Ramulus Mori (Sangzhi) Alkaloids Attenuate Diet- Induced Obesity by Promoting White Adipose Browning and Enhancing Brown Fat Thermogenesis

Ramulus Mori (Sangzhi) Alkaloids Attenuate Diet- Induced Obesity by Promoting White Adipose Browning and Enhancing Brown Fat Thermogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ramulus Mori (Sangzhi) Alkaloids Attenuate Diet- Induced Obesity by Promoting White Adipose Browning and Enhancing Brown Fat Thermogenesis Ruining Zhang, Ge Peng, Xiaohui Pan, Nanwei Tong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8094331/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Obesity is a chronic metabolic disorder characterized by excessive adipose accumulation and is closely associated with type 2 diabetes mellitus (T2DM), cardiovascular diseases, and metabolic dysfunction associated steatotic liver disease. Enhancing the thermogenic capacity within adipose tissues has emerged as a promising therapeutic strategy to counteract obesity and its related metabolic complications. Ramulus Mori (Sangzhi) alkaloids (SZ-A), a natural alkaloid complex derived from Morus alba L. (mulberry twig), have been clinically approved for the T2DM treatment and exhibit multiple metabolic regulatory properties. However, the precise anti-obesity mechanisms of SZ-A remain largely unclear. In this study, male C57BL/6 mice were fed a high-fat diet (HFD) for 14 weeks to induce obesity and subsequently treated with SZ-A (200 or 600 mg/kg) for 6 weeks. SZ-A markedly attenuated HFD-induced weight gain independent of food intake, improved glucose tolerance and insulin sensitivity, and alleviated dyslipidemia and hepatic steatosis. Furthermore, SZ-A reduced adipose tissue mass and upregulated key thermogenic regulators and beige adipocyte markers in white adipose tissue (WAT). It also restored brown adipose tissue (BAT) thermogenic activity by enhancing uncoupling protein 1 (UCP1) expression. In vitro, SZ-A promoted the expression of thermogenic and mitochondrial biogenesis-related genes in 3T3-L1 adipocytes, facilitating a beige-like phenotype. Transcriptomic analysis revealed that SZ-A significantly activated fatty acid catabolism and oxidation pathways, along with enrichment in the brown adipocyte differentiation pathway. Collectively, these findings demonstrate that SZ-A exerts potent anti-obesity effects through the dual mechanism of promoting WAT browning and enhancing BAT thermogenesis. Given its established clinical safety in T2DM, SZ-A represents a promising therapeutic candidate for adipose-based obesity and associated metabolic disorders. Biological sciences/Biochemistry Health sciences/Diseases Health sciences/Endocrinology Biological sciences/Molecular biology Biological sciences/Physiology Sangzhi alkaloids Obesity Browning 3T3-L1 cells HFD-induced obese mice Thermogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Obesity has become a major global health concern, with its prevalence continuing to rise across all age groups and regions. Clinical obesity is defined as a chronic, systemic illness characterized by alterations in the function of tissues, organs, the entire individual, or a combination thereof, due to excess adiposity. It can lead to severe end-organ damage, causing life-altering and potentially life-threatening complications such as myocardial infarction, stroke, and renal failure[ 1 ]. Based on morphological and functional characteristics, adipose tissue is generally categorized into WAT, BAT, and beige adipose tissue (BeAT) [ 2 ]. WAT primarily stores energy in the form of unilocular lipid droplets and mobilizes fatty acids during energy demand. In contrast, BAT is characterized by multilocular lipid droplets and abundant mitochondria, with high expression of UCP1, which mediates non-shivering thermogenesis by dissipating energy as heat to increase whole-body energy expenditure and maintain body temperature [ 3 ]. In obese individuals, BAT mass and thermogenic activity are markedly reduced or even absent [ 4 ], whereas WAT exhibits hypertrophy and excessive accumulation. Notably, the boundary between WAT and BAT can become functionally ambiguous under certain circumstances. Upon cold exposure [ 5 ] or β-adrenergic receptor agonists [ 6 ], adipocytes of WAT can exhibit brown fat-like features, including increased number of mitochondria and elevated UCP1 expression—a process known as “browning”. The resulting beige adipocytes have been identified in both humans and rodents [ 7 ]. Conversely, BAT can acquire white fat-like characteristics such as enlarged lipid droplets, reduced mitochondrial content, and downregulated UCP1 expression, referred to as “whitening”, which commonly occurs in obese individuals and worsens with obesity severity. Given the limited BAT mass in adults, promoting WAT browning is considered a promising strategy to enhance thermogenic potential, improve energy metabolism, and combat obesity and its associated metabolic complications [ 8 ]. SZ-A are a group of natural alkaloid compounds derived from Morus alba L. (mulberry twig), accounting for more than 50% of the total extract. The major bioactive constituents include 1-deoxynojirimycin (1-DNJ), fagomine (FA), and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB). SZ-A has been clinically approved for the treatment of T2DM in China since 2020 [ 9 ]. Previous studies have shown that SZ-A is rapidly absorbed and distributed to multiple metabolic target tissues, where it exerts broad metabolic benefits, including improved glucose homeostasis, reduced systemic inflammation, and regulated lipid metabolism [ 10 – 14 ]. Adipose tissue is the primary target of anti-obesity treatment. Several natural compounds have been reported to induce WAT browning, thereby exerting anti-obesity effects [ 4 , 15 – 23 ]. However, it remains unclear whether SZ-A can promote WAT browning and enhance BAT thermogenic activity, either directly or indirectly. In this study, we systematically evaluated the anti-obesity potential of SZ-A and its regulatory effects on adipose tissue browning both in vivo and in vitro. Our results demonstrate that SZ-A effectively promoted WAT browning and enhanced BAT thermogenic activity, ameliorated HFD-induced obesity and metabolic disturbances beyond food intake. Materials and Methods Chemicals and Reagents SZ-A powder was kindly provided by Beijing Wehand-Bio Pharmaceutical Co., Ltd. (Beijing, China). For experimental use, SZ-A was dissolved in saline for oral gavage in animals or in cell culture medium to prepare designated concentrations for in vitro studies. Animals and Treatment Male C57BL/6 mice (7 weeks old, 20–25 g) were purchased from Chengdu Dossy Biological Technology Co., Ltd. (Chengdu, China). Mice were housed under controlled conditions (n = 3 per Cage, 23 ± 2°C, 12 h light/dark cycle, 50–60% humidity) with free access to food and water. After one-week acclimatization period, mice were randomly divided into two groups: standard diet (SD; 10% fat, 20% protein, 70% carbohydrate; n = 6) or high-fat diet (HFD; 60% fat, 20% protein, 20% carbohydrate; n = 18) (XTHF60 vs XTCON50J, Xietong Shengwu, Jiangsu, China) to induce obesity. After 14 weeks of feeding, HFD-fed mice were further randomized into three groups (n = 6 per group): HFD control (HFD), low-dose SZ-A group (HFD + SZL; 200 mg/kg/day), and high-dose SZ-A group (HFD + SZH; 600 mg/kg/day). The SD and HFD control groups received equivalent volumes of normal saline by oral gavage, while the SZ-A groups were administered high/low dose of SZ-A daily for 6 weeks. Body weight and food intake were recorded weekly. At the end of the experiment, all animals were anesthetized with isoflurane (RWD Life Science, China) delivered via inhalation. Mice were placed in an induction chamber and exposed to 5% isoflurane in oxygen for approximately 10 seconds until deep anesthesia was achieved, as confirmed by the absence of righting and pedal withdrawal reflexes. Euthanasia was performed under anesthesia in all animals. Specifically, mice were euthanized by cervical dislocation in accordance with the institutional guidelines and the AVMA Guidelines for the Euthanasia of Animals. Death was confirmed by the complete cessation of heartbeat, which was assessed visually and by gentle palpation of the thoracic area. Following confirmation of death, organs and tissue samples were dissected, weighed, immediately frozen in liquid nitrogen, and stored at − 80°C until further analysis. The study was carried out in compliance with the ARRIVE guidelines ( https://arriveguidelines.org ). All animal procedures were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (Approval No. 20240926002). Glucose Tolerance Test and Insulin Tolerance Test Tolerance tests were performed within 3 days after the completion SZ-A administration. Before intraperitoneal glucose tolerance test (ipGTT) or insulin tolerance test (ipITT), mice were fasted for 12 h. On the day of the experiment, mice were weighed and injected intraperitoneally with glucose (1 g/kg, dissolved in sterile saline) or insulin (0.5 U/kg, dissolved in sterile saline). Blood glucose levels were measured from tail vein samples at 0, 15, 30, 60, 90, and 120 min after injection using glucometers (Roche, Germany). Cell Culture and Adipogenic Differentiation Mouse 3T3-L1 preadipocytes were purchased from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37°C in a humidified incubator containing 5% CO₂. For adipogenic differentiation, 3T3-L1 preadipocytes were cultured until full confluence and maintained for 2 days to achieve contact inhibition. Differentiation was then induced by incubation in DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, 1 µM rosiglitazone, and 2 µg/mL insulin for 3 days. The medium was then replaced with DMEM containing 10% FBS, 1 µM rosiglitazone, and 2 µg/mL insulin for 4 days, followed by maintenance in DMEM with 10% FBS for another 2 days to complete differentiation. Cell Viability Assay 3T3-L1 preadipocytes were seeded into 96-well plates at a density of 4–6 × 10⁴ cells/mL in 100 µL complete medium per well. After incubation for 12 h at 37°C and 5% CO₂ atmosphere, the medium was replaced with 200 µL of complete medium containing various concentrations of SZ-A (6.25, 12.5, 