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. 2025 Feb 14;17(3):488–499. doi: 10.1016/j.chmed.2025.02.003

Anthraquinones of Cassiae Semen alleviate lipid accumulation in obesity by regulating brown adipose tissue and liver function

Yijie Li a, Ruiyu Wu b, Xin Li b, Jianan Li a, Yinhao Zhang a, Yanbo Huang c, Guifang Fan b, Xiaojiaoyang Li a,⁎
PMCID: PMC12301913  PMID: 40734907

Abstract

Objective

Cassiae Semen (CS, Juemingzi in Chinese) is a widely used traditional Chinese medicine with a variety of pharmacological effects. This study aimed to investigate the potential therapeutic effects and molecular mechanisms of anthraquinones of CS (AQS) for adiposity.

Methods

The chemical components of the AQS were determined using high-performance liquid chromatography (HPLC). Network pharmacology analysis was used to predict potential anti-obesity targets of action for AQS. We constructed high fat with high sugar water diet-induced obese mice and observed their body weight and whole-body lipid metabolism to evaluate the efficacy of AQS in promoting lipid metabolism. Subsequently, the epidermal temperature at the brown adipose tissue (BAT) before and after cold stimulation was observed and the expression of lipid metabolism-related genes in the liver and BAT tissues was detected to clarify the mechanism of action of AQS.

Results

Network pharmacology analysis showed that AQS was involved in the regulation of liver and adipose tissue function under obesity. Pathological and biochemical results showed that AQS reduced lipid accumulation in the liver and adipose tissue induced by an unhealthy diet. With the increase of cold tolerance, the volume and weight of BAT were increased by AQS, suggesting that it regulated the body heat production dominated by BAT. After AQS treatment, the levels of genes related to uncoupling protein1 (UCP1)-mediated adaptive thermogenesis in BAT tissues and lipid metabolism in the liver were also increased, which further proved that AQS activated BAT function to promote lipid metabolism in the whole body.

Conclusion

This study revealed the pharmacological effects of AQS, thereby providing a scientific basis for regulating BAT thermogenesis and liver lipid metabolism to alleviate obesity and providing clues for further exploring the application of natural active ingredients in the treatment of metabolism-related diseases.

Keywords: adiposity, anthraquinones, brown adipose tissue, Cassiae Semen, liver, lipid metabolism, thermogenesis

1. Introduction

Obesity, as a serious public health problem worldwide, has continued to increase and now affects about ten percent adults that poses a major challenge to overall population health (Mohanty & Mohanty, 2021). In 2030, the number of overweight and obese adults will reach 1.35 billion and 573 million, respectively (Kelly et al., 2008). The high prevalence of obesity has led to a dramatic increase in the incidence of metabolic liver disease, type 2 diabetes mellitus, cardiovascular disease, cancer, etc., and inevitably, has imposed a heavy economic burden on society (The Lancet Gastroenterology, 2021). Obesity is characterized by hypertrophy or hyperplasia of adipocytes and abnormalities of systemic lipid metabolism, which is often viewed as an imbalance of multiple factors such as excessive energy intake (Hall & Guo, 2017), physical inactivity (Carbone et al., 2019) and genetic susceptibility (Albuquerque et al., 2017). When the rate of adipose tissue expansion exceeds the angiogenic capacity, it will result in continuous cellular hypoxia. Hypoxia-induced apoptosis recruits macrophages and other immune cells to infiltrate adipose tissue to remove necrotic adipocytes, generating an inflammatory response, further accelerating insulin resistance and impairing the metabolic function of adipose tissue (Saltiel & Olefsky, 2017). Meanwhile, the persistence of chronic inflammation will induce a fibrotic response in the tissues, with deposition of collagen and extracellular matrix proteins limiting the hypertrophic capacity of the adipocytes, resulting in reduced adipose tissue lipid storage and metabolism, exacerbating adipose tissue dysfunction. The spilled lipids enter the systemic circulation and continue to cause lipotoxicity and inflammation in other tissues, ultimately leading to systemic metabolic disorders (Kawai et al., 2021).

