Inhibitory effects of the flavonoids extracted from Pollen Typhae on palmitic acid-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism

Abstract

Background

Pollen Typhae (PT), a traditional Chinese medicine herb utilized in diabetes management, exerts anti-inflammatory effects through its flavonoids, yet the active constituents and mechanisms remain unclear.

Methods

PT total flavone (PTF) was extracted from PT and identified the compounds by UHPLC-MS. Network pharmacology and molecular docking were used to predict the underlying targets and anti-inflammatory mechanisms of PTF. The prediction was validated in RAW264.7 macrophages. IL-1β and IL-18 in culture supernatants were analyzed by ELISA. The protein and gene expression were checked by western blotting and Real-time PCR, respectively. Intracellular ROS production was detected by DCFH-DA method. Intracellular lipids were analyzed by ELISA and Enzyme assay. The Caspase-1 activity was evaluated by bioluminescent method.

Results

PTF was identified 47 flavonoid compounds, including typhaneoside (TYP). Network pharmacology and molecular docking indicated that the flavonoid compounds might regulate inflammatory response, fatty acid metabolism, and the NOD-like receptor, AMPK pathways. PTF and TYP inhibited palmitic acid (PA)-induced NLRP3 inflammasome activation in lipopolysaccharide-primed RAW264.7 macrophages, leading to decreased secretion of IL-1β and IL-18. Furthermore, PTF and TYP improved intracellular lipid metabolism in PA-induced macrophages, indicating decreased free fatty acid and triglyceride contents, reduced protein expression of CD36, PPARγ, FAS, DGAT1, and CPT-1, as well as declined ROS with increased ATP production. Additionally, PTF and TYP increased the p-AMPK/AMPK ratio and upstream p-LKB1/LKB ratio. Activated AMPK, in turn, ameliorated lipid metabolism dysfunction, thus abolishing PA-induced ROS production and NLRP3 inflammasome activation. Antioxidant and improving lipid metabolism by suppressing ACC also inhibited NLRP3 inflammasome activation, respectively. Importantly, AMPK inhibition attenuated or abolished the inhibitory effects of PTF or TYP on ROS production, IL-1β and IL-18 secretion, and Caspase-1 activity.

Conclusion

The findings highlight the ability of PTF and its active component TYP to inhibit PA-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism, implying the potential use of PT flavonoid compounds as anti-diabetic inflammation lead compounds.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-025-05024-4.

Introduction

The nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is a critical protein complex involved regulating the immune response by controlling the maturation and secretion of interleukin-1β (IL-1β) and IL-18. Its core constituents consist of NLRP3, Caspase-1, an effector protein, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), a bipartite adaptor protein. NLRP3 inflammasome mainly resides in the immune system such as monocytes/macrophages, neutrophils, lymphocytes, and non-immune system organizations. Canonical activation of NLRP3 inflammasome needs to undergo a priming step followed by an activation process [1]. During the priming step, upon stimulation with microbial infections, bacterial lipopolysaccharide (LPS) or endogenous damage stress, NLRP3, pro-IL-1β and pro-IL-18 are transcriptionally expressed through activation of the nuclear factor κB (NF-κB) pathway [2]. In the subsequent activation step, NLRP3 is liberated from self-inhibition, and then clusters and recruits ASC to scaffold pro-Caspase-1 in the present of various stimulus including pathogens, extracellular adenosine triphosphate (ATP), cholesterol crystals, potassium efflux, and reactive oxygen species (ROS) [1, 3]. This leads to the cleavage of pro-Caspase-1 into its active form, which then processes pro-IL-1β and pro-IL-18 into their mature forms IL-1β p17 and IL-18, facilitating their release and triggering inflammatory responses to counteract microbial invasion and endogenous damage [1, 3]. However, excessive activation of NLRP3 inflammasome results in severe inflammation and subsequent injury to cells and tissues.

Type 2 diabetes is often accompanied by chronic inflammation, manifesting abnormal activation of NLRP3 inflammasome and continuously increased serum or local inflammatory cytokines [4, 5], which in turn deteriorates diabetes by impairing insulin sensitivity and pancreatic β cells [6]. Inhibiting NLRP3 inflammasome-mediated inflammation has been shown to improve progression of type 2 diabetes [7]. Macrophages, distributed in various tissues including fatty tissues, liver, pancreatic islets, and brain, are believed to originate from blood monocytes and play a key role in regulating inflammation. Increasing evidences show that there are much more infiltrated macrophages in fatty tissues, liver, pancreatic islets, and kidney of diabetic patients than those of non-diabetic ones [8–10]. Notably, monocytes/macrophages isolated from obese and type 2 diabetic patients have elevated NLRP3 inflammasome activity with systemic inflammation [5, 11]. Macrophage depletion alleviates local and systemic inflammation with low expression of NLRP3 [10, 12]. Currently, anti-inflammatory treatment has become an important strategy for diabetes management.

