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BACKGROUND: "Gansui Banxia decoction" (GBD) is a classical traditional Chinese medicine formula for treating abnormal accumulation of fluid, such as malignant ascites (MA). Although GBD has shown definite water-expelling effects, its exact underlying mechanism has not been elucidated. PURPOSE: This study aimed to investigate the drug effects of GBD on MA rats and its underlying mechanisms. METHODS: The main chemical composition was determined by ultra-high performance liquid chromatography. The drug effects of GBD was evaluated in the established cancer cell-induced MA rat model. The symptoms were analyzed, and biological samples were collected for detecting immune and inflammation-related indicators by enzyme-linked immunosorbent assays, western blot, and flow cytometry. RESULTS: GBD increased urine discharge, decreased ascites production, and alleviated cachexia. After GBD treatment, the expression of TLR4, MyD88, and NF-кB and the release of pro-inflammatory cytokines such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α were reduced. In addition, GBD increased G1 phase arrest and inhibit excessive proliferation of cells in bone marrow while alleviating G1 phase arrest and increasing proliferation of cells in the thymus. Correspondingly, the development and maturation of T cells also changed. GBD increased the proportion of mature T-cells (CD4+CD8- and CD4-CD8+ single-positive (SP) T-cells), and decrease the proportion of immature cells (CD4+CD8+ double-positive (DP) T-cells and CD4-CD8- double-negative (DN) T-cells) in the blood or tumor microenvironment (TME, the ascites microenvironment). Finally, we further analysis of immune cell subsets, GBD decreased the proportion of immunosuppressive T-cells in the blood (CD4+CD25+Foxp3+T-cells) and TME (CD8+CD25+Foxp3+T-cells), and increased the proportion of anti-tumor immune cells (CD8+CD28+T-cells and NK cells) in the TME. CONCLUSION: These findings indicated that the drug effects of GBD were attributed to regulating the immune-inflammatory homeostasis, thereby mitigating the destruction of cancer cells and reducing the generation of ascites, which provided theoretical support for the clinical rational application and extended the scientific connotation of "water-expelling" of GBD.

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ETHNOPHARMACOLOGICAL RELEVANCE: Haizao Yuhu decoction (HYD) is a classic Chinese herbal formula described in the surgical monographs of the Ming Dynasty "Waikezhengzong." It has been widely used to treat goiter for approximately 500 years and found to be particularly effective. HYD contains glycyrrhiza and sargassum. This pair of herbs belongs to "18 incompatible medicaments" of traditional Chinese medicine theory. Although these two herbs are opposite, our preliminary study proved that they have superior effect when added into HYD at 2 times the dose of Chinese Pharmacopoeia. However, the species of glycyrrhiza in HYD that are the most effective have not been recorded in ancient Chinese medical texts. According to the Chinese Pharmacopoeia, glycyrrhiza is divided into the following three species: Glycyrrhiza uralensis Fish., G. glabra L., and G. inflata Bat. The effect of HYD containing different species of glycyrrhiza and their mechanisms remain to be further explored. AIM OF THE STUDY: To investigate the effect of HYD containing three species of glycyrrhiza on goiter, and to elucidate the molecular mechanism using network pharmacology combined with RNA sequencing (RNA-seq). MATERIALS AND METHODS: A rat model of goiter was established by 14 days of intragastric gavage of propylthiouracil (PTU), and the rats were treated for 4 weeks with HYD containing three different species of glycyrrhiza. The body weight and rectal temperature of rats were tested weekly. At the end of the experiment, the serum and thyroid tissues of rats were collected. The effect of the three HYDs was assessed based on general observations (including body weight, rectal temperature, and living status of rats), absolute/relative thyroid weight, thyroid function (including triiodothyronine, thyroxine, free triiodothyronine, free thyroxine, and thyroid-stimulating hormone levels), and thyroid tissue pathology. Next, we explored their pharmacological mechanisms using network pharmacology combined with RNA-seq and validated key targets using real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blotting (WB), and immunofluorescence (IF) assays. RESULTS: The three HYDs reduced the absolute/relative weights of thyroid tissues and improved the pathological structure, thyroid function, and general findings of rats with goiter. Overall, the effect of HYD-G. uralensis Fish. (HYD-U) was better. Results from network pharmacology and RNA-seq jointly suggested that both the pathogenesis of goiter and the mechanism of action of HYD for goiter were related to the phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) pathway. We validated the key targets in the pathway, namely, vascular endothelial growth factor (VEGF) A, VEGF receptor 2, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and its encoded protein PI3K (p85), AKT serine/threonine kinase 1 (AKT1), phospho-AKT and cyclin D1 using RT-qPCR, WB, and IF assays. The PI3K-Akt pathway was hyperactivated in rats with PTU-induced goiter, whereas the three HYDs could inhibit the pathway. CONCLUSION: This study confirmed the definite effect of the three HYDs in the treatment of goiter, and HYD-U was found to be more effective. The three HYDs inhibited angiogenesis and cell proliferation in goiter tissue by inhibiting the PI3K-Akt signaling pathway.

