| Purpose:By making EAT liver stagnation and spleen deficiency syndrome combination model and giving Ruanjian Xiaoying granule treatment,observe the general state、objective indicators and thyroid autoantibody changes of EAT rats with liver stagnation and spleen deficiency syndrome after the application of Ruanjian Xiaoying granule,to evaluate the curative effect,and to explore the pathogenesis essence of Hashimoto’s thyroiditis with liver stagnation and spleen deficiency syndrome;By observing the metabolomic changes of Ruanjian Xiaoying granule on rats with liver depression and spleen deficiency syndrome of EAT,find the characteristic metabolites and metabolic pathway of Hashimoto’s thyroiditis.Then analyze the change law of metabolites,and explore the therapeutic mechanism of Ruanjian Xiaoying granule.By observing the changes of intestinal flora of rats with EAT who have liver depression and spleen deficiency syndrome treated with Ruanjian Xiaoying granule,find the characteristics of Hashimoto thyroiditis flora.Analyze the unique flora and the changes of intestinal flora in each group,and discuss the therapeutic mechanism of Ruanjian Xiaoying Granule.Materials and methods:First of all,48 SPF grade SD female rats weighing 200±20g were selected.After adaptive feeding for 1 week,the rats were randomly divided into 12normal group and 36 model group.The rats in the normal group were not subjected to any factors.The other rats were fed with high iodine drinking water combined with subcutaneous injection of thyroglobulin to establish Hashimoto thyroiditis rat model,and the method of"chronic restraint stress+excessive fatigue+eating disorder"was used to create the model of liver depression and spleen deficiency.After 8 weeks,2 ml of whole blood of rats in each group was taken by retrobulbar venous plexus puncture.The serum thyroid function and the TGAb and TPOAb were detected by ELISA to evaluate whether success of Hashimoto thyroiditis model.Observe the general state of rats,including hair color,food intake,body weight,,fecal water content,open field experiment,compare the differences between the groups,and evaluate whether success of the model of liver depression and spleen deficiency.The unsuccessful model rats were excluded,and the remaining model rats were randomly divided into three groups:normal group（9）,model group（9）,Ruanjian Xiaoying group（9）and positive control group（9）.The dosage of rats was calculated according to the equivalent dose conversion method of body surface area between human and rats.Rats in normal group and model group were given 2ml distilled water by gavage twice a day for 12 weeks;The rats in the positive control group were given Jinshuibao aqueous solution by oral gavage at the dose of 0.45g/kg·D-1;The rats in Ruanjian Xiaoying granule group were given Ruanjian Xiaoying granule by oral gavage according to the crude drug dosage of 12.96g/kg·D-1 for 12weeks.After 12 weeks of treatment,the hair color,state,food intake,weight gain and fecal characteristics of rats were observed,and the serum and feces of rats were taken to detect relevant indexes.The levels of FT3,FT4,TSH,TGAb and TPOAb in serum of rats in each group were detected by ELISA;The serum of rats in each group was detected by LC-MS（liquid chromatography tandem mass spectrometry）.The original data were converted into mz XML format by proteowizard,and then the peak alignment,retention time correction and peak area extraction were carried out by XCMS program.Metabolite structure identification adopts the methods of accurate mass number matching（<25 ppm）and secondary spectrum matching to retrieve the self built database of the laboratory.After Pareto scaling preprocessing,multidimensional statistical analysis was carried out.The stability of the model was verified by partial least squares discriminant analysis（PLS-DA）and orthogonal partial least squares discriminant analysis（OPLS-DA）,the authenticity and reliability of the data were judged by hotellings T2 analysis,and the different metabolites and different metabolic pathways of each group were analyzed to speculate the therapeutic mechanism of Ruanjian Xiaoying granule;Fecal samples were detected by 16Sr DNA high-throughput sequencing technology.According to the characteristics of amplified 16S region,a small fragment library was constructed and sequenced by paired end based on the sequencing platform.Through reading splicing filtration,OTUs（operational taxonomic units）clustering,species annotation and abundance analysis,we can reveal the species composition of samples,excavate the differences between samples,screen the different flora,further explore the therapeutic mechanism of Ruanjian Xiaoying granule.Analyze the experimental data to provide new ideas and theoretical basis for the treatment of Hashimoto’s thyroiditis by soothing the liver and strengthening the spleen.Result:1.Model evaluation:compared with the normal group,there was no significant difference in FT3 and FT4,TSH decreased significantly,and the levels of serum thyroid antibodies TGAb and TPOAb increased significantly（P<0.01）;Compared with the normal group,food ration,weight growth rate,total distance of exercise and the number of perforations in the central area in the open field experiment of the model rats were significantly reduced,and the fecal water content was significantly increased（P<0.05）.No abnormality was found in the appearance and behavior of rats in the normal group.The model rats had the symptoms of liver depression and spleen deficiency,dark and rough hair color with no luster.At the early stage of modeling,model rats were irritable and had a strong response to the stimulation of bondage.they struggled and resisted.After the bondage was removed,they quickly avoided and the back hair stood up.At the later stage of modeling,the response