Pollen Typhae (PT), the yellow dry pollen harvested from plants of the genus Typhaceae in summer such as Typha orientalis Presl. and Typha angustifolia L., has been applied as an herbal medicine for more than two thousand years, and was recognized as one of functional foods by Ministry of Health of China in 2002. Recent pharmacological studies have identified different kinds of chemical components in PT, containing flavonoids, polyphenols, polysaccharides, organic acids, amino acids, and nucleosides [13–15]. Notably, carbonized PT, characterized by its black or brown appearance after stir-fry, promotes the endogenous and exogenous blood coagulation pathways, attributed to active flavonoids such as quercetin-3-O-glucoside, quercetin, and kaempferol [15, 16]. The ethanolic extracts of PT are rich in flavonoids containing typhaneoside (TYP) and isorhamnetin-3-O-neohesperidoside (I3ON) [13, 17]. TYP and I3ON exhibit high activity in regulating antioxidant enzymes [17]. Moreover, TYP inhibits mitochondrial ROS production and improves mitochondrial function in human kidney cells induced by ischemia-reperfusion [18], implying the potential effects on energy metabolism. Besides, TYP dose-dependently decreases serum IL-6 and TNF-α in a rat with heart failure followed by myocardial infarction [19]. But the anti-inflammatory mechanism of TYP remains to be elaborated.

PT has been used for the treatment of type 2 diabetes in clinical practice. Recent studies have shown that PTF promotes glucose consumption and transportation in 3T3-L1 adipocytes [20], suggesting potential anti-diabetes effects. In further research, PTF, containing TYP, has been found to ameliorate blood glucose and lipid levels, enhance insulin sensitivity and insulin secretion function in type 2 diabetic rats induced by high fat diet plus streptozotocin [21–23]. The anti-diabetes mechanisms of PTF involve increasing glucose consumption and glucose uptake, improving palmitic acid (PA)-caused insulin resistance in C2C12 myotubes through the regulation of the β-arrestin-2-mediated pathway [21], and promoting GPR40 signaling to relieve PA-caused impairment of glucose-stimulated insulin secretion in INS-1 pancreatic β cells [23]. Furthermore, PTF has been shown to decrease blood IL-6 levels in type 2 diabetic rats [24], and to inhibit the extracellular regulated protein kinases 1/2 pathway and subsequent expression of IL-1β and tumor necrosis factor-alpha (TNF-α) in LPS-triggered macrophages [13]. These suggested that the anti-diabetes action of PT flavonoids is at least partially attributed to the improvement of inflammation. Nonetheless, the active components and mechanisms by which PT improves inflammation are not fully understood. The present study was designed to evaluate the effects of PTF and its active components on NLRP3 inflammasome activation in PA-induced macrophages, and to reveal the potential mechanisms involved.

Methods

Preparation of PT flavonoids and analysis of chemical compounds

The pollen of Typha angustifolia L. (Puhuang in Chinese) was harvested in Hubei, China, and provided by Xi’an Salao Biotechnology Co., Ltd. (Xi’an, China). The pollen was validated by Chunling Wang (College of Pharmacy, Guangxi University of Chinese Medicine, Nanning, China). The extraction of PTF from the pollen was carried out following a previously reported method with some modification [21]. Initially, the dried pollen was extracted with 85% ethanol in four cycles. The resulting solution was then filtered and concentrated under low-temperature, vacuum conditions. Subsequently, the extracts were purified by macroporous resin adsorption method. The effluent was concentrated under vacuum and dried by spray drying. The dried power, with an extraction rate of 0.8%, was analyzed for flavonoid contents by ultraviolet spectrophotometry, revealing a content of 90.25%. Table S1 showed the chemical physical and microbiology control for the extract. Voucher specimens, including the pollen (No. HB-2107) and PTF (No. 20220328), were deposited in the Guangxi Key Laboratory of Chinese Medicine Foundation Research, Institute of Traditional Chinese and Zhuang-Yao Ethnic Medicine, Guangxi University of Chinese Medicine (Nanning, China).

Then, 1.0 g PTF powder sample was dissolved in 40 ml of 80% methanol and ultrasonicated for 30 min. The sample solution was centrifuged for 10 min (4 °C, 12000 rpm). Then the supernatant was pipetted into an autosampler vial. A Vanquish Flex ultra-high performance liquid chromatograph (UHPLC, Thermo Fisher Scientific, Waltham, MA, USA) was installed with an ACQUITY UPLC HSS T3 column (2.1 mm ×100 mm, 1.7 μm) and used for chromatographic separation at 40 °C. The elution was carried out at a flow rate of 0.3 ml/min using 0.1% formic acid in water (A) and acetonitrile (B) as the following procedure: 0 ~ 1 min, 98% A; 1 ~ 14 min, 70% A; 14 ~ 28 min, 0 A; 28.1 ~ 30 min, 98% A. After that, a hybrid quadrupole orbitrap high resolution mass spectrometer (Q Exactive, Thermo Fisher Scientific, Waltham, MA, USA) was applied to analyze the chemical components. The following parameters were set in this experiment: positive and negative ion source voltages were 3.7 kV and 3.5 kV, respectively. Heated capillary temperature was set at 320 °C. The sheath gas, the auxiliary gas and the collision gas was nitrogen, with pressures of 30 psi, 10 psi and 1.5 mTorr, respectively. The solvent was evaporated at 300 °C. The raw data were obtained in Full scan/dd-MS2 mode according to the following parameters. Full scan: resolution 70,000, auto gain control target 1 × 10⁶, the m/z scanning range 100 ~ 1500, maximum interval time 50 ms. The dd-MS2: resolution 17,500, auto gain control target 1 × 10⁵, isolation window m/z2, maximum interval time 50 ms, collision energy 10 V, 30 V, 60 V and intensity threshold 1 × 10⁵. The mass spectrometer (MS) data were analyzed using Progenesis QI 3.0 (Waters Corp., MA, USA). The compounds were determined using the reference substance database (TCM Pro 2.0, Beijing Hexin Technology Co., Ltd) and theoretical database based on literatures and public databases.