White adipose tissue (WAT) is the most abundant adipocyte in humans and is found in subcutaneous tissues and around organs, storing energy mainly in the form of triglyceride (TG). Brown adipose tissue (BAT) contains a large number of mitochondria, where high levels of the uncoupling protein1 (UCP1) uncouples mitochondrial oxidative respiratory from TG and glucose and then consumes these substrates in non-shivering thermogenesis (Chouchani et al., 2019). Also, liver is another important metabolic organ in the body and is responsible for regulating the conversion of major nutrients including lipid. After released from adipose tissue, plasma TG and free fatty acids (FFA) are taken up by hepatocytes and accumulated in the liver, which will be further eliminated by intracellular oxidation or secreted into plasma rich in very low-density lipoproteins (Grabner et al., 2021). Physiologically, the liver stores only small amounts of fatty acids as TG and is able to oxidize lipids, as well as synthesizes excess lipids that are secreted into the blood and stored in other tissues (Alves-Bezerra & Cohen, 2017). In obesity, elevated blood lipid and altered hepatic fatty acid metabolism usually result in the accumulation of intracellular TG in cytoplasmic lipid droplets (Kawano & Cohen, 2013). Collectively, disturbed lipid metabolism that happened in almost 90% of obese patients can induces the development of simple fatty liver in healthy livers, which becomes the most common obesity-associated hepatic metabolic abnormality (Suriawinata & Fiel, 2004). With the development of obesity-associated fatty liver, exorbitant build-up and subsequent injury of fat in the liver also generate multiple signals that may dysregulate cellular metabolism and will deal a “second blow” to the adipose tissue, resulting in a vicious circle of damage between tissues (Yip et al., 2022).

Traditional Chinese medicine (TCM) has the advantages of multi-dimensional, multi-target, low adverse effects and good long-term efficacy. Cassiae Semen (CS, Juemingzi in Chinese) is the dried mature seed of Cassia obtusifolia L. or Cassia tora L., which belongs to the Leguminosae family. The seeds are widely used in China, Japan and Korea for improving visual acuity, and having laxative, antioxidant, neuroprotective and anti‑bacterial effects, in addition to lowering the blood pressure (Chen et al., 2023, Dong et al., 2021). Meng et al. showed that the ethanolic extract of CS reduced the serum levels of phospholipids (PL), total cholesterol (TC) and TG in rats. They suggested that CS regulated the plasma post heparin lipolytic activity (PHLA) and lipoprotein lipase (LPL) activity to inhibit cholesterol acyltransferase activity, which in turn inhibited cholesterol biosynthesis to exerted hypolipidemic effect (Meng et al., 2019). Another study found that the water-soluble fraction of CS showed better efficacy in mitigating dyslipidemia compared to ether soluble fraction (Yuen et al., 2021). Consistently, our recent study revealed that CS water extract significantly reduced liver and serum TG levels and significantly attenuated oleic acid-palmitic acid (OAPA)-induced lipid accumulation in hepatocytes by increasing activated protein kinase-mediated autophagy flux and inhibiting fatty acid synthase (FASN) level (Ding et al., 2023). Besides, the effect of different effective parts of CS on the improvement of obesity has gradually attracted attention. For instance, water-soluble polysaccharide from CS showed a significant inhibitory effect on α-amylase and pancreatic lipase, and was able to reduce the binding of bile acids and thus reduce cholesterol absorption (Huang et al., 2012). Meanwhile, it is worthy to note that anthraquinones of CS (AQS) has got promising therapeutic effects on hepatic steatosis by increasing the expression of peroxisome proliferator activated receptor (PPAR)-γ (Luo et al., 2011). We also found that aurantio-obtusin, a representative component of AQS, promoted BAT thermogenesis by activating PPARα and its downstream genes (Meng et al., 2019). Although all of the above studies have pointed out the modulatory effects of CS on lipids, whether and how AQS improves the function of BAT and liver in obesity-related metabolic disorders has not been well elucidated.

Here, we aim to elucidate that AQS significantly activated lipid metabolism on BAT and liver tissues in diet-induced obese mice. We first evaluated the lipid accumulation in multiple organs and examined the levels of lipid-related products in serum, BAT and liver. The potential mechanisms and regulatory networks underlying the therapeutic effects of AQS were extensively investigated by network pharmacology analysis. Taken together, our results suggested that AQS improved obesity and systemic lipid metabolism disorders by modulating the BAT-hepatic lipid metabolism network.

2. Materials and methods

2.1. Materials

CS was purchased from Beijing Tongrentang (Group) Co., Ltd. (2209064, Beijing, China). Rubrofusarin 6-O-β-gentiobioside (content ≥ 98%, HPLC), aurantio-obtusin (AO) (content ≥ 98%, HPLC), rhein (content ≥ 98%, HPLC), emodin (content ≥ 98%, HPLC) and cassiaside C (content ≥ 98%, HPLC) were prepared from Yuanye Bio-Technology Co., Ltd (Shanghai, China). Acetonitrile, methanol and ethanol (content ≥ 98%, HPLC) were obtained from Merck KGaA (Darmstadt, Germany). High-fat diet was obtained from Harlan Laboratories, Inc. (TD.88137, Indianapolis, USA).

2.2. Preparation of AQS (anthraquinones of CS)

CS was authenticated by Dr. Bing Xu from the Department of Pharmacognosy, School of Chinese Materia Medica, Beijing University of Chinese Medicine. CS was pulverized and then soaked in 100% methanol. The mixture was then sonicated for 30 min at 65°C for complete extraction of AQS in methanol. The anthraquinone content of the sample was verified using the color development experiment of 1,8-hydroxyanthraquinone with magnesium acetate.

