| Purpose:The main purpose of this study is to develop the external traditional Chinese medicine of Yingzhongxiao into an hospital preparation in the form of ointment.It is necessary to determine the preparation process of Yingzhongxiao,to establish quality standards and to investigate the stability of Yingzhongxiao.First of all,we have analysed the therapeutic mechanism of Yingzhongxiao ointment in the treatment of thyroncus from the perspective of autophagy and apoptosis,and then we have appraised the Non-clinical safety of it through toxicity test.At the end,a randomized controlled trial was conducted to evaluate the efficacy of Yingzhongxiao Point-application in the treatment of nodular goiter.Material and method:First part:This part focused on pharmaceutical technology of Yingzhongxiao:The Weighted scoring method was used to investigate every compound ingredients,penetration enhancer and the others.Then we have confirmed the stability and investigation of pharmaceutical pulverization process about the Yingzhongxiao,from the result of pilot test.Second part:This part focused on quality standard and stability of Yingzhongxiao:With HPLC and Chromatography,the distinctive compound of Yingzhongxiao was found.The differences of Rhubarb officinale,Fructus Gardeniae,Indigo Naturalis,Prunella vulgaris,Thunberg Fritillaria Bulb and Zedoary were identified by thin layer chromatography about the interference of negative fluid.The stability of three batches of samples were all re-examined by room temperature sample retention method and accelerated heating method.Third part:This part is the pharmacodynamics study of Yingzhongxiao ointment:704-week-old Wistar rats,half male and half female,were selected from SPF grade.10 rats were be randomly selected as normal group,and the others were preparated of goiter model by gavage,0.01g/（kg·d）PTU for 14d,after a week of adaptive feeding.60 rats were randomly divided into 6 groups:Model group,Low dose group（equal to 0.45g/kg·d of dried herbs）,Middle dose group（equal to 0.9g/kg·d of dried herbs）,High dose group（equal to 1.8g/kg·d of dried herbs）,Placebo group（only ointment base without crude drug）and LT4 group（0.2μg/kg Euthyrox aqueous solution）,10 rats in each group.From the fifteenth day,only the rats in normal group and model group were raised normally.At the same time other rats were removed hair from their necks with 6%sodium sulfide.And then they were given Yingzhongxiao ointment to the neck per day for 28 continuous days,the thickness of Yingzhongxiao ointment coverage is about 0.2cm.And the drug was wrapped with medical gauze,and it was fixed with non-irritating tape.After 28 days of consecutive intervention,serum and thyroid tissue of each rat were preserved.Using ELISA to detect serum thyroid hormones content.Using HE staining method to observe the morphological changes of thyroid morphology.Using quantitative PCR to detect PI3K-Akt-mTOR signal transduction pathway.Using Western blot to detect Beclin-1,LC3 protein expression in thyroid tissue of each group.The expressions of Fas、Fasl in rat thyroid were detected by means of immunohistochemical method.The initial pharmacodynamics were completed after this part of experiment.Forth part:This part is the toxicity experiment of Yingzhongxiao ointment:Acute toxicity experiment of 20 New Zealand White Rabbits,weight:2±0.2kg,half male and female.The skin of the two sides of the spinal column of each rabbit was shaved with 6%sodium sulfide aqueous solution after one week of adaptive feeding.Twenty rabbits were randomly divided into 5 groups（stratified according to their sex and weight）,with 4 in each group.The five groups were:the excipient control group,intact skin high dose group,intact skin low dose group,damaged skin high dose group and damaged skin low dose group.Each rabbit skin of damaged skin group was rubbed with#400 sand paper,to the degree of bleeding.The excipient control group was only treated with ointment base.Two high dose groups were treated with high dose Yingzhongxiao ointment equal to 0.3g/g of dried herbs.At the same time two low dose groups were treated with low dose Yingzhongxiao ointment equal to0.1g/g of dried herbs.Body weight,skin,hair,eye and mucous membrane changes,respiration,central nervous system,extremities activity and other toxic manifestations were observed and recorded in each group,at 1 h,24 h,48 h,72 h to 7 d after drug removal.At last the results were used to evaluate the toxic reactions of rabbit skin exposed to the tested substances for a short period of time.Long-term toxicity test of Yingzhongxiao ointment:Thirty New Zealand white rabbits,weight 2±0.2kg and half of the male and female,were selected for this experiment.The skin of the two sides of the spinal column of each rabbit was shaved with 6%sodium sulfide aqueous solution after one week of