The reference compounds TYP (purity by HPLC, 99.61%; Lot: MUST-19090902 and MUST-22081314) and I3ON (purity by HPLC, 99.79%; Lot: MUST-20082104) were extracted from pollen of Typha angustifolia L. and provided by Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). They were authenticated by HPLC and NMR (Fig. S1 and S2).

Network pharmacology analysis

The SMILES information for the identified flavonoid compounds from PT was obtained from the PubChem databases (https://pubchem.ncbi.nlm.nih.gov/). The data were subjected to predict the potential ingredient targets of the flavonoids using the Swiss Target Prediction (www.swisstargetprediction.ch/) and the Comparative Toxicogenomics Database (http://ctdbase.org/). GeneCards (http://www.genecards.org/) was applied to retrieve the disease-related gene targets for type 2 diabetes. The intersecting targets between the flavonoid compounds and the disease were identified to construct Venn diagrams using Venny software (http://www.bioinformatics.com.cn/). Subsequently, the intersecting targets were typed into the STING Database (https://cn.string-db.org/) to draw a protein-protein interaction (PPI) network and then determined the key targets using Cytoscape 3.9.1 software. The data involving the flavonoid compounds, the key targets and the disease were performed to produce flavonoid compound-target-disease network using Cytoscape 3.9.1 software. Finally, the intersecting targets were input into the Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) to display the target functions and molecular pathways using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, respectively.

Cell culture

RAW264.7 macrophages were acquired from the Cell Bank of Type Culture Collection of the Chinese Academy of Science (Shanghai, China), and grown in high-glucose DMEM medium containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 110 mg/L sodium pyruvate (Sigma, St. Louis, MO, USA). The cells were incubated at 37˚C in a humidified incubator with 5% CO₂. The medium was changed every two days. The passage number of the cells used for the experiments was no more than 15 from the original batch.

Preparation for PA solution

PA (Sigma, St. Louis, MO, USA) was dissolved according to the method we reported with some modification [23]. Briefly, 100 mM PA solution was obtained by dissolving PA in 0.1 M NaOH at 70˚C. After that, the solution was mixed with 10% FFA-free bovine serum albumin (BSA, Sigma, St. Louis, MO, USA)-DMEM medium at the ratio of 1 to 19 at 55˚C. After filtration sterilization, the solution was stored as aliquots at -80˚C. The stock solution was thawed in a water bath at 55˚C for 15 min, and then equilibrated to room temperature before use.

Cell viability assay

CCK-8 method was conducted to analyze cell viability. RAW264.7 macrophages were plated at 1 × 10⁴ cells/well and grown in 96-well plates, and then interfered with 1% FFA-free BSA-DMEM medium containing different concentrations of PTF (0.0, 0.03125, 0.0625, 0.125, 0.25, 0.5 mg/ml), TYP (0, 5, 10, 20, 40, 80, 100 µM) or I3ON (0, 5, 10, 20, 40, 80, 100 µM) for 16–20 h. Subsequently, the culture supernatants were replaced by the CCK-8 working solution (Dojingdo, Japan). After incubation for 1.5 h at 37˚C, the cell viability was evaluated through reading the absorbance at a wavelength of 450 nm using a Biotek microplate reader (Epoch2).

ELISA assay for IL-1β and IL-18

RAW264.7 macrophages were pretreated with LPS (1 ng/ml, Sigma, St. Louis, MO, USA) or/and PTF (0.03125, 0.0625, 0.125, 0.25, 0.5 mg/ml), TYP (10, 20, 40, 80, 100 µM), I3ON (10, 20, 40, 80, 100 µM), Compound C (10 µM), NAC (10 mM, Sigma, St. Louis, MO, USA) for 3 h followed by exposure to 0.5 mM PA for 16 h, the culture supernatants were collected to analyze IL-1β and IL-18 using commercial ELISA kits for mouse IL-1β (Boster Biological Technology Co. Ltd., Wuhan, China) and IL-18 (Cusabio, Wuhan, China) according to the manufacturers’ manual.