2.3. High-performance liquid chromatography (HPLC) for AQS

The HPLC system (Waters, MA, United States) comprised a Waters 2695 separation unit, a 2996 diode array detector, and a 2420 evaporative light scattering detector. In addition, the temperature was settled at 30°C and the chromatographic column (100 mm × 2.1 mm, 1.7 μm) used was a Williams’ Medium E with L-glutamine (Elite, Dalian, China). The UV spectra were 284 nm, and the mobile phases were a mixture of acetonitrile (A) and water (B). The flow rate was 0.2 mL/min and the gradient program was used: 0−90 min, 5%−100% A. The rubrofusarin 6-O-β-gentiobioside, AO, rhein, emodin and cassiaside C in AQS were qualified using standard calibration curves.

2.4. Network pharmacology analysis

The potential effective compounds in CS were identified with the name of herbs as keywords and the thresholds of oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18 in TCMSP Database. From the screened effective compounds, those belonging to compounds of anthraquinone were selected as the potential effective compounds of AQS in this study. The targets of AQS were searched using the TCMSP Database and Swiss Target Prediction Database. The adiposity-related targets were searched from Genecards databases by using “adiposity” as the keyword. The intersection targets between the adiposity-related genes and the predicted AQS targets were obtained and then were imported into the STRING and Metascape database for GO analysis.

2.5. Animal study

Male C57BL/6J mice (8-week-old) were purchased from Vital River Laboratory Animal technology (Beijing, China) and were kept under 12 h dark and 12 h light cycle at constant temperature [(22 ± 2) °C]. After 1-week adaptive feeding, mice were randomly divided into five group (n = 6): (1) control group (chow diet); (2) high fat with high sugar water (HFHS) diet group [western diet (42% Kcal from fat and 0.2% cholesterol) with a high sugar water (D-fructose: 23.1 g/L and D-glucose: 18.9 g/L) in drinking water]; (3) HFHS diet treated with AQS low dose group (1 g/kg) (AQSL); (4) HFHS diet treated with AQS medium dose group (2 g/kg) (AQSM); (5) HFHS diet treated with AQS high dose group (4 g/kg) (AQSH). Groups (1) were accepted chow diet for 8 weeks while mice in group (2–5) were first fed with HFHS diet for 4 weeks to induce adiposity followed by gavage different doses of AQS for another 4 weeks. After 8 weeks treatment, mice were sacrificed and collected serum, adipose and liver tissues for further studies. The experiments were performed according to the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine (No. BUCM-4-2020083001-3011).

2.6. Body temperature measurement and infrared thermography

Each mouse was placed in separate cages and only provided water for 5 h at 4°C to test cold exposure. The core body temperature was detected using a rectal probe for mice (Omron Digital Thermometer, USA). Mice were shaved on the interscapular area and then infrared images were photographed by Fluke TiS60+ (FLUKE, Washington, USA) before and after cold exposure.

2.7. Hematoxylin and eosin (H&E) staining

The histopathological examination of adipose and liver tissues was performed using H&E staining. Adipose and liver samples were put in a 10% formaldehyde solution, dehydrated in an ethanol gradient, embedded in paraffin, and cut into 4 μm sections. After deparaffinization, the sections were stained with hematoxylin (G1005-1; Servicebio, China) and eosin (G1005-2; Servicebio, China) and then mounted and observed under a microscope (Olympus, Tokyo, Japan).

2.8. Oil red O staining

Oil red O (ORO) staining was performed as previously described (Li et al., 2023). Pieces from the main liver lobes and BATs were embedded in optimal cutting temperature (O.C.T.) compound (Tissue-Plus, Scigen, 4583) and frozen at −20 °C. The thick sections (14 μm) were cut at −20 °C in a Cryos tat chamber. Unfixed tissue slides were stained with ORO and images were taken with a Zeiss VivaTome microscope (Zeiss, Thornwood, NY, USA).

2.9. Detection of biochemical indicators

Serum was collected by centrifugation at 14 800 r/min for 15 min at 4 °C. Liver was homogenized in the saline and centrifuged at 12 000 r/min for 15 min at 4 °C. Serum and liver alanine aminotransferase (ALT) and aspartate transaminase (AST) were assessed by ALT and AST assay kits (Nanjing Jiancheng Bioengineering Co., Ltd., Nanjing, China) and were analyzed according to the instructions.

2.10. Detection of lipid metabolism indicators

Serum was collected by centrifugation at 14 800 r/min for 15 min at 4 °C. BAT and liver were homogenized in the saline and centrifuged at 12 000 r/min for 15 min at 4 °C. The concentrations of TC, TG and FFA in serum, BAT and liver were analyzed using relative commercial kits (Nanjing Jiancheng, Bioengineering Co., Ltd., Nanjing, China).