25, 50, 100, 200, and 400 µg/mL). Normal control and blank wells were included, with 6 replicates per group. After 24, 48, and 72 h of incubation, 100 µL of CCK-8 working solution (GlpBio, USA) was added to each well and incubated for 1 h at 37°C. The optical density (OD) was then measured at 450 nm using a multifunctional microplate reader (BioTek, USA), and cell viability was calculated based on OD values relative to the control group. Oil Red O Staining The Oil Red O stock solution was prepared by dissolving 300 mg of Oil Red O powder (Sigma, USA) in 100 mL of 100% isopropanol and stored at 4°C in the dark. The working solution (0.2%) was freshly prepared by mixing the stock solution with ultrapure water at a ratio of 3:2, followed by filtration. After differentiation, 3T3-L1 adipocytes were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. The fixed cells were washed with ultrapure water, incubated with 60% isopropanol for 2 min, and then stained with 0.2% Oil Red O working solution at room temperature for 10 min. Following staining, cells were washed thoroughly with water until the rinse became clear. Stained lipid droplets were observed under an inverted microscope (Olympus BX53M, Japan). For quantitative analysis, the retained dye was eluted with isopropanol, and absorbance was measured at 520 nm using a spectrophotometer. Quantitative PCR Analysis Total RNA was extracted using TRIzol reagent (Oriscience, China) and reverse-transcribed into cDNA using a reverse transcription kit (Vazyme, China). Quantitative PCR was performed using SYBR Green Master Mix (Vazyme, China) on a real-time PCR system. β-actin served as the internal control for normalization. Primer sequences are listed in S1 Table. Histological and Immunohistochemical Analysis BAT and WAT samples were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned. For histological evaluation, tissue sections were deparaffinized and stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed to detect UCP1 expression in BAT and WAT using an anti-UCP1 antibody (Abcam, UK). Hepatic lipid accumulation was assessed by Oil Red O staining. Liver tissues were rinsed with PBS, embedded in OCT compound, and sectioned at a thickness of 8–10 µm. The frozen sections were fixed with 4% paraformaldehyde, stained with Oil Red O working solution, counterstained with hematoxylin, rinsed, and mounted. Images were acquired using the SLIDEVIEW VS200 scanner (Olympus, Japan) and quantitatively analyzed with ImageJ software. Western Blotting Adipose tissues were lysed in RIPA buffer (Beyotime Biotech, China) containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Biotech, China). Equal amounts of protein samples were separated by 4–12% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies, including β-tubulin (Affinity Biosciences, China) and UCP1 (HuaAn Biotechnology, China). After washing, the membranes were incubated for 1 h at room temperature with secondary antibodies—anti-rabbit IgG (Bioswamp, China) or anti-mouse IgG (Proteintech, USA). Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Oriscience, China) and analyzed with a Chemi-Doc imaging system (E-Blot, USA). RNA Sequencing and Data Analysis Tanscriptomic sequencing of epididymal WAT (eWAT) was performed by Novogene Co., Ltd. (Beijing, China). Total RNA was extracted from eWAT using TRIzol reagent (Invitrogen, USA). RNA concentration and integrity were examined with the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) to ensure that the samples met the quality requirements for library construction. Poly(A)-tailed mRNA was enriched from total RNA using oligo(dT)-attached magnetic beads, followed by fragmentation and cDNA synthesis. The obtained cDNA fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, MA, USA) and amplified by PCR to generate the final sequencing libraries. Library quality was assessed using the Agilent 2100 Bioanalyzer and quantitative real-time PCR (qRT-PCR). High-quality libraries were sequenced on the Illumina NovaSeq platform to generate 150 bp paired-end reads. Clean reads were aligned to the Ensembl mouse reference genome (mm10/GRCm38) using HISAT2 software (version 2.0.5). Differential expression analysis was conducted using the DESeq2 R package (version 1.20.0), and p-values were adjusted by the Benjamini–Hochberg method to control the false discovery rate (FDR). Genes with p adj ≤ 0.05 and |log₂(fold change)| ≥ 1 were defined as differentially expressed genes (DEGs). Furthermore, gene set enrichment analysis (GSEA) was performed based on the identified DEGs to evaluate the transcriptional regulation of lipid metabolism and adipose tissue browning pathways following SZ-A treatment. The GSEA analysis was carried out by Novogene (Beijing, China). Statistical Analysis All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 10. For normally distributed data, comparisons between two groups were made using Student’s t-test, while one-way analysis of variance (ANOVA) followed by post-hoc tests was used for multiple-group comparisons. Repeated-measures ANOVA was used for time-course data. A value of p < 0.05 was considered statistically significant. Results SZ-A reduces body weight gain and adipose accumulation induced by HFD During the 20-week feeding experiment, mice in the HFD group exhibited a significantly higher body weight compared with those fed a standard diet (SD) (p < 0.0001) (Fig. 1 A). Following 6 weeks of SZ-A administration starting from week 14, body weight gain in HFD-fed mice was markedly suppressed in a dose-dependent manner, with the high-dose SZ-A group (600 mg/kg) showing the more pronounced effect (p < 0.0001) (Fig. 1 A–B). Importantly, SZ-A treatment did not significantly affect the average weekly food intake (p = 0.0573) (Fig. 1 C), indicating that SZ-A exerts a significant anti–adipose-based obesity effect and this action occurs beyond the regulation of food intake. To determine whether the attenuation of body weight gain was associated with reduced adipose accumulation, the mass of various adipose depots was measured. Compared with the SD group, HFD feeding significantly increased the fat/body weight ratio of eWAT, inguinal WAT (iWAT), and BAT (p < 0.0001), whereas SZ-A administration markedly reduced the relative weights of these adipose depots (p < 0.05) (Fig. 1 D–F). Moreover, SZ-A treatment significantly reduced the weight of liver and kidneys (p < 0.05) (Fig. 1 G–H), consistent with the overall trend of body weight reduction. Previous studies have demonstrated that HFD commonly leads to hepatic lipid accumulation and steatosis [ 24 ]. Consistent with these findings, Oil Red O staining revealed a marked increase in both the number and size of lipid droplets in hepatocytes of HFD-fed mice (p < 0.0001). In contrast, SZ-A treatment notably alleviated hepatic lipid accumulation in a dose-dependent manner, with the high-dose group showing the most prominent improvement (p < 0.0001) (Fig. 1 I–J). These findings indicate that SZ-A effectively protects against HFD-induced hepatic steatosis and excessive fat deposition. SZ-A improves glucose and lipid metabolism disorders in HFD-fed mice To further evaluate the metabolic effects of SZ-A, parameters related to glucose and lipid homeostasis were assessed. The ipGTT demonstrated that HFD-fed mice exhibited significantly higher blood glucose levels after glucose challenge and an increased area under the curve (AUC) compared with SD group (p < 0.0001), indicating impaired glucose tolerance. In contrast, SZ-A administration reduced the blood glucose peak, accelerated glucose clearance, and significantly decreased the AUC (p < 0.05) (Fig. 2A–B), suggesting that SZ-A effectively ameliorated HFD-induced glucose intolerance. Similarly, results from the ipITT revealed that HFD-fed mice displayed blunted responses to insulin stimulation, as evidenced by a smaller glucose reduction and a significantly higher AUC (p < 0.0001), indicating insulin resistance. High-dose SZ-A treatment led to a greater decline in blood glucose and a significantly reduced AUC (p < 0.01) (Fig. 2C–D), demonstrating enhanced insulin sensitivity in obese mice. Consistent with the reduction in liver mass and alleviation of fat accumulation, high-dose SZ-A treatment significantly lowered fasting blood glucose levels (p < 0.05) (Fig. 2E). In terms of lipid metabolism, serum levels of TC, TG, LDL-C, and HDL-C were all significantly elevated in HFD-fed mice relative to SD group (p < 0.05), indicating pronounced dyslipidemia (Fig. 2F–H). High-dose SZ-A administration significantly reduced these elevated lipid parameters (p < 0.01) (Fig. 2F–H), suggesting a corrective effect on HFD-induced lipid abnormalities. Collectively, these results demonstrate that SZ-A exerts systemic regulatory effects on glucose and lipid metabolism in HFD-fed mice by improving insulin resistance and enhancing insulin sensitivity, thereby highlighting its therapeutic potential in the prevention and treatment of obesity and related metabolic syndromes. SZ-A promotes browning of WAT in HFD-fed mice To further investigate the effects of SZ-A on adipose tissue, histological and molecular analyses were performed on iWAT and eWAT samples. H&E staining revealed that adipocytes in the HFD group displayed markedly enlarged lipid droplets and reduced cell numbers per unit area compared with the SD group (p < 0.0001). In contrast, SZ-A treatment significantly attenuated adipocyte hypertrophy in both iWAT and eWAT, with a more pronounced reduction observed in the high-dose group (p < 0.001) (Fig. 3 A–B). These findings indicate that SZ-A effectively prevents HFD-induced adipocyte enlargement. Regarding thermogenic activity, IHC staining showed a substantial reduction in the UCP1-positive area in both iWAT and eWAT of HFD-fed mice (p < 0.001). High-dose SZ-A administration significantly increased UCP1 staining intensity and positive area (p < 0.05) (Fig. 3 A, 3 C), suggesting enhanced browning of WAT upon SZ-A treatment. At the molecular level, HFD feeding resulted in a significant downregulation of most thermogenic and beige adipocyte marker genes—including Ucp1, peroxisome proliferator-activated receptor gamma (Pparγ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α), PR domain