Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 2025 Sep 30;77(5):181. doi: 10.1007/s10616-025-00848-x

Guizhi fuling decoction inhibits pyroptosis to improve high-fat diet-induced obesity by up-regulating ACE2

Wei Zhang 1, Jia He 1,✉
PMCID: PMC12484488  PMID: 41040130

Abstract

Obesity has become a global public problem. Guizhi Fuling Decoction (GZFLD) is a traditional Chinese medicine decoction. This study aims to investigate the therapeutic effects of GZFLD on high-fat diet (HFD)-induced obesity in mice. The obese mouse model was induced by HFD. The effects of GZFLD on obesity were assessed by recording body weight (BW) and inguinal white adipose tissue (Ing-WAT) weight of mice. Histological changes in adipose and liver tissues were evaluated by hematoxylin–eosin staining. Enzyme-linked immunosorbent assay was exploited to detect the expression of blood lipid indexes and inflammatory factors. The expression of pyroptosis-related proteins was detected by western blot and immunohistochemistry. Transcriptome sequencing was executed to explore the mechanism of GZFLD on obese mice and screen the pyroptosis-related genes. Furthermore, the effect of Angiotensin-converting enzyme 2 (ACE2) on obese mice was verified by ACE2 inhibitor-DX600. In HFD-induced mice, with the increased dose of GZFLD, GZFLD significantly improved obesity, hepatic steatosis and dyslipidemia, with decreased BW, Ing-WAT weight, Ing-WAT/BW ratio, levels of triglycerides, cholesterol low-density lipoprotein, and increased high-density lipoprotein levels. Hematoxylin–eosin staining exhibited that GZFLD improved steatosis and adipocyte enlargement induced by HFD. Additionally, GZFLD suppressed the levels of lactate dehydrogenase, inflammation factors, and pyroptosis-related proteins. RNA-sequencing identified ACE2 as a key gene target. The ACE2 inhibitor DX600 attenuated the inhibitory effects of GZFLD on hepatic steatosis, dyslipidemia, inflammation, and pyroptosis in HFD-induced obese mice. Collectively, GZFLD had a protective effect on HFD-induced obesity by alleviating dyslipidemia, and inhibiting inflammation and pyroptosis, which was achieved by up-regulating ACE2.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10616-025-00848-x.

Keywords: Guizhi fuling decoction, Obesity, Pyroptosis, ACE2, RNA-sequencing

Introduction

With the progress and development of society, the obesity rate among the global population has significantly increased. Sedentary lifestyles and unhealthy diets have become important factors contributing to this serious problem (Green et al. 2020). Obesity, characterized by excessive adipose tissue expansion, is far more than a condition of caloric imbalance; it is a state of chronic low-grade inflammation and metabolic dysfunction that predisposes individuals to a spectrum of comorbidities, including type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), cardiac complications, and hypertension, thus leading to a decline in the quality of life and life expectancy (Blüher 2019; Ferguson et al. 2020). Obesity is usually caused by many factors, including genetic factors, high-fat diet (HFD), gut microbiota, and other factors (Fan et al. 2023). Among them, obesity caused by high-fat intake is relatively common. Therefore, it is necessary to explore relevant treatment methods for improving HFD-induced obesity in society.

The Guizhi Fuling prescription is a traditional Chinese medicine prescription, consisting of Guizhi, Fuling, Shaoyao, Tanpi, and Danpi (Liu et al. 2024). It is usually developed into different dosage forms, such as pills, decoctions, capsules, and granules, which play an important role in the clinical treatment of certain diseases and tumors (Liu et al. 2024). The Guizhi Fuling Pill has a remarkable therapeutic effect in treating endometriosis (Wang et al. 2021), and can also relieve liver fibrosis in rats by inhibiting the PTEN/AKT/mTOR signaling pathway (Yao et al. 2024). Guizhi Fuling capsules inhibit neuroinflammation to alleviate memory deficits through blocking the JAK2/STAT3 pathway (Yang et al. 2023a, b). However, the Guizhi Fuling Decoction (GZFLD) has not been studied in HFD-induced obesity.

Persistent low-level inflammation is fueled not only by adipocyte hypertrophy and immune cell infiltration but also by a specific form of pro-inflammatory programmed cell death—pyroptosis (Cinti 2023). Pyroptosis is distinctively driven by inflammasome complexes, most notably the NOD-like receptor protein 3 (NLRP3) inflammasome, which orchestrates the cleavage of caspase-1 and the subsequent maturation and release of potent interleukin (IL)-1β and IL-18 (Miao et al. 2022). Crucially, in obesity, nutrient overload and metabolic stress (e.g., lipotoxicity, oxidative stress) can activate the NLRP3 inflammasome within adipocytes and macrophages. This activation triggers pyroptosis, creating a vicious cycle: the rupture of pyroptotic cells releases damage-associated molecular patterns (DAMPs) and inflammatory cytokines into the microenvironment, further amplifying local and systemic inflammation and exacerbating insulin resistance and tissue dysfunction (Miao et al. 2022). Certain traditional Chinese medicines (TCM) have demonstrated potential in modulating the progression of obesity-related diseases by regulating pyroptosis (Yin et al. 2021; Qiu et al. 2023). For instance, Xiezhuo Tiaozhi Decoction has been found to inhibit hepatocyte pyroptosis, liver injury, and lipid accumulation, thereby ameliorating HFD-induced NAFLD progression (Qiu et al. 2023). However, it remains unknown whether GZFLD affects pyroptosis in obesity-related diseases.