adaptive feeding.Thirty rabbits were randomly divided into 5 groups（stratified according to their sex and weight）,with 6 in each group.The five groups were:the excipient control group,intact skin high dose group,intact skin low dose group,damaged skin high dose group and damaged skin low dose group.Each rabbit skin of damaged skin group was rubbed with#400 sand paper,to the degree of bleeding.The excipient control group was only treated with ointment base twice per day.The other groups were given Yingzhongxiao ointment twice a day for 6 hours,for continuous administration for 12 weeks.The body weight,skin condition,whole body symptom,poisoning manifestation and recovery degree of each group were observed and recorded.Four animals in each group were randomly selected.After 24 hours of fasting water,the animals were killed.The results of pathological sections of animal organs and serum biochemistry were analyzed,after the 12th weekend administration.The remaining animals were observed 2 weeks after drug withdrawal,the observation index was the same as above.The remaining 10 rabbits were sacrificed under intraperitoneal anesthesia after 24 h of fasting water,and the main arteries of the abdominal aorta and the organs of the rabbits were taken.Automatic blood cell analyzer was used to detect the changes of blood cells in each group of rabbits.And then the changes of Liver Function,Renal Function,Blood Glucose and Blood Lipid of each rabbit in each group were observed by automatic biochemistry analyzer.The organs and skin of the tested rabbit were stained by HE staining method.Pathological slices were used to observe under the microscope.Fifth part:This part is the clinical study on the treatment of nodular goiter with Yingzhongxiao Point-application:nodular goiter patients defined as the fire-transmission with the stagnation of Qi,phlegm and blood in TCM syndrome were recruited from March 2018 to March 2019 in the First Affiliated Hospital of Liaoning University of TCM in endocrine out-patient that were were randomly divided into two groups:treatment group and control group.The treatment group was treated with Yingzhongxiao Point-application（5g twice a day,3 hours each time）for 8 weeks on the swollen side of the thyroid,at Futu Point and Shuitu Point.The control group was in negative control and no treatment was taken to them,all of them were only observed within 8 weeks.The anteroposterior diameter of left lobe of thyroid gland,anteroposterior diameter of right lobe of thyroid gland,thyroid isthmus thickness and the maximum diameter of thyroid nodule were all compared between the two groups before and after the trial.Two groups of patients were scored before and after the trial,by thyroid color ultrasound and TCM syndrome score table formulated before.In order to evaluate the effective rate of Yingzhongxiao Point-application in the treatment of nodular goiter by the result of thyroid color ultrasound and TCM syndrome score table,which were based on the guiding principles of clinical new drugs of traditional Chinese medicine.Paired-sample t test or non-parametric test was used in comparison of changes before and after the trial between the two different groups.Independent-sample t test or nonparametric test or chi-square test was used in comparison of efficacy between the two groups.Results:1.The preparation process of this preparation is as follows:Break Prunella Vulgaris,Rheum officinale,Gardenia,Thunberg Fritillary Bulb,Curcuma zedoary and Mentha haplocalyx into fine powder,and then fine the borneol.Mix the medicinal powder and add indigo naturalis,and weigh it next.Concentrate the powder nine times and then cool it down to 40℃.The ingredients of the ointment matrix are as follows:Octadecanoic acid 400g,atolein 500g,vaseline 500g,glycerin monostearate 400g and azone 400g as oil-phase.Glycerol 600g,triethanolamine 100g,ethyl p-hydroxybenzoate 10g,proper distilled water as.The compounds were evenly divided into two containers,heated to 80℃,melted and mixed,stirring continuously to make ointment bases,total 10000g.2.Three batches of Yingzhongxiao sample loading tests,particle size tests and microbiological tests all meet the requirements of Pharmacopoeia.The results of TLC identification in Rhubarb officinale,Fructus Gardeniae,Indigo Naturalis,Prunella vulgaris,Thunberg Fritillaria Bulb and Zedoary showed that only Rhubarb officinale,Fructus Gardeniae,Indigo Naturalis were writed in the standard because of the repeatable and specialization.The test results of Gardenia jasminoides glycoside content,determined by high performance liquid chromatography（HPLC）,meet the requirements.The content of jasminoidin,in Yingzhongxiao ointment,should not be limited less than 0.3mg/g.Based on the above results,a draft quality standard for Yingzhongxiao ointment was drawn up.The characters,qualitative