Western blotting

The macrophages were pre-exposed to LPS (1 ng/ml) or/and PTF (0.5 mg/ml), TYP (100 µM), AICAR (2 mM, Beyotime, Shanghai, China), MCC950 (10 µM, Cell Signaling Technology, Danvers, MA, USA), PF-05175157 (10 µM, GlpBio Technology, Montclair, CA, USA) for 3 h before treatment with PA (0.5 mM) for 16 h. The cells were harvested to obtain total protein. The protein expression was conducted to analyze by western blotting as previously described [25]. The primary antibodies used were as follows: anti-NLRP3 (dilution, 1: 800), Caspase-1 (dilution, 1: 1,000), IL-1β (dilution, 1: 1,000), CD36 (dilution, 1: 1,200), fatty acid synthase (FAS; dilution, 1: 1,100), and ATP synthase alpha-subunit (Atp5a1; dilution, 1: 1,000) antibodies (Cell Signaling Technology, Danvers, MA, USA), anti-AMP-activated protein kinase (AMPK; dilution, 1: 1,000) antibody (Abcam, Cambridge, MA, USA), anti-GAPDH antibody (Cell Signaling Technology, and Afffnity Biosciences, Changzhou, China; dilution, 1: 1,000 and 1: 5,000, respectively), anti-diacylglycerol-O-acyltransferase 1 (DGAT1; dilution, 1: 1,000), phospho-AMPK (p-AMPK; dilution, 1: 1,000), NF-κB p65 (dilution, 1: 1,000), phospho-NF-κB p65 (p-NF-κB p65; dilution, 1: 1,000), phospho-inhibitor of NF-kappaB (p-IκBα; dilution, 1: 1,000), IκBα (dilution, 1: 1,000), and liver kinase B1 (LKB1; dilution, 1: 500) antibodies (Beyotime, Shanghai, China), anti-phospho-LKB1 (p-LKB1; dilution, 1: 500) antibody (Afffnity Biosciences), anti-carnitine palmitoyltransferase 1 (CPT-1; dilution, 1: 300) antibody (Santa Cruz, CA, USA), and anti-peroxisome proliferator-activated receptor gamma (PPARγ; dilution, 1: 1,000) antibody (BIOSS, Beijing, China). Secondary antibodies (dilution, 1: 2,000) against rabbit and mouse were obtained from Cell Signaling Technology and Zhongshan Golden Bridge Biotechnology (Beijing, China).

Real-time quantitative PCR

After pretreatment with LPS (1 ng/ml) or/and PTF (0.5 mg/ml), TYP (100 µM) for 3 h followed by the treatment with PA (0.5 mM) for 16 h, total RNA was isolated from the macrophages using a TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturers’ protocol. Total RNA was then transcribed into complementary DNA, the gene expression was measured by Real-time PCR according to our previous report [25]. The primers used were shown in Table S2.

Molecular docking analysis

The two-dimensional structure of the component TYP was acquired from PubChem databases and then converted to 3D structure using ChemBio 3D software. The Protein Data Bank database (https://www.rcsb.org/) provides the crystal structures of the target proteins. TYP and the target proteins, regarded as a ligand and receptors, respectively, were docked using AutoDock Tools (version 1.5.7). Finally, PyMOL software (version 2.5.2) was conducted to display the docking results. Generally, the binding activity between ligand and receptor is negatively correlated with binding energy. The binding energy which is less than or equal to -7 kcal/mol indicates strong binding activity between ligand and receptor.

Intracellular ROS assay

The macrophages were seeded in 96-well plates, and then pretreated with LPS (1 ng/ml) or/and PTF (0.5 mg/ml), TYP (100 µM), Compound C (10 µM), NAC (10 mM), AICAR (2 mM) for 3 h before stimulation with PA (0.5 mM) for 16 h. Intracellular ROS generation was quantified using a ROS assay kit (DCFH-DA method, Beyotime, Shanghai, China) according to the manufacturers’ instructions.

Intracellular lipid assay

After treatment, the cells were lysed and then centrifuged to collect supernatants. The supernatants were used to check intracellular TG, FFA, DAG, MAG and LEC contents using a Cellular TG Enzyme assay kit (Beijing APPLYGEN Technology Co., LTD., Beijing, China) and mouse FFA, DAG, MAG and LEC ELISA kits (Shanghai YSRIBIO Industrial Co., LTD., Shanghai, China) following the manufacturers’ instructions.

Intracellular ATP content assay

After treatment, the macrophages were lysed in cold ATP detection lysis buffer for 30 min. Supernatants were acquired through centrifugation the lysate (12,000 g for 5 min, 4˚C), and intracellular ATP content was determined using an ATP Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s manual. In brief, each supernatant or ATP standard solution was added to ATP detection working solution at the ratio of 1 to 5, and the luminescence was checked using a fluorescence microplate reader (VICTOR Nivo, PerkinElmer, USA). ATP content was normalized according to the protein concentration.