The angiotensin II (ANG II)-ANG II type 1 receptor axis plays a crucial role in obesity-related pathologies. Angiotensin-converting enzyme 2 (ACE2), a membrane-bound carboxypeptidase encoded by the ACE2 gene, is capable of hydrolyzing angiotensin peptides (Kuriakose et al. 2021). Studies have demonstrated that ACE2 can regulate lipid-metabolism-related genes, thereby reducing hepatic lipid accumulation (Cao et al. 2016), and regulate intramuscular fat by enhancing mitochondrial function (Cao et al. 2019), which is of great significance in the regulation of lipid deposition. In this study, through in vivo models, we investigated the effects of GZFLD on HFD-induced obese mice and screened the pyroptosis-related hub gene ACE2 via RNA sequencing. It was discovered that GZFLD ameliorated obesity by regulating pyroptosis through up-regulating ACE2, thus providing a new treatment approach for HFD-induced obesity.

Materials and methods

Herbal ingredients and preparation

GZFLD was made up of Ramulus Cinnamomi (Guizhi, 15 g), Poria (Fuling, 15 g), Paeonia lactiflora Pall. (Shaoyao, 10 g), Persicae Semen (Taoren, 15 g), and Paeonia suffruticosa Andr. (Danpi, 15 g). The drugs were purchased from the Chinese Pharmacy of Traditional Chinese Medicine Miscellaneous Disease Hospital in Yuexiu District, Guangzhou. The above Chinese herbal medicines were soaked in water and boiled to 400 mL.

Construction of an obese mouse model

Male C57BL/6 J mice aged 6 weeks (18–20 g) were purchased from SiPeiFu Biotechnology Co., Ltd (Beijing, China). Mice were housed in a specific pathogen-free (SPF) environment with ad libitum access to water and food under a 12-h light/12-h dark cycle at 22 ± 2℃, 50 ± 5% humidity. All mice were randomly divided into the Control group (fed normal diet), the HFD group, HFD + L-GFB (low dose of GZFLD, 22.5 mL/kg/day), HFD + H-GFB (high dose of GZFLD, 45 mL/kg/day), and HFD + H-GFB + DX600 (intraperitoneally injected with 5 μg/kg/day) (Yousif et al. 2012). Except the Control group, mice in the other groups were fed with HFD (containing 60% fat, 20% protein, and 20% carbohydrate) (Yang et al. 2023a, b). After 4 weeks of feeding, mice received GZFLD administration by gavage and continued for 4 weeks, during which the body weight of mice was measured daily. Finally, mice were anesthetized by inhalation of 1–1.5% isoflurane-oxygen mixture and euthanized by cervical dislocation. Blood samples were collected from the heart. Serum, liver and adipose tissue were collected and stored at − 80℃. The study was approved by the local Ethical Committee and conducted in accordance with “China’s Regulations for the Use of Laboratory Animals”.

Hematoxylin–eosin (H&E) staining

The frozen liver and adipose tissue sections were rewarmed at room temperature and dried for 10 min. The slices were fixed in acetone at − 20 °C for 10 min and then rinsed in water for 1–2 min. The slices were stained in hematoxylin for 3–5 min and then washed. Subsequently, slices were differentiated in hydrochloric acid-alcohol for 1–3 s and rinsed with water for 15 min for blue-reversing. Then the slices were stained in eosin for 40 s-1 min and dehydrated in gradient alcohol and xylene. Finally, the slices were sealed with neutral balsam, and the staining results were observed and photographed under an optical microscope.

Immunohistochemistry (IHC)

Adipose tissue samples were fixed in 4% paraformaldehyde, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections were cut at a thickness of 4 μm using a microtome. The sections were then baked at 65 °C for 30 min, deparaffinized in xylene, and rehydrated through a descending ethanol series to distilled water. For antigen repair, the sections were incubated in citrate buffer and heated appropriately. Endogenous peroxidase activity was quenched by treating the sections with 3% hydrogen peroxide for 10 min at room temperature. Non-specific binding sites were blocked by incubating the sections with 3% bovine serum albumin (BSA) for 30 min at room temperature in a humidified chamber. The sections were subsequently incubated with primary antibodies against apoptosis-associated speck-like protein (ASC) (1/400, ab309497, Abcam, Cambridge, UK) and NLRP3 (1/400, 30,109–1-AP, Proteintech, Chicago, USA) at 37 °C for 1 h. After washing with phosphate-buffered saline, the sections were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1/200, Abcam) at 37 °C for 30 min. Immunoreactivity was visualized using a 3,3′-diaminobenzidine. The sections were then counterstained with hematoxylin for 2 min, differentiated in 1% acid alcohol, and blued in lithium carbonate solution for 30 s. Finally, the sections were dehydrated through an ascending ethanol series, cleared in xylene, and mounted with neutral balsam. Stained sections were examined and imaged under a light microscope. Image analysis was performed using ImageJ software to quantify the positive staining area.

Enzyme-linked immunosorbent assay (ELISA)

According to the instruction of manufacturer, the levels of inflammatory factors tumor necrosis factor-α (TNF-α, EK20220), IL-18 (EK11385), and IL-1β (EK20533), lactate dehydrogenase (LDH, EK20034), triglycerides (TG, EK20481), total cholesterol (TC, EK20539), and low-density lipoprotein (LDL, EK20540), and high-density lipoprotein (HDL, EK20541) in mice serum were detected by corresponding ELISA kits (Esebio, Shanghai, China), and the optical density (OD) was measured at a wavelength of 450 nm using a microplate reader. The concentration in the sample was calculated through the standard curve.

Western blotting (WB)

Total proteins were extracted from adipose tissues using RIPA lysis buffer. After being separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight at 4 °C with primary antibodies (1/1000, Abcam) against Caspase-1 (ab138483), GSDMD-N (ab209845), GSDME (ab215191), ACE2 (ab108252), IL-1β (ab234437), PRTN3 (ab270441), MELK (ab108529), KIF23 (ab174303) and GAPDH (ab9485). Afterwards, the membrane was incubated with goat anti-rabbit secondary antibody (1/5000, Abcam) for 1 h. Subsequently, the PVDF membranes were washed with Tris-buffered saline with Tween-20, placed on the exposure plate of the Tanon 5200 chemiluminescence imaging system (Tanon, Shanghai, China), and developed using ECL solution. The expression levels of these proteins were quantified using Image J.