containing 16 (Prdm16), cluster of differentiation 137 (Cd137), and cell death-inducing DFFA-like effector A (Cidea)—in iWAT and eWAT (p < 0.001), whereas transmembrane protein 26 (Tmem26) expression was not significantly altered compared with SD controls. SZ-A treatment significantly reversed these changes, with the high-dose group exhibiting the most pronounced upregulation, particularly for the key transcriptional regulators Ucp1, Pgc-1α, and Prdm16 (p < 0.05) (Fig. 3 D–E). SZ-A improves BAT morphology and enhances thermogenic activity in HFD-fed mice Under certain conditions, BAT can acquire cellular characteristics of WAT, characterized by mitochondrial dysfunction and lipid droplet accumulation—a phenomenon known as BAT “whitening” [ 25 – 27 ]. To further investigate the role of SZ-A in adipose tissue energy metabolism, histological and molecular analyses were performed on BAT samples from mice. Histologically, H&E staining revealed that BAT from HFD-fed mice displayed markedly enlarged lipid droplets, loose cellular organization, and disrupted tissue architecture, indicative of a whitening phenotype. In contrast, SZ-A treatment markedly reduced lipid accumulation and improved tissue morphology, with the high-dose group showing a more pronounced effect (Fig. 4 A). IHC staining of UCP1 demonstrated a significant reduction in UCP1-positive areas in the HFD group, whereas SZ-A administration notably restored UCP1 expression and positive staining regions (Fig. 4 A), suggesting recovery of BAT thermogenic activity. At the molecular level, qPCR analysis showed that the expression of several thermogenic and browning-related genes (Ucp1, Pgc-1α, Pparγ, Prdm16, Cidea, and Cd137) was significantly downregulated in BAT of HFD mice compared with the SD group (p < 0.05). SZ-A treatment, particularly at high doses, markedly reversed these changes (p < 0.05) (Fig. 4 B). Furthermore, Western blot analysis confirmed that UCP1 protein levels were significantly elevated in the high-dose SZ-A group (600 mg/kg) compared with HFD group (Fig. 4 C). SZ-A promotes browning of 3T3-L1 adipocytes in vitro To further determine whether SZ-A exerts similar browning effects in vitro, 3T3-L1 preadipocytes were used as a cellular model. The potential cytotoxicity of SZ-A was first evaluated using the CCK-8 assay after 72-hour exposure to increasing concentrations (6.25–400 µg/mL). As shown in Fig. 5 A, SZ-A exhibited no cytotoxicity up to 50 µg/mL, whereas cell viability significantly decreased at 100 µg/mL and above (p < 0.05). Therefore, concentrations of 50 and 100 µg/mL were selected as safe and effective doses for subsequent experiments. Oil Red O staining demonstrated that lipid droplet accumulation was reduced in SZ-A–treated adipocytes in a dose-dependent manner (Fig. 5 B). Quantitative analysis further confirmed that 100 µg/mL SZ-A significantly decreased Oil Red O staining intensity compared with the control group (p < 0.05) (Fig. 5 C), indicating reduced intracellular lipid storage. At the molecular level, qPCR analysis revealed that treatment with 100 µg/mL SZ-A significantly upregulated the expression of beige adipocyte marker genes—Cidea and cytochrome c oxidase subunit 8B (Cox8b)—as well as thermogenic regulators Ucp1, Pgc-1α, and Pparγ (p < 0.05) (Fig. 5 D). The expression of Prdm16 showed an increasing trend but did not reach statistical significance. Transcriptomic profiling and pathway enrichment analysis of eWAT To further elucidate the metabolic regulatory mechanisms of SZ-A, RNA sequencing was performed on eWAT from HFD and high-dose SZ-A–treated HFD mice (n = 5 per group). Principal component analysis (PCA) revealed a clear separation between the two groups (Fig. 6A), indicating substantial transcriptional reprogramming in response to SZ-A treatment. Hierarchical clustering analysis further confirmed pronounced transcriptomic divergence (Fig. 6B), while Venn diagram analysis identified 669 genes uniquely expressed in the HFD + SZ-A group (Fig. 6C). Volcano plot analysis identified 1,604 upregulated and 1,703 downregulated genes (p adj ≤ 0.05, |log₂FC| ≥ 1) (Fig. 6D). GSEA demonstrated significant upregulation of multiple lipid metabolism–related pathways, including fatty acid catabolism, fatty acid oxidation, and lipid oxidation (NES ≈ 1.8–2.0, FDR < 0.2), suggesting that SZ-A enhances fatty acid degradation and mitochondrial oxidative metabolism (Fig. 6E). In addition, a positive enrichment trend was observed for the brown fat cell differentiation pathway (NES = 1.61, FDR = 0.21), implying that SZ-A may promote the browning of white adipose tissue. Collectively, these transcriptomic findings are consistent with histological and molecular evidence, supporting that SZ-A exerts anti-obesity effects by simultaneously promoting lipid metabolism and adipose tissue browning. Discussion The American Association of Clinical Endocrinology (AACE) redefined obesity in 2017 as an adiposity-based chronic disease (ABCD), recognizing it as a chronic, heterogeneous neurohormonal and metabolic disorder. Its hallmark feature is excess or abnormally distributed adipose tissue that disrupts metabolic balance and impairs organ function. The development of obesity arises from complex interactions among genetic, environmental, and neuroendocrine factors, which collectively disturb energy homeostasis and drive pathological fat accumulation [ 28 ]. In recent years, enhancing adipose thermogenic capacity has emerged as a promising therapeutic approach. Both BAT activation and WAT browning dissipate energy through UCP1–mediated non-shivering thermogenesis, thereby counteracting lipid accumulation and improving systemic metabolic homeostasis [ 29 ]. Because obese individuals often exhibit diminished or absent BAT activity [ 4 ], natural compounds capable of inducing WAT browning under obese conditions have gained considerable interest as potential anti-obesity agents. In the present study, we demonstrated that SZ-A exerts potent anti-obesity effects in a HFD–induced mouse model. SZ-A treatment effectively suppressed body weight gain and fat accumulation in a dose-dependent manner, improved lipid profiles and glucose tolerance, and these effects were achieved primarily through enhanced energy expenditure rather than reduced energy intake. Combined evidence from histological, immunohistochemical, and molecular analyses revealed that SZ-A exerts dual regulatory effects on both WAT and BAT [ 30 ]. In WAT, SZ-A markedly reduced adipocyte hypertrophy, increased cell density, and upregulated thermogenic genes including Ucp1, Pgc-1α, and Prdm16, together with beige adipocyte markers Cidea, Cd137, and Tmem26. In BAT, SZ-A reversed HFD-induced “whitening,”restored compact cellular morphology, and significantly enhanced UCP1 expression. These findings suggest that SZ-A establishes a dual energy-dissipating mechanism by promoting WAT browning and alleviating BAT whitening, thereby mitigating obesity and related metabolic dysfunction. Mechanistically, the metabolic regulatory effects of SZ-A likely involve coordinated modulation of lipogenic and thermogenic pathways, consistent with reports on other plant-derived bioactive compounds [ 10 ]. Previous studies have shown that SZ-A confers broad metabolic benefits, including protection of pancreatic β-cells, improvement of insulin resistance and chronic inflammation, reshaping of gut microbiota, promotion of glucagon-like peptide-1 secretion, and reduction of hepatic lipid accumulation, with potential cardiovascular and renal protection [ 10 – 14 ]. Interestingly, SZ-A has also been reported to promote adipogenesis under specific conditions, suggesting that its effects are context-dependent and may act bidirectionally to maintain energy homeostasis[ 13 ]. In our study, SZ-A improved dyslipidemia characterized by elevated TG, TC, and LDL-C levels, alleviated hepatic lipid accumulation, and reduced liver mass. Concurrently, it significantly upregulated thermogenic and beige adipocyte markers in WAT—Ucp1, Cidea, and Tmem26—as well as key transcriptional regulators Pgc-1α and Prdm16. PGC-1α, a master regulator of mitochondrial biogenesis, acts synergistically with PRDM16 to drive the expression of brown/beige adipocyte-specific genes and activate UCP1-mediated thermogenesis, thereby enhancing energy expenditure [ 31 – 33 ]. Moreover, the browning-promoting effects of SZ-A were confirmed in vitro. In differentiated 3T3-L1 adipocytes, non-cytotoxic concentrations of SZ-A (≤ 100 µg/mL) significantly increased the expression of thermogenic genes (Ucp1, Pgc-1α, Pparγ) and beige markers (Cidea, Cox8b), indicating that SZ-A directly induces adipocyte conversion toward a metabolically active, beige-like phenotype, independent of systemic effects. The consistent in vivo and in vitro results reinforce the robustness of SZ-A’s browning and anti-obesity effects. Collectively, these findings suggest that SZ-A may activate adipose thermogenesis through the PRDM16–PGC-1α–UCP1 signaling axis, enhancing mitochondrial activity and energy dissipation in both WAT and BAT. Furthermore, transcriptomic analysis revealed that SZ-A profoundly remodeled the eWAT gene expression landscape. GSEA showed significant activation of pathways related to fatty acid catabolism, β-oxidation, and lipid oxidation, along with a positive enrichment of the brown adipocyte differentiation pathway. These results suggest that SZ-A enhances mitochondrial oxidative metabolism and promotes adipose browning at the transcriptional level, providing mechanistic insight into its ability to improve insulin resistance and enhance insulin sensitivity. Compared with other natural compounds, SZ-A offers a distinct translational advantage—its clinical approval for T2DM provides a well-established safety and pharmacological foundation. Given the shared pathological basis of T2DM and obesity, including adipose dysfunction and impaired energy metabolism, our findings that SZ-A simultaneously improves glucose tolerance and lipid profiles suggest a multifaceted mechanism integrating inhibition of adipogenesis, activation of thermogenesis, and restoration of energy balance. Thus, SZ-A emerges as a promising multi-target therapeutic candidate for metabolic syndrome and adipose-based