Research On Professor Lin Lan’s Clinical Experience In The Treatment Of Hyperthyroidism And The Mechanism Of JiaKangning Capsule In Regulating Of ERK Pathway In FRTL-5 Cells

Hyperthyroidism (hereinafter referred to as Jia Kang) is one of the most co mmon thyroid diseases.At present,the underlying pathogenesis are still poorly und erstood.There is still a lack of targeted therapy,especially for the recurrence of h yperthyroidism.Conventional antithyroid drugs can not solve the problem of exces sive proliferation of thyroid cells and recurrence of hyperthyroidism.With more th an 50 years of clinical experience, Professor Lin Lan has a profound understandi ng of the treatment of hyperthyroidism and have developed a number of effectiv e traditional Chinese medicine prescriptions.Traditional Chinese Medicine Jia Kan gning capsule is her essence of the clinical experience of the prevention of hype rthyroidism.In this paper, first of all, we made a systematic study on the experie nce of the prevention and treatment of hyperthyroidism by Professor Lin Lan.By means of the FRTL-5 thyroid cells cultured in vitro, with the interference and over expression of ERK1/2,we studied the effect of Jia Kangning capsule on the signal transduction pathway of ERK to investigate the possible molecular mecha nism of the effect of Jia Kangning on the regulation of the growth of thyroid c ells by inhibiting the proliferation and promoting the apoptosis of thyroid cells.Ⅰ. Clinical ResearchObjectives:To conduct data analysis on the effective clinical cases of hyperthyr oidism treatment by Professor Lin Lan and summarize the application laws of tr aditional Chinese medicine for hyperthyroidism by Professor Lin Lan.Combined with communication with my tutor,we tyied to summarize the clinical experience of treating diseases such as hyperthyroidism complicated with liver damage,hypert hyroidism complicated with leucopenia,hyperthyroidism complicated with exophtha lmos and hyperthyroidism complicated with emmeniopathy.To provide evidence-ba sed basis for refining and optimizing the hyperthyroidism prevention and cure la ws of traditional Chinese medicine.Methods:160 cases of patients with hyperthyroidism and totally 437 visits diag nosed by Professor Lin Lan in the endocrine clinic of Guang’anmen hospital fro m December 2013 to December 2015 were input EXCEL2007 spreadsheet.The S PSS19.0 software,weka3.7 data mining software and Liquorice mining software w ere used and the methods such as frequency analysis,cluster analysis and associat ion rule were adopted to analyze the prescription application laws of Professor L in Lan.Through communicating with my tutor,we tried to sort out her clinical ex perience of treating diseases such as hyperthyroidism,hyperthyroidism complicated with liver damage, hyperthyroidism complicated with leucopenia,hyperthyroidism complicated with exophthalmos and hyperthyroidism complicated with emmeniop athy.Results:(1) According to the cases inclusion and exclusion criteria,totally 160 qualified cases were selected and 167 kinds of traditional Chinese medicine invol ved in this disease treatment were obtained with the total frequency of 9032, am ong which the frequencies of 64 kinds in total were above 10 times with the tot al frequency of 8634.Among them,the medicines such as Keel,Calcined magnet,N acre,Ophiopogon japonicus,Semen Ziziphi Spinosae,Schisandra chinensis,Prunella v ulgariS,Pinellia tuber,Mountain arrowhead and so on were the core drugs for the treatment of hyperthyroidism by Professor Lin Lan. (2) By collecting all the trad itional Chinese medicine prescriptions used for the treatment of 160 patients with hyperthyroidism of 437 times by Professor Lin Lan,we sorted all the traditional Chinese medicine in the prescriptions according to their total frequencies and ta ked the drugs with the use frequency above 10 as the high frequency traditional Chinese medicine by statistics,69 kinds of traditional Chinese medicines were sel ected in total.Referring to the teaching material of the seventh edition of Science of Chinese