Caspase-1 activity assay

Caspase-1 activity in culture supernatants and the macrophages was detected using a Caspase-Glo^® 1 Inflammasome Assay kit (Promega Corporatton, Madison, WI, USA) according to the protocols. After treatment in white 96-well plates with clear bottom, the culture supernatants (50 µl/well) were transferred to another new plate followed by the addition of Caspase-Glo^® 1 Reagent (50 µl/well). Then, the macrophages were incubated with new FBS-free DMEM medium (100 µl/well) and Caspase-Glo^® 1 Reagent (100 µl/well). After that, the plates were shook at 300 rpm for 30 s, and kept in dark at room temperature for 60 min. The luminescence was checked using a fluorescence microplate reader.

Statistical analysis

Data analysis was conducted using SPSS 16.0 software, and the results are presented as means ± SD. The data was conducted to normality testing before comparative analysis. To compare the differences between three or more groups, one-way ANOVA followed by LSD test was employed. The student’s t-test was applied to analyze the differences between two groups. Statistical significance was considered when the P value was less than 0.05 (P < 0.05).

Results

Identification of chemical compounds of PTF

Firstly, the flavonoid contents of PT extracts (dried powder) were quantified, revealing a high flavonoid content (purity, 90.25%). Then, the chemical compounds of the extracts, referred to as PTF, were identified by UHPLC in conjunction with MS. The chromatograms for both positive and negative ion modes were observed in Fig. 1. A total of ninety-four chemical compounds were identified (Table 1), with 47 of them being flavonoid compounds (including flavonoids and isoflavonoids) such as TYP, I3ON, kaempferol-3-O-rutinoside, astragalin, quercetin, naringenin, kaempferol, isorhamnetin, among others.

Fig. 1.

Analysis of chemical compounds of PTF by UHPLC-MS. (A) Positive ion chromatogram. (B) Negative ion chromatogram

Table 1.