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ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza and sargassum are among the 18 incompatible medicaments according to traditional Chinese medicine (TCM) theory. Although it contains glycyrrhiza and sargassum, Haizao Yuhu decoction (HYD) is a classic prescription widely used as TCM to treat goiter. According to the Chinese Pharmacopoeia, glycyrrhiza is divided into three varieties: Glycyrrhiza uralensis Fish., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat. Whether the three varieties of glycyrrhiza have different efficacy or toxicity when applied in the HYD is unknown. AIM OF THE STUDY: To explore whether the HYDs comprising three varieties of glycyrrhiza have different efficacy or toxicity when used to treat goiter in rats and the underlying mechanisms of these HYDs. MATERIALS AND METHODS: For two weeks, the goiter model was replicated by intragastric propylthiouracil (PTU) administration. Samples were divided into the control group, model group, euthyrox group, HYD with glycyrrhiza uralensis (HYD-U) group, HYD with glycyrrhiza glabra (HYD-G) group, and HYD with glycyrrhiza inflata (HYD-I) group. After four weeks of treatment, body weight, rectal temperature, thyroid/liver/kidney coefficient, thyroid/liver/kidney function, thyroid/liver/kidney histomorphology, and thyroid ultrastructure were evaluated. Then, real-time quantitative reverse-transcription polymerase chain reaction (RTqPCR), Western blot, and immunofluorescence analyses were performed to detect genes and proteins affecting autophagy and apoptosis in thyroid cells in the AMP-activated Protein Kinases (AMPK)/Mammalian target of rapamycin (mTOR) pathway. RESULTS: All three HYDs increased thyroid hormones (THs) levels, relieved thyroid pathological tissue and ultrastructure, and activated vital proteins and genes in the AMPK/mTOR pathway. Comparisons among the efficacy of the three HYDs indicated that HYD-U restored the THs most effectively; however, no difference in the anti-goiter effect was observed. Moreover, the three HYDs resulted in no toxicity and promoted the recovery of impaired liver and kidney function caused by PTU. Comparisons among the recovery effects of the three HYDs on the liver and kidney were the same. CONCLUSION: Our experiments demonstrated that the three HYDs had outstanding anti-goiter effects and protected liver and kidney function. Their anti-goiter effects were attributed to AMPK/mTOR pathway-induced autophagy and apoptosis. HYD-U resulted in the best THs recovery. It was further indicated that in our present study, glycyrrhiza and sargassum were compatible in the three HYDs, thereby suggesting their safety of compounding in HYD and providing a basis for the research of the 18 incompatible medicaments.

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In traditional Chinese medicine, Glycyrrhiza and Sargassum are one pair of the "18 incompatible medicaments," which in theory cannot be used together. However, since ancient times, many reports have described using compounds containing both Glycyrrhiza and Sargassum to treat diseases. Haizao Yuhu Decoction (HYD), which contains both ingredients, is mainly used to treat goiter. Chinese Pharmacopoeia officially recorded three varieties of Glycyrrhiza: Glycyrrhiza uralensis, Glycyrrhiza inflata, and Glycyrrhiza glabra. These three varieties have certain differences in chemical composition and pharmacological effects. The purpose of the present study was to investigate whether the HYD containing different varieties of Glycyrrhiza and Sargassum had different therapeutic effects in rats with goiter and to elucidate the underlying mechanism of any difference. In this study, propylthiouracil (PTU) was used to replicate the goiter model, then HYDs containing different varieties of Glycyrrhiza were used for treatment for four weeks, and then the relevant indicators were tested. The results demonstrated that HYD had antigoiter effects, alleviated the pathological changes in the thyroid tissue, and restored the abnormal serum levels of hormones related to thyroid function induced by PTU. HYD containing Glycyrrhiza uralensis had the best therapeutic effect in rats with PTU-induced goiter. The antigoiter effect of HYD may function through the hypothalamic-pituitary-thyroid (HPT) axis, inhibit the expression of the Tg and NIS genes, and regulate the synthesis of thyroid hormones, thereby reducing the excessive stimulation of TSH in thyroid cells. In addition, HYD also prevented goiter by promoting thyroid cell apoptosis and inhibiting the ERK/RSK1 pathway of cell proliferation. In conclusion, three types of HYD had different therapeutic effects in rats with goiter, which might be caused by the compatibility of different varieties of Glycyrrhiza and Sargassum.