2.11. Quantitative real-time RT-PCR (RT-qPCR)

Total RNA was isolated by Fast Pure Cell/Tissue Total RNA isolation kit (RC101-01, Vazyme Biotech, Nanjing, China) and was reverse-transcribed by HiScript III RT SuperMix (R323-01, Vazyme Biotech). AceQ Universal SYBR qPCR Master Mix (Q511-02, Vazyme Biotech) was used to detect mRNA expression of related genes. Primers for RT-qPCR are listed in Table S1.

2.12. Western blot experiment

Protein extraction, quantification, preparation and Western blot experiment were performed as previously described (Ding et al., 2023). Briefly, tissue samples were homogenized in radio-immunoprecipitation assay (RIPA) buffer, estimated by BCA Protein Assay Kit, separated by SDS-PSGE gels, blotted onto PVDF membranes and blocked with 5% non-fat milk. The membrane was incubated overnight with UCP1 (23673-1-AP, Proteintech Group, Inc., Wuhan, China), FASN (sc-48357, Santa Cruz Biotechnology, Inc., Dallas, USA), CPT1a (15184-1-AP, Proteintech), SREBP1 (14088-1-AP, Proteintech) and β-actin (66009-1-lg, Proteintech) at 4 °C. Membranes incubated with relative secondary antibodies and visualized by ChemiDocTM Touch Imaging System (Bio-Rad, Hercules, CA).

2.13. Statistical analysis

All date was presented by mean ± SEM and analyzed by One-way ANOVA analysis using GraphPad Prism Software 8.0 (GraphPad, La Jolla, CA, USA). P value ≤ 0.5 was considered statistically significant.

3. Results

3.1. AQS chemical characterization by HPLC

HPLC was used to separate and detect a variety of compounds with high accuracy and sensitivity. Being obtained by methanol and ultrasound, AQS extraction was analyzed by HPLC. The polarity of AQS was identified and the retention time of five representative chemical components by injection was determined. On the basis of the HPLC pattern, rubrofusarin 6-O-β-gentiobioside, cassiaside C, aurantio-obtusin, rhein and emodin were eluted at 26.2, 28.4, 62.7, 71.1 and 77.1 min, respectively (Fig. 1A). The results showed that AQS we prepared matched five representative chemical components in HPLC (Fig. 1B).

Fig. 1.

Fig. 1

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Main component in AQS detected by HPLC. (A) HPLC identification of AQS. (B) HPLC identification of references of (1) rubrofusarin 6-O-β-gentiobioside, (2) cassiaside C, (3) aurantio-obtusin, (4) rhein and (5) emodin.

3.2. Network pharmacology analysis of AQS

According to pharmacokinetic characteristics (OB ≥ 30% and DL ≥ 0.18) and ADME information, five active components of AQS were selected from ingredients of CS in TCMSP database. The TCMSP and Swiss Target Prediction databases were used to determine the pharmacological targets of the AQS. Eventually, 149 potential targets were identified after removing duplicates. Using “Adiposity” as the search term, disease-targets were obtained from GeneCards database and 4 236 related-adiposity potential targets were finally collected. Applying a Venn diagram, 82 common targets were found overlapped between AQS targets and adiposity-related targets (Fig. 2A). Next, we analyzed 82 potential therapeutic targets by using the STRING database to obtain a PPI network (Fig. 2B). Biological functions of these target genes were further analyzed and screened the top ranked GO terms (Fig. 2C). A total of 82 potential therapeutic targets mainly concentrated in the response to stimulus, biological regulation and metabolic process were found (Fig. 2D). Prediction of related diseases was closely associated with diabetes mellitus and fatty liver disease, which was consistent with our previous experimental results (Ding et al., 2023) (Fig. 2E). Interestingly, liver and adipose cells were predicted target organs and cells, respectively, suggesting that adipose tissue and liver might participate in and communicate with the treatment of obesity with AQS (Fig. 2F). In AQS potential therapeutic targets, we found that the inhibition of fat mass and obesity (FTO) associated gene promoted UCP1 transcription by activating forkhead box protein o1 (FoxO1). Secondly, FTO inhibition activated mechanistic target of rapamycin (mTOR) to promote PPARγ coactivator 1 α (PGC1A) expression and PPARα or PPARγ transcription, which together promoted UCP1 transcription, thus AQS may dually affect thermogenesis. Subsequently, FTO may interfere with fatty acid oxidation through sterol-regulatory element binding protein (SREBP) and carnitine palmitoyltransferase 1 (CPT1) and simultaneously regulates the generation of FASN pathway through activating transcription factor 4 (ATF4). Therefore, we speculated that AQS had a potential pharmacological role in regulating BAT thermogenesis and livr lipid metabolism (Fig. 2G).

Fig. 2.

Fig. 2

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Network pharmacology analysis of AQS in adiposity. (A) Venn graph of co-differentially expressed genes (DEGs) in both SAB and IPF. (B) PPI network of co-DEGs targets by STRING database. (C, D) GO functional annotation of co-DEGs. (E, F) Prediction of co-DEGs possible diseases and corresponding associated tissues or cells. (G) Candidate gene pathways.

