernatant of the model group were significantly higher.Compared with the model group,the levels of IL-1? and IL-18 in each experimental drug group were significantly decreased.There was no significant difference in the levels of IL-1?(P = 0.216 > 0.05)and IL-18(P = 0.530 > 0.05)between the middle dose group,high dose group and the prednisone group.4.5 Detect Caspase-1,ELAVL-1,GSDMD-N,NLRP-3 protein expression level by western blot: Compared with the blank group,the expression level of Caspase-1,ELAVL-1,GSDMD-N,NLRP-3 protein in the model group was significantly increased,and the expression level of each index protein in each experimental drug group was decreased in different degrees compared with the model group.Among them,the protein expression level of Caspase-1(P = 0.019 < 0.05),ELAVL-1(P = 0.016 < 0.05),GSDMD-N(P = 0.011 <0.05),NLRP-3(P = 0.006 < 0.01)in the middle dose TCM group was significantly higherthan that in the prednisone group.4.6 Detect Tg,TPO,Caspase-1,NLRP-3 and ELAVL-1 m RNA by Real-time PCR: Compared with the blank group,the m RNA levels of Tg,TPO,Caspase-1,NLRP-3 and ELAVL-1 in the model group were significantly higher,and the m RNA levels in each experimental drug group were decreased to varying degrees compared with the model group.In the middle dose group,Tg(P = 0.000 < 0.01),TPO(P = 0.000 < 0.01),Caspase-1(P = 0.000 < 0.01),ELAVL-1m RNA(P = 0.000 < 0.01)and NLRP-3(P = 0.000 < 0.01)were significantly lower than those in the prednisone group.4.7 Cells Annexin V-FITC PI staining: Compared with the blank group,the proportion of annexin V(+ / PI(+)and annexin V(+ / PI(-)cells in the model group increased significantly.The ratio of annexin V(+ / PI(+)and annexin V(+ / PI(-)cells in each experimental drug group was decreased in different degrees compared with that in the model group.The ratio of annexin V + / PI(-)cells in the middle dose group and high dose group was significantly lower than that in the prednisone group,but there was no significant difference between the ratio of annexin V + / PI(-)cells and that in the prednisone group.Conclusion:1.Based on the theory of “dissipating excessive depression of fire”,the method of “ascending Yang and clearing away heat” is an effective and safe treatment for SAT in the acute stage with the syndrome of excessive heat and toxin.Professor Gao Tianshu created the traditional Chinese medicine compound Yingtongyin,which has the effect of ascending Yang and clearing away heat,detoxifying and eliminating goiter,and is in line with the pathogenesis of treating the acute stage of SAT with the syndrome of excessive heat and toxin.2.“Ascending Yang and clearing away heat” method in the SAT treatment can relieve the pain,shorten the fever time,shrink the goiter,restore the thyroid function,control the inflammatory(ESR)reaction in the acute stage of SAT,and the incidence of hypothyroidism and the recurrence rate of SAT are much lower than the prednisone.According to the Meta analysis,the method of “ascending Yang and clearing away heat” based on the theory of“dissipating excessive depression of fire” is an effective and safe treatment for SAT.3.“Ascending Yang and clearing away heat” method in the SAT treatment can inhibit thepyroptosis process of adenovirus-induced Nthy-roi3-1 cell infection,mainly by descending the expression and transcription level of ELEVL-1,inhibiting the activation of NLRP3 inflammatory body,descending the activation level of Caspase-1,inhibiting the expression of inflammatory cytokines IL-1 ? and IL-18,and inhibiting the expression of GSDMD-N. |
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