Abstract Herbs with a “hot” properties are frequently used to treat cold symptoms in TCM. However, the underlying mechanisms of the herbs with “hot” properties on hypothyroidism have not been investigated. This study aimed to explore four typical “hot” and “cold” property herb on hypothyroidism. Firstly, the difference efficacy between the four typical “hot” property herbs and the four typical “cold” property herbs was assessed by physical signs, thyroid function, and the metabolic profile using multivariate statistical analysis. The influence of the four typical “hot” property herbs on hypothyroidism was validated pathologically. The impact mechanism of the four typical “hot” property herbs on hypothyroidism was investigated through a metabolomics method combined with network analysis. Na^+/K^+-ATP, ACC1 enzyme, UCP-1, and the PI3K-Akt pathway were used to confirm the metabolite pathways and target-associated metabolites. The results showed that the four typical “hot” property herbs could significantly improve physical signs, thyroid function, and the metabolic profile in hypothyroidism rats, the four typical “cold” property herbs did not show any benefit. Moreover, the four typical “hot” property herbs could improve lipid metabolism, energy metabolism, and thyroid hormone levels by the PI3K-Akt signaling pathway, Ca^2+- AMPK signaling pathways, purine metabolism, and tryptophan metabolism. Additionally, the levels of UCP-1, Na+/K + -ATP enzyme, and ACC1 were ameliorated by the four typical “hot” property herbs in hypothyroidism rats. Therefore, a metabolomics strategy combined with network analysis was successfully performed and interpreted the mechanism of the four typical “hot” property herbs on hypothyroidism based on the theory of “cold and hot” properties of TCM well. Keywords: Typical “hot” property herbs, metabolomics, hypothyroidism, network analysis, metabolic mechanism, cold-hot medicine properties Introduction Hypothyroidism is on of the most common thyroid diseases affecting people worldwide, particularly during pregnancy and childhood ([34]Taylor et al., 2018). Obesity, cold limbs, depression, hyperlipidemia, negative emotions, reduced lipolysis, and gluconeogenesis are the most essential clinical manifestations ([35]Meier and Kaplan, 2002). Currently, thyroid hormone replacement therapy is the main treatment for hypothyroidism ([36]Clarke and Kabadi, 2004). However, during hypothyroidism therapy, the level of thyroid hormones is difficult to regulate, and over-medication was prevalent, increasing the risk of cardiovascular disease ([37]Flynn et al., 2010), osteoporosis ([38]La Vignera et al., 2008), and subclinical liver damage ([39]Beckett et al., 1985). Therefore, there is an urgent need to seek new strategies for the management of hypothyroidism. According to the traditional Chinese medicine (TCM) theory, hypothyroidism is classified as a “cold” syndrome, and “treating cold syndrome with hot herbs and treating heat syndrome with cold herbs” is a fundamental medication principle of Chinese medicine ([40]Xiao et al., 2017). The “cold and hot” properties of TCM refer to the properties that can cause a certain type of reaction in the body, and are used to treat diseases. However, pharmacology is modern science that studies the regularity of interaction between drugs and the body and the mechanism of the drug effect. Although we have reports suggesting that the “hot” drugs have a significant improvement influencing hypothyroidism ([41]Cheng et al., 2016), there are no reports concerning the typical “hot and cold” property herb influencing hypothyroidism. Therefore, it is necessary to further study the effects of the four typical “hot” property herbs and four typical “cold” property herbs on hypothyroidism to provide a basis for treating hypothyroidism, as well as reveal the scientific essence for the medication principle. TCM has been applied in preventing and treating the diseased for thousands of years ([42]Feng et al., 2006). Complex-component, multi-target, and multi-path are the typical characteristics of Chinese herbs, which can be used to holistically treat diseases ([43]Pan et al., 2020). One of the major challenges facing TCM is explaining the mechanisms underlying the efficacy of medicines used in TCM ([44]Peng et al., 2018). Therefore, a novel research method consistent with TCM characteristics is crucial. Metabolomics is a high-throughput qualitative and quantitative analysis approach focusing on all small endogenous molecule metabolites in the body to reveal the pathological and physiological status at the overall level ([45]Carneiro et al., 2019). Metabolomics with the systematic biological characteristics is consistent with the theory of TCM and provides a powerful tool for the research on TCM ([46]Cheng et al., 2018). Recently, the network pharmacology approach offered a new understanding of Chinese medicine research from a system perspective and at the molecular level