Pharmacology and Chinese Material Medica,we categorized the 167 kinds of high frequency traditional Chinese medicine according to their efficacy and added up the use frequency of all the medicines in each category of traditio nal Chinese medicine.The statistical results showed that the tonifying drugs and t ranquilization medicine were the most used for treatment of hyperthyroidism by Professor Lin Lan,followed by the heat-clearing drugs,drugs for invigorating bloo d circulation and eliminating stasis,drugs for suppressing hyperactive liver for cal ming endogenous wind,drugs for inducing astringency,drugs for reducing phlegm,r elieving cough and preventing asthma,and drugs for regulating qi.When using the tonifying medicines,the priority was given to the hemopoietics and yin tonics and then the drugs for tonifying qi and yang.The traditional Chinese medicines with the functions of nourishing yin and suppressing hyperactive yang and calming th e nerves are the most commonly used drugs for treatment of hyperthyroidism by Professor Lin. (3) Using SPSS19.0 statistical software to conduct cluster analysis on high frequency traditional Chinese medicine based on the effect,the results sh owed that the prescriptions used by Professor Lin Lan in the treatment of hypert hyroidism were divided into nine categories:Herbaleonuri,Cortex moutan,Fuctuscor ni,dried human placenta and angelica; Stir-baked cortex eucommiae with salt soluti on,Alisma,fried Atractylodes macrocephala Koidz,Raspberry,Fructus Alpiniae Oxyp hyllae,Rhizomacyperi,Rhizomacoptidis,Radix aucklandiae,Safflower,Rhizomaligustici wallichii,Sandalwood,Raw radix astragalis,Processed radix polygalae,Cassia seed;Ca pillary artemisia,Cortex phellodendri,Raw oyster,Nacre;Radix paeoniae alba,Crude ossadraconis,Fructusschisandrae,Selfheal;Radix ophiopogonis,Radix pseudostellariae, Semen boitae,Semen Ziziphi Spinosae;Prunella vulgaris,Fructusarctii;medlar,Calcine d magnetitum,uncaria;Radix curcumae,Corydalis tuber;Pinelliaternata,Fructus Aurant ii Immaturus.(4)Clustering analysis of the high frequency herbal medicines was p erformed with the data mining software Liquorice based on efficacy.The results s howed that Professor Lin Lan’s core prescription with nourishing yin and suppres sing yang, phlegm scattered nodes,functions of replenishing qi and nourishing yin for characteristics.Raw keel,Calcined magnet,Prunella vulgaris,Medlar,Uncaria,Radi x Pseudostellariae,Ophiopogon root,White peony root,Schisandra,Prepared rhizome of rehmannia,Chinese arborvitae and Wild jujube benevolence formed the mostco mmon prescription combination.(5)Using weka3.7 data mining software and adopt ing the method of association rules,the results showed that the commonly used c ombinations of couplet medicines by Professor Lin Lan in the clinical treatment are as follows:combination for suppressing yang to extinguish wind-crude was m ade of Ossadraconis andCalcined magnetitum;combination for reducing phlegm an d resolving masses-indian was made of iphigenia bulb,fructusarctii,fritillary bulb a nd phillyrin;combination for soothing the liver and regulating qi-radix was made of bupleuriand radix paeoniae alba;Combination for regulating qi for eliminating phlegm was made of pinellia ternata,fructus aurantii immaturus,radix curcumae an d corydalistuber;combination for tonifying spleen andeliminating dampness was m ade of rhizoma atractylodis and magnolia bark;combination fortranquilization was made of crude ossadraconis,raw oyster,calcined magnetitum,nacre,semen zizyphis pinosae and semen boitae;combination for invigorating the spleen for eliminating dampness was made of poriacocos and alisma;combination for nourishing liver and kidney was made of raspberry,Fructus Alpiniae Oxyphyllae and eucommiabar k;combination for nourishing the liver to improve visual acuity was made of w olfberry fruit,cassia seed;combination for dispelling pathogenic wind for improvin g eyesight was made of semen celosiae and buddleja officinalis.