obesity. In conclusion, this study systematically demonstrates that SZ-A counteracts HFD-induced obesity and metabolic disturbances through a dual mechanism involving inhibition of lipid accumulation and activation of thermogenic pathways. By promoting WAT browning and enhancing BAT thermogenesis, SZ-A effectively improves energy metabolism and insulin sensitivity. Considering its established clinical use in T2DM, SZ-A holds strong translational potential as a safe and effective therapeutic agent for the integrated management of adipose-based obesity and metabolic syndrome. Conclusion In summary, SZ-A exerts potent anti–adipose-based effects in HFD–induced obese mice, characterized by suppressed body weight gain, reduced adipose and liver mass, and improved hepatic lipid accumulation, lipid profiles, and glucose tolerance. Mechanistically, SZ-A enhances adipose metabolic activity by promoting WAT browning and preventing BAT whitening during obesity progression, accompanied by morphological improvements and upregulation of key thermogenic markers. In vitro, SZ-A directly activated the expression of genes associated with browning and energy expenditure in adipocytes, thereby enhancing thermogenic capacity. Transcriptomic profiling further revealed that SZ-A profoundly remodeled the WAT gene expression landscape, significantly activating pathways related to fatty acid metabolism, lipid oxidation, and brown adipocyte differentiation. These findings provide strong transcriptomic evidence supporting its adipose-targeted anti-obesity effects, which collectively contribute to improved insulin resistance and enhanced insulin sensitivity. Given its established clinical application in the treatment of T2DM, SZ-A represents a promising multitarget therapeutic candidate for adipose-based obesity and associated metabolic disorders. Future investigations integrating comprehensive energy metabolism analyses, mechanistic pathway validation, and clinical evaluation of tissue- and organ-level protection are warranted to further elucidate its molecular basis and long-term efficacy. Declarations Acknowledgments We would like to express our gratitude to Public Laboratory Platform of West China Hospital and Animal Experiment Center of West China Hospital for their assistance and support throughout the course of this research. Author contributions R.Z. and N.T. conceived and designed the study. R.Z. performed the animal and cell experiments, analyzed the data, and drafted the main manuscript text. G.P. assisted with data visualization and prepared Figures 6. X.P. and N.T. supervised the project and contributed to funding acquisition. R.Z., G.P., and X.P. revised the manuscript. All authors reviewed and approved the final version of the manuscript. Funding This research was funded by the grants from the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (No. ZYGD 18017) and Natural Science Foundation of Sichuan Province, Grant No. 2024NSFSC1620. Data Availability Statement The data supporting the findings of this study are available from the corresponding author upon request. Conflicts of Interest The authors declare no conflicts of interest. References Rubino, F. et al. Definition and diagnostic criteria of clinical obesity. Lancet Diabetes Endocrinol. 13 , 221–262. https://doi.org/10.1016/S2213-8587(24)00316-4 (2025). Chait, A. & den Hartigh, L. J. Adipose Tissue Distribution, Inflammation and Its Metabolic Consequences, Including Diabetes and Cardiovascular Disease. Front. Cardiovasc. 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Biol. 17 , 480–495. https://doi.org/10.1038/nrm.2016.62 (2016). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 Jan, 2026 Reviews received at journal 09 Jan, 2026 Reviews received at journal 27 Dec, 2025 Reviewers agreed at journal 20 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers invited by journal 06 Dec, 2025 Editor assigned by journal 06 Dec, 2025 Editor invited by journal 01 Dec, 2025 Submission checks completed at journal 27 Nov, 2025 First submitted to journal 27 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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16:33:07","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108178,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/60108cc41378ff26be535de0.html"},{"id":97987169,"identity":"d406d28d-81c6-4814-aeba-4ffdfe86abfc","added_by":"auto","created_at":"2025-12-11 13:57:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":345998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of SZ-A on HFD-induced body weight gain, fat accumulation, and hepatic lipid deposition. (A) Body weight changes over 20 weeks in mice fed SD, HFD, HFD + SZ-A (200 mg/kg), and HFD + SZ-A (600 mg/kg); (B) Total body weight gain; (C) Average weekly food intake; (D–F) Ratios of eWAT, iWAT, and BAT to body weight; (G–H) Liver and kidney weights; (I) Representative images of Oil Red O staining in liver sections (scale bar = 100 μm); (J) Quantitative analysis of hepatic lipid deposition area. Data are expressed as mean ± SEM (n = 5–6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Significance levels: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 compared with HFD; ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/82a99908e04c26c208692ebc.png"},{"id":98423405,"identity":"a0319123-ceb6-4845-9712-0c90c2dce3fd","added_by":"auto","created_at":"2025-12-17 16:32:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZ-A improves HFD-induced glucose intolerance and dyslipidemia. (A) ipGTT blood glucose curves; (B) AUC of ipGTT; (C) ipITT blood glucose curves; (D) AUC of ipITT; (E) Fasting blood glucose levels; (F) Serum HDL levels; (G) Serum LDL levels; (H) Serum TC levels; (I) Serum TG levels. Data are presented as mean ± SEM (n = 5–6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Significance levels: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 compared with HFD; ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/891348502cbc26ad4560d838.png"},{"id":97987168,"identity":"63bacc65-139b-47a8-ab0c-844e0deefb9c","added_by":"auto","created_at":"2025-12-11 13:57:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZ-A alleviates HFD-induced adipocyte hypertrophy and promotes the expression of thermogenesis-related genes in white adipose tissue. (A) H\u0026amp;E staining and UCP1 immunohistochemistry of iWAT and eWAT (scale bar = 200 μm); (B) Quantitative analysis of lipid droplet area in eWAT and iWAT; (C) Percentage of UCP1-positive staining area in eWAT and iWAT; (D) Relative mRNA expression levels of thermogenic and browning-related genes (Cidea, Tmem26, Cd137, Ucp1, Pgc-1α, Pparγ, Prdm16) in eWAT; (E) Relative mRNA expression levels of thermogenic and browning-related genes in iWAT. Data are presented as mean ± SEM (n = 5–6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison correction. Significance levels: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 compared with HFD; ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/f1250ef26a556e2f0f4df2be.png"},{"id":98425401,"identity":"7a3bfe88-98d7-412c-9958-bfb244a47405","added_by":"auto","created_at":"2025-12-17 16:34:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":384893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSZ-A ameliorates HFD-induced metabolic abnormalities in BAT and regulates the expression of browning-related genes. (A) IHC staining of UCP1 and H\u0026amp;E staining of BAT (scale bar = 100 μm); (B) Relative mRNA expression levels of thermogenic and browning-related genes (Cidea, Cd137, Ucp1, Pgc-1α, Pparγ, Prdm16) in BAT; (C) Western blot analysis of UCP1 protein expression in BAT, with β-tubulin as the loading control. Bands were obtained from the same membrane but were non-adjacent; boundaries are indicated by dividing lines. Data are presented as mean ± SEM (n = 5–6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison correction. Significance levels: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 compared with HFD; ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/420cc8ffa8e7e2120c56c8d9.png"},{"id":98424231,"identity":"76b3b799-d47c-415a-a437-2e56f289cb27","added_by":"auto","created_at":"2025-12-17 16:33:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":311487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrowning effects of SZ-A in 3T3-L1 adipocytes. (A) CCK-8 assay showing the effect of SZ-A treatment for 72 h on cell viability; (B) Oil Red O staining of control and SZ-A-treated 3T3-L1 adipocytes (scale bar = 50 μm); (C) Quantification of lipid levels in control and SZ-A-treated 3T3-L1 adipocytes; (D) Relative mRNA expression levels of thermogenic and browning-related genes (Cidea, Cox8b, Ucp1, Pgc-1α, Pparγ, Prdm16). Data are presented as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison correction. Significance levels: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 compared with HFD; #p \u0026lt; 0.05, ##p \u0026lt; 0.01 compared with 0 μg/ml group; ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/1b4389bb3014e70c4fe1c582.png"},{"id":98424792,"identity":"e574e720-4929-4e47-8a91-800537c80e1a","added_by":"auto","created_at":"2025-12-17 16:33:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":163680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic and GSEA analyses of eWAT from HFD and high-dose SZ-A–treated HFD mice. (A) PCA showing distinct clustering between HFD (blue) and high-dose SZ-A–treated HFD (red) groups. (B) Heatmap of differentially expressed genes DEGs between the two groups. Red and green colors represent upregulated and downregulated genes, respectively. (C) Venn diagram displaying the overlap and unique gene expression profiles in HFD and high-dose SZ-A–treated HFD groups. (D) Volcano plot illustrating the distribution of DEGs between high-dose SZ-A–treated HFD and HFD mice (p\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eadj\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e ≤ 0.05, |log₂FC| ≥ 1), with red and green dots indicating upregulated and downregulated genes, respectively. (E) GSEA bubble plot showing significantly enriched pathways related to lipid metabolism and adipose tissue browning.