Transcriptome sequencing

TRIZOL reagent (Invitrogen, CA, USA) was used to extract RNA from adipose tissue samples of the HFD group and HFD + H-GFB group for transcriptome sequencing (n = 3). The concentration and purity of RNA were detected by Nano-300 (ALLSHENG, Hangzhou, China), and then the sequencing libraries were conducted. Transcriptome sequencing was performed on Illumina Novaseq 6000 sequencing platform (Illumina, San Diego, CA, USA). The screening condition of differentially expressed genes (DEGs) were set as |log2FoldChange|> 1 and P-value ≤ 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were performed. Pyroptosis-related genes were obtained from Genecards databases (https://www.genecards.org/), and intersected by Evenn (http://www.ehbio.com/test/venn/) to obtain DEGs related to pyroptosis (Yang et al. 2024a, b).

Analysis of hub genes

The Search Tool for the Retrieval of Interaction Gene (STRING) database (https://www.string-db.org/) was utilized to analyze 30 pyroptosis-related DEGs, predicting the interaction relationships among the proteins encoded by genes that played important roles after GZFLD treatment. For the significance standard, the confidence interaction score was set at 0.400. Subsequently, Cytoscape software (www.cytoscape.org/) was used to visualize the Protein–Protein Interaction (PPI) network, and the Cytoscape plugin MCODE was operated to analyze sub-networks and select hub genes. According to the expression data of hub genes in samples, R language was applied to perform Principal Component Analysis (PCA). A matrix heatmap was drawn for visualization to reveal the correlation of the selected genes.

Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR)

The total RNA of tissues was extracted with TRIZOL reagent (Invitrogen). RNA (1 μg) was taken to measure the RNA concentration on the Nano-300 (ALLSHENG). The synthesis of cDNA was completed using the SweScript All-in-One First-Stand cDNA Synthesis Supermix kit (Servicebio Co., Wuhan, China). A SYBR Green Master Mix and Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to perform qRT-PCR detection on cDNA samples. The reaction procedure was: 95 °C for 30 s, followed by 95 °C for 10 s and 60 °C for 30 s for 40 cycles. GAPDH was chosen as an internal reference. The obtained Ct values were analyzed by the 2−ΔΔCt method, and the experiment was repeated three times. The primer sequences for RT-qPCR were shown in Table S1.

Statistical analysis

All data were processed using GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA) and presented in the form of mean ± standard deviation. The t-test was used for comparison between two groups, One-Way ANOVA was used for comparison among multiple groups, and Tukey’s multiple comparisons test was used for pairwise comparison after ANOVA analysis. P < 0.05 indicated that the difference had significant statistical significance.

Results

GZFLD improves obesity and dyslipidemia in HFD-induced mice

To investigate the therapeutic effects of GZFLD on obesity in mice, C57BL/6 J mice were divided into four groups: Control, HFD, HFD + L-GFB, and HFD + H-GFB. Compared to the Control group, the HFD group exhibited a significant increase in body weight. In contrast, both the HFD + L-GFB and HFD + H-GFB groups showed a significant reduction in body weight with increasing doses of GZFLD compared with the HFD group (Fig. 1A). Additionally, compared to the Control group, the HFD group had a significant increase in the weight of inguinal white adipose tissue (Ing-WAT) and the ratio of Ing-WAT and body weight (Ing-WAT/BW). However, both the HFD + L-GFB and HFD + H-GFB groups demonstrated a significant decrease in these parameters with increasing doses of GZFLD in contrast with the HFD group (Fig. 1B-C). Histological examination of the liver via H&E staining was performed to assess hepatic steatosis. As shown in Fig. 1D, liver sections from the Control group displayed normal hepatic architecture with hepatocytes radially arranged around the central vein, uniform cytoplasm without vacuoles, and central, regularly shaped nuclei. In contrast, the HFD group exhibited severe hepatic steatosis, characterized by numerous lipid droplets of varying sizes appearing as clear vacuoles within the hepatocytes. Treatment with GZFLD markedly attenuated this HFD-induced steatosis in a dose-dependent manner, as evidenced by a reduction in vacuolation and improved tissue architecture in both the HFD + L-GFB and HFD + H-GFB groups, with the latter showing a more potent effect. Furthermore, compared to the Control group, the HFD group displayed severe degeneration of white and brown adipose tissues and a marked increase in adipocyte size. Conversely, both low and high doses of GZFLD significantly inhibited the degeneration of white and brown adipose tissues and reduced adipocyte size (Fig. 1D). Biochemical indexes were measured by ELISA. Compared with the Control group, the levels of serum TG, TC, and LDL in HFD-induced mice were significantly increased, while the level of HDL was significantly decreased. Compared with the HFD group, the levels of serum TG, TC, and LDL were significantly decreased in the HFD + L-GFB group and the HFD + H-GFB group, and the HDL level was significantly elevated as the increasing dose of GZFLD (Fig. 1E).

Fig. 1.

Fig. 1

Open in a new tab

Guizhi Fuling Decoction improves obesity and dyslipidemia in high-fat diet (HFD)-induced mice. A. Mice weight. B. Weight of inguinal white adipose tissue (Ing-WAT). C. Ratio of Ing-WAT and body weight (BW). D. Hematoxylin–Eosin staining of liver (magnification: × 200, scale: 100 μm) and adipose tissues (magnification: × 400, scale: 100 μm). E. Enzyme-linked immunosorbent assay (ELISA) detection of lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels. (n ≥ 6) **P < 0.01 vs. Control group, #P < 0.05 ##P < 0.01 vs. HFD group