3.3. AQS attenuated multi-organ lesions in diet-induced obese mice

After four weeks of HFHS feeding, the body weight gain of mice was significantly increased compared with normal diet, suggesting the emergence of obesity (Fig. 3A). After four weeks of treatment with different doses of AQS, the body weight gain of AQS-treatment mice began to be significantly reduced compared with HFHS-fed mice (Fig. 3B). There was no difference in caloric intake between groups, suggesting that the reduction in body weight gain of AQS-treated mice was not associated with reduction in food intake (Fig. 3C). To clarify whether the suppression of weight gain caused by AQS was due to the reduced accumulation of adipose tissue, individual adipose depot masses were measured. As shown in Fig. 3D and E, WAT depot masses (containing epididymal adipose tissue, mesenteric adipose tissue and perirenal adipose tissue) were dramatically lower in AQS-treated mice than in obese mice. Consistent with this, the body fat percentage of obese mice was significantly reduced after AQS treatment (Fig. 3F). Interestingly the mass and volume of BAT significantly increased after AQS treatment (Fig. 3G and H). As the liver is an important organ for lipid metabolism, observation of its weight and lipid accumulation is significant in assessing the degree of obesity and the occurrence of metabolic diseases. We found a slight decrease in the weight of liver and spleen after AQS treatment (Fig. 3I). Also, liver representative pictures showed that the liver of obese mice appeared yellowish due to lipid accumulation, which was reversed by AQS treatment (Fig. 3J). Based on these experimental results, AQS could alleviate systemic metabolism centered on adipose tissue and liver, suggesting that AQS was involved in lipid metabolic processes in multiple organs, especially for BAT and liver.

Fig. 3.

Fig. 3

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AQS alleviated lipid accumulation in WAT and liver of obese mice and promoted proliferation of BAT (mean ± SEM, n = 6). (A) Body weight of mice after feeding chow or diet HFHS diet for 4 weeks. (B) Body weight gain during AQS or vehicle treatment. (C) Caloric intake per mouse per day during AQS or vehicle treatment. (D) WAT weights (Epdi: epididymal adipose tissue; Mes: mesenteric adipose tissue; Per: perirenal adipose tissue) and (E) representative images of WAT. (F) Adipose index. (G) BAT weights and (H) representative images of BAT. (I) liver index and spleen index. (J) Representative images of liver. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, compared between groups.

3.4. AQS activated BAT thermogenesis to attenuate systemic lipid metabolism

In contrast to WAT that stores excess energy, BAT dissipates energy and produces heat as a defense against obesity in mammals. To assess whether AQS exerted a modulatory effect on BAT thermogenesis, we subjected mice to a 5 h cold exposure. Infrared thermal images showed that the temperature at the BAT site was higher in AQS-treated mice in a dose-dependent manner (Fig. 4A). Anal temperature is generally used to represent mouse core temperature, so we examined anal temperature every hour to quantify changes in body temperature. Consistent with the thermographic image results, mice core temperature rebounded significantly after AQS stimulation compared to obese mice (Fig. 4B), suggesting that BAT thermogenesis could be activated by AQS. The histopathological results visualized the multiple pathological changes in different organs. H&E staining results showed that BAT and WAT of obese mice contained more adipocytes with larger lipid droplets. Accompanying this phenomenon, the vacuolar degeneration of liver was significantly increased in HFHS diet-induced mice compared to normal mice (Fig. 4C). After four weeks of AQS treatment, the above pathological changes in these tissues resolved significantly. ORO specifically strongly combined with neutral fats such as TG and then visualizes neutral fat in red. Our previous results showed that AQS promoted body heat production, so we applied ORO staining in BAT instead of WAT. Since hepatic lipid accumulation is a key pathological manifestation of fatty liver disease, we also assessed the liver lipid accumulation. We found that the red staining area in BAT and liver was significantly reduced, suggesting that lipid overaccumulation due to the HFHS diet could be significantly alleviated by AQS (Fig. 4D). Thus, the effect of AQS in reducing systemic lipid accumulation might be exerted by activating heat production in BAT.

Fig. 4.

Fig. 4

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AQS activated BAT thermogenesis to reduce lipid accumulation in multiple tissues (mean ± SEM, n = 6). (A) Representative infrared images of interscapular temperature. (B) Rectal temperature of mice after 5 h acute cold stress. (C) H&E images of BAT, WAT and liver. Magnification is × 400, scale bar = 30 μm. (D) Images of Oil Red O staining for sections of BAT and livers. Magnification is × 400, scale bar = 30 μm. Statistical significance: *P < 0.05, compared between groups.