to external stimuli was slow,the struggle and resistance were weakened,and the duration was significantly reduced activity,they piled up and arched back,reduced food intake,slow weight growth,and even occasional loose stools.From the changes of subjective and objective indexes,it shows that the rat model of eat liver depression and spleen deficiency is successful.2.Efficacy evaluation:after treatment,compared with the model group,there was no significant difference in FT3 and FT4 in the Ruanjian Xiaoying group,TSH increased,approached to the normal group,TGAb and TPOAb decreased significantly（P<0.01）,and the difference was statistically significant.At the same time,the hair color of the Ruanjian Xiaoying group was slightly shiny,the amount of activity increased,the eating was ok,and there was no loose stool.It shows that Ruanjian Xiaoying granule has a certain therapeutic effect.After treatment,compared with the model group,TSH in the positive control group rose and approached the normal group,TGAb and TPOAb decreased significantly（P<0.01）,the difference was statistically significant,the activity of the positive control group increased slightly,and the loose stools of rats improved.It shows that Jinshuibao capsule has a certain therapeutic effect.Compared with the positive control group,there was no significant（P>0.05）difference in TGAb and TPOAb between Ruanjian Xiaoying group and the positive control group,indicating that the curative effects of the two groups were equivalent.3.To explore the therapeutic mechanism from the perspective of metabolomics:The results of QC sample TIC show that the variation caused by instrument error is small in the whole process of this experiment.The principal component analysis（PCA）of the overall samples shows that the QC samples in the positive and negative ion mode are closely gathered together,indicating that the experiment of this project has good repeatability.Hotellings T2analysis found that most of the observed samples were within the 99%confidence interval,indicating that the sample data were reliable.Compared with the normal group,19 potential biomarkers such as mannose,taurine and L-carnitine were detected in the model group.Mainly involving three metabolic pathways:cancer central carbon metabolism,ABC transporter and protein metabolism and absorption.Compared with the model group,Ruanjian Xiaoying granule can regulate 17 metabolites such as mannose,taurine and L-carnitine,and has a significant callback effect on the metabolic pathways such as central carbon metabolism,ABC transporter and protein metabolism and absorption of cancer.Compared with the model group,the positive control group could regulate 16 metabolites such as anthranilic acid,acetylcarnitine and sphingomyelin.It plays a significant role in three metabolic pathways:protein metabolism and absorption,ABC transporter and aminoacyl t RNA anabolism.Both Ruanjian Xiaoying granule group and positive control group can regulate ABC transporter pathway and the contents of mannose and phenylalanine,but for other metabolic pathways and products,they have no common mechanism,and their mechanism needs to be further studied.4.To explore the therapeutic mechanism from the perspective of intestinal flora chang e:Compared with the normal group,the intestinal flora in the model group increased in Firmicutesc,decreased in Proteobacteria,increased in Clostridia,decreased in Gram maproteobacteriabing,increased in Clostridiales,decreased in Pseudomonadales,decreased in Moraxellaceae,increased in Ruminococcaceae,decreased in the number of Escheric hia and Myroides,increased in Prevotella,decreased in coli species.Ruanjian Xiaoyin g granule can regulate the number of Firmicutesc,Proteobacteria,Clostridia,Grammapr oteobacteriabing,Clostridiales,Pseudomonadales,Moraxellaceae,Ruminococcaceae,Escheric hia,Myroides,Prevotella,coli species close to the normal group.It shows that Ruanji an Xiaoying granule applied according to the method of soothing the liver and strengt hening the spleen can regulate the intestinal flora.Jinshuibao has no regulatory effect on the above flora.It is considered to treat Hashimoto’s thyroiditis with liver depressi on and spleen deficiency through other mechanisms,which is also in line with the the ory of"same disease and different treatment"of traditional Chinese medicine.Conclusion:1.High iodine water combined with immune-mediated,chronic restraint,excessive fatigue and eating disorder can successfully establish eat rat model of liver depression and spleen deficiency syndrome.2.The thyroid autoantibody of rats with EAT liver depression and spleen deficiency syndrome increased significantly.Ruanjian Xiaoying granule can reduce the thyroid antibody and play a therapeutic role in the disease.Jinshuibao can also reduce thyroid antibodies,and the curative effects of the two are equivalent.3.Ruanjian Xiaoying granule may play a therapeutic role in Hashimoto’s thyroiditis with liver depression and spleen deficiency syndrome by regulating 17 metabolites such as mannose,taurine and L-carnitine and metabolic pathways such as central carbon metabolism,ABC transporter,protein metabolism and absorption of cancer.Jinshuibao can also regulate the ABC transporter pathway and the contents of mannose and phenylalanine,but the other metabolites and pathways are different.4.Ruanjian Xiaoying granule may regulate the imbalance of intestinal flora and play a therapeutic role in Hashimoto’s thyroiditis by regulating the number of Firmicutesc,Proteobacteria,Clostridia,Grammaproteobacteriabing,Clostridiales,Pseudomonadales,Moraxellaceae,Ruminococcaceae,Escherichia,Myroides,Prevotella,coli species and so on.No regulation effect of Jinshuibao on all of the above flora was found based on the current research. |
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