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/0f0dfc06b03949774532d5a7.png"},{"id":105755991,"identity":"70b8de44-2e4f-404e-8efb-b1a9ec15859d","added_by":"auto","created_at":"2026-03-30 16:33:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3873929,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8094331/v1/fba6b8e4-51fc-47af-9794-51ffc51deb55.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ramulus Mori (Sangzhi) Alkaloids Attenuate Diet- Induced Obesity by Promoting White Adipose Browning and Enhancing Brown Fat Thermogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity has become a major global health concern, with its prevalence continuing to rise across all age groups and regions. Clinical obesity is defined as a chronic, systemic illness characterized by alterations in the function of tissues, organs, the entire individual, or a combination thereof, due to excess adiposity. It can lead to severe end-organ damage, causing life-altering and potentially life-threatening complications such as myocardial infarction, stroke, and renal failure[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Based on morphological and functional characteristics, adipose tissue is generally categorized into WAT, BAT, and beige adipose tissue (BeAT) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. WAT primarily stores energy in the form of unilocular lipid droplets and mobilizes fatty acids during energy demand. In contrast, BAT is characterized by multilocular lipid droplets and abundant mitochondria, with high expression of UCP1, which mediates non-shivering thermogenesis by dissipating energy as heat to increase whole-body energy expenditure and maintain body temperature [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In obese individuals, BAT mass and thermogenic activity are markedly reduced or even absent [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], whereas WAT exhibits hypertrophy and excessive accumulation.\u003c/p\u003e\u003cp\u003eNotably, the boundary between WAT and BAT can become functionally ambiguous under certain circumstances. Upon cold exposure [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] or β-adrenergic receptor agonists [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], adipocytes of WAT can exhibit brown fat-like features, including increased number of mitochondria and elevated UCP1 expression\u0026mdash;a process known as \u0026ldquo;browning\u0026rdquo;. The resulting beige adipocytes have been identified in both humans and rodents [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Conversely, BAT can acquire white fat-like characteristics such as enlarged lipid droplets, reduced mitochondrial content, and downregulated UCP1 expression, referred to as \u0026ldquo;whitening\u0026rdquo;, which commonly occurs in obese individuals and worsens with obesity severity. Given the limited BAT mass in adults, promoting WAT browning is considered a promising strategy to enhance thermogenic potential, improve energy metabolism, and combat obesity and its associated metabolic complications [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSZ-A are a group of natural alkaloid compounds derived from \u003cem\u003eMorus alba L.\u003c/em\u003e (mulberry twig), accounting for more than 50% of the total extract. The major bioactive constituents include 1-deoxynojirimycin (1-DNJ), fagomine (FA), and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB). SZ-A has been clinically approved for the treatment of T2DM in China since 2020 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Previous studies have shown that SZ-A is rapidly absorbed and distributed to multiple metabolic target tissues, where it exerts broad metabolic benefits, including improved glucose homeostasis, reduced systemic inflammation, and regulated lipid metabolism [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Adipose tissue is the primary target of anti-obesity treatment. Several natural compounds have been reported to induce WAT browning, thereby exerting anti-obesity effects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20 CR21 CR22\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, it remains unclear whether SZ-A can promote WAT browning and enhance BAT thermogenic activity, either directly or indirectly.\u003c/p\u003e\u003cp\u003eIn this study, we systematically evaluated the anti-obesity potential of SZ-A and its regulatory effects on adipose tissue browning both in vivo and in vitro. Our results demonstrate that SZ-A effectively promoted WAT browning and enhanced BAT thermogenic activity, ameliorated HFD-induced obesity and metabolic disturbances beyond food intake.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eChemicals and Reagents\u003c/h2\u003e\n \u003cp\u003eSZ-A powder was kindly provided by Beijing Wehand-Bio Pharmaceutical Co., Ltd. (Beijing, China). For experimental use, SZ-A was dissolved in saline for oral gavage in animals or in cell culture medium to prepare designated concentrations for in vitro studies.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAnimals and Treatment\u003c/h3\u003e\n\u003cp\u003eMale C57BL/6 mice (7 weeks old, 20\u0026ndash;25 g) were purchased from Chengdu Dossy Biological Technology Co., Ltd. (Chengdu, China). Mice were housed under controlled conditions (n\u0026thinsp;=\u0026thinsp;3 per Cage, 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 12 h light/dark cycle, 50\u0026ndash;60% humidity) with free access to food and water. After one-week acclimatization period, mice were randomly divided into two groups: standard diet (SD; 10% fat, 20% protein, 70% carbohydrate; n\u0026thinsp;=\u0026thinsp;6) or high-fat diet (HFD; 60% fat, 20% protein, 20% carbohydrate; n\u0026thinsp;=\u0026thinsp;18) (XTHF60 vs XTCON50J, Xietong Shengwu, Jiangsu, China) to induce obesity. After 14 weeks of feeding, HFD-fed mice were further randomized into three groups (n\u0026thinsp;=\u0026thinsp;6 per group): HFD control (HFD), low-dose SZ-A group (HFD\u0026thinsp;+\u0026thinsp;SZL; 200 mg/kg/day), and high-dose SZ-A group (HFD\u0026thinsp;+\u0026thinsp;SZH; 600 mg/kg/day). The SD and HFD control groups received equivalent volumes of normal saline by oral gavage, while the SZ-A groups were administered high/low dose of SZ-A daily for 6 weeks. Body weight and food intake were recorded weekly. At the end of the experiment, all animals were anesthetized with isoflurane (RWD Life Science, China) delivered via inhalation. Mice were placed in an induction chamber and exposed to 5% isoflurane in oxygen for approximately 10 seconds until deep anesthesia was achieved, as confirmed by the absence of righting and pedal withdrawal reflexes. Euthanasia was performed under anesthesia in all animals. Specifically, mice were euthanized by cervical dislocation in accordance with the institutional guidelines and the AVMA Guidelines for the Euthanasia of Animals. Death was confirmed by the complete cessation of heartbeat, which was assessed visually and by gentle palpation of the thoracic area. Following confirmation of death, organs and tissue samples were dissected, weighed, immediately frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis. The study was carried out in compliance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003c/span\u003e). All animal procedures were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (Approval No. 20240926002).\u003c/p\u003e\n\u003ch3\u003eGlucose Tolerance Test and Insulin Tolerance Test\u003c/h3\u003e\n\u003cp\u003eTolerance tests were performed within 3 days after the completion SZ-A administration. Before intraperitoneal glucose tolerance test (ipGTT) or insulin tolerance test (ipITT), mice were fasted for 12 h. On the day of the experiment, mice were weighed and injected intraperitoneally with glucose (1 g/kg, dissolved in sterile saline) or insulin (0.5 U/kg, dissolved in sterile saline). Blood glucose levels were measured from tail vein samples at 0, 15, 30, 60, 90, and 120 min after injection using glucometers (Roche, Germany).\u003c/p\u003e\n\u003ch3\u003eCell Culture and Adipogenic Differentiation\u003c/h3\u003e\n\u003cp\u003eMouse 3T3-L1 preadipocytes were purchased from Wuhan Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China). Cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37\u0026deg;C in a humidified incubator containing 5% CO₂. For adipogenic differentiation, 3T3-L1 preadipocytes were cultured until full confluence and maintained for 2 days to achieve contact inhibition. Differentiation was then induced by incubation in DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 \u0026micro;M dexamethasone, 1 \u0026micro;M rosiglitazone, and 2 \u0026micro;g/mL insulin for 3 days. The medium was then replaced with DMEM containing 10% FBS, 1 \u0026micro;M rosiglitazone, and 2 \u0026micro;g/mL insulin for 4 days, followed by maintenance in DMEM with 10% FBS for another 2 days to complete differentiation.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eCell Viability Assay\u003c/h2\u003e\n \u003cp\u003e3T3-L1 preadipocytes were seeded into 96-well plates at a density of 4\u0026ndash;6 \u0026times; 10⁴ cells/mL in 100 \u0026micro;L complete medium per well. After incubation for 12 h at 37\u0026deg;C and 5% CO₂ atmosphere, the medium was replaced with 200 \u0026micro;L of complete medium containing various concentrations of SZ-A (6.25, 12.5, 25, 50, 100, 200, and 400 \u0026micro;g/mL). Normal control and blank wells were included, with 6 replicates per group. After 24, 48, and 72 h of incubation, 100 \u0026micro;L of CCK-8 working solution (GlpBio, USA) was added to each well and incubated for 1 h at 37\u0026deg;C. The optical density (OD) was then measured at 450 nm using a multifunctional microplate reader (BioTek, USA), and cell viability was calculated based on OD values relative to the control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eOil Red O Staining\u003c/h3\u003e\n\u003cp\u003eThe Oil Red O stock solution was prepared by dissolving 300 mg of Oil Red O powder (Sigma, USA) in 100 mL of 100% isopropanol and stored at 4\u0026deg;C in the dark. The working solution (0.2%) was freshly prepared by mixing the stock solution with ultrapure water at a ratio of 3:2, followed by filtration. After differentiation, 3T3-L1 adipocytes were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. The fixed cells were washed with ultrapure water, incubated with 60% isopropanol for 2 min, and then stained with 0.2% Oil Red O working solution at room temperature for 10 min. Following staining, cells were washed thoroughly with water until the rinse became clear. Stained lipid droplets were observed under an inverted microscope (Olympus BX53M, Japan). For quantitative analysis, the retained dye was eluted with isopropanol, and absorbance was measured at 520 nm using a spectrophotometer.