Identification of chemical compounds of PTF by UHPLC-MS

No.	Ion Mode	Retention time (min)	Adducts	Experimental m/z	Theoretical m/z	Mass Error (ppm)	Fragments	Formula	Compounds	Class	Identification by Reference Substance
1	POS	0.79	[M + H]+	175.1190	175.1190	0.26	70.0664, 175.1189, 159.9982, 60.0571, 116.071, 159.0139, 130.0977, 158.0922, 62.9831, 117.9882	C6H14N4O2	Arginine	Carboxylic acids and derivatives	Yes
2	POS	0.85	[M + H]+	118.0866	118.0863	2.54	118.0867, 59.0744, 58.0666	C5H11NO2	Betaine	Carboxylic acids and derivatives	Yes
3	POS	0.92	[M + H]+	144.1018	144.1019	-0.74	144.1019, 99.0448	C7H13NO2	Stachydrine	Carboxylic acids and derivatives	Yes
4	POS	0.94	[M + H]+	153.0406	153.0407	-0.95	130.9821, 152.057, 131.9743, 113.9643, 72.9384, 153.041, 90.9487, 110.0355, 128.9868, 116.9667	C5H4N4O2	Xanthine	Imidazopyrimidines	Yes
5	POS	3.26	[M + H]+	127.0392	127.0390	1.29	109.029, 127.0393, 81.0346, 53.0401	C6H6O3	5-hydroxymethylfurfural	Organooxygen compounds	Yes
6	POS	3.83	[M + H]+	249.1958	249.1962	-1.62	249.1964, 98.985, 142.9659	C15H24N2O	Sophoridine	Lupin alkaloids	Yes
7	NEG	5.14	[M-H]−	353.0880	353.0878	0.57	191.0549, 179.0332, 353.0881, 135.043	C16H18O9	Neochlorogenic acid	Organooxygen compounds	Yes
8	POS	6.21	[M + H]+	247.1438	247.1441	-1.40	188.0709, 60.0823, 146.06, 247.144, 493.2811, 144.0808, 453.9435, 340.2359, 175.5119, 79.9084	C14H18N2O2	Hypaphorine	Carboxylic acids and derivatives	Yes
9	NEG	6.74	[M-H]−	353.0880	353.0878	0.70	191.0549, 353.0874	C16H18O9	Chlorogenic acid	Organooxygen compounds	Yes
10	NEG	7.05	[M-H]−	353.0881	353.0878	0.80	173.044, 135.043, 179.0334, 353.0875, 191.0548, 93.0322, 85.0271, 161.0228	C16H18O9	Cryptochlorogenic acid	Organooxygen compounds	Yes
11	POS	7.06	[M + H]+	179.0338	179.0339	-0.88	179.0337, 137.9869, 114.9714, 123.0444, 133.0287, 108.0451, 139.9817, 155.0021, 152.0705, 116.966	C9H6O4	Esculetin	Coumarins and derivatives	Yes
12	POS	8.04	[M]+	342.1695	342.1700	-1.68	192.1018, 342.17, 177.0782	C20H24NO4+	Phellodendrine	Protoberberine alkaloids and derivatives	Yes
13	POS	8.22	[M + H]+	423.0916	423.0922	-1.42	273.039, 303.0496, 423.0926, 405.0807, 327.0496, 299.0526, 257.044, 351.0486, 339.0495, 387.0706	C19H18O11	Mangiferin	Benzopyrans	Yes
14	NEG	8.85	[M + FA-H]−	525.1616	525.1614	0.37	121.0272, 525.1619, 479.1564, 91.575	C23H28O11	Albiflorin	Prenol lipids	Yes
15	POS	8.85	[M + H]+	137.0596	137.0597	-0.79	136.0216, 137.0598, 114.9713, 81.071, 95.0864, 56.9663, 136.0618, 91.0551, 93.0708, 109.0652	C8H8O2	4-hydroxyacetophenone	Organooxygen compounds	Yes
16	POS	9.15	[M + H]+	165.0544	165.0546	-1.24	147.044, 100.0251, 118.0353, 119.0495, 142.9673, 91.0551, 116.9666, 139.9822, 120.0328, 165.0543	C9H8O3	P-coumaric acid	Cinnamic acids and derivatives	Yes
17	NEG	9.39	[M + FA-H]−	525.1613	525.1614	-0.05	121.0273, 449.1463, 525.1628, 165.0538, 479.1517, 327.1079	C23H28O11	Paeoniflorin	Prenol lipids	Yes
18	POS-NEG	9.60	[M + H]+	595.1650	595.1658	-1.28	617.148, 309.0385, 331.1002	C27H30O15	Kaempferol − 3-O-neohesperidoside	Flavonoids	NO
19	POS-NEG	9.62	[M + H]+	449.1071	449.1079	-1.65	303.0496, 449.1077, 287.0553	C21H20O11	Quercetin − 3-alpha-l-rhamnofuranoside	Flavonoids	NO
20	POS	9.78	[M + H]+	163.0386	163.0390	-2.23	100.0251, 163.0389, 118.0353, 139.9821, 116.9666, 80.9462, 82.0148, 162.0911, 107.0498, 119.0496	C9H6O3	7-hydroxycoumarin	Coumarins and derivatives	NO
21	NEG	9.86	[M-H]−	415.1036	415.1034	0.51	253.0499, 415.1043, 252.0424, 117.0323, 135.0068, 224.045, 119.0479	C21H20O9	Daidzin	Isoflavonoids	Yes
22	POS	9.89	[M + H]+	565.1543	565.1552	-1.52	565.1529, 379.0815, 547.1469, 325.0704, 307.0589, 295.0692, 349.07, 397.0919, 433.0937, 337.0725	C26H28O14	Schaftoside	Flavonoids	Yes
23	NEG	10.30	[M + FA-H]−	435.1294	435.1297	-0.59	227.0707, 185.0595, 389.125, 143.0482	C20H22O8	Piceid	Stilbenes	Yes