identification,content determination and related examination of 3batches of Yingzhongxiao ointment are in accordance with the regulations,by normal temperature sample retention method and heating accelerated test method,along the six-month study period.3.Pharmacodynamics results showed that:（1）There was no significant difference in body weight among the seven groups（P>0.05）.Compared with normal group,thyroid weight and relative weight of rats in any other group increased significantly（P<0.01）.Compared with model group,there was no statistical difference of thyroid weight and relative weight in placebo group（P>0.05）.The absolute weight and relative weight of thyroid in LT4 group and each dose group were significantly decreased model group（P<0.01）.Compared with LT4group,there was no statistical difference of thyroid weight and relative weight in low dose group（P>0.05）.The absolute weight（P<0.05）and relative weight of thyroid（P<0.01）in middle dose group and high dose group were both decreased.Compared with low dose group,the absolute weight and relative weight of thyroid in middle dose group and high dose group were lighter（P<0.05）.There was no difference in the absolute weight and relative weight of thyroid between middle dose group and high dose group（P>0.05）.（2）The thyroid follicles in the normal group were medium size,the epithelial cells were cubic or flat,arranged regularly,and the inner parts were filled with glia.There was no abnormal hyperplasia of loose connective tissue between thyroid follicles,and a small number of capillaries could be seen around the thyroid follicles in the normal group.In the model group,the thyroid follicles were overdilated,the size of thyroid follicles were obviously different,the arrangement was irregular,the content of colloid in the cavity was obviously reduced,the epithelial cells were highly proliferative phase,the boundary between follicles was blurred,and the follicles were separated by different number of fibrous tissues.Inflammatory cell infiltration can be seen in the model group.Compared with the model group,the morphology of placebo group is similar to it.Thyroid tissue morphology and follicular structure were observed to recover in each dose group and LT4 group.（3）Serum TT3,TT4,TSH concentration in each group of rats:There was no significant difference in serum TT3 level among the seven groups（P>0.05）.Compared with normal group,the serum TT4 of rats in any other group decreased significantly（P<0.01）.Compared with model group,the level of serum TT4 in LT4 group was increased（P<0.05）,and there was no significant difference in any of the other group（P>0.05）.Compared with LT4 group,the level of serum TT4 in low dose group、high dose group and placebo group were all in low concentration（P<0.05）.There was no significant difference in serum TT4 level among each group（P>0.05）.Compared with normal group,the concentration of serum TSH among any other group was higher（P<0.01）.Compared with model group,the concentration of serum TSH in LT4 group was significantly decreased（P<0.01）.But there was no statistical difference between any other group and model group（P>0.05）.Compared with LT4 group,the concentrations of serum TSH among three different dose groups were all significantly increased（P<0.01）.And there was no significant difference among three different dose groups（P>0.05）.（4）Compared with normal group,there was higher expression of PI3K mRNA in any other group（P<0.01）.Compared with model group,there is no statistical change about the mRNA expression of PI3K in placebo group（P>0.05）.But it was significantly decreased（P<0.01）in each different dose group or LT4 group.Compared with LT4 group,there was no statistically different expression of PI3K mRNA in low dose group（P>0.05）.The middle dose and high dose group expressed less PI3K mRNA than that in LT4 group（P<0.01）.Compared with low dose group,the mRNA expression of PI3K in middle dose group and high dose group were both significantly decreased（P<0.01）.Compared with normal group,there was higher expression of Akt mRNA in any other group（P<0.01）.Compared with model group,there is no statistical change about the mRNA expression of Akt in placebo group（P>0.05）.But it was significantly decreased（P<0.01）in each different dose group or LT4 group.Compared with LT4 group,there was no statistically different expression of Akt mRNA in low dose group（P>0.05）.The middle dose and high dose group expressed less Akt mRNA than that in LT4 group（P<0.01）.Compared with low dose group,the mRNA expression of Akt in middle dose group and high dose group were both significantly decreased（P<0.01）.Compared with normal group,the expression of mTOR mRNA increased in each group.There were statistically increased in middle dose group and high dose group（P<0.05）.And the expression of mTOR mRNA in normal group was