\u003c/p\u003e\n\u003ch3\u003eQuantitative PCR Analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent (Oriscience, China) and reverse-transcribed into cDNA using a reverse transcription kit (Vazyme, China). Quantitative PCR was performed using SYBR Green Master Mix (Vazyme, China) on a real-time PCR system. \u0026beta;-actin served as the internal control for normalization. Primer sequences are listed in S1 Table.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eHistological and Immunohistochemical Analysis\u003c/h2\u003e\n \u003cp\u003eBAT and WAT samples were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned. For histological evaluation, tissue sections were deparaffinized and stained with hematoxylin and eosin (H\u0026amp;E). Immunohistochemistry (IHC) was performed to detect UCP1 expression in BAT and WAT using an anti-UCP1 antibody (Abcam, UK). Hepatic lipid accumulation was assessed by Oil Red O staining. Liver tissues were rinsed with PBS, embedded in OCT compound, and sectioned at a thickness of 8\u0026ndash;10 \u0026micro;m. The frozen sections were fixed with 4% paraformaldehyde, stained with Oil Red O working solution, counterstained with hematoxylin, rinsed, and mounted. Images were acquired using the SLIDEVIEW VS200 scanner (Olympus, Japan) and quantitatively analyzed with ImageJ software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern Blotting\u003c/h2\u003e\n \u003cp\u003eAdipose tissues were lysed in RIPA buffer (Beyotime Biotech, China) containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Biotech, China). Equal amounts of protein samples were separated by 4\u0026ndash;12% SDS\u0026ndash;polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight at 4\u0026deg;C with primary antibodies, including \u0026beta;-tubulin (Affinity Biosciences, China) and UCP1 (HuaAn Biotechnology, China). After washing, the membranes were incubated for 1 h at room temperature with secondary antibodies\u0026mdash;anti-rabbit IgG (Bioswamp, China) or anti-mouse IgG (Proteintech, USA). Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Oriscience, China) and analyzed with a Chemi-Doc imaging system (E-Blot, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA Sequencing and Data Analysis\u003c/h2\u003e\n \u003cp\u003eTanscriptomic sequencing of epididymal WAT (eWAT) was performed by Novogene Co., Ltd. (Beijing, China). Total RNA was extracted from eWAT using TRIzol reagent (Invitrogen, USA). RNA concentration and integrity were examined with the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) to ensure that the samples met the quality requirements for library construction. Poly(A)-tailed mRNA was enriched from total RNA using oligo(dT)-attached magnetic beads, followed by fragmentation and cDNA synthesis. The obtained cDNA fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, MA, USA) and amplified by PCR to generate the final sequencing libraries. Library quality was assessed using the Agilent 2100 Bioanalyzer and quantitative real-time PCR (qRT-PCR). High-quality libraries were sequenced on the Illumina NovaSeq platform to generate 150 bp paired-end reads. Clean reads were aligned to the Ensembl mouse reference genome (mm10/GRCm38) using HISAT2 software (version 2.0.5). Differential expression analysis was conducted using the DESeq2 R package (version 1.20.0), and p-values were adjusted by the Benjamini\u0026ndash;Hochberg method to control the false discovery rate (FDR). Genes with p\u003csub\u003eadj\u003c/sub\u003e \u0026le; 0.05 and |log₂(fold change)| \u0026ge; 1 were defined as differentially expressed genes (DEGs). Furthermore, gene set enrichment analysis (GSEA) was performed based on the identified DEGs to evaluate the transcriptional regulation of lipid metabolism and adipose tissue browning pathways following SZ-A treatment. The GSEA analysis was carried out by Novogene (Beijing, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 10. For normally distributed data, comparisons between two groups were made using Student\u0026rsquo;s t-test, while one-way analysis of variance (ANOVA) followed by post-hoc tests was used for multiple-group comparisons. Repeated-measures ANOVA was used for time-course data. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eSZ-A reduces body weight gain and adipose accumulation induced by HFD\u003c/h2\u003e\n \u003cp\u003eDuring the 20-week feeding experiment, mice in the HFD group exhibited a significantly higher body weight compared with those fed a standard diet (SD) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following 6 weeks of SZ-A administration starting from week 14, body weight gain in HFD-fed mice was markedly suppressed in a dose-dependent manner, with the high-dose SZ-A group (600 mg/kg) showing the more pronounced effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;B). Importantly, SZ-A treatment did not significantly affect the average weekly food intake (p\u0026thinsp;=\u0026thinsp;0.0573) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating that SZ-A exerts a significant anti\u0026ndash;adipose-based obesity effect and this action occurs beyond the regulation of food intake.\u003c/p\u003e\n \u003cp\u003eTo determine whether the attenuation of body weight gain was associated with reduced adipose accumulation, the mass of various adipose depots was measured. Compared with the SD group, HFD feeding significantly increased the fat/body weight ratio of eWAT, inguinal WAT (iWAT), and BAT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas SZ-A administration markedly reduced the relative weights of these adipose depots (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;F). Moreover, SZ-A treatment significantly reduced the weight of liver and kidneys (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG\u0026ndash;H), consistent with the overall trend of body weight reduction.\u003c/p\u003e\n \u003cp\u003ePrevious studies have demonstrated that HFD commonly leads to hepatic lipid accumulation and steatosis [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consistent with these findings, Oil Red O staining revealed a marked increase in both the number and size of lipid droplets in hepatocytes of HFD-fed mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, SZ-A treatment notably alleviated hepatic lipid accumulation in a dose-dependent manner, with the high-dose group showing the most prominent improvement (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026ndash;J). These findings indicate that SZ-A effectively protects against HFD-induced hepatic steatosis and excessive fat deposition.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eSZ-A improves glucose and lipid metabolism disorders in HFD-fed mice\u003c/h2\u003e\n \u003cp\u003eTo further evaluate the metabolic effects of SZ-A, parameters related to glucose and lipid homeostasis were assessed. The ipGTT demonstrated that HFD-fed mice exhibited significantly higher blood glucose levels after glucose challenge and an increased area under the curve (AUC) compared with SD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating impaired glucose tolerance. In contrast, SZ-A administration reduced the blood glucose peak, accelerated glucose clearance, and significantly decreased the AUC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;2A\u0026ndash;B), suggesting that SZ-A effectively ameliorated HFD-induced glucose intolerance. Similarly, results from the ipITT revealed that HFD-fed mice displayed blunted responses to insulin stimulation, as evidenced by a smaller glucose reduction and a significantly higher AUC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating insulin resistance. High-dose SZ-A treatment led to a greater decline in blood glucose and a significantly reduced AUC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;2C\u0026ndash;D), demonstrating enhanced insulin sensitivity in obese mice. Consistent with the reduction in liver mass and alleviation of fat accumulation, high-dose SZ-A treatment significantly lowered fasting blood glucose levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;2E).\u003c/p\u003e\n \u003cp\u003eIn terms of lipid metabolism, serum levels of TC, TG, LDL-C, and HDL-C were all significantly elevated in HFD-fed mice relative to SD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating pronounced dyslipidemia (Fig.\u0026nbsp;2F\u0026ndash;H). High-dose SZ-A administration significantly reduced these elevated lipid parameters (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;2F\u0026ndash;H), suggesting a corrective effect on HFD-induced lipid abnormalities.