| Objective:To explore pharmacological mechanisms of HYD acting on iodine-deficient goiter by combining microarray gene expression profiling,network analysis and experimental validations,and to provide evidence for potential roles of herb pair HZ and GC in HYD.Methods:Part 1 Gene expression profiling1 Goiter rat model construction and grouping For microarray analysis,20 male/female rats were divided into five groups with the equal number of 4: normal control group(con),goiter model group(mod),goiter rats treated with HYD formula(HYD-treatment group),goiter rats treated with HYD formula without HZ(Non-HZ group)and goiter rats treated with HYD formula without GC(Non-GC group).Goiter rat model caused by iodine deficiency wasconstructed by oral administration of propylthiouracil ata dosage of 0.01 g/(kg.day)for 14 days.Drug treatmentof goiter was delivered by oral administration for 28 days.The dosage selection for each treated groups were set at0.9 g/(kg.day)on the basis of clinical dosage for human.Rats in the control and goiter model groups were alladministrated by an equal volume of saline.The serum levels of T3,T4 and TSH in different groups were determined by enzyme-linked immunosorbent assay(ELISA).2 Microarray gene expression profiling By gene expression profile chip(ROA2.1)technology,testing thyroid tissue gene expression profiles,and differential expression data analysis.Part 2 Network analysis1 Candidate targets of HYD Chemical compounds containing in HYD were obtained from TCM Database@Taiwan.The putative targets of HYD's chemical compounds were predicted based on the following hypothesis: drugs with similar chemical structure usually bind functionally related targets and exert similar therapeutic effects.2 Chemical compounds of HYD Liquid chromatography triple quadrupole-tandemmass spectrometry(LC-QQQ-MS/MS)was performedto identify the chemical compounds of each herb containing in HYD.3 Known goiter-related genes Known goiter-related genes were obtained from the following two databases: Drug Bank and KEGG.4 Candidate targets of HYD acting on goiter To shed light on the pharmacological mechanisms of HYD acting on goiter,we constructed disease-relatedgenes-drug-regulating genes network based on the interactions among hub goiter deregulated genes,known goiter related genes,HYD regulating genes and putative targets of HYD.A total of hub nodes,the degree values of which are more than the two fold ofmedian degree of all nodesin the disease-related genes-drug-regu-lating genes network,were identified.Part 3 Experimental validation based on independent samples Goiter rat model construction and grouping For independent experimental validation,32 male/female rats were also divided into four groups with the equal number of 8:normal control group(con),goiter model group(mod),goiter rats treated with HYD formula(HYD-treatmentgroup)and goiter rats treated with HYD formula without HZ and GC(Non-HZ/GC group).Goiter rat model caused by iodine deficiency was constructed by oral administration of propylthiouracil ata dosage of 0.01 g/(kg.day)for 14 days.Drug treatmentof goiter was delivered by oral administration for 28 days.The dosage selection for each treated groups were set at 0.9 g/(kg.day)on the basis of clinical dosage for human.Rats in the control and goiter model groups were all administrated by an equal volume of saline.The serum levels of T3,T4 and TSH in different groups were determined by enzyme-linked immunosorbent assay(ELISA).After the tissue collection,the thyroid tissues were divided into tree categories.One was fixed in 4% paraformaldehyde and embedded in paraffin.Then,4um-thick tissue sections were prepared and stained with hematoxylin and eosin(H&E)for histological studies.The other part was immediately stored in liquid nitrogen at-80? for real-time PCR and western-blottinganalysis.Results:Part 1 Gene expression profiling1 Goiter rat model construction and grouping Compared to the normal rats,the serum levelsof T3 and T4 were both significantly decreased,whilethe serum levels of TSH were dramatically increasedin rats with oral administration of propylthiouracil for14 days,suggesting thesuccessful construction of goiter rat model caused byiodine deficiency.2 Microarray gene expression profiling Microarray gene expression profiling expressed gene(DEG)screening,there were 139 hub goiter deregulated genes in thyroidtissues of goiter rats compared to the normal rats.The DEG screening identified 426 HYD regulati-nggenes inthyroid tissues of goiter rats treated with