GZFLD inhibits inflammation and pyroptosis in HFD-induced mice

The levels of TNF-α, IL-18, and IL-1β in mice serum were measured by ELISA, indicating that compared to the Control group, the HFD group exhibited a significant increase in the levels of TNF-α, IL-18, and IL-1β. In contrast, both the HFD + L-GFB and HFD + H-GFB groups showed a significant reduction in these cytokine levels with increasing doses of GZFLD, compared to the HFD group (Fig. 2A). Additionally, LDH level was assessed by ELISA, demonstrating that the HFD group had significantly elevated LDH level compared to the Control group, which significantly decreased with increasing doses of GZFLD (Fig. 2B). WB revealed that compared to the Control group, the HFD group displayed a significant increase in the protein expression of Caspase-1, GSDMD-N, and GSDME. Conversely, both low and high doses of GZFLD significantly reduced the protein expression of Caspase-1, GSDMD-N, and GSDME in HFD-induced mice (Fig. 2C). To further validate the inhibition of pyroptosis in adipose tissue, IHC staining for key pyroptosis-related proteins (NLRP3 and ASC) was performed. As shown in Fig. 2D, IHC analysis revealed scant expression of NLRP3 and ASC in the Control group. The HFD group exhibited a significant increase in the positive staining areas for both proteins. Treatment with GZFLD, at both low and high doses, ameliorated this increase, with the high-dose group (HFD + H-GFB) showing a more substantial reduction in NLRP3-positive area compared to the low-dose group (HFD + L-GFB).

Fig. 2.

Fig. 2

Open in a new tab

Guizhi Fuling Decoction inhibits inflammation and pyroptosis in HFD-induced mice. A. ELISA was executed to detect tumor necrosis factor-α (TNF-α), interleukin-18 (IL-18), and interleukin-1β (IL-1β) levels. B. LDH level was detected by ELISA. C. Western blot was used to detect the expression of Caspase-1, GSDMD-N, and GSDME. D. Representative immunohistochemical staining images (magnification: × 400, scale: 50 μm) and quantitative analysis of NOD-like receptor protein 3 (NLRP3) and apoptosis-associated speck-like protein (ASC) positive areas in white adipose tissue. (n ≥ 6) **P < 0.01 vs. Control group, #P < 0.05 ##P < 0.01 vs. HFD group

Identification of DEGs

Three samples of adipose tissue from the HFD group (OB) and the HFD + H-GFB group (TCM) were sent for transcriptome sequencing, and a total of 3611 DEGs were obtained, among which 2360 genes were up-regulated and 1251 genes were down-regulated. The heatmap and volcano plot of DEGs were shown in Fig. 3A-B. Sample TCM_3 did show relatively lower correlation with the other two replicates (TCM_1 and TCM_2), and this variability could potentially arise from inherent biological differences between individual animals, which could not affect the results. The intersection of the DEGs and pyroptosis-related genes was taken, and 30 pyroptosis-related DEGs were obtained, including 11 up-regulated genes (Fig. 3C) and 19 down-regulated genes (Fig. 3D).

Fig. 3.

Fig. 3

Open in a new tab

Identification of differentially expressed genes (DEGs). A. Heatmap of DEGs. B. Volcano plot of DEGs. C. Venn diagram of pyroptosis-related up-regulated DEGs. D. Venn diagram of pyroptosis-related down-regulated DEGs

GO and KEGG enrichment analysis

GO and KEGG pathway enrichment analyses were performed on 30 pyroptosis-related DEGs. The GO analysis revealed that these DEGs were primarily involved in biological processes (BP) such as regulation of inflammatory response, negative regulation of immune system processes, and negative regulation of cellular secretion (Fig. 4A). In terms of molecular functions (MF), they exhibited activities related to phospholipase inhibitor, single-stranded DNA refolding, and calcium binding protein A (Fig. 4B). Regarding cellular components (CC), the enriched terms included membrane rafts, microdomains, and inflammasomes (Fig. 4C). KEGG analysis showed that DEGs were mainly enriched in signaling pathways such as programmed necrosis, cytoplasmic DNA-sensing pathway, and NOD-like receptor signaling pathway (Fig. 4D).

Fig. 4.

Fig. 4

Open in a new tab

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of pyroptosis-related DEGs. A. Enriched biological processes of GO analysis. B. Enriched molecular functions of GO analysis. C. Enriched cellular components of GO analysis. D. Enriched KEGG pathways

PPI network

STING was utilized to screen for proteins interacting with the 30 pyroptosis-related DEGs, and the Cytoscape software was employed to construct a PPI network. The PPI network comprised 24 nodes and 38 edges (Fig. 5A). Using the MCODE plugin, two functional modules were identified, which included nine hub genes: ACE2, CASP1, IL-1B, PRTN3, AGER, MELK, KIF23, MKI67, and Cep55 (Fig. 5B-C). Based on the expression data of these hub genes in the samples, PCA was conducted using R language, indicating that the top two principal components, PC1 and PC2, together provided an 87% explanation rate for the variance between the HFD group and the HFD + H-GFB group (Fig. 5D). Furthermore, to reveal the correlation among the selected genes within the sample data, a matrix heatmap was generated for visualization (Fig. 5E).

Fig. 5.

Fig. 5

Open in a new tab

Construction of protein–protein interaction (PPI) network and analysis of hub genes. A. PPI network. B. Function module 1 in PPI network. C. Function module 2 in PPI network. D. Principal component analysis of hub genes. E. Matrix heatmap of hub genes

Identification of hub genes

The hub genes ACE2, CASP1, IL-1B, PRTN3, MELK, and KIF23 were selected and validated through RT-qPCR and WB, which indicated that compared to the HFD group, the mRNA and protein expression levels of ACE2 and PRTN3 were increased in the adipose tissue of the HFD + H-GFB group, whereas the levels of CASP1, IL-1B, MELK, and KIF23 were reduced (Fig. 6A-B), consistent with the RNA-sequencing results.

Fig. 6.