\u003c/p\u003e\n \u003cp\u003eCollectively, these results demonstrate that SZ-A exerts systemic regulatory effects on glucose and lipid metabolism in HFD-fed mice by improving insulin resistance and enhancing insulin sensitivity, thereby highlighting its therapeutic potential in the prevention and treatment of obesity and related metabolic syndromes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eSZ-A promotes browning of WAT in HFD-fed mice\u003c/h2\u003e\n \u003cp\u003eTo further investigate the effects of SZ-A on adipose tissue, histological and molecular analyses were performed on iWAT and eWAT samples. H\u0026amp;E staining revealed that adipocytes in the HFD group displayed markedly enlarged lipid droplets and reduced cell numbers per unit area compared with the SD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, SZ-A treatment significantly attenuated adipocyte hypertrophy in both iWAT and eWAT, with a more pronounced reduction observed in the high-dose group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B). These findings indicate that SZ-A effectively prevents HFD-induced adipocyte enlargement. Regarding thermogenic activity, IHC staining showed a substantial reduction in the UCP1-positive area in both iWAT and eWAT of HFD-fed mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). High-dose SZ-A administration significantly increased UCP1 staining intensity and positive area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC), suggesting enhanced browning of WAT upon SZ-A treatment. At the molecular level, HFD feeding resulted in a significant downregulation of most thermogenic and beige adipocyte marker genes\u0026mdash;including Ucp1, peroxisome proliferator-activated receptor gamma (Ppar\u0026gamma;), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1\u0026alpha;), PR domain containing 16 (Prdm16), cluster of differentiation 137 (Cd137), and cell death-inducing DFFA-like effector A (Cidea)\u0026mdash;in iWAT and eWAT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas transmembrane protein 26 (Tmem26) expression was not significantly altered compared with SD controls. SZ-A treatment significantly reversed these changes, with the high-dose group exhibiting the most pronounced upregulation, particularly for the key transcriptional regulators Ucp1, Pgc-1\u0026alpha;, and Prdm16 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;E).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eSZ-A improves BAT morphology and enhances thermogenic activity in HFD-fed mice\u003c/h2\u003e\n \u003cp\u003eUnder certain conditions, BAT can acquire cellular characteristics of WAT, characterized by mitochondrial dysfunction and lipid droplet accumulation\u0026mdash;a phenomenon known as BAT \u0026ldquo;whitening\u0026rdquo; [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. To further investigate the role of SZ-A in adipose tissue energy metabolism, histological and molecular analyses were performed on BAT samples from mice.\u003c/p\u003e\n \u003cp\u003eHistologically, H\u0026amp;E staining revealed that BAT from HFD-fed mice displayed markedly enlarged lipid droplets, loose cellular organization, and disrupted tissue architecture, indicative of a whitening phenotype. In contrast, SZ-A treatment markedly reduced lipid accumulation and improved tissue morphology, with the high-dose group showing a more pronounced effect (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). IHC staining of UCP1 demonstrated a significant reduction in UCP1-positive areas in the HFD group, whereas SZ-A administration notably restored UCP1 expression and positive staining regions (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), suggesting recovery of BAT thermogenic activity.\u003c/p\u003e\n \u003cp\u003eAt the molecular level, qPCR analysis showed that the expression of several thermogenic and browning-related genes (Ucp1, Pgc-1\u0026alpha;, Ppar\u0026gamma;, Prdm16, Cidea, and Cd137) was significantly downregulated in BAT of HFD mice compared with the SD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). SZ-A treatment, particularly at high doses, markedly reversed these changes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Furthermore, Western blot analysis confirmed that UCP1 protein levels were significantly elevated in the high-dose SZ-A group (600 mg/kg) compared with HFD group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eSZ-A promotes browning of 3T3-L1 adipocytes in vitro\u003c/h2\u003e\n \u003cp\u003eTo further determine whether SZ-A exerts similar browning effects in vitro, 3T3-L1 preadipocytes were used as a cellular model. The potential cytotoxicity of SZ-A was first evaluated using the CCK-8 assay after 72-hour exposure to increasing concentrations (6.25\u0026ndash;400 \u0026micro;g/mL). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, SZ-A exhibited no cytotoxicity up to 50 \u0026micro;g/mL, whereas cell viability significantly decreased at 100 \u0026micro;g/mL and above (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, concentrations of 50 and 100 \u0026micro;g/mL were selected as safe and effective doses for subsequent experiments. Oil Red O staining demonstrated that lipid droplet accumulation was reduced in SZ-A\u0026ndash;treated adipocytes in a dose-dependent manner (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Quantitative analysis further confirmed that 100 \u0026micro;g/mL SZ-A significantly decreased Oil Red O staining intensity compared with the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating reduced intracellular lipid storage. At the molecular level, qPCR analysis revealed that treatment with 100 \u0026micro;g/mL SZ-A significantly upregulated the expression of beige adipocyte marker genes\u0026mdash;Cidea and cytochrome c oxidase subunit 8B (Cox8b)\u0026mdash;as well as thermogenic regulators Ucp1, Pgc-1\u0026alpha;, and Ppar\u0026gamma; (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). The expression of Prdm16 showed an increasing trend but did not reach statistical significance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eTranscriptomic profiling and pathway enrichment analysis of eWAT\u003c/h2\u003e\n \u003cp\u003eTo further elucidate the metabolic regulatory mechanisms of SZ-A, RNA sequencing was performed on eWAT from HFD and high-dose SZ-A\u0026ndash;treated HFD mice (n\u0026thinsp;=\u0026thinsp;5 per group). Principal component analysis (PCA) revealed a clear separation between the two groups (Fig.\u0026nbsp;6A), indicating substantial transcriptional reprogramming in response to SZ-A treatment. Hierarchical clustering analysis further confirmed pronounced transcriptomic divergence (Fig.\u0026nbsp;6B), while Venn diagram analysis identified 669 genes uniquely expressed in the HFD\u0026thinsp;+\u0026thinsp;SZ-A group (Fig.\u0026nbsp;6C). Volcano plot analysis identified 1,604 upregulated and 1,703 downregulated genes (p\u003csub\u003eadj\u003c/sub\u003e \u0026le; 0.05, |log₂FC| \u003cstrong\u003e\u0026ge;\u003c/strong\u003e 1) (Fig. 6D). GSEA demonstrated significant upregulation of multiple lipid metabolism\u0026ndash;related pathways, including fatty acid catabolism, fatty acid oxidation, and lipid oxidation (NES\u0026thinsp;\u0026asymp;\u0026thinsp;1.8\u0026ndash;2.0, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.2), suggesting that SZ-A enhances fatty acid degradation and mitochondrial oxidative metabolism (Fig. 6E). In addition, a positive enrichment trend was observed for the brown fat cell differentiation pathway (NES\u0026thinsp;=\u0026thinsp;1.61, FDR\u0026thinsp;=\u0026thinsp;0.21), implying that SZ-A may promote the browning of white adipose tissue. Collectively, these transcriptomic findings are consistent with histological and molecular evidence, supporting that SZ-A exerts anti-obesity effects by simultaneously promoting lipid metabolism and adipose tissue browning.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe American Association of Clinical Endocrinology (AACE) redefined obesity in 2017 as an adiposity-based chronic disease (ABCD), recognizing it as a chronic, heterogeneous neurohormonal and metabolic disorder. Its hallmark feature is excess or abnormally distributed adipose tissue that disrupts metabolic balance and impairs organ function. The development of obesity arises from complex interactions among genetic, environmental, and neuroendocrine factors, which collectively disturb energy homeostasis and drive pathological fat accumulation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In recent years, enhancing adipose thermogenic capacity has emerged as a promising therapeutic approach. Both BAT activation and WAT browning dissipate energy through UCP1\u0026ndash;mediated non-shivering thermogenesis, thereby counteracting lipid accumulation and improving systemic metabolic homeostasis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Because obese individuals often exhibit diminished or absent BAT activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], natural compounds capable of inducing WAT browning under obese conditions have gained considerable interest as potential anti-obesity agents.\u003c/p\u003e\u003cp\u003eIn the present study, we demonstrated that SZ-A exerts potent anti-obesity effects in a HFD\u0026ndash;induced mouse model. SZ-A treatment effectively suppressed body weight gain and fat accumulation in a dose-dependent manner, improved lipid profiles and glucose tolerance, and these effects were achieved primarily through enhanced energy expenditure rather than reduced energy intake. Combined evidence from histological, immunohistochemical, and molecular analyses revealed that SZ-A exerts dual regulatory effects on both WAT and BAT [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In WAT, SZ-A markedly reduced adipocyte hypertrophy, increased cell density, and upregulated thermogenic genes including Ucp1, Pgc-1α, and Prdm16, together with beige adipocyte markers Cidea, Cd137, and Tmem26. In BAT, SZ-A reversed HFD-induced \u0026ldquo;whitening,\u0026rdquo;restored compact cellular morphology, and significantly enhanced UCP1 expression. These findings suggest that SZ-A establishes a dual energy-dissipating mechanism by promoting WAT browning and alleviating BAT whitening, thereby mitigating obesity and related metabolic dysfunction.