(6)Through the c ommunication with my tutor,she suggested the diagnosis and treatment of hyperth yroidism divided into four types, i.e.,qi stagnation and phlegm coagulation,yin de ficiency and yang hyperactivity,fire excess from yin deficiency and deficiency of both qi and yin,and she also attached importance to reducing phlegm and invig orating the circulation of blood and emphasized the western medicine running thr ough the treatment.When treating the hyperthyroidism complicated with exophthal mos,my tutor emphasized the diagnosis and treatment by distinguishing the prima ry symptom.When treating the hyperthyroidism complicated with liver damage,my tutor advocated treatment by stages.When treating the hyperthyroidism complicate d with leukopenia,the tutor taked tonifying qi and yin as the primary treatment method.When treating the hyperthyroidism complicated with emmeniopathy, the tu tor used the artificial cycle therapy of traditional Chinese medicine combined wit h the thyroid drugs for maintenance treatment.Conclusions:This research used the data mining technology such as frequency analysis,cluster analysis and association rules to conduct analysis on the clinical medication laws in the treatment of hyperthyroidism by Professor Lin Lan for th e first time and combined with oral teaching that inspires true understanding wit hin,summarized the prescription medication rules and treatment experience of hyp erthyroidism,hyperthyroidism exophthalmos,hyperthyroidism complicated with liver damage,hyperthyroidism complicated with leukopenia and hyperthyroidism complic ated with emmeniopathy by Professor Lin Lan.II. Experimental StudyObjective:To investigate the molecular mechanism of Jia Kangning Capsule in th e treatment of hyperthyroidism based on the ERK signal transduction pathway.Methods:This study taked the FRTL-5 cells of primary culture as the research object,and CCK-8 method was used to select theoptimal concentration for traditio nal Chinese medicine Jia Kangning Capsule to inhibit the cell proliferation ofFR TL-5 as the drug concentration for the intervention of FRTL-5 cells.Eight groups were set for the experiment,i.e.,control group (Con),Jia Kangning group,Jia Kang ning plus ERKl/2-siRNA group(Jia Kangning collaborative group),siRNA negativ e sequence group (NC), siRNAinterference group, overexpression oflentivirus gro up, Jia Kangning plus overexpression of lentivirus group and emptyvector group (NC).The method of CCK-8 was adopted to detect the effect of JiaKangning on the cell proliferation of each group;radioimmunoassay was used to detect the a mount of cAMP in supernatant,AV/PI double staining method was used to the d etermination of apoptosis rate,and Western Blot and RT-qPCR method were used to observe the changes of proliferation and apoptosis related genes,factors (c-Ra f,p-ERKl/2,ERK1/2,Caspase-9,cyclinDl,Bcl-2,Bax) in protein and gene level.To c ompare the intervention effects oftraditional Chinese medicine Jia Kangning Caps ule on each group of cells from two aspects of interference and overexpression.Results:(1)The primary culture of FRTL-5 cells was adopted in this experiment, and the cells were frozen and stored enough,which laid the foundation for the su bsequent experimental study.(2)The CCK-8 method was used to detect the prolife ration of FRTL-5 cells with different concentrations of Jia Kangning at different intervention time, and after repeated experiments, finally determines the 5.438mg/ ml of F12 complete medium containing traditional Chinese medicine Jia Kangnin g at the 24th hours of intervention has the most appropriate intervention concent ration for FRTL-5 cells.