significantly different from that in model group,placebo group,low dose group and LT4 group（P<0.01）.Compared with model group,the mTOR mRNA expression of each group decreased except placebo group（P>0.05）,and the low dose group and LT4 group had statistical difference（P<0.05）.The difference between the other two different dose group and model group was significant（P<0.01）.Compared with LT4 group,there was no statistically different expression of mTOR mRNA in low dose group（P>0.05）.The middle dose and high dose group expressed less mTOR mRNA than that in LT4 group（P<0.05）.Compared with low dose group,the mRNA expression of mTOR mRNA in middle dose group and high dose group were both significantly decreased（P<0.05）.There was no significant difference between middle dose group and high dose group（P>0.05）.（5）Compared with normal group,Beclin-1protein expression was similar in LT4 group（P>0.05）.Compared with the normal group,the expression of Beclin-1 protein in placebo group was increased（P<0.05）,and the other groups had significantly higher expression than the normal group（P<0.01）.Compared with model group,there is no statistical change about Beclin-1 protein expression in placebo group（P>0.05）.The protein content of Beclin-1 in LT4 group was higher than that in normal group（P<0.05）,and the expression of each dose group was significantly increased（P<0.01）.Compared with LT4 group,the Beclin-1 protein content of each dose group was significantly increased（P<0.01）.Compared with low dose group,the expression of Beclin-1 in middle dose group and high dose group increased（P<0.05,P<0.01）.Compared with normal group,the expression of LC3II/I protein in each group was significantly decreased（P<0.01）.Compared with model group,the expression of LC3II/I protein in each group were significantly increased（P<0.01）except placebo group（P>0.05）.Compared with LT4 group,there was no statistical difference in low dose group（P>0.05）,as the expression of LC3II/I increased in middle and high dose groups（P<0.05）.Compared with low dose group,the expression of LC3II/I in middle dose group and high dose group increased（P<0.05）.（6）Compared with normal group,the expression of Fas in model group was increased（P<0.05）,and the other groups all increased significantly（P<0.01）.Compared with model group,there was no difference in placebo group（P>0.05）.The expression of Fas in thyroid tissue in LT4 group was enhanced（P<0.05）,and the other dose groups were significantly enhanced（P<0.01）.Compared with LT4 group,the expression was significantly increased in each dose group（P<0.01）.Compared with low dose group,the Fas expression was significantly increased in the other two dose groups（P<0.01）.Compared with normal group,the expression of FasL in the other groups all increased significantly（P<0.01）.Compared with model group,there was no significant difference between placebo group,low dose group and LT4group（P>0.05）about the mean optical density of FasL,and the expression of FasL was significantly increased in middle and high dose groups（P<0.01）.Compared with LT4 group,the positive expression of FasL was significantly increased in each dose group（P<0.01）.Compared with low dose group,the expression of FasL in middle dose group and high dose group was significantly enhanced（P<0.01）.4.（1）Acute toxicity test of Yingzhongxiao ointment:After intervention by the ointment,all the animals in each group survived,no abnormal changes in body weight,normal behavior,bright hair,no abnormal changes in eyes and mucous membranes,no significant changes in diet and urination and defecation at each observation timing.There were no erythema and edema in each rabbit of intact skin group.In the damaged skin group,there was no erythema and edema in the scab,and the wound returned to normal.（2）Long-term toxicity test of Yingzhongxiao ointment:All animals in each group survived,no abnormal changes in body weight,normal behavior,moist hair,no abnormal changes in eyes and mucous membranes,and no significant changes in diet,drinking and urination and defecation were observed during the period of medication and the period of observation.There were no erythema and edema in each rabbit of intact skin group.In the damaged skin group,there was no erythema and edema in the scab,and the wound returned to normal.Compared with the excipient control group,the intervention of different doses of intact skin and damaged skin had no significant effect on the body weight of the tested animals.Compared with the excipient control group,the intervention of different doses of intact skin and damaged skin had no significant effect on the peripheral blood cells of rabbits（P>0.05）,such as WBC,NEUT,BASO,MONO,LYMPH,RBC,HGB,HCT,MCV,MCH,MCHC,RDW-CV,RDW-SD,PLT,PCT,MPV,PDW and P-LCR,at the observation period after 12 weeks of intervention or 2 weeks after withdrawal.Compared