3.5. AQS reduced systemic lipid accumulation represented by adipose tissue and liver

In order to better quantify the accumulation of lipids in adipose tissue and the liver, we next measured the levels of a variety of lipids. TC is one of the most important raw materials for the formation of cell membranes, as well as for the formation of vitamin D, estrogens, androgens and glucocorticoids. TG is the body's “energy bank” and serves to store calories. Excessive increase of TC and TG declares abnormal lipid in the body, indicating the occurrence of pathological state of obesity. Our results showed that HFHS diet significantly increased serum and liver TC levels, with less effect on BAT. After AQS treatment, the levels of TC in serum and BAT were slightly decreased (Fig. 5A). TG levels were significantly elevated in serum and liver of obese mice, which were markedly alleviated by AQS treatment, especially for the low and medium dosages. In contrast, BAT, as an energy-consuming organ, was not significantly treated by AQS (Fig. 5B). FFA are one of the substances into which neutral fats are broken down, and their high levels usually indicate hyperlipidemia and endocrine disorders. We found that the level of FFA in the liver was significantly decreased after AQS treatment compared to obese mice (Fig. 5C). High FFA level leads to a high generation of ROS, which initiates oxidative stress mechanisms causing tissue damage. Therefore, we examined the levels of ALT and AST in serum and liver to assess whether the liver was damaged. We found that the levels of ALT and AST in serum did not change significantly in different groups (Fig. 5D). The emergence of severe obesity-induced liver injury requires long-term stimulation, so the liver function of obese mice was not significantly altered. And the level of ALT but not AST in the liver was significantly increased after fed with this unhealthy diet but slightly decreased after the AQS intervention, which suggests that the liver injury was alleviated to some extent (Fig. 5E).

Fig. 5.

Fig. 5

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AQS influenced lipid metabolism between liver and BAT to reduce hyperlipidemia and potential liver damage (mean ± SEM, n = 6). TG (A), TC (B) and FFA (C) levels of serum, liver and BAT. (D) ALT and AST levels in serum. (E) ALT and AST levels in liver. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, compared between groups.

3.6. AQS activated BAT thermogenesis-related genes and liver lipid metabolism-related genes to promote lipid metabolism

UCP1 dissipates the proton gradient generated by the electron transport chain, leading to uncoupled respiration and thermogenesis. BAT possess the efficient metabolic heat-producing pathway due to its high expression of UCP1. Furthermore, we applied network pharmacology analysis and indicated that AQS might promote UCP1 expression by FTO and FoxO1 pathways. Thus, we examined mRNA levels of Ucp1 in BAT and found that AQS significantly reversed the reduction in Ucp1 due to HFHS dietary stimulation (Fig. 6A). Consistent with this, AQS also increased protein levels of UCP1 in BAT of obese mice (Fig. 6B). Previous study reported that the binding of PR domain containing 16 (PRDM16) to UCP1 promoter and inhibitory effect of recombinant FoxO1 on transcription factors could promote UCP1 transcription (Liu et al., 2016, Seale et al., 2007). Our results showed a significant decrease in FoxO1 mRNA expression and a slight elevation in Prdm16 mRNA expression after AQS treatment (Fig. 6C). Based on the predicted results of network pharmacology in Fig. 2G, the mRNA levels of other potential target genes were also detected. We found that AQS slightly increased mRNA levels of Pparg, Pgc1a and mTor, revealing a potential regulatory pathway for elevation of UCP1 mRNA expression (Fig. 6D). Some studies have found that these above genes promote mitochondrial genesis, so we next examined mitochondrial DNA (mt-Nd3) and cytochrome C, a marker for mitochondria. We found that a HFHS diet significantly reduced the mRNA levels of CytoC and slightly affected the mRNA levels of mt-Nd3. The mRNA level of these two genes increased after AQS treatment, suggesting that AQS may alleviate obesity-induced mitochondrial damage to some extent (Fig. 6E).

Fig. 6.

Fig. 6

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AQS alleviated obesity by activating thermogenic genes in BAT and lipid synthesis- and catabolism-related genes in liver (mean ± SEM, n = 6). Relative mRNA levels of (A) Ucp1, (C) Foxo1, Prdm16, (D) Pparg, Pgc1a, mTor, (E) mt-Nd3 and CytoC in BAT. (B) Protein levels of UCP1 in BAT determined by Western blot using β-actin as a loading control. Relative mRNA levels of (F) Atf4, Fasn, (G) Ppara, Srebp, Cpt1a, (H) Tgfb and Col1a1 in liver. Hprt1 was used as an internal reference. (I) Protein levels of FASN, CPT1a and SREBP1 in the liver were determined by Western blot using β-actin as a loading control. Statistical significance: *P < 0.05, **P < 0.01, compared between groups.