24	POS	10.32	[M + H]+	193.0493	193.0496	-1.40	193.0496, 133.0285, 177.0545, 128.0197, 145.0283, 111.9688, 192.0657, 178.0266, 129.979, 117.0337	C10H8O4	Scopoletin	Coumarins and derivatives	Yes
25	POS	10.35	[M + H-H2O]+	177.0543	177.0546	-2.00	145.0285, 177.0546, 117.034, 89.0396, 149.0598, 134.0365	C10H10O4	Ferulic acid	Cinnamic acids and derivatives	Yes
26	POS-NEG	10.41	[M + H]+	611.1591	611.1607	-2.63	303.0495, 85.0294, 465.1031, 71.0503	C27H30O16	Rutin	Flavonoids	Yes
27	NEG	10.60	[M-H]−	405.1189	405.1191	-0.49	243.0659, 405.1191	C20H22O9	2,3,5,4'-tetrahydroxy stilbene-2-O-β-d-glucoside	Stilbenes	Yes
28	POS	10.77	[M + H]+	771.2321	771.2343	-2.78	317.0652, 463.123, 302.0417, 85.0294, 71.0504, 625.1762	C34H42O20	Typhaneoside	Flavonoids	Yes
29	POS-NEG	10.83	[M-H]−	463.0883	463.0882	0.35	300.027, 463.088, 271.0243, 255.0298, 151.0018	C21H20O12	Hyperoside	Flavonoids	Yes
30	NEG	11.13	[M-H]−	623.1618	623.1617	0.02	314.0437, 623.1629, 299.0199, 271.025, 243.0294, 301.0356, 285.0403, 255.03, 151.0017	C28H32O16	Isorhamnetin-3-O-neohespeidoside	Flavonoids	Yes
31	POS	11.20	[M + H-H2O]+	177.0543	177.0546	-1.60	145.0284, 177.0544, 117.034, 149.0597, 89.0395, 91.0551, 125.003, 149.0233, 137.987, 154.9691	C10H10O4	Isoferulic acid	Cinnamic acids and derivatives	Yes
32	POS-NEG	11.40	[M-H]−	755.2049	755.2040	1.25	300.0276, 755.205, 271.0252, 315.0515, 255.03, 285.0406, 151.0018, 299.0195, 243.0298, 609.1488	C33H40O20	Manghaslin	Flavonoids	NO
33	POS-NEG	11.67	[M-H]−	593.1512	593.1512	0.00	285.0403, 593.1523, 284.0328, 255.0299, 227.0344, 229.0497	C27H30O15	Nicotiflorin	Flavonoids	Yes
34	POS	11.69	[M + H]+	449.1069	449.1079	-2.15	287.0548, 85.0295	C21H20O11	Cynaroside	Flavonoids	Yes
35	NEG	11.76	[M-H]−	579.1720	579.1719	0.17	271.0614, 151.0016, 579.1752, 119.0477, 300.0275, 107.0117, 285.0409	C27H32O14	Narirutin	Flavonoids	Yes
36	POS	11.80	[M + H]+	265.0965	265.0972	-2.41	265.097, 206.0837, 247.0864, 219.0914, 207.09	C16H12N2O2	Perlolyrine	Harmala alkaloids	Yes
37	NEG	11.87	[M + FA-H]−	507.1145	507.1144	0.12	299.056, 461.1092, 284.0326, 255.0297, 283.0252, 507.1152, 298.0474, 446.085, 297.0404	C22H22O11	Tectoridin	Isoflavonoids	Yes
38	NEG	12.06	[M-H]−	447.0934	447.0933	0.23	447.0937, 285.0406, 151.0018, 255.03, 287.0564, 284.0327, 227.0343, 300.0284, 301.0349, 135.043	C21H20O11	Astragalin	Flavonoids	Yes
39	POS-NEG	12.15	[M-H]−	431.0984	431.0984	0.07	431.0989, 268.0379, 267.0301, 239.0343	C21H20O10	Sophoricoside	Isoflavonoids	Yes
40	POS-NEG	12.21	[M-H]−	579.1721	579.1719	0.32	151.0018, 579.1729, 271.0615, 119.048, 107.0115, 459.1163, 65.0007, 177.0179, 83.0113	C27H32O14	Naringin	Flavonoids	Yes
41	POS-NEG	12.26	[M-H]−	577.1569	577.1563	1.07	577.1569, 268.0379	C27H30O14	Rhoifolin	Flavonoids	Yes
42	NEG	12.59	[M-H]−	515.1197	515.1195	0.41	173.044, 179.0331, 135.0431, 515.1099, 191.0554, 353.0885, 93.0322, 198.3915, 217.4645, 151.0015	C25H24O12	Isochlorogenic acid a	Organooxygen compounds	Yes
43	POS-NEG	12.95	[M + H]+	463.1225	463.1235	-2.12	301.0703, 286.0469, 463.1219, 303.0484, 258.0521	C22H22O11	Isorhamnetin-3-alpha-l-rhamnofuranoside	Flavonoids	NO
44	POS	13.08	[M + H-H2O]+	177.0544	177.0546	-1.42	145.0283, 177.0543, 117.0339, 89.0396, 149.0597, 176.071, 91.0553, 155.9759, 149.0235, 121.1014	C10H10O4	Methyl caffeate	Cinnamic acids and derivatives	Yes
45	POS	13.92	[M + H]+	447.0913	447.0922	-2.15	303.0495, 271.0597, 447.0911	C21H18O11	Baicalin	Flavonoids	Yes
46	POS-NEG	13.97	[M + FA-H]−	475.1249	475.1246	0.58	267.0664, 252.0426, 475.1288	C22H22O9	Ononin	Isoflavonoids	Yes
47	POS	14.13	[M + H]+	419.1327	419.1337	-2.43	257.0804, 137.0234, 147.0439, 119.0497	C21H22O9	Isoliquiritin	Flavonoids	Yes
48	POS	14.19	[M + H]+	625.1751	625.1763	-2.05	317.0656, 85.0295, 71.0505, 479.1189	C28H32O16	Isorhamnetin-3-O-rutinoside	Flavonoids	NO
