Exploring the effect and mechanism of Haizao Yuhu decoction containing three variants of glycyrrhiza on goiter using an integrated strategy of network pharmacology and RNA sequencing.

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.

Haizao Yuhu decoctions including three species of glycyrrhiza protected against propylthiouracil-induced goiter with hypothyroidism in rats via the AMPK/mTOR pathway.

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.

Association between miR-27a rs895819 polymorphism and breast cancer susceptibility: Evidence based on 6118 cases and 7042 controls.

BACKGROUND: Polymorphism in miR-27a rs895819 has been associated with breast cancer (BC) risk, but studies have reported inconsistent results. This meta-analysis investigated the possible association between miR-27a rs895819 polymorphism and BC risk. METHODS: PubMed, EMBASE, Google Scholar, and the Chinese National Knowledge Infrastructure (CNKI) databases were systematically searched to identify relevant studies in English and Chinese. Meta-analyses were performed to examine the association between miR-27a rs895819 and BC susceptibility. RESULTS: A total of 16 case-control studies involving 6118 cases and 7042 controls were included. Analysis using five genetic models suggested no significant association between miR-27a rs895819 polymorphism and BC risk in the total population, or specifically in Asian or Chinese subpopulations. In the Caucasian subpopulation, however, the G-allele and AG genotype at rs895819 were significantly associated with decreased BC risk according to the allelic model (OR 0.90, 95% CI 0.84-0.97, P = .004) and heterozygous model (OR 0.89, 95% CI 0.81-089, P = .02), while the wild-type AA genotype was significantly associated with increased BC risk according to the dominant model (OR 1.13, 95% CI 1.03-1.24, P = .007). CONCLUSION: These results indicate that among Caucasians, the wild-type AA genotype at rs895819 may confer increased susceptibility to BC, while the G-allele and AG genotype may be protective factors. These conclusions should be verified in large, well-designed studies.