with the excipient control group,the intervention of different doses of intact skin and damaged skin group had no significant effect on the TP,ALB,GLOB,A/G,TBIL,CHOL,GLU of the tested animals（P>0.05）,at the observation period after 12 weeks of intervention or 2 weeks after withdrawal.Compared with the excipient control group,the intervention of different doses of intact skin group and damaged skin group had no significant effect on the liver function（ALP,ALT,AST,AST/ALT）of the tested animals（P>0.05）,at the observation period after 12 weeks of intervention or 2 weeks after withdrawal.Compared with the excipient control group,the intervention of different doses of intact skin group and damaged skin group had no significant effect on the renal function（UREA,CREA）of the tested animals（P>0.05）,at the observation period after 12 weeks of intervention or 2 weeks after withdrawal.Compared with the excipient control group,there was no significant difference in the brain,heart,lung,liver,spleen,adrenal gland,gonad and other important organ coefficients between the intact skin group and the damaged skin group（P>0.05）,at the observation period after 12 weeks of intervention or 2 weeks after withdrawal.Compared with the excipient control group,there were no obvious inflammatory changes in the important organs and skin of rabbits in other groups,whether with visual inspection or by microscope in HE staining method.There were no changes of pathology or acute or chronic toxic injuries.5.Clinical observation results:Forty-four patients with nodular goiter were recruited in this Clinical research,22 in the treatment group and 22 in the control group.The intergroup baseline is comparable.The results of thyroid color ultrasound before and after the trial in two groups of patients showed that:The anterior and posterior diameter of left lobe of thyroid gland and the anterior and posterior diameter of right lobe of thyroid gland decreased significantly（P<0.05）,after the treatment group was treated.There was no significant difference in thyroid isthmus thickness and maximal nodule diameter before and after treatment（P>0.05）.There was no significant difference be found between the anterior and posterior diameter of the left lobe of the thyroid,the anterior and posterior diameter of the right lobe,the thickness of the isthmus and the maximum nodule diameter（P>0.05）,before and after the trial in the control group.The malignant score of thyroid nodules in the treatment group was significantly lower than that in the control group（P<0.05）.There was no significant difference in the malignant score of thyroid nodules before and after the test in the control group（P>0.05）.The results of TCM syndrome score before and after the trial in the two groups showed that the TCM syndrome score in the treatment group was significantly decreased after treatment（P<0.01）,and the difference was statistically significant（P<0.01）.The scores of TCM syndrome in the control group decreased before and after treatment,but there was no significant difference（P>0.05）.According to the result of thyroid color ultrasound,the efficiency was 45.45%in the treatment group and 13.64%in the control group.There was statistically difference between the two groups（P<0.05）aboout the efficiency.The result based on the integral of TCM syndrome score table of two groups shows that:The efficiency was 77.27%in the treatment group and 9.09%in the control group.There was statistical significance difference in the total efficiency about the TCM syndrome integral between the two groups（P<0.01）.There was no significant difference in the changes of Thyroid function,Liver function and Renal function between the two groups before and after treatment.Conclusion:1.The preparation technology of Yingzhongxiao ointment has reached the standard of quality.The finished product has good stability.Yingzhongxiao is a kind of safe external ointment preparation,which is suitable for the development of hospital preparation.2.It can reduce the thyroid volume of rats,restore the shape of thyroid gland,and have no significant effect on the thyroid function of rats,by external therapy of Yingzhongxiao ointment.The mechanism of its therapeutic effect may be related to the down-regulation of PI3K/Akt/mTOR signaling pathway gene expression in goiter rats and promote the expression of autophagy-associated protein Beclin-1,LC3 and apoptosis-related protein Fas,FasL in goiter rats.3.The thyroidvolume and TCM syndrome integral ofnodulargoiter patients were effectively lessened by 8 weeks treatment with Yingzhongxiao Point-application.It reduces the anteroposterior diameter of the thyroid gland availably.It has no obvious effect on thyroid function,liver function and renal function,and is safe and effective to be popularized. |
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