\u003c/p\u003e\u003cp\u003eMechanistically, the metabolic regulatory effects of SZ-A likely involve coordinated modulation of lipogenic and thermogenic pathways, consistent with reports on other plant-derived bioactive compounds [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous studies have shown that SZ-A confers broad metabolic benefits, including protection of pancreatic β-cells, improvement of insulin resistance and chronic inflammation, reshaping of gut microbiota, promotion of glucagon-like peptide-1 secretion, and reduction of hepatic lipid accumulation, with potential cardiovascular and renal protection [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Interestingly, SZ-A has also been reported to promote adipogenesis under specific conditions, suggesting that its effects are context-dependent and may act bidirectionally to maintain energy homeostasis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our study, SZ-A improved dyslipidemia characterized by elevated TG, TC, and LDL-C levels, alleviated hepatic lipid accumulation, and reduced liver mass. Concurrently, it significantly upregulated thermogenic and beige adipocyte markers in WAT\u0026mdash;Ucp1, Cidea, and Tmem26\u0026mdash;as well as key transcriptional regulators Pgc-1α and Prdm16. PGC-1α, a master regulator of mitochondrial biogenesis, acts synergistically with PRDM16 to drive the expression of brown/beige adipocyte-specific genes and activate UCP1-mediated thermogenesis, thereby enhancing energy expenditure [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, the browning-promoting effects of SZ-A were confirmed in vitro. In differentiated 3T3-L1 adipocytes, non-cytotoxic concentrations of SZ-A (\u0026le;\u0026thinsp;100 \u0026micro;g/mL) significantly increased the expression of thermogenic genes (Ucp1, Pgc-1α, Pparγ) and beige markers (Cidea, Cox8b), indicating that SZ-A directly induces adipocyte conversion toward a metabolically active, beige-like phenotype, independent of systemic effects. The consistent in vivo and in vitro results reinforce the robustness of SZ-A\u0026rsquo;s browning and anti-obesity effects. Collectively, these findings suggest that SZ-A may activate adipose thermogenesis through the PRDM16\u0026ndash;PGC-1α\u0026ndash;UCP1 signaling axis, enhancing mitochondrial activity and energy dissipation in both WAT and BAT.\u003c/p\u003e\u003cp\u003eFurthermore, transcriptomic analysis revealed that SZ-A profoundly remodeled the eWAT gene expression landscape. GSEA showed significant activation of pathways related to fatty acid catabolism, β-oxidation, and lipid oxidation, along with a positive enrichment of the brown adipocyte differentiation pathway. These results suggest that SZ-A enhances mitochondrial oxidative metabolism and promotes adipose browning at the transcriptional level, providing mechanistic insight into its ability to improve insulin resistance and enhance insulin sensitivity.\u003c/p\u003e\u003cp\u003e Compared with other natural compounds, SZ-A offers a distinct translational advantage\u0026mdash;its clinical approval for T2DM provides a well-established safety and pharmacological foundation. Given the shared pathological basis of T2DM and obesity, including adipose dysfunction and impaired energy metabolism, our findings that SZ-A simultaneously improves glucose tolerance and lipid profiles suggest a multifaceted mechanism integrating inhibition of adipogenesis, activation of thermogenesis, and restoration of energy balance. Thus, SZ-A emerges as a promising multi-target therapeutic candidate for metabolic syndrome and adipose-based obesity.\u003c/p\u003e\u003cp\u003eIn conclusion, this study systematically demonstrates that SZ-A counteracts HFD-induced obesity and metabolic disturbances through a dual mechanism involving inhibition of lipid accumulation and activation of thermogenic pathways. By promoting WAT browning and enhancing BAT thermogenesis, SZ-A effectively improves energy metabolism and insulin sensitivity. Considering its established clinical use in T2DM, SZ-A holds strong translational potential as a safe and effective therapeutic agent for the integrated management of adipose-based obesity and metabolic syndrome.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, SZ-A exerts potent anti\u0026ndash;adipose-based effects in HFD\u0026ndash;induced obese mice, characterized by suppressed body weight gain, reduced adipose and liver mass, and improved hepatic lipid accumulation, lipid profiles, and glucose tolerance. Mechanistically, SZ-A enhances adipose metabolic activity by promoting WAT browning and preventing BAT whitening during obesity progression, accompanied by morphological improvements and upregulation of key thermogenic markers. In vitro, SZ-A directly activated the expression of genes associated with browning and energy expenditure in adipocytes, thereby enhancing thermogenic capacity. Transcriptomic profiling further revealed that SZ-A profoundly remodeled the WAT gene expression landscape, significantly activating pathways related to fatty acid metabolism, lipid oxidation, and brown adipocyte differentiation. These findings provide strong transcriptomic evidence supporting its adipose-targeted anti-obesity effects, which collectively contribute to improved insulin resistance and enhanced insulin sensitivity.\u003c/p\u003e\u003cp\u003eGiven its established clinical application in the treatment of T2DM, SZ-A represents a promising multitarget therapeutic candidate for adipose-based obesity and associated metabolic disorders. Future investigations integrating comprehensive energy metabolism analyses, mechanistic pathway validation, and clinical evaluation of tissue- and organ-level protection are warranted to further elucidate its molecular basis and long-term efficacy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to Public Laboratory Platform of West China Hospital and Animal Experiment Center of West China Hospital for their assistance and support throughout the course of this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.Z. and N.T. conceived and designed the study. R.Z. performed the animal and cell experiments, analyzed the data, and drafted the main manuscript text. G.P. assisted with data visualization and prepared Figures 6. X.P. and N.T. supervised the project and contributed to funding acquisition. R.Z., G.P., and X.P. revised the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the grants from the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (No. ZYGD 18017) and Natural Science Foundation of Sichuan Province, Grant No. 2024NSFSC1620.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRubino, F. et al. 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Biol.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 480\u0026ndash;495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrm.2016.62\u003c/span\u003e\u003cspan address=\"10.1038/nrm.2016.62\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sangzhi alkaloids, Obesity, Browning, 3T3-L1 cells, HFD-induced obese mice, Thermogenesis","lastPublishedDoi":"10.21203/rs.3.rs-8094331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8094331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObesity is a chronic metabolic disorder characterized by excessive adipose accumulation and is closely associated with type 2 diabetes mellitus (T2DM), cardiovascular diseases, and metabolic dysfunction associated steatotic liver disease. Enhancing the thermogenic capacity within adipose tissues has emerged as a promising therapeutic strategy to counteract obesity and its related metabolic complications. Ramulus Mori (Sangzhi) alkaloids (SZ-A), a natural alkaloid complex derived from \u003cem\u003eMorus alba L.\u003c/em\u003e (mulberry twig), have been clinically approved for the T2DM treatment and exhibit multiple metabolic regulatory properties. However, the precise anti-obesity mechanisms of SZ-A remain largely unclear. In this study, male C57BL/6 mice were fed a high-fat diet (HFD) for 14 weeks to induce obesity and subsequently treated with SZ-A (200 or 600 mg/kg) for 6 weeks. SZ-A markedly attenuated HFD-induced weight gain independent of food intake, improved glucose tolerance and insulin sensitivity, and alleviated dyslipidemia and hepatic steatosis. Furthermore, SZ-A reduced adipose tissue mass and upregulated key thermogenic regulators and beige adipocyte markers in white adipose tissue (WAT). It also restored brown adipose tissue (BAT) thermogenic activity by enhancing uncoupling protein 1 (UCP1) expression. In vitro, SZ-A promoted the expression of thermogenic and mitochondrial biogenesis-related genes in 3T3-L1 adipocytes, facilitating a beige-like phenotype. Transcriptomic analysis revealed that SZ-A significantly activated fatty acid catabolism and oxidation pathways, along with enrichment in the brown adipocyte differentiation pathway. Collectively, these findings demonstrate that SZ-A exerts potent anti-obesity effects through the dual mechanism of promoting WAT browning and enhancing BAT thermogenesis. Given its established clinical safety in T2DM, SZ-A represents a promising therapeutic candidate for adipose-based obesity and associated metabolic disorders.\u003c/p\u003e","manuscriptTitle":"Ramulus Mori (Sangzhi) Alkaloids Attenuate Diet- Induced Obesity by Promoting White Adipose Browning and Enhancing Brown Fat Thermogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 13:57:02","doi":"10.21203/rs.3.rs-8094331/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-14T17:44:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-09T16:53:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T21:00:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235108439433421807875573705090489913388","date":"2025-12-21T00:21:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297564125929765232493936028549128479747","date":"2025-12-09T04:19:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-07T01:06:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-06T09:53:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-01T11:58:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-27T09:15:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-27T08:50:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c00b6ee0-aed2-4355-bae0-5805b2bb1059","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59359035,"name":"Biological sciences/Biochemistry"},{"id":59359036,"name":"Health sciences/Diseases"},{"id":59359037,"name":"Health sciences/Endocrinology"},{"id":59359038,"name":"Biological sciences/Molecular biology"},{"id":59359039,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-03-30T16:28:19+00:00","versionOfRecord":{"articleIdentity":"rs-8094331","link":"https://doi.org/10.1038/s41598-026-45462-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-27 16:11:53","publishedOnDateReadable":"March 27th, 2026"},"versionCreatedAt":"2025-12-11 13:57:02","video":"","vorDoi":"10.1038/s41598-026-45462-9","vorDoiUrl":"https://doi.org/10.1038/s41598-026-45462-9","workflowStages":[]},"version":"v1","identity":"rs-8094331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8094331","identity":"rs-8094331","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}