Fig. 6

Open in a new tab

The mRNA and protein expression levels of hub genes were evaluated by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) (A) and western blot (B). (n ≥ 6) **P < 0.01 vs. HFD group

DX600 suppresses the therapeutic effects of GZFLD on HFD-induced mice

DX600, an ACE2 inhibitor, was used to validate the effect of ACE2 on the therapeutic efficacy of GZFLD in obese mice. Compared to the Control group, HFD significantly induced an increase in body weight, Ing-WAT, and the Ing-WAT/BW ratio in mice. The high dose of GZFLD significantly inhibited the HFD-induced increases in body weight, Ing-WAT, and the Ing-WAT/BW ratio, whereas compared to the HFD + H-GFB group, DX600 notably reversed the inhibitory effects of GZFLD on body weight, Ing-WAT, and the Ing-WAT/BW ratio in obese mice (Fig. 7A-C).

Fig. 7.

Fig. 7

Open in a new tab

The ACE2 inhibitor DX600 suppresses the therapeutic effects of Guizhi Fuling Decoction on HFD-induced mice. A. Mice weight. B. Weight of Ing-WAT. C. Ratio of Ing-WAT and BW. D. RT-qPCR was carried out to detect the mRNA level of ACE2. E. Hematoxylin–Eosin staining of liver (magnification: × 200, scale: 100 μm) and adipose tissue (magnification: × 400, scale: 100 μm). F. ELISA was performed to measure blood biochemical indicators (TG, TC, LDL, and HDL). (n ≥ 6) **P < 0.01 vs. Control group, ##P < 0.01 vs. HFD group, $$P < 0.01 vs. HFD + H-GFB group

Additionally, RT-qPCR results demonstrated that compared to the Control group, the expression of ACE2 was significantly reduced in the HFD group, while a high dose of GZFLD significantly enhanced ACE2 expression. Compared to the HFD + H-GFB group, DX600 significantly suppressed ACE2 expression (Fig. 7D).

H&E staining revealed that a high dose of GZFLD significantly attenuated the degeneration of white and brown adipose tissues in HFD-induced obese mice, with a notable reduction in adipocyte size. In contrast, compared to the HFD + H-GFB group, DX600 exhibited severe degeneration of both white and brown adipose tissues, with a significant increase in adipocyte size (Fig. 7E). Furthermore, the therapeutic effect of high-dose GZFLD (HFD + H-GFB) on hepatic steatosis was significantly attenuated by co-administration of the ACE2 inhibitor DX600, as indicated by an increase in lipid vacuoles compared to the HFD + H-GFB group (Fig. 7E).

ELISA indicated that HFD led to a significant increase in serum levels of TG, TC, and LDL, while HDL level was significantly decreased, compared to the Control group. The high dose of GZFLD notably inhibited the levels of TG, TC, and LDL, and enhanced HDL levels, while DX600 significantly reversed these effects (Fig. 7F). Taken together, the ACE2 inhibitor DX600 can suppress the therapeutic effects of GZFLD on obese mice.

DX600 promotes inflammation and pyroptosis in HFD-induced mice

The levels of inflammatory cytokines TNF-α, IL-18, and IL-1β were measured using ELISA, showing that compared to the Control group, HFD significantly elevated the levels of TNF-α, IL-18, and IL-1β, while high dose of GZFLD treatment notably inhibited the HFD-induced increase in these cytokines. However, this effect was mitigated by DX600 (Fig. 8A). A similar pattern was observed with LDH levels. DX600 attenuated the inhibitory effect of a high dose of GZFLD on LDH levels in HFD-induced mice (Fig. 8B).

Fig. 8.

Fig. 8

Open in a new tab

ACE2 inhibitor DX600 promotes inflammation and pyroptosis in HFD-induced mice. A. ELISA was performed to detect the levels of inflammatory factors (TNF-α, IL-18, and IL-1β). B. ELISA was carried out to measure LDH levels. C. Western blot was executed to measure the expression levels of pyroptosis-related proteins (Caspase-1, GSDMD-N, and GSDME). D. Representative immunohistochemical staining images (magnification: × 400, scale: 50 μm) and quantitative analysis of NLRP3 and ASC positive areas in white adipose tissue. (n ≥ 6) **P < 0.01 vs. Control group, ##P < 0.01 vs. HFD group, $$P < 0.01 vs. HFD + H-GFB group

Additionally, WB revealed that compared to the Control group, pyroptosis-related proteins Caspase-1, GSDMD-N, and GSDME were significantly upregulated in HFD-induced mice, whereas a high dose of GZFLD reduced their expression. Compared to the HFD + H-GFB group, DX600 significantly enhanced the protein expression of Caspase-1, GSDMD-N, and GSDME (Fig. 8C). IHC staining further demonstrated that the suppressive effect of high-dose GZFLD on HFD-induced expression of NLRP3 and ASC was partially abolished by co-administration of the ACE2 inhibitor DX600 (Fig. 8D).

Discussion

Obesity is one of the important causes of other related diseases, such as hypertension, diabetes, and so on (Litwin and Kułaga 2021; Ruze et al. 2023), and has become a public problem. In our study, we discovered that GZFLD significantly improved HFD-induced obesity, hepatic steatosis and dyslipidemia, and inhibited inflammatory responses and pyroptosis. Moreover, through RNA sequencing, we identified the key pyroptosis gene ACE2 in obesity. Inhibition of ACE2 could significantly suppress the therapeutic effect of GZFLD on obese mice and promote inflammation and pyroptosis, indicating that GZFLD inhibited pyroptosis by up-regulating ACE2, thereby improving HFD-induced obesity.