HYD compared to the goiter model rats.Part 2 Network analysis1 Candidate targets of HYD There were 565 chemical compounds containing in HYD were obtained from TCM Database@Taiwan There were 426 HYD regulating genesand 142 putative targets of HYD.2 Chemical compounds of HYD There were 22 chemical compounds of each herbcontaining in HYD was performedto identify by LC-QQQ-MS/MS.3 Known goiter-related genes After deleting redundancy,there were 10 known goiter related genes collected in this study.4 Candidate targets of HYD acting on goiter According to the pathway enrichment analysis,the candidate targets of HYD acting on goiter were significantly associated with thyroid hormone synthesis,in which there were nine HYD candidate targets involved,includ-ing Adcy1,Adcy2,Creb1,Hspa5,Pdia4,Plcb1,Prkca,Prkcb and Tpo,implying that HYD might exert its therapeutic effectson iodine-deficient goiter via regulating thyroid hormonesynthesis.To further clarify pharmacological mechanisms of herb pair HZ and GC acting on goiter,we constructed goiter-related genes-HZ-GC-regulating genes networkbased on the interaction-s among 139 hub goiter deregulated genes,10 known goiter related genes,56HZ-GC-regulating genes and 28 putative targets of HZ and GC.According to the pathway enrichment analysis,the 181 nodes were most significantly associated with thyroid hormone synthesis.There are 8 genes,including Adcy2,Atp1a2,Gsr,Iyd,Pdia4,Plcb1,Tg and Tpo,involved into thyroid hormone synthesis.Part 3 Experimental validation based on independent samples In line with findings based on samples of microarray analysis,the reduced levels of T3 and T4 in sera of goiter rats could be efficiently increased by the administration of HYD,while the elevated serum of TSH in goiter rats were significantly decreased in HYD treatment group(P<0.01).The weight of thyroid tissues and the thyroid weight/body weight ratio(TBR)in different groups were also measured at a macroscopic level.There were obvious increases in the weight of thyroid tissues and TBR in goiter model group compared to the normal group(both P<0.001).After the treatment of HYD,the two parameters were both significantly lower than those in goiter model rats(P<0.05,P<0.01).The observations of histological analysis show that normal thyroid tissues were characterized of round or oval thyroid follicular cavity,cuboidal follicular epithelial cells and a small amount of blood capillaries around the follicle,which were changed into hyperplasia and hypertrophy of parenchyma,collapsed follicles,scanty colloid contained as well as serious mononuclear cell infiltration in thyroidtissues of goiter model group.Following HYD treatment,the number of follicular cells and height of epithelial cells were markedly increased,and the severity of mononuclear cell infiltration and the blood capillaries reduction were efficiently relieved.The real-time PCR analysis based on independent samples showed that the m RNA levels of Adcy1(P<0.05),Adcy2(P<0.05),Creb1(P<0.05),Hspa5,Pdia4,Plcb1(P<0.05),Prkca(P<0.05),Prkcb(P<0.05)and Tpo(P<0.05)expression in thyroid tissues of goiter rats were all lower than those in normal rats,while the administration of HYD could markedly enhance their expression(except for Hspa5 and Pdia4 genes,all the other genes had differences with statistical significance: all P<0.05),which were all consistent with the findings of Western blot analysis(except for Hspa5 and Pdia4 proteins,all the other proteins had differences with statistical significance: all P<0.05).The results of real-time PCR and western blot analyses indicated that both the m RNA and protein levels of Adcy2(P<0.05),Atp1a2(P<0.05),Gsr(P<0.01),Iyd(P<0.01),Pdia4,Plcb1(P<0.05),Tg(P<0.05)and Tpo(P<0.05)expression in thyroid tissues of goiter rats were all lower than those in normal rats,but could be effectively increased by HYD treatment.Notably,when the herb pair HZ-GC was deleted from HYD,its reversing effects on these genes were markedly decreased.Conclusion:This integrative study combining microarray gene expressi-on profiling,network analysis and experimental validations offers the convi-ncing evidence that HYD may alleviate iodine-deficient goiter via regulating thyroid hormone synthesis and explains the necessity of the herb pair HZ and GC in HYD.Our work provides a novel and powerful means to clarify themechanisms of action for multi-component drugs suchas herbal formulae in a holistic way,which may improve drug development and applications. |
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