As the liver is a core organ for lipid metabolism and the results of network pharmacology, we next examined the expression of genes related to hepatic lipid synthesis and catabolism. We found that AQS increased the mRNA expression of lipid catabolism-related genes (Ppara and Cpt1a), and decreased lipid synthesis-related genes (Srebp, Atf4 and Fasn), suggesting that hepatic lipid metabolism was activated by AQS treatment (Fig. 6F and G). Whereas prolonged lipid accumulation in liver lipids can lead to the development of hepatic inflammation and fibrosis, we therefore examined the levels of mRNA for Tgf-b and Col1a1. We found that AQS elevated the level of Tgf-b and decreased the level of Col1a1, which may be due to the activation of inflammatory repair in the organism by AQS (Fig. 6H). Subsequently, we performed Western blotting analysis on the liver. We found that AQS decreased the protein expression of FASN and SREBP1 and elevated the protein expression of CPT1a, which was consistent with the qPCR results (I). The above results suggested that AQS promoted mitochondria heat production in BAT and activated lipid metabolism in liver to alleviate liver injury and systemic lipid metabolism abnormalities.

4. Discussion

Numerous studies have reported that natural active ingredients ameliorate obesity by promoting BAT thermogenesis and hepatic lipid metabolism to modulate adipose-liver communication (Chen et al., 2021, Chen et al., 2021, Ma et al., 2022, Wang et al., 2021). In the present study, AQS may affect liver and adipose tissue function in obesity based on predictions of network pharmacology. We demonstrated that AQS reduced adipose mice weight gain, blood lipid concentrations, and lipid accumulation in multiple organs. In addition, AQS promoted thermogenesis by increasing UCP1 and regulating PGC1a and FoxO1 mRNA expression in BAT to reduce lipid droplet accumulation, and exerted dual therapeutic effects on hepatic lipid disorders by increasing the mRNA expression levels of genes related to lipid synthesis and decreasing genes related to lipid catabolism. Thus, our study mainly revealed that AQS may be involved in lipid exchange among multiple organs to alleviate systemic lipid metabolism syndrome.

BAT is a specialized tissue that consumes energy by producing heat. During respiration, an electron transport chain located in the inner mitochondrial membrane pumps protons from the stroma to the membrane interstitium, establishing a proton gradient, which drives the rotation of ATP synthase to produce ATP. UCP1 is the uncoupler between proton delivery and ATP synthesis in the mitochondrial electron transport chain (Cannon and Nedergaard, 2004, Chouchani et al., 2019). It has been previously shown that sympathetic activation triggered by cold exposure, diet and drugs can induce BAT activation (Scheele & Wolfrum, 2020). In BAT, AQS stimulation leads to lipolysis, which increased the concentration of FFA (Fig. 5C). When activated by FFA, UCP1 catalyzes proton leakage across the inner mitochondrial membrane, causing mitochondria in BAT to generate heat and consume energy (Wu et al., 2006). It has been reported that acting as an “on/off switch” in adipose differentiation, PRDM16 is a transcription factor that regulates fat and muscle metabolism and regulates the transcription of various target genes (e.g., Ucp1) involved in the transformation of WAT-BAT and muscle-BAT (Ohno et al., 2012). Our results also showed that AQS increased the mRNA level of Prdm16 in BAT (Fig. 6C). Thus, AQS may increase thermogenesis in BAT by increasing the mRNA levels of Ucp1 and Prdm16. The results of our network pharmacological analysis suggest that FTO and its downstream thermogenic genes may be potential targets for AQS to relieve obesity. As a transcription factor, FoxO1 regulates adipocyte differentiation and insulin function in a variety of metabolically active cells. When FoxO1 is silenced, TFEB transcript and protein levels dramatically drop, thus boosting the expression of UCP1. As expected, AQS significantly inhibited FoxO1 mRNA levels. Associating with PPARγ, PGC1a that be upregulated by mTOR is responsible for the interaction between mitochondria and nucleus as well as the activation of its downstream regulators, nuclearfactor erythroidderived 2-like 2 (NRF1/2), and mitochondrial transcription factor A (TFAM), to promote the transcription of the mitochondrial genome to modulate mitochondrial biogenesis (Fan et al., 2021). AQS slightly increased the mRNA level of Pgc1a, mTor and Pparg in BAT, which was consistent with the changes in the mRNA levels of mitochondrial gene mt-Nd3 and nuclear gene CytoC (Fig. 6D and E), suggesting that AQS may play a role in enhancing BAT thermogenesis by promoting mitochondrial proliferation to a certain extent.