TCM and its proprietary formulations have emerged as promising strategies for the treatment of obesity (Chen et al. 2023). TCM formulations like GZFLD have shown promise in managing obesity-related conditions. The components of GZFLD play an active role in inhibiting inflammation and lipid accumulation. The mixture of Cinnamomi Ramulus extract and two lactic acid bacteria can regulate the gut microbiota and alleviate HFD-induced obesity (Jeung et al. 2019). In addition, Cinnamomi Ramulus is also a widely applied anti-cancer drug, which inhibits the growth of cancer cells by inducing G2/M arrest (Li et al. 2023). Poria cocos polysaccharide suppresses the pyroptosis of small intestinal macrophages and reduces the destruction of the intestinal vascular barrier to alleviate HFD-induced NAFLD (Ye et al. 2022). Paeonia lactiflora inhibits oxidative stress and apoptosis by inhibiting the PKC-ERK pathway, thus alleviating acute liver injury (Li et al. 2024). Paeonol, a bioactive compound derived from Paeonia Suffruticosa Andr., can reduce lipid accumulation and play a protective role in anti-atherosclerosis (Gao et al. 2024). Our study demonstrated that GZFLD significantly attenuated HFD-induced increases in body weight, adipose tissue mass, and adipocyte hypertrophy. Furthermore, it markedly improved dyslipidemia, as evidenced by reduced serum levels of TG, TC, and LDL and increased HDL. These beneficial effects are likely attributable to the synergistic actions of its constituents.

Emerging evidence highlights pyroptosis, a highly inflammatory form of programmed cell death, as a critical mechanism in obesity-related metabolic dysfunction (Giordano et al. 2013). Recent studies suggest that TCM and their active components may ameliorate obesity and its complications by targeting this pathway. For instance, ginseng components have been shown to inhibit NLRP3 inflammasome activation and suppress caspase-1/GSDMD activity, thereby reducing inflammation and improving insulin sensitivity in diabetic models (Sun, Liu et al. 2025). Similarly, Wogonin is found to alleviate obesity-induced lipid metabolism disorders and cardiac injury by suppressing pyroptosis and deactivating the IL-17 signaling pathway (Zhou and Yin 2022). Furthermore, the potential of TCM compounds to modulate pyroptosis is often linked to broader metabolic benefits, including improved adipose tissue function and enhanced gut microbiota composition (Tang, Zhao et al. 2025). In this study, GZFLD suppressed inflammatory responses and pyroptosis in obese mice, suggesting that the inhibition of pyroptosis may represent a key molecular mechanism underlying its therapeutic effects.

Research has demonstrated that ACE2 and its receptor expression are significantly involved in the pathophysiology of conditions such as hypertension and obesity (Perez et al. 2021; Sun et al. 2021). Cao et al. report that ACE2 is highly expressed in brown adipose tissue, and the activation of the ACE2 pathway can improve glucose and lipid metabolism, making it a potential therapeutic target for metabolic disorders (Cao et al. 2022). Exosomal ACE2 inhibits the activation of the NLRP3 inflammasome and suppresses pyroptosis to protect islet function, providing new insights into the prevention and treatment of obesity and diabetes (Yang et al. 2024a, b). ACE2 exerts an anti-obesity effect by stimulating brown adipose tissue and inducing browning of white adipose tissue (Kawabe et al. 2019). Chen et al. report that ACE2 improves symptoms of obesity-related glomerulopathy by activating the Nrf2 pathway, reducing fat deposition and inflammatory responses (Chen et al. 2022). Additionally, ACE2 deficiency leads to damage to the pancreatic microvascular system, worsening islet function in HFD-induced mice (Yuan et al. 2013). Our transcriptomic analysis revealed that GZFLD modulated the expression of 30 pyroptosis-related genes, indicating a broad impact on pyroptosis beyond the core inflammasome components (Caspase-1, IL-1β). Pathway enrichment analysis indicated these genes were significantly involved in the cytoplasmic DNA-sensing pathway and NOD-like receptor signaling pathway. Among these DEGs, ACE2 was identified as a high-confidence hub gene through PPI network analysis, suggesting a potentially central regulatory role within the pyroptosis network altered by GZFLD. This, combined with our functional validation confirming that ACE2 inhibition abolished the anti-pyroptotic effects of GZFLD, providing strong evidence that ACE2 was a key mediator through which GZFLD regulated pyroptosis in obesity.

While this study identifies the ACE2-pyroptosis axis as a key mechanism for GZFLD, some limitations remain. The precise mechanistic link between GZFLD administration and increased ACE2 remains to be fully determined. It is unclear whether active compounds within GZFLD directly bind to the promoter of the ACE2 gene to enhance its transcription, or if this upregulation is an indirect consequence of modulating upstream signaling pathways that converge on ACE2, such as those involved in inflammation, metabolic stress, or epigenetic regulation. For instance, some herbal constituents are known to activate nuclear receptors like PPARγ (Ge et al. 2025; Yang et al. 2025), which has been implicated in the regulation of ACE2 expression (Vallée et al. 2021). Therefore, identifying the direct molecular targets of GZFLD’s active components and delineating the specific pathway leading to ACE2 upregulation constitute essential and exciting directions for future research.

In conclusion, this study verified the effects of GZFLD on HFD-induced obese mice through in vivo assays, which demonstrated that GZFLD can inhibit inflammation and pyroptosis, as well as relieve hepatic steatosis and dyslipidemia, thereby alleviating and improving obesity. Moreover, through RNA-sequencing, an important pyroptosis-related gene ACE2 was discovered, and it was uncovered that GZFLD could improve obesity in mice by up-regulating ACE2 to inhibit pyroptosis. This study provides a scientific basis for supporting the clinical application of GZFLD in the therapy of HFD-induced obesity and also supplies a reliable reference for exploring the pharmacological mechanism of GZFLD.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 2274 KB) (2.2MB, pdf)
Supplementary file2 (DOCX 12 KB) (12.4KB, docx)

Acknowledgements

Not applicable.

Author contributions

Wei Zhang: Substantial contributions to conception and design, data acquisition, drafting the article; Jia He: data acquisition, drafting the article; All the authors took part in the experiment. All the authors read and approvaled the manuscript.

Funding

Not applicable.