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INTRODUCTION: The fundamental theory of traditional Chinese medicine (TCM) implies that when different diseases have the same pathogen, the syndromes of these individual diseases will be the same. "Treating different diseases with the same method" is a TCM principle suggesting that when different diseases have similar pathological changes during different stages of their development, the same method of treatment can be applied. Our study aims to analyze the concept "treating different diseases with the same method" from a molecular perspective, in order to clarify its biological basis and to objectively standardize future TCM syndrome research. OBJECTIVE: The TCM syndromes Qi deficiency and blood stasis have similar pathogenesis in relation to coronary heart disease (CHD) and stroke. We aim to use big data technology and complex network theory to mine the genes specifically relevant to these TCM syndromes. This study aims to explore the correlation between the biological indicators of CHD and stroke from a scientific perspective. METHODS: Mining the relevant neuroendocrine-immune (NEI) genes by means of gene entity recognition, complex network construction, network integration, and decomposition to categorize relevant syndrome terms and establish a digital dictionary of gene specifically related to individual diseases. We analyzed the biological basis of "treating different diseases with the same method" from a molecular level using the TCMIP v2.0 platform in order to categorize the TCM syndromes most relevant to CHD and stroke. RESULTS: We found 46 genes were involved in the TCM syndromes of Qi deficiency and blood stasis of CHD and stroke. The same genes and their molecular mechanism also appeared to be in close relation to inflammatory response, apoptosis, and proliferation. CONCLUSION: By using information extraction and complex network technology, we discovered the biological indicators of the TCM syndromes Qi deficiency and blood stasis of CHD and stroke. In the era of big data, our results can provide a new method for the researchers of TCM syndrome differentiation, as well as an effective and specific methodology for standardization of TCM.

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Sargassum species combined with Glycyrrhiza uralensis is a famous herbal pair in traditional Chinese medicine, as one of the so-called "eighteen antagonistic medicaments." In the Chinese Pharmacopoeia, two different species of Sargassum, Sargassum pallidum and Sargassum fusiforme, are recorded but they are not clearly differentiated in clinical use. In this study, we aimed to determine whether the two species of Sargassum could result in different effects when combined with G. uralensis in Haizao Yuhu Decoction (HYD), which is used for treating thyroid-related diseases, especially goiter. HYD containing S. pallidum or S. fusiforme was administered to rats with propylthiouracil-induced goiter. After 4 weeks, pathological changes in the thyroid tissue and the relative thyroid weight indicated that HYD containing S. pallidum or S. fusiforme protected thyroid tissues from propylthiouracil damage. Neither species increased the propylthiouracil-induced decrease in serum levels of thyroid hormones. However, there were some differences in their actions, and only HYD containing S. fusiforme abated the propylthiouracil-induced elevation of serum thyroid-stimulating hormone levels and activated thyroglobulin mRNA expression.

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[This corrects the article DOI: 10.1371/journal.pone.0174095.].

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BACKGROUND: Haizao Yuhu Decoction has been widely used to treat thyroid-related diseases especially goiter with few side effects in traditional Chinese medicine (TCM), including herb pair Sargassum (HZ) and Glycyrrhizae Radix et Rhizoma (GC), as one of "eighteen antagonistic medicaments." The two different species of Sargassum, Sargassum fusiforme (Sf) and Sargassum pallidum (Sp), are not clearly differentiated in clinical use, so that herb pair Sf-GC and Sp-GC could show different effect and toxicity. METHODS: We investigated the antigoitrous effect and toxicity and clarified the potential underlying mechanism of the two different species of Sargassum in HYD (HYDf and HYDp) in PTU-reduced goiter rats. RESULTS: The results demonstrated that both HYDf and HYDp could exhibit antigoitrous effect through alterations in hypothalamus-pituitary-thyroid (HPT) axis and inhibition of the TPO gene expression; there is no difference in the antigoitrous effects between the two different species of Sargassum application in HYD. CONCLUSION: This study evaluated the safety and efficacy of herb pair HZ-GC applied in HYD in goiter rats at molecular, cellular, and whole level and compared the two species of Sargassum further. We provide a reliable way to clarify the possible mechanism of the antagonistic medicament herb pair HZ-GC for its application.

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OBJECTIVES: To evaluate whether urinary iodine concentration (UIC) can predict goiter among school-age children, and to assess the association between UIC and goiter prevalence. METHODS: We searched the MEDLINE, EMBASE, Cochrane Library (Cochrane Database of Systematic Reviews), Web of Science, CNKI, VIP, and Wan Fang databases for relevant reports in both English and Chinese up to August 25, 2016. The mean differences (MD) and 95% confidence intervals (CI) were calculated for the UIC and goiter prevalence assessments. Pooled odds ratios and 95% CIs were used to compare the prevalences of goiter in the different UIC groups. RESULTS: We identified 11 case-control studies, and found that children with goiter had lower UIC values, compared to children without goiter (MD: -1.82, 95% CI: -3.24, -0.40, p < 0.05). An increased risk of goiter was associated with UIC values of < 20 µg/L or > 200 µg/L. CONCLUSION: The results of our meta-analysis suggest that lower UIC values were associated with an increased risk of goiter, and that iodine deficiency may lead to an increased risk of goiter. Furthermore, we observed U-shaped relationships between UIC and the prevalence of goiter, which suggests that both severe iodine deficiency and excessive iodine intake may lead to increased risks of goiter.

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