Hepatic lipid metabolism includes lipid uptake and production, export and oxidation, and hepatic lipid content is affected by (1) delivery of FFA from adipose tissue sources to the liver; (2) uptake from the diet; (3) intrahepatic de novo lipid synthesis (DNL); (4) lipid oxidation and secretion, with the first three being the major sources of intrahepatic lipids and the latter being the major destination of intrahepatic lipids (Jones, 2016). In addition to BAT, the results of network pharmacology also suggested AQS may affecte hepatic lipid metabolism by FTO its downstream genes. Notably, FASN regulates hepatic fat DNL and is one of the most attractive targets in the treatment of hepatic lipid metabolism disorders (Jones & Infante, 2015). We found that AQS slightly reduced Atf4 mRNA expression and remarkably reduced Fasn mRNA expression (Fig. 6F). The two pathways of fatty acid oxidative catabolism are mitochondrial β-oxidation and peroxisomal β-oxidation. PPARα regulates a variety of metabolic processes in the liver, mainly including mitochondrial fatty acid oxidation, TG synthesis and catabolism, fatty acid binding and activation, and lipoprotein metabolism (Li et al., 2021). Our results revealed that AQS partially promoted the mRNA expression of Ppara (Fig. 6G). PPARα activates the expression of CPT1, which stimulated the entry of acyl-coenzyme A into the mitochondria and the subsequent tricarboxylic acid cycle (Schlaepfer & Joshi, 2020). AQS increased the mRNA level of Cpt1a in the liver of obese mice (Fig. 6H). SREBP is a key nuclear transcription factor involved in the synthesis and absorption of cholesterol and fatty acid synthesis, and regulates the level of lipid metabolism. As expected, AQS decreased the mRNA level of Srebp (Fig. 6G). Thus, AQS attenuated hepatic lipid accumulation by regulating hepatic lipid synthesis and catabolism.

With advances in molecular biology, adipose tissue is no longer considered a passive energy storage depot, but a complex endocrine and immune organ that actively secretes many bioactive substances. Since the first cloning and discovery of leptin (a protein synthesized and secreted mainly by adipose tissue) in 1994 adipose tissue has been able to secrete more than 600 bioactive molecules collectively known as adipocytokines (Zorena et al., 2020). These adipocytokines are secreted into the bloodstream and travel throughout the body to regulate the function of target organs including the brain, liver, pancreas, muscles, blood vessels and gastrointestinal tract (F. Villarroya et al., 2017). Recent studies have found that interleukin-6 (IL-6) produced by brown adipose tissue reprograms hepatic metabolism and promotes hepatic gluconeogenesis under conditions of external stress stimuli, thereby maintaining hyperglycemia in the body under stress, which exacerbates the inflammatory response (Qing et al., 2020). Scheja et al. found that BAT cells can regulate the function of target organs including the brain, pancreas, muscles, blood vessels and gastrointestinal tract through the massive synthesis and secretion of insulin-like growth factor 1 (IGF1) that reduced plasma glucagon levels and regulate hepatic metabolic processes (Scheja & Heeren, 2016). In addition to secreting the above adipocytokines, adipose tissue also releases other substances, including lipids, metabolites, non-coding RNAs and extracellular vesicles (EVs) (Scheja & Heeren, 2016; J. Villarroya et al., 2013). It has been found that EVs from brown adipose intravenously significantly increase the expression of thermogenic genes such as Pgc1a, Cidea and Ucp1 in mice and induce browning of white adipose, improving systemic energy expenditure, glucose homeostasis, insulin sensitivity and adipose tissue inflammation in obese individuals (Thomou et al., 2017). Therefore, the mutual exchange between BAT and liver in obesity may be a focus. The results of network pharmacology analysis also imply that the target of action of AQS involves the liver and BAT. Meanwhile, we found a significant decrease in serum lipid levels after AQS treatment and a slight increase in FFA levels in BAT (Fig. 5C). These results suggested a material exchange between adipose tissues and the liver with the presence of AQS, which may be the manifestation of the multi-targeted action of herbal medicines but still needs to be further explored.

5. Conclusion

In the present study, we demonstrated that AQS accelerated body lipid metabolism by regulating the expression of thermogenesis-related genes in the BAT and lipid catabolism- and synthesis-related genes in the liver, thereby alleviating multi-organ lipid accumulation in the HFHS-diet obese model. Meanwhile, AQS may modulate lipid exchange between BAT and liver to alleviate potential liver injury. Our study not only reveals the effects of AQS in promoting lipid metabolism, but also provides a basis for the development of small molecule components isolated from AQS as innovative drug candidates for treating obesity and related complications.

CRediT authorship contribution statement

Yijie Li: Data curation, Methodology, Writing – original draft. Ruiyu Wu: Data curation, Methodology. Xin Li: Data curation. Jianan Li: Methodology. Yinhao Zhang: Methodology. Yanbo Huang: Methodology. Guifang Fan: Methodology. Xiaojiaoyang Li: Conceptualization, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by grants from the Fundamental Research Funds for the Central Universities (No. 2023-JYB-JBZD-046); National Key Research, Development Program on Modernization of Traditional Chinese Medicine (No. 2022YFC3502100); the National High-Level Talents Special Support Program; High-level traditional Chinese medicine key subjects construction project of National Administration of Traditional Chinese Medicine-Chinese Medicine Epidemic Disease (No. zyyzdxk-2023264).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chmed.2025.02.003.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary Data 1
mmc1.docx (27.3KB, docx)

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