Data availability

The data used and/or analyzed during the current study are available from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/bioproject/1310747).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The study was approved by the Animal Ethics Committee of Zhujiang Hospital of Southern Medical University (No. LAEC-2024-282) and conducted in accordance with “China’s Regulations for the Use of Laboratory Animals”.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Blüher M (2019) Obesity: global epidemiology and pathogenesis. Nat Rev Endocrinol 15:288–298 [DOI] [PubMed] [Google Scholar]
  2. Cao X, Yang F, Shi T, Yuan M, Xin Z, Xie R, Li S, Li H, Yang JK (2016) Angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis. Sci Rep 6:21592 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cao X, Lu XM, Tuo X, Liu JY, Zhang YC, Song LN, Cheng ZQ, Yang JK, Xin Z (2019) Angiotensin-converting enzyme 2 regulates endoplasmic reticulum stress and mitochondrial function to preserve skeletal muscle lipid metabolism. Lipids Health Dis 18:207 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cao X, Shi TT, Zhang CH, Jin WZ, Song LN, Zhang YC, Liu JY, Yang FY, Rotimi CN, Xu A, Yang JK (2022) ACE2 pathway regulates thermogenesis and energy metabolism. Elife. 10.7554/eLife.72266 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen YY, Hong H, Lei YT, Zou J, Yang YY, He LY (2022) ACE2 deficiency exacerbates obesity-related glomerulopathy through its role in regulating lipid metabolism. Cell Death Discov 8:401 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen YK, Liu TT, Teia FKF, Xie MZ (2023) Exploring the underlying mechanisms of obesity and diabetes and the potential of Traditional Chinese Medicine: an overview of the literature. Front Endocrinol (Lausanne) 14:1218880 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cinti S (2023) Obese adipocytes have altered redox homeostasis with metabolic consequences. Antioxidants. 10.3390/antiox12071449 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fan S, Chen S, Lin L (2023) Research progress of gut microbiota and obesity caused by high-fat diet. Front Cell Infect Microbiol 13:1139800 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ferguson EC, Stewart EK, Hannah C, Elder CJ (2020) Obesity: unrecognised or avoided? We are missing opportunities to “make every contact count.” Arch Dis Child 105:310–311 [DOI] [PubMed] [Google Scholar]
  10. Gao M, Dong L, Yang Y, Yan J, Liang Y, Ma X, Zhou M, Wu H, Liu Y, Dai M (2024) The anti-atherosclerotic effect of Paeonol against the lipid accumulation in macrophage-derived foam cells by inhibiting ferroptosis via the SIRT1/NRF2/GPX4 signaling pathway. Biochem Biophys Res Commun 708:149788 [DOI] [PubMed] [Google Scholar]
  11. Ge X, Fan YZ, Deng Y, Chen Y, Gao J, Zhu G, Liu Y, Li Y, Ge G, Liu HX (2025) Pectolinarigenin mitigates 5-FU-induced intestinal mucositis via suppressing ferroptosis through activating PPARγ/GPX4 signaling. Phytomedicine 143:156843 [DOI] [PubMed] [Google Scholar]
  12. Giordano A, Murano I, Mondini E, Perugini J, Smorlesi A, Severi I, Barazzoni R, Scherer PE, Cinti S (2013) Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis. J Lipid Res 54:2423–2436 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Green M, Arora K, Prakash S (2020) Microbial medicine: prebiotic and probiotic functional foods to target obesity and metabolic syndrome. Int J Mol Sci. 10.3390/ijms21082890 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jeung WH, Nam W, Kim HJ, Kim JY, Nam B, Jang SS, Lee JL, Sim JH, Park SD (2019) Oral administration of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 with Cinnamomi Ramulus extract reduces diet-induced obesity and modulates gut microbiota. Prev Nutr Food Sci 24:136–143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kawabe Y, Mori J, Morimoto H, Yamaguchi M, Miyagaki S, Ota T, Tsuma Y, Fukuhara S, Nakajima H, Oudit GY, Hosoi H (2019) ACE2 exerts anti-obesity effect via stimulating brown adipose tissue and induction of browning in white adipose tissue. Am J Physiol Endocrinol Metab 317:E1140-e1149 [DOI] [PubMed] [Google Scholar]
  16. Kuriakose J, Montezano AC, Touyz RM (2021) ACE2/Ang-(1–7)/Mas1 axis and the vascular system: vasoprotection to COVID-19-associated vascular disease. Clin Sci (Lond) 135:387–407 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li J, Huang HY, Lin YC, Zuo H, Tang Y, Huang HD (2023) Cinnamomiramulus inhibits cancer cells growth by inducing G2/M arrest. Front Pharmacol 14:1121799 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li Y, Deng X, Hu Q, Chen Y, Zhang W, Qin X, Wei F, Lu X, Ma X, Zeng J, Efferth T (2024) Paeonia lactiflora Pall. ameliorates acetaminophen-induced oxidative stress and apoptosis via inhibiting the PKC-ERK pathway. J Ethnopharmacol 329:118107 [DOI] [PubMed] [Google Scholar]
  19. Litwin M, Kułaga Z (2021) Obesity, metabolic syndrome, and primary hypertension. Pediatr Nephrol 36:825–837 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu X, Chen L, Sun P, Zhan Z, Wang J (2024) Guizhi fuling formulation: a review on chemical constituents, quality control, pharmacokinetic studies, pharmacological properties, adverse reactions and clinical applications. J Ethnopharmacol 319:117277 [DOI] [PubMed] [Google Scholar]
  21. Miao P, Ruiqing T, Yanrong L, Zhuwen S, Huan Y, Qiong W, Yongnian L, Chao S (2022) Pyroptosis: a possible link between obesity-related inflammation and inflammatory diseases. J Cell Physiol 237:1245–1265 [DOI] [PubMed] [Google Scholar]