(3)The inhibition effect of Jia Kangning on the proliferati on of FRTL-5 cells was confirmed with the CCK-8 method. (4)The AV/PI Anne xin double staining method was used to detect the effect of Jia Kangning on the apoptosis of FRTL-5 cells transfected with ERKl/2-siRNA and infected with E RK1/2 overexpression of lenti virus group,the results showed that compared with t he control group, cell apoptosis was detected in Jia Kangning group (P<0.01). After the interference of ERK1/2-siRNA,the apoptosis occurred to the FRTL-5 ce lls and the cell apoptosis of the Jia Kangning cooperative group becomed more obvious(P<0.05).Compared with the control group, the apoptosis rates of the ER K1/2-siRNA group and ERKl/2-siRNA negative sequence group was higher but were not statistically significant.Compared with Jia Kangning group,there was no significant difference in the apoptosis rate of Jia Kangning collaborative group and Jia Kangning plus overexpression of lentivirus group.Compared with ERK1/2-siRNA negative sequence group, apoptosis rate increased dsignificantly in Jia Ka ngning cooperative group (P<0.01).Compared with the empty virus group, apopt osis rate of Jia Kangning Collaborative group and Jia Kangning group was signi ficantly increased (P<0.01).(5)The release of cAMP in the supernatant of each gr oup was determined by radioimmunoassay and compared with the control group, there was no significant differences between the cAMP release volumes of ERK 1/2-siRNA group and ERK1/2-siRNA negative sequence group,and the cAMP rel ease volumes of the rest of groups all reduced compared with the control group. The differences were of statistical significance(P<0.05 or P<0.01) and among the m,the cAMP release of the Jia Kangning group reduced most significantly.Compa red with the Jia Kangning group,the differences of the cAMP release between th e control group, ERK1/2-siRNA group and ERKl/2-siRNA negative sequence gro up were of statistical significance (P<0.05 or P<0.01).Compared with the ERKl/2-siRNA negative sequence group,the amount of cAMP in the ERKl/2-siRNA neg ative sequence group reduced obviously (P< 0.01).And compared with the ERK1/ 2 lentivirus negative control group,the cAMP release of the control group was in creased (P<0.05).(6)RT-qPCR results showed that compared to control group, mR NA expression of c-raf,ERK1,ERK2,CyclinDl and Bcl-2 in Jia Kangning group,E RKl/2-siRNA group,Jia KangNing cooperative group were significantly decreased (P<0.01).The expression of bax mRNA and Caspase-9 mRNA in the Jia Kangni ng group was higher but not statistically significant,while it was significantly hig her in the ERK1/2-siRNA group and the Jia Kangning Collaborative Group(P<0. 01).Compared with the control group, the mRNA expressions of c-Raf,ERK1,ER K2 and bax in the overexpression of lentivirus group and the Jia Kangning plus overexpression of lentivirus group were significantly increased (P<0.01).While th e expression of CyclinDl mRNA and bcl-2 mRNA decreased significantly (P<0.0 1).(7)The results of Western Blot showed that the protein expressions of c-Raf, p-ERKl/2,ERK1/2,cyclinD1 and Bcl-2 were significantly down-regulated (P<0.01)i n Jia Kangning group,ERK1/2-siRNA group,and the Jia Kangning Collaborative g roup.The protein expression levels of Caspase-9 were increased in the Jia Kangni ng group(P<0.01),and the protein expressions of Bax and Caspase-9 were signific antly up-regulated (P<0.01) in the ERK1/2-siRNA group and the Jia Kangning Collaborative group.Compared with the control group,protein expressions of c-Raf, p-ERKl/2,ERK1/2 and Bax in the overexpression of lentivirus group and the Ji a Kangning plus overexpression of lentivirus group were significantly higher (P< 0.01),while the cyclinD1 mRNA and the Bcl-2 protein expression was significantl y decreased (P<0.01).Conclusions:ERK1/2-siRNA inhibited the proliferation and promoted the apop tosis of FRTL-5 cells induced by Chinese herbal Jia Kangning.Over-expression of ERKl/2 enabled the FRTL-5 cells to obtain higher capacities in proliferation an d anti-apoptotic ability,as well as the drug resistance of Jia Kangning is significa ntly increased.By blocking the phosphorylation of ERKl/2,Chinese herbal Jia Kan gning regulated the expression of c-AMP, c-Raf,cyclinDl,Bax,Bc1-2,caspase-9 and cyclinD1 in FRTL-5 cells, and inhibited the proliferation of FRTL-5 cells and p romoted their apoptosis.Thereby,Jia Kangning improved cellular dynamics and reg ulated the growth of thyroid cells, and then reached the destination of treatment of hyperthyroidism and prevention of recurrence.