by predicting the interactions between multiple targets of compounds in herbs and/or multiple genes related to diseases ([47]Fang et al., 2017). The metabolic process of the body is mainly catalyzed by enzymes, which are inseparable (metabolites are regulated by proteins, and the activity of proteins is also changed by metabolites, such as described in KEGG). Metabolomics can be used to identify specific molecular markers in certain physiological and pathological conditions ([48]Barnes et al., 2016). Network pharmacology emphasizes the regulation of multiple targets by signal pathways to improve the therapeutic effect of drugs and reduce the toxicity and side effects ([49]Yuan et al., 2017), both of them provide insights into the disease at the molecular and systemic levels. These approaches have been widely applied in the assessment of the therapeutic effects and mechanisms of TCM in recent years ([50]Buriani et al., 2012). Therefore, an integrated metabolomic strategy combined with network analysis methods was applied to reveal the effects of four typical “hot” property herbs and four typical “cold” property herbs on hypothyroidism. According to the records in ancient books of past dynasties, pharmacopoeia of the People’s Republic of China and modern research in the “cold/hot” property, the four typical “hot” property herbs ([51]Jia et al., 2017; [52]Committee, 2020), including Aconitum carmichaeli Debeaux (FZ), Zingiber officinale Roscoe (GJ), Cinnamomum cassia (L.) J. Presl (RG), and Euodia ruticarpa (A. Juss.) Benth. (WZY), were selected. Meanwhile, the four typical “cold” property herbs ([53]Wang et al., 2016; [54]Committee, 2020), including Scutellaria baicalensis Georgi (HQ), Coptis chinensis Franch. (HL), Gardenia jasminoides J. Ellis (ZZ), and Rheum palmatum L. (DH) were also selected as a contrasting experiment to explore the medication principles of the “cold/hot” property. Finally, the results of biochemistry, metabolomics, and the network analysis were integrated to clarify the effect of the four typical “hot” property herbs on hypothyroidism. Materials and methods Chemicals and reagents Acetonitrile (HPLC grade, United States) and formic acid (LC-MS grade, United States) were purchased from Fisher Chemical. The four typical “cold” property herbs: Scutellaria baicalensis Georgi (HQ), Coptis chinensis Franch. (HL), Gardenia jasminoides J. Ellis (ZZ), and Rheum palmatum L. (DH), and the four typical “hot” property herbs: Aconitum carmichaeli Debeaux (FZ), Zingiber officinale Roscoe (GJ), Cinnamomum cassia (L.) J. Presl (RG), and Euodia ruticarpa (A. Juss.) Benth. (WZY), which were purchased from Beijing Tongrentang Co., Ltd (Beijing, China). The herbs were authenticated by Professor Zhenyue Wang (Heilongjiang University of Chinese Medicine, Harbin, China) and all voucher specimens were preserved at Heilongjiang University of Chinese Medicine. 6-propyl-2-thiouracil (PTU) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany, batch Number: BCBR87087), and L-thyroxine (L-T4) was purchased from Aladdin Biochemical Technology Co., Ltd., (Shanghai, China, batch Number: H2014187). The ELISA kits for rat triiodothyronine (T3, batch Number: C0384010396), thyroxine (T4, batch umber: C0346030349), and thyroid-stimulating hormone (TSH, batch Number: C0373050316) were purchased from Wuhan Huamei Biotechnology Co., Ltd., (Wuhan, China). The ELISA kits for the Na+/K + -ATP enzyme (batch Number: 202109) and Acetyl CoA carboxylase 1 (ACC1, batch Number: 202109) were purchased from Jiangsu Meimian Industry Co., Ltd. (Jiangsu, China). Herb extraction The four typical “hot” property herbs (FZ, GJ, RG, and WZY) and the four typical “cold” property herbs (HQ, HL, ZZ, and DH) were extracted by decocting with water three times [mass (g): volume (v) = 10:1, 8:1, and 8:1; 0.5 h each]. Then, the combined decoctions were concentrated and dried in a vacuum to obtain a FZ/GJ/RG/WZY/HQ/HL/ZZ/DH crude extract. Animals Sprague–Dawley rats (200 ± 10 g, male) were obtained from Liaoning Changsheng Biotechnology Co., Ltd., [Liaoning, China; Certificate No. SCXK (Liao) 2020–0001]. All the rats were housed under a 12 h/12 h light/dark cycle, at a temperature of 20 ± 3°C, with 60% ± 10% relative humidity. They had free access to standard food and sterile-filtered water. All the animal experiments were approved by the Experimental Animal Ethics Committee of the Heilongjiang University of Chinese Medicine and performed in accordance with relevant guidelines. After adaptive breeding for 1 week, all rats were randomly divided into 11 groups: control group (control, n = 7), hypothyroidism model group (Hypo, n = 8), positive drug group (Hypo + T4, n = 8), and treatment group by the four typical “hot” property herbs, including Hypo + FZ (n = 8), Hypo + GJ (n = 8), Hypo + RG (n = 8), and Hypo + WZY (n = 8), as well as the contrasting experimental group by the four typical “cold” property herbs, including Hypo + HQ (n = 8), Hypo + HL (n = 8), Hypo + ZZ (n = 8), and Hypo + DH (n = 8). With the exception of the rats in the control group, the hypothyroidism model was established in all rats by intraperitoneal injection PTU (10 mg/kg/d) for 28 days consecutively ([55]Baltaci and Mogulkoc, 2018). The positive drug group was intragastrically administered T4 (0.3 mg/kg/d) from day 14 to the end. The treatment group and contrasting experimental group FZ (7 g/kg/d), GJ (4.7 g/kg/d), RG (2.4 g/kg/d), WZY (2.4 g/kg/d), HQ (4.7 g/kg/d), HL (2.4 g/kg/d), ZZ (4.7 g/kg/d), DH (7 g/kg/d) were respectively continuous intra-gastrically administered for 28 days, respectively (The dose was calculated according to the Chinese Pharmacopoeia). Sample collection and preparation After the last administration, the rats were placed alone in metabolic cages and the collection tube in a box with ice was kept to reduce bacterial contamination, for 12 h urine collection, which was centrifuged for 10 min, at 12,000 rpm. The supernatant was stored at −80°C until analysis. After euthanasia with pentobarbital sodium by intraperitoneal injection, the serum was collected by centrifugation at 3,000 rpm for 10 min and stored at −80°C until further analyses. The live, brown adipose tissue (BAT), the thyroid gland were obtained and fixed in 4% paraformaldehyde for histological examinations. Moreover, approximately 0.1 g of the liver tissue was homogenized at a 0.9% saline solution, which was used to detect the Na^+/K^+-ATPase and ACC l activities and frozen at −80°C before use. The urine sample was mixed with four-fold cold acetonitrile, vortexed for 2 min, and centrifugation at 4°C at 13,000 rpm for 10 min. After high-speed centrifugation, the prepared supernatant was transferred into a fresh 2 ml LC-MS glass vial. In addition, the quality control (QC) sample was prepared by mixing an equal amount of every sample from an identical experiment group. Finally, 2 μL of the supernatant for each sample was injected into the UPLC-Q-TOF/MS system for metabolomic analysis. UPLC-Q-TOF/MS analysis An ACQUITY UPLC system (Waters, Milford, United States) in tandem with a Q-TOF synapt G2-SI mass spectrometer (Waters, Milford, United States) was acquired for LC-MS/MS analyses with an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm, Waters, Milford, United States). The chromatography separation was performed at an ambient temperature of 40°C. The mobile phase included acetonitrile with 0.1 % formic acid (A), and deionized water with 0.1% formic acid (B), and the gradient of eluent was used: 0–15 min, 5%–68 % (A); 15–18 min, 68 %–95 % (A); 18–19 min, 95 %–5 % (A); and 19–20 min, 5 % (A). The flow rate was set at 0.3 ml/min, and the injection volume was 2 µL. The key parameters of Q-TOF/MS were optimized as follows: scan type: positive and negative, acquire mass over the range 100–1300 Da, scan time: 0.25s, collision energy: 25–35 V, cone voltage: 40 V. the electrospray capillary voltage was 3.2 kV in the positive and negative ionization modes, the ion source temperature was 320°C, and the auxiliary heater temperature was 350°C. Data processing and metabolism profile analysis After the UPLC-Q-TOF/MS analysis, the raw data were imported into the Progenesis QI software (version 2.0, Nonlinear Dynamics, Waters, United States) for peak detection, alignment, deconvolution, and normalization. The resultant data matrices were linked to EZ info (version 2.0, Waters Corporation) for the principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The list of ions that contributed to the grouping was obtained from loading the S-plot and variable importance plot (VIP). The biomarkers were identified by MS/MS fragment ion and accurate mass by searching reliable online biochemical databases such as the Human metabolome database (HMDB) ([56]http://www.hmdb.ca/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ([57]https://www.kegg.jp/kegg/) in the Progenesis QI software ([58]Ren et al., 2016). Hematological analyses, biochemical parameters, and real-time -PCR assay On day 28, the thyroid tissue was soaked in 4% paraformaldehyde, embedded in paraffin, cut into 5-mm sections, and stained with Hematoxylin and Eosin (H&E). The sections were visualized using light microscopy (Nikon, Tokyo, Japan), and digital images were captured and analyzed. A commercial ELISA kit (Wuhan Huamei Biotechnology Co., Ltd., Wuhan, China) was used to analyze the levels of T3, T4, and TSH in the serum in accordance with the manufacturer’s protocol. ACC1 and Na^+/K^+-ATP enzymes in the liver were detected by a commercial ELISA kit (Meimian, Jiangsu, China) in accordance with the manufacturer’s protocols. The rectal temperature of the rats was measured by an electronic standard rectal thermometer and the bodyweight of the rats was weighed by electronic scales at days 1, 7, 14, and 28. The extracted total RNA from BAT, the primers designed, and the cDNA synthesis were references reported in the literature, which was