  22. Perez A, Naljayan M, Shuja I, Florea A, Reisin E (2021) Hypertension, obesity, and COVID-19: a collision of pandemics. Curr Hypertens Rep 23:36 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Qiu J, Chen L, Zhang L, Xu F, Zhang C, Ren G, Chang K, He G, Du Z, Le Y, Yu Z, Li S, Liu Q, Dou X (2023) Xie Zhuo Tiao Zhi formula modulates intestinal microbiota and liver purine metabolism to suppress hepatic steatosis and pyroptosis in NAFLD therapy. Phytomedicine 121:155111 [DOI] [PubMed] [Google Scholar]
  24. Ruze R, Liu T, Zou X, Song J, Chen Y, Xu R, Yin X, Xu Q (2023) Obesity and type 2 diabetes mellitus: connections in epidemiology, pathogenesis, and treatments. Front Endocrinol (Lausanne) 14:1161521 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sun J, Chen L, Xiao XA, Jia MQ, Wang XY, Jiao H, Gao Y (2021) Intestinal expression of ACE2 in mice with high-fat diet-induced obesity and neonates exposed to maternal high-fat diet. Nutrition 90:111226 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sun M, Liu GC, Wang MY, Wang WT, Zhang ZH, Yu H, Sun HM, Wang CM, Liu W (2025) Panax ginseng C. A. Mey. Targeting pyroptosis for treatment of diabetes related metabolic diseases: a review of potential therapeutic strategies. Phytother Res. 10.1002/ptr.70047 [DOI] [PubMed] [Google Scholar]
  27. Tang H, Zhao Y, Xu J, Tian Y (2025) Bai Mao tea polysaccharides induce adipose tissue browning and ameliorate diet-induced obesity by modulating butyrate production and gut microbiota composition. J Agric Food Chem. 10.1021/acs.jafc.5c06964 [DOI] [PubMed] [Google Scholar]
  28. Vallée A, Lecarpentier Y, Vallée JN (2021) Interplay of opposing effects of the WNT/β-catenin pathway and PPARγ and implications for SARS-CoV2 treatment. Front Immunol 12:666693 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wang X, Shi Y, Xu L, Wang Z, Wang Y, Shi W, Ma K (2021) Traditional Chinese medicine prescription Guizhi Fuling Pills in the treatment of endometriosis. Int J Med Sci 18:2401–2408 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yang B, Zhao Y, Luo W, Zhu W, Jin L, Wang M, Ye L, Wang Y, Liang G (2023a) Macrophage DCLK1 promotes obesity-induced cardiomyopathy via activating RIP2/TAK1 signaling pathway. Cell Death Dis 14:419 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yang G, Tong Y, Wang X, Zhao C, Ba Z, Ahelijiang R, Liu X, Gao W, Zhao Y, Gu Y, Yang J, Xu Y (2023b) Guizhi fuling capsule relieves memory deficits by inhibition of microglial neuroinflammation through blocking JAK2/STAT3 pathway in presenilin1/2 conditional double knockout mice. Front Immunol 14:1185570 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang M, Chen T, Liu YX, Huang L (2024a) Visualizing set relationships: EVenn’s comprehensive approach to Venn diagrams. Imeta 3:e184 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yang S, Cao J, Wang Y, Chen Q, Li F, Gao Y, Li R, Yuan L (2024b) Small intestinal endocrine cell derived exosomal ACE2 protects islet β-cell function by inhibiting the activation of NLRP3 inflammasome and reducing β-cell pyroptosis. Int J Nanomedicine 19:4957–4976 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yang S, Duan H, Zeng J, Yan Z, Niu T, Ma X, Zhang Y, Hu J, Zhang L, Zhao X (2025) Luteolin modulates macrophage phenotypic switching via the AMPK-PPARγ pathway to alleviate ulcerative colitis in mice. J Ethnopharmacol 339:119157 [DOI] [PubMed] [Google Scholar]
  35. Yao H, He Q, Xiang L, Liu S, Yang Z, Li X, Liu W, Huang C, Wang B, Xie Q, Gao Y, Zheng C, Li X (2024) Guizhi fuling wan attenuates tetrachloromethane-induced hepatic fibrosis in rats via PTEN/AKT/mTOR signaling pathway. J Ethnopharmacol 334:118593 [DOI] [PubMed] [Google Scholar]
  36. Ye H, Ma S, Qiu Z, Huang S, Deng G, Li Y, Xu S, Yang M, Shi H, Wu C, Li M, Zhang J, Zhang F, Qin M, Huang H, Zeng Z, Wang M, Chen Y, Lin H, Gao Z, Cai M, Song Y, Gong S, Gao L (2022) Poria cocos polysaccharides rescue pyroptosis-driven gut vascular barrier disruption in order to alleviates non-alcoholic steatohepatitis. J Ethnopharmacol 296:115457 [DOI] [PubMed] [Google Scholar]
  37. Yin K, Zhou X, Jiang W, Wang L, Dai Z, Tang B (2021) Jiangzhi ligan decoction inhibits GSDMD-mediated canonical/noncanonical pyroptosis pathways and alleviates high-fat diet-induced nonalcoholic fatty liver disease. Dis Markers 2021:9963534 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yousif MH, Dhaunsi GS, Makki BM, Qabazard BA, Akhtar S, Benter IF (2012) Characterization of angiotensin-(1–7) effects on the cardiovascular system in an experimental model of type-1 diabetes. Pharmacol Res 66:269–275 [DOI] [PubMed] [Google Scholar]
  39. Yuan L, Wang Y, Lu C, Li X (2013) Angiotensin-converting enzyme 2 deficiency aggravates glucose intolerance via impairment of islet microvascular density in mice with high-fat diet. J Diabetes Res 2013:405284 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhou C, Yin X (2022) Wogonin ameliorated obesity-induced lipid metabolism disorders and cardiac injury via suppressing pyroptosis and deactivating IL-17 signaling pathway. Am J Chin Med 50:1553–1564 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (PDF 2274 KB) (2.2MB, pdf)
Supplementary file2 (DOCX 12 KB) (12.4KB, docx)

Data Availability Statement

The data used and/or analyzed during the current study are available from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/bioproject/1310747).

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES