Abstract
Chronic inflammation induced by obesity plays a crucial role in the pathogenesis of insulin resistance. The infiltration of macrophages into adipose tissues contributes to adipose tissue inflammation and insulin resistance. Kaempferol, a flavonoid present in various vegetables and fruits, has been shown to possess remarkable anti-inflammatory properties. In this study, we used leptin receptor-deficient obese mice (db/db) as an insulin-resistant model and investigated the effects of kaempferol treatment on obesity-induced insulin resistance. Our findings revealed that the administration of kaempferol (50 mg/kg/day, for 6 weeks) significantly reduced body weight, fat mass, and adipocyte size. Moreover, it effectively ameliorated abnormal glucose tolerance and insulin resistance in db/db mice. In the adipose tissue of obese mice treated with kaempferol, we observed a reduction in macrophage infiltration and a downregulation of mRNA expression of M1 marker genes TNF-α and IL-1β, accompanied by an upregulation of Arg1 and IL-10 mRNA expression. Additionally, kaempferol treatment significantly inhibited the STING/NLRP3 signaling pathway in adipose tissue. In vitro experiments, we further discovered that kaempferol treatment suppressed LPS-induced inflammation through the activation of NLRP3/caspase 1 signaling in RAW 264.7 macrophages. Our results suggest that kaempferol may effectively alleviate inflammation and insulin resistance in the adipose tissue of db/db mice by modulating the STING/NLRP3 signaling pathway.
Introduction
Obesity is commonly linked to an elevated prevalence of morbidity and mortality in individuals with metabolic syndrome, encompassing type 2 diabetes, hypertension, and atherosclerosis (1, 2, 3). Individuals with obesity display heightened levels of circulating inflammatory markers such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-1β, and monocyte chemoattractant protein 1 (MCP-1), as well as decreased plasma levels of anti-inflammatory cytokines (adiponectin, IL-10) (4). Chronic inflammation in obese adipose tissues is characterized by a substantial infiltration of macrophages (5). In the context of prolonged positive energy homeostasis, adipose tissue undergoes expansion to accommodate increased energy. Adipose tissue expresses various factors, including leptin, adiponectin, and resistin, collectively referred to as adipokines, with adiponectin serving as an anti-inflammatory factor in obesity (6, 7). Adipose tissue macrophages can be classified into two main phenotypes: classical M1 macrophages and alternatively activated macrophages M2. Although both M1 and M2 macrophages increase in adipose tissue during expansion, M1 macrophages tend to dominate, aggregating around apoptotic adipocytes to form a characteristic feature known as a crown-like structure (CLS) (8). M1 macrophages in adipose tissue are considered instigators of insulin resistance due to the secretion of multiple pro-inflammatory cytokines, including TNFα (9). Chronic inflammation advances through paracrine interaction between enlarged adipocytes and adipose tissue macrophages (10).
The inflammasome NOD-like receptor 3 (NLRP3) is a recently identified protein complex that is responsible for the activation of inflammatory reactions (11). NLRP3 orchestrates the cleavage and maturation of IL-1β and IL-18, setting off an intricate network of cellular responses that culminate in both local and systemic inflammation (12). In the context of obesity, NLRP3 emerges as a potential key player in chronic inflammation. The activation of the NLRP3 inflammasome induced by obesity, coupled with alterations in its upstream and downstream signaling molecules, including NF-κB, has been implicated in the onset of insulin resistance in individuals with obesity (13).
Stimulator of interferon genes (STING, encoded by TMEM173) is critical for regulating type I interferons (IFNs) and pro-inflammatory cytokines upon sensing (14, 15). Mounting evidence underscores the pivotal role of STING as a signaling molecule in immunity and inflammation, activated by various stress signals, including viral infections. Upon cytoplasmic DNA binding, cyclic GMP-AMP synthase (cGAS) facilitates the generation of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) by interacting with STING (16, 17). Subsequently, STING colocalizes with TBK1 and IRF3, inducing type I IFNs, and recruits TRAF6 and TBK1 or TRAF3 and IKKα to activate the NF-κB pathway (18, 19, 20). Recent studies have highlighted the involvement of STING in cytosolic DNA-induced NLRP3 inflammasome activation in human myeloid cells. Additionally, in mice bone marrow-derived macrophages (BMDMs), the cGAS–STING pathway is essential for Chlamydia trachomatis-induced inflammasome activation and IL-1β secretion (21). In a model of LPS-induced cardiac injury, STING activation by LPS was found to trigger NLRP3 activation in a reactive oxygen species (ROS)-dependent manner. Furthermore, the protective effects of STING knockdown in LPS-induced cardiomyocyte injury were abrogated by the overexpression of NLRP3 via adenovirus (22). A recent study demonstrated that STING activation occurred in adipose tissue from mice with diet-induced obesity, implicating its role in diet-induced adipose tissue inflammation and insulin resistance (23). Consequently, we investigated whether the activation of the STING/NLRP3 signaling pathway in adipose tissue contributes to adipose tissue inflammation and insulin resistance.
Kaempferol, a type of flavonoid, is widely present in various vegetables and fruits (24, 25). Numerous studies have highlighted its diverse beneficial effects, including anti-diabetic, anti-inflammatory, antioxidant, antitumor, anti-atherosclerotic, hypoglycemic, and hypolipidemic effects (26, 27). Kaempferol is also known to regulate lipid metabolism, mitigating insulin resistance, and reducing lipotoxicity (28). This study aimed to investigate the impact of chronic kaempferol administration on adipose tissue morphology, chronic inflammation, and insulin resistance in diabetic animals. The detailed mechanisms underlying kaempferol's protective effect on inflammation and its associated signaling pathway in the adipose tissue of obese mice remain unexplored. Here, our objective is to evaluate whether kaempferol can suppress adipose tissue inflammation and alleviate insulin resistance by modulating the STING/NLRP3 signaling pathway in obese db/db mice.
Materials and methods
Animals and drug administration
All animal experiments were approved by the Research Animal Care Committee of Nanjing University of Chinese Medicine and were conducted in accordance with the Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. In total, 24 male C57BLKS db/db mice (6–8 weeks; weight, 45–50 g) and 7 male db/m mice (6–8 weeks; weight, 22–28 g) were purchased from GemPharmatech Co. Ltd. Nanjing, China. The mice were housed in a specific pathogen-free environment, maintaining controlled temperature and humidity conditions (22 ± 2°C, 50 ± 5% humidity), with a standard 12 h light:12 h darkness cycle. They were provided unrestricted access to food and water. Following a 1-week acclimatization period, the db/db mice were randomly divided into three groups: db/db group, db/db + kaempferol group (50 mg/kg b.wt/day; dissolved in 1% CMC-Na), and db/db + metformin group (0.15 g/kg). Administration was carried out via gavage for 6 weeks, as per previous protocols (26, 27, 29). Both the db/m group and db/db model group received an equivalent volume of 1% CMC-Na solution to minimize the potential impact of gavage procedure. Changes in body weight were recorded weekly during the experimental period.
Glucose and insulin tolerance tests
After 6 weeks of administration, mice underwent an insulin tolerance test, wherein 1 unit/kg human insulin was intraperitoneally injected following a 4-h fast (30). Blood samples were collected from the tail vein at 0 (just before insulin injection), 15, 30, 60, 90, and 120 min to assess glucose levels.
For the glucose tolerance test (GTT), mice were subjected to a 12-h fast. Subsequently, following intragastric administration of glucose (1g/kg), blood samples were collected from the tail vein at 0 (just before glucose administration), 15, 30, 60, 90, and 120 min for the measurement of glucose levels (31).
Adipose tissue preparation
Following the oral glucose tolerance test, the mice were euthanized under anesthesia. Subsequently, blood samples were obtained from the heart and transferred to microcentrifuge tubes for serum extraction. The subcutaneous and epididymal white adipose tissues were promptly excised from the sacrificed mice and weighed. A portion of each epididymal adipose tissue was preserved in liquid nitrogen for homogenization and subsequent protein and RNA extraction. Another portion of each epididymal adipose tissue was fixed in 4% paraformaldehyde for 24 h, followed by dehydration, paraffin embedding, and sectioning into 6 μm thick sections. These sections underwent hematoxylin/eosin and immunohistochemical staining.
Histopathological analysis and immunohistochemistry
The epididymal adipose tissue collected from the animals underwent a series of procedures. Initially, it was dissected, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into 4 μm transverse sections. These sections were subsequently deparaffinized and rehydrated using standard technique. For histological assessment of epididymal adipose tissue changes, the sections were stained with H&E. In the immunohistochemical (IHC) analysis, the sections were incubated with primary antibody against F4/80 (1:200; cat. no. 28463-1-AP, Proteintech Group) at 4°C overnight. Following washing, the sections were incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (cat. Ab6721, Abcam) at a dilution of 1:1000 for 1 h. The F4/80 signal was detected using the DAB method. Finally, the sections were photographed with a Nikon fluorescence microscope (×400) magnification, ECLIPSE, Ts2R-FL, Tokyo, Japan). Image-Pro Plus 6.0 software was employed to collect and analyze the pixels in the images, and the change of F4/80 positive pixel ratio was calculated (10).
Real-time PCR
Total RNA from the frozen adipose tissue and cells was extracted using TRIzol reagent (Invitrogen). Subsequently, cDNA was synthesized using PrimeScript reverse transcriptase (TaKaRa) following the manufacturer’s instructions. After the synthesis of cDNA using a cDNA synthesis kit, individual cDNAs underwent real-time PCR with SYBR Green dye on the ABI Prism 7500 Sequence Detection System (Applied Biosystems). The results were normalized to the quantity of β-actin RNA and calculated by the 2−∆∆CT method. The primers employed are detailed in Supplementary Table 1 (see section on supplementary materials given at the end of this article).
Western blot analysis
The adipose tissue was homogenized, and cells were lysed in ice-cold RIPA buffer. Protein concentration was quantified with a BCA protein quantification kit (Beyotime, Shanghai, China). The supernatant was combined with a buffer (25% glycerol, 2% SDS, 0.01% bromophenol blue, and Tris–HCl, pH 6.8) and heated at 100°C for 5 min. Subsequently, the samples underwent 10% SDS-PAGE, followed by transfer onto a PVDF membrane (Millipore). The membranes were incubated overnight with the specified primary antibodies. HRP-conjugated secondary antibodies were then applied to bind and visualize the primary antibodies. Quantitative analysis was performed by AlphaEaseFC software (version 3.1.2 Alpha Innotech Corporation, CA, USA). Antibodies against GAPDH (cat. no. 60004-1-Ig, 1:1000 dilution), IL-1β (cat. no. 16806-1-AP, 1:1000 dilution) were obtained from Proteintech Group. Antibodies against NLRP3 (cat. no. A5652, 1:1000 dilution) and caspase 1 (cat. no. A0964, 1:1000 dilution) were obtained from ABclonal Technology.
Cell viability assay and drug treatment in RAW 264.7 macrophages
Murine RAW 264.7 macrophages were purchased from the American Type Culture Collection. These cells were incubated in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin in humidified 5% carbon dioxide (CO2) at 37°C (32).
To evaluate the impact of kaempferol on the viability of RAW 264.7 macrophages, a CCK-8 assay was conducted. Briefly, cells (5 × 103 cells/well) were seeded into 96-well plates for 24 h before exposure to varying concentrations of kaempferol (0, 5, 10, 25, 50, and 100 μM) in humidified 5% CO2 at 37°C for 24 h. Subsequently, a reagent containing CCK-8 reagent (10 μL) was added to each well, and after 2 h, the absorbance of each well was measured at 450 nm using a microplate reader.
For further experiment, RAW 264.7 macrophages were seeded into 96-well plates and pretreated with or without kaempferol at a concentration of 10 μM for 24 h. Following this, LPS (1 μg/mL) was added for an additional 24 h. After collecting the supernatant, the cells were lysed using an RNA/Protein Isolation Kit.
Statistical analysis
Statistical analyses were performed using GraphPad Prism software (version 8.0.2 OriginLab Corporation, MA, USA). The data are presented as the means ± s.d. Group comparisons were conducted through one-way or two-way ANOVA for multiple data groups. Statistical significance was considered achieved when P < 0.05.
Results
Effects of kaempferol administration on body weight, glucose tolerance test, and insulin tolerance test
To investigate the effect of kaempferol on glucose metabolism in an insulin-resistant model, mice were subjected to daily gavage with 50 mg/kg of kaempferol over a 6-week period to evaluate its potential protective effects against insulin resistance. Metformin (0.15 g/kg) was used as a positive control. Throughout the treatment, body weight was significantly higher in db/db mice compared to control mice. Remarkably, kaempferol treatment resulted in a significant decrease in body weight (Fig. 1A). Db/db mice exhibited a substantial impairment in both glucose and insulin tolerance compared with the control mice. However, impaired glucose and insulin tolerance were effectively alleviated by kaempferol treatment (Fig. 1B and C).
Chronic administration of kaempferol demonstrates a pronounced effect on improving insulin sensitivity in db/db mice. (A) body weight, (B) glucose tolerance test (GTT), and (C) insulin tolerance test (ITT). Data are presented as mean ± s.e.m. (n = 7−8), ***P < 0.001 vs db/dm, #P < 0.05, ##P < 0.01, ###P < 0.001 vs db/db.
Citation: Endocrine Connections 13, 5; 10.1530/EC-23-0379
Effects of kaempferol administration on fat accumulation and adipocyte hypertrophy in db/db mice
Accumulation of fat and enlargement of adipocytes elicit the infiltration of macrophages and chronic inflammation, which is the underlying pathophysiology of insulin resistance. In comparison to the control mice, db/db mice displayed notably higher amounts of epididymal and subcutaneous fat, while kaempferol administration alleviated the increases in fat mass and adiposity in the db/db mice (Fig. 2A, B, C, and D). H&E staining further illustrated that kaempferol alleviated the enlargement of adipocytes in the epididymal adipose tissue (Fig. 2E and F).
Kaempferol treatment improves HFD-induced obesity and fat accumulation in mice. (A) Representative images of epididymal adipose tissue from the mice. (B) Weights of eWAT from the mice. (C) Representative images of subcutaneous adipose tissue from the mice. (D) Weights of sWAT from the mice. H&E staining of eWAT (E). Scale bar, 100 mm. (F) Quantitative analyses of adipocyte size. Data are presented as mean ± s.e.m. (n = 4–5), ***P < 0.001 vs db/dm, #P < 0.05, ##P < 0.01, ###P < 0.001 vs db/db.
Citation: Endocrine Connections 13, 5; 10.1530/EC-23-0379
Kaempferol treatment alleviates the inflammation state in the epididymal adipose tissue of db/db mice
We investigated the effect of kaempferol on systemic and adipose tissue inflammation in db/db mice. To identify and quantify macrophages within epididymal white adipose tissue (eWAT), we immunohistochemically stained sections for the F4/80 antigen, a specific marker for mature macrophages. The percentage of F4/80-expressing cells in db/db mouse eWAT was calculated. Our data revealed that oral administration of kaempferol reduced the percentage of F4/80-positive cells within eWAT in db/db mice (Fig. 3A and B). Specifically, M1 macrophage mRNA expression of TNF-α and IL-18 was significantly upregulated, while M2 macrophage mRNA expression of Arg1 and IL-10 was downregulated in db/db mice. Kaempferol administration effectively reversed the M1-specific macrophage and M2-specific macrophage mRNA expressions in the eWAT (Fig. 3C, D, E, and F). These findings indicate that kaempferol treatment can substantially inhibit adipose tissue inflammation in db/db mice.
Kaempferol alleviates inflammation in epididymal adipose tissue of db/db mice. (A) F4/80 immunohistochemical analysis of eWAT; scale bar = 100 μm (n = 4–5). (B) The ratio of all F4/80-positive pixels was counted in each fraction of eWAT (n = 4–5). (C, D) The expression of macrophage and M1 pro-inflammatory genes in eWAT was quantified by RT-PCR (n = 4–5). (E, F) The relative mRNA levels of M2 anti-inflammatory genes in eWAT of mice. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs db/dm, #P < 0.05, ##P < 0.01 vs db/db.
Citation: Endocrine Connections 13, 5; 10.1530/EC-23-0379
Kaempferol reverses the activation of STING/NLRP3-caspase1 pathway in adipose tissue
To further elucidate the potential mechanism by which kaempferol alleviates the inflammation state in adipose tissue, we investigated its effect on the STING/NLRP3-caspase 1 pathway. Immunohistochemical analysis revealed elevated levels of STING in db/db mice compared to dm/dm mice. Kaempferol administration effectively reversed the expression of STING in the eWAT (Fig. 4A and B). Protein expressions of NLRP3, caspase 1, and IL-1β were assessed by Western blotting. We observed a significant upregulation of NLRP3, caspase 1, and IL-1β in the adipose tissue of db/db mice compared to control mice (Fig. 4C). Remarkably, this activation was significantly reversed by kaempferol treatment.
Effects of kaempferol on the STING/NLRP3-caspase1 pathway in adipose tissue. (A) STING immunohistochemical analysis of eWAT; scale bar = 20 μm (n = 4–5). (B) The ratio of all STING-positive pixels was counted in each fraction of eWAT (n = 6–8). (C) Western blot analysis of NLRP3, caspase 1, and IL-1β (n = 4–5). (D–G) Relative densitometric values. The data are presented as the means ± s.e.m. **P < 0.01, ***P < 0.001 vs db/dm, #P < 0.05, ##P < 0.01, ###P < 0.001 vs db/db.
Citation: Endocrine Connections 13, 5; 10.1530/EC-23-0379
Effects of kaempferol on LPS-induced inflammatory cytokine secretion and NLRP3 inflammasome in RAW 264.7 macrophages
Considering the pivotal role of adipose tissue macrophages in the development of insulin resistance, we finally examined the effect of kaempferol on macrophage function in vitro. To assess the effect of kaempferol on cell viability, we utilized the CCK-8 kit. Initially, we determined the safe dose of the drug, revealing that kaempferol had no adverse effect on cell viability at concentrations ranging from 0 to 10 μM. Consequently, 10 μM kaempferol was selected for subsequent experiments (Fig. 5A). In RAW 264.7 macrophages, LPS significantly increased the mRNA and expressions of IL-18, IL-1β, and TNF-α compared to the control group. However, coincubation with kaempferol mitigated the expression of these pro-inflammatory mediators in RAW 264.7 macrophages (Fig. 5B). Additionally, LPS induced a substantial increase in protein (Fig. 5C) and mRNA (Fig. 5D and E) expressions of NLRP3, caspase 1, and IL-1β in RAW 264.7 macrophages. Notably, treatment with kaempferol decreased the expression of NLRP3, caspase 1, and IL-1β when coincubated with LPS in RAW 264.7 macrophages. These findings indicate that kaempferol suppresses LPS-induced inflammatory cytokine secretion and NLRP3 inflammasome activation in RAW 264.7 macrophages.
Kaempferol represses LPS-induced inflammatory cytokine secretion and NLRP3 inflammasome in RAW 264.7 macrophages. (A) Viability of cells after kaempferol treatment determined by the CCK-8 method. (B) Inflammatory cytokine mRNA expression levels quantified by RT-qPCR. (C, D) The protein and mRNA expressions of NLRP3, caspase 1, and IL-1β were measured by western blot and RT-qPCR analysis, respectively. Data were collected from three independent experiments and expressed as the mean ± s.e.m. *P < 0.05,**P < 0.01,***P < 0.001 vs control, ##P < 0.01, ###P < 0.001 vs LPS group.
Citation: Endocrine Connections 13, 5; 10.1530/EC-23-0379
Discussion
Obesity and diabetes are intricately linked to low-grade chronic inflammation, particularly within adipose tissue. Adipose tissue macrophages (ATMs) play a pivotal role in inflammation associated with obesity and insulin resistance (33). In this study, we have demonstrated for the first time that kaempferol treatment effectively improved the impaired glucose and insulin tolerance, ameliorated adipose tissue inflammation, at least partially via the STING/NLRP3 signaling pathway. Furthermore, in vitro findings also supported the inhibitory effect of kaempferol on LPS-induced inflammation and activation of the NLRP3/caspase 1 signaling in RAW 264.7 macrophages.
The natural product kaempferol, extracted from plants, has emerged as the focus in various studies. Kaempferol, a dietary flavonoid, is abundantly present in human diets, making a substantial contribution to daily flavonoid intake. Notably, it orchestrates numerous metabolic processes to safeguard against obesity and related disorders (34). In a study involving high-fat diet (HFD) obese mice, the administration of kaempferol in daily doses of 50 mg/kg significantly improved blood glucose control, associated with reduced hepatic glucose production and improved whole-body insulin sensitivity (26). In the present study, we demonstrated that oral administration of kaempferol (50 mg/kg daily) for 6 weeks mitigated body weight gain and improved the impaired glucose and insulin tolerance in db/db mice. Hence, this study indicates that this natural flavonoid can be exploited as a cost-effective and safe compound for the treatment and prevention of obesity-induced insulin resistance in db/db mice.
Adipose tissue plays a crucial role in insulin resistance (IR). An augmented adipocyte population translates to increased lipid accumulation and the release of inflammatory cytokines, thereby fostering the progression of IR (35). Increased hypertrophy is a hallmark of adipose tissue enlargement in obesity, correlating with metabolic alterations, pro-inflammatory response, and increased risk of developing T2DM (36). In our study, a notable augmentation in adipocyte size, accompanied by a disrupted arrangement, was observed in the adipose tissue of diabetic mice. Furthermore, the administration of kaempferol for 6 weeks effectively alleviated the increases in fat mass and adipocyte size. These findings underscore the potential of kaempferol in ameliorating obesity, fat accumulation, and adipocyte hypertrophy in db/db mice.
It is well known that hypertrophic adipocytes secrete chemokines, attracting various inflammatory cells and contributing to chronic inflammation in adipose tissue. In obesity, macrophages emerge as the predominant inflammatory cells within adipose tissue. Adipose tissue and adipose tissue macrophages serve as major sources of pro-inflammatory molecules (9). The endocrine-mediated cross talk among adipose tissue, the liver, and skeletal muscle leads to both local and systemic inflammation, along with insulin resistance, through autocrine and paracrine signaling. The recruitment of macrophages into adipose tissue marks an initial event and a primary contributor to inflammation in obesity. A strong correlation exists between the phenotypic transformation of adipose tissue macrophages (ATMs) from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype and the ensuing inflammation and insulin resistance in adipose tissue. Previous studies have reported that inhibiting the infiltration of macrophages into adipose tissue can ameliorate insulin resistance in obese mice (37). It has been demonstrated that alternatively activated M2 macrophages contribute to maintaining insulin sensitivity by expressing and secreting anti-inflammatory cytokines, while classically activated M1 macrophages induce insulin resistance through the expression and secretion of pro-inflammatory cytokines. In obesity, metabolic inflammation in adipose tissue precedes inflammation in the liver, indicating that the liver does not play a role in the initial development of metabolic inflammation. Moreover, hepatic inflammation is of lesser importance in the development of insulin resistance compared to adipose tissue inflammation (38). Therefore, our study focused on adipose tissue inflammation and insulin resistance. In our study, we discovered that kaempferol rescued macrophage recruitment and reversed M1 macrophage polarization in the adipose tissues of db/db mice. Our data demonstrated that kaempferol treatment reduced the presence of macrophages in the epididymal white adipose tissue (eWAT) of db/db mice, as confirmed by immunohistochemical staining and RT-PCR analysis. Furthermore, kaempferol suppressed the expression of TNF-α and IL-18, which are marker genes for M1 macrophages, while also increasing the mRNA levels of marker genes for M2 macrophages in the epididymal white adipose tissue (eWAT). In vitro, we also confirmed that kaempferol inhibited LPS-induced macrophage inflammatory responses and the expression of inflammatory cytokines. These results indicate that kaempferol may improve insulin resistance by alleviating the inflammatory state in adipose tissue.
The protein STING, consisting of 379 amino acids, is expressed across various cell types and plays essential roles in regulating infection and inflammation. Early studies revealed STING’s essential role in the immune response to bacterial and viral invasion (39). Recent studies have also uncovered that STING signaling can be triggered by self-DNA in necrotic cells, initiating autoinflammatory diseases (40). Cytosolic DNA binds to cGAS, generating cyclic dinucleotides (CDNs). Upon binding to these CDNs, STING forms a complex with TBK1, initiating signal transduction that ultimately activates IRF3 and NF-κB (41). Kaempferol has been shown to alleviate cardiac injury induced by cisplatin via inhibiting the STING/NF-κB-mediated inflammatory response (42). As a crucial molecule for cellular innate immunity and inflammatory responses, STING stimulates NLRP3 activation and promotes the release of mature inflammatory cytokines. In the LPS-induced model of cardiac injury, STING activation by LPS leads to NLRP3 activation, dependent on ROS. Moreover, NLRP3 overexpression by adenovirus abrogates the protective effects of STING knockdown in LPS-induced cardiomyocytes. STING is involved in cytosolic DNA-induced NLRP3 inflammasome activation in human myeloid cells, and in mice BMDMs, STING is required for pathogen-induced inflammasome activation and IL-1β secretion (43). Accumulating evidence suggests that various cellular factors contribute to NLRP3 inflammasome stimulation, playing a central role in adipose tissue inflammation, subsequent tissue damage associated with obesity, and metabolic dysfunction. Kaempferol has been reported to inhibit NLRP3 inflammasome activation (24). To elucidate the mechanism of kaempferol treatment in db/db mice, we investigated whether kaempferol improves the obesity-associated adipose tissue inflammation and insulin resistance through the STING/NLRP3 signaling pathway. Our study observed a reduction in the expression of NLRP3, caspase 1, and IL-1β in the adipose tissue of db/db mice following kaempferol administration. Additionally, kaempferol inhibited activation of the NLRP3/caspase-1 signaling pathways in RAW 264.7 macrophages. Furthermore, kaempferol administration reversed the elevated expression of STING in the epididymal white adipose tissue (eWAT). Therefore, these findings may suggest that kaempferol mitigates systemic inflammation and insulin resistance in the adipose tissue of db/db mice through the STING/NLRP3 signaling pathway.
In conclusion, our data implied that kaempferol's anti-obesity effect alleviates insulin resistance, primarily through its anti-inflammatory effects on the adipose tissue. This beneficial impact is likely exerted by inactivating the STING–NLRP3 inflammasome pathway. The present study provides a new mechanistic basis of the therapeutic effect of kaempferol in addressing obesity-associated insulin resistance. However, a limitation of this study is the absence of mice treated with STING agonist to confirm the underlying molecular mechanism. In future experiments, we plan to administer a STING agonist to investigate whether kaempferol improves insulin resistance by regulating NLRP3-mediated adipose tissue inflammation through the STING pathway, both in vivo and in vitro. Future research should place greater emphasis on exploring the potential protective mechanisms of kaempferol in the treatment of chronic inflammatory-related metabolic diseases.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EC-23-0379.
Declaration of interest
‘The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the study reported.
Funding
This study was supported by grants from the Jiangsu Provincial Administration of Traditional Chinese Medicine, project name: Traditional Chinese Medicine Science and Technology Development Plan Project (No. YB2020034), and the Nanjing Municipal Health Commission, project name: Traditional Chinese Medicine Science and Technology Special (No. ZYYB202215).
Author contribution statement
H Zhai and D Wang performed experiments and analyzed data. They contributed equally to this paper. Y Wang and H Gu assisted the experiments. J Jv, L Yuan, and C Wang critically reviewed the manuscript. L Chen wrote the manuscript.
Acknowledgements
The authors would like to give sincere appreciation to the reviewers for their helpful comments on the article.
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