The Effect And Mechanism Of The Anti-drug Combination Of Haizao Yuhu Decoction Under Specific Dosage Conditions For Anti-goiter

Background:Sargassum(HZ)and Glycyrrhiza(GC)are one combination of the Eighteen antagonistic medicaments.Haizao Yuhu decoction(HYD),first recorded by Chen Shigong of Ming Dynasty,contains HZ and GC are one combination of antagonistic medicaments.It is a classic prescription commonly used in clinic.It is often used to treat goiter,gallbladder polyp,ovarian cyst,hyperplasia of mammary gland,infantile scrofula,etc.However,the use of antagonistic medicaments combination involves the safety and rationality of clinical medication,which is related to the national economy and people's livelihood.Up to now,Although more and more researchers attach importance to it and participate on the research in the compatibility of antagonistic drug combinations,and achieved some results,Whether they can be used together is still controversialThe National Program on Key Basic Research Project(973 Program)participated by the research group stulied Haizao Yuhu decoction containing antagonistic medicaments combination of HZ and GC from the perspective of combination of prescriptions and drugs,and studied HZ and GC from different compatibility ratio,different dosage,different processing varieties and other aspects,to explore the characteristics and rules of its medication On the basis of previous experimental research the following NSFC funded projects were divided into three dose groups:the base limit,the high limit and 2 times higher limit of the Pharmacopoeia,according to the dosage of GC and HZ specified in the Chinese Pharmacopoeia 2015.From the perspective of dose toxicity effect this studies whether different varieties of HZ and different compatibility doses of HZ and GC are the factors that influence the compatibility of HZ and GC.The results show that there are differences in the effects of various compatibility dose groups on the thyroid function indexes and the thyroid tissue morphology of rats with PTU induced goiter.The conditions with relatively obvious"effect" were screened out:HZ was selected from Haihaozi species,HZ and GC were selected twice of the high limit dose of Pharmacopoeia;under these conditions,the indexes of heart,liver and kidney of model rats were not significantly different compared with blank group,and no obvious toxicity was found.However,the material basis and mechanism of its action need to be further explored.Objective:Based on the previous research of the research group,this experiment further explored the application of HZ and GC in Haizao Yuhu Decoction under the condition that the dosage of HZ and GC is twice of the high limit dosage of Pharmacopoeia(HZ 24g,GC 20g).It studied the material basis,the regulatory effect of hypothalamus pituitary thyroid axis,cell apoptosis and proliferation,trying to uncover the mechanism of antithyroid ism was shown in order to provide experimental basis for improving the compatibility of HZ and GC and guiding clinical application.Methods:1 Material Basis:Select the common components of the medicine contained in the compound and the components that have a greater impact on the dissolution content in combination with the antagonistic drug combination mentioned in the literature report:hesperidin(HD,the common components of Qingpi and Chenpi);ferulic acid(FA,the common components of Chuanxiong,Danggui and Duhuo);forsythin(PN);glycyrrhizin(LG)and glycyrrhizic acid(LA)(the anti drug combination mentioned in the literature report have greater impact on the dissolution content ingredients).HPLC was used as the detection method to compare the dissolution content of the same active ingredients among HYD,HYD-G,HYD-H,HYD-GH,HZ and GH(HZandGC).2 Mechanism Research:Healthy adult Wistar rats,half male and half female,weighing 200±20g,were divided into blank group,model group,positive drug group(euthanasia),HYD,HYD-G,HYD-H,HYD-GH.Through the changes of thyroid pathological tissue morphology(light microscopy observation),the detection of thyroid function blood biochemical indicators,the preliminary analysis of the model and the e ffectiveness of the drug,and then through the observation of the morphological changes of its microstructure under the electron microscope and TUNEL detection of apoptosis index,further through the detection of protein and gene,To explore the mechanism of anti goitre effect.At the end of the animal experiment,the rats were perfused with drugs 1 hour before taking the materials.The mechanism related indexes of antithyroid ism were detected by thyroid gland,half fixed and half frozen in liquid nitrogenPathological morphology of thyroid:the changes of pathological morphology of thyroid were detected by light and electron microscopy.Serum:T3,T4,FT3,FT4 reflect the hormone level of thyroid function;thyroid hormone synthesis indicators Tg,TPO,TSHR level;thyroid hormone transport indicators TBG,ALB;thyroid function regulation related indicators TRH,TSH level.Thyroid related protein and gene expression:TUNEL method was used to detect apoptosis cells in thyroid tissue.The effect of HZ combined with GC on the expression of Caspase-8 and Caspase-9 in thyroid gland of rats was studied by protein immunob lotting.The effect of HYD and its decoctions on the levels of Fas mRNA and Bcl-2 mRNA in thyroid tissue of rats with goiter was studied by real-time fluorescence quantitative detection(qPCR).Results:1 The effect of HZ and GC in HYD on the dissolution content of the main active componentsThe HPLC method is simple,sensitive,accurate and reproducible.It is suitable for the study of the compatibility of HZ and GC and the change of the dissolution content of the active ingredients after compatibility in HYD.The results showed that:the dissolution content of LG and LA in each group wasHYD-H>HYD>GC>GH;the dissolution content of PN,HD and FA in each group was HYD-H>HYD-GH>HYD-G>HYD.2 The effect of the antagonistic medicinal combination in HYD on the HPT axis in the rat model of goiterCompared the thyroid coefficient of each group,observed the morphological changes of the thyroid tissue under the light microscope,and counted the number of nuclei and the area of follicles in the sections.It was found that the improvement of the pathological morphology of the thyroid tissue among the groups:HYD>HYD-GH>HYD-H>HYD-G.Detection of T3,T4,FT3 and FT4 of thyroid hormone,the results showed that HYD had a significant effect on the level of thyroid hormone in the rats with goiter,the callback conditions from high to low are:HYD,HYD-G,HYD-H,HYD-GH;The levels of Tg,TPO and TSHR in HYD and HYD-G were higher than those in model group.The levels of TBG and ALB in HYD and its disassembled groups were higher than those in model group The levels of TRH and TSH were decreased in each group,with statistical difference.3 The effect of the antagonistic medicinal combination in HYD on the apoptosis of rat model of goiter and its apoptotic pathwayCompared with the model group,HYD showed the characteristics of cell overall shrinkage,nuclear pyknosis or disintegration and dispersion in the cytoplasm,chromatin concentration and marginalization;rough endoplasmic reticulum decreased,the number of mitochondria decreased,the volume decreased,the number of lysosomes increased,a large number of typical autophagy vesicles and other apoptotic characteristics.HYD-G,HYD-H and HYD-GH had typical apoptosis characteristic changes,but the degree of apoptosis was lower than HYD.Compared with HYD,HYD-G had the strongest degree of apoptosis andHYD-GH had the weakest degree of apoptosis.TUNEL method was used to detect the apoptosis of thyroid cells and calculate the apoptosis index.It was found that compared with the model group,the number of apoptosis cells in HYD was significantly increased,while compared with the model group,there was no significant change in HYD-G and HYD-GH,and the number of apoptosis cells in HYD-H group was reduced obviously.Compared with blank group through Western blot,the level of Caspase-8 and Caspase-9 in the model group had no significant change.Compared with the model group,the level of Caspase-8 and Caspase-9 in the treated groups had no significant difference;the expression of Fas mRNA and Bcl-2 mRNA in the model group was detected by PCR,and the expression of Fas in the model group was no significant change in Fas content compared with the blank group;the model group was treated by HYD and its disassembled formula After that,there was no significant change in Fas content compared with the model group;there was no significant change in Bcl-2 level compared with the blank group;there was no significant difference in Bcl-2 gene level in the treated groups compared with the model group.Conclusion:1 When HZ is combined with GC alone,the dissolution of glycyrrhizin and glycyrrhizic acid in GC decreases.When they were used in HYD,the content of dissolution was almost the same as GC group.Compared with HYD,the decocting group without the combination of HZ and GC showed the results of increasing the content of glycyrrhizin,glycyrrhizic acid,forsythin,hesperidin and ferulic acid,which can provide reference for the clinical application of HYD and the compatibility application of the combination of HZ and GC.2 HYD can improve the thyroid hormone level and tissue morphology of rats with goiter.The improvement of thyroid histopathology was HYD>HYD-GH>HYD-H>HYD-G;the recovery of thyroid hormone was HYD>HYD-G>HYD-H>HYD-GH.In the aspect of improving goiter,the effect of a antagonistic medicinal combination in HYD is better than that subtract of those.The mechanism of improving goiter is related to the regulation of the synthesis and transport of thyroid hormone and the regulation of hypothalamus pituitary thyroid axis.3 It was found that HYD could promote the apoptosis ofthyroid cells in the goiter model rats,and the level of promoting apoptosis in the HYD was higher than that in the disassembled recipe group.It was found that HYD and its decoctions had no significant correlation with Caspase-8,Caspase-9 protein,Fas and Bcl-2,and there was no statistical difference.