49	POS-NEG	14.30	[M + H]+	255.0645	255.0652	-2.61	255.065, 199.0752, 137.0233	C15H10O4	Daidzein	Isoflavonoids	Yes
50	NEG	14.40	[M-H]−	255.0662	255.0663	-0.36	119.048, 255.0662, 135.0065, 153.0174, 91.0164	C15H12O4	Liquiritigenin	Flavonoids	Yes
51	POS	15.09	[M + H]+	303.0489	303.0500	-3.37	303.0495, 153.0183, 229.0494	C15H10O7	Quercetin	Flavonoids	Yes
52	POS-NEG	15.13	[M + H]+	287.0544	287.0550	-2.34	287.0549, 285.0753, 153.0183	C15H10O6	Luteolin	Flavonoids	Yes
53	NEG	15.30	[M + FA-H]−	965.4982	965.4963	2.01	919.4932, 757.4401, 101.0221, 71.0114, 595.382	C45H76O19	Timosaponin bii	Steroids and steroid derivatives	Yes
54	POS-NEG	15.31	[M + H]+	593.1851	593.1865	-2.29	285.0755, 593.1872, 447.1288, 270.0518	C28H32O14	Linarin	Flavonoids	Yes
55	POS-NEG	15.46	[M + H]+	285.0751	285.0758	-2.51	305.0548, 263.0808, 281.0919, 206.0834, 306.2057	C16H12O5	Calycosin	Isoflavonoids	Yes
56	POS-NEG	15.51	[M-H]−	593.1308	593.1300	1.26	285.0407, 284.0328, 255.0297, 593.1293, 227.0345, 285.0765, 145.0284, 229.0505, 257.0453	C30H26O13	Tiliroside	Flavonoids	Yes
57	POS-NEG	15.53	[M-H]−	431.0984	431.0984	0.23	431.0987, 285.0403, 151.0018, 284.0329, 257.0452, 107.0115	C21H20O10	Kaempferol rhamnoside	Flavonoids	NO
58	POS-NEG	16.43	[M + FA-H]−	867.2952	867.2928	2.68	659.2356, 366.1106, 351.0875, 323.0926	C39H50O19	Epimedin c	Flavonoids	Yes
59	NEG	16.63	[M-H]−	271.0613	271.0612	0.61	271.0614, 135.043, 151.0018, 119.048, 269.0447, 107.0114, 135.0067, 153.0174, 65.0007	C15H12O5	Naringenin	Flavonoids	Yes
60	POS	16.64	[M + H]+	677.2428	677.2440	-1.86	369.1334, 313.0702, 531.1869, 85.0294, 71.0503, 677.2394, 231.0375, 375.1018, 323.9519, 114.1489	C33H40O15	Icariin	Flavonoids	Yes
61	POS-NEG	16.78	[M-H]−	269.0455	269.0455	-0.22	269.0457	C15H10O5	Genistein	Isoflavonoids	Yes
62	POS	17.05	[M + H]+	303.0852	303.0863	-3.85	153.0182, 303.0857, 303.0497, 177.0546, 145.0284, 301.0698, 286.0472, 117.034, 171.0288, 179.0338	C16H14O6	Hesperetin	Flavonoids	Yes
63	POS-NEG	17.08	[M-H]−	285.0403	285.0404	-0.60	285.0406	C15H10O6	Kaempferol	Flavonoids	Yes
64	POS	17.45	[M + H]+	317.0646	317.0656	-3.16	317.0655, 302.0419, 153.0183	C16H12O7	Isorhamnetin	Flavonoids	Yes
65	POS	17.48	[M + H]+	301.0699	301.0707	-2.56	301.0703, 286.0469, 258.0518, 153.0182	C16H12O6	Diosmetin	Flavonoids	Yes
66	NEG	17.62	[M + FA-H]−	695.4022	695.4012	1.40	487.343, 695.4026	C36H58O10	Pedunculoside	Prenol lipids	Yes
67	NEG	17.71	[M-H]−	269.0456	269.0455	0.10	269.0455, 228.992	C15H10O5	Baicalein	Flavonoids	Yes
68	POS-NEG	17.88	[M + H]+	257.0801	257.0809	-2.81	257.0806, 137.0233, 147.0439, 256.2632, 119.0496	C15H12O4	Isoliquiritigenin	Linear 1,3-diarylpropanoids	Yes
69	POS-NEG	18.24	[M + H]+	269.0799	269.0809	-3.49	269.0806, 213.0909, 226.0623	C16H12O4	Formononetin	Isoflavonoids	Yes
70	NEG	18.53	[M-H]−	821.3980	821.3965	1.88	821.3984, 351.0572, 113.0221, 85.0269, 71.0112, 193.034, 72.9905, 75.0062, 59.0113, 99.0063	C42H62O16	Glycyrrhizic acid	Prenol lipids	Yes
71	POS	18.53	[M + NH4]+	812.4779	812.4791	-1.37	141.0182, 95.0864, 421.3465, 439.3571, 85.0295, 163.06, 109.1018, 81.071, 91.0399, 107.0861	C42H66O14	Chikusetsusaponin iva	Prenol lipids	Yes
72	NEG	18.89	[M + FA-H]−	515.1922	515.1923	-0.06	469.1871, 515.1932, 229.1245, 96.9579, 264.9881, 312.9982, 214.3481, 68.583, 217.0731	C26H30O8	Limonin	Prenol lipids	Yes
73	POS	19.15	[M + H-H2O]+	439.3559	439.3570	-2.63	421.3463, 95.0864, 109.1018, 119.0859, 133.1012, 107.0862, 145.1012, 159.1168, 81.071, 121.1015	C30H48O3	Betulic acid	Prenol lipids	NO
74	POS	19.17	[M + H]+	301.0696	301.0707	-3.48	283.06, 301.0697, 227.0704	C16H12O6	Kaempferide	Flavonoids	Yes
75	POS	19.26	[M + H-H2O]+	423.3609	423.3621	-2.95	441.3728, 95.0863, 107.0861, 423.3614, 109.1019, 81.071, 93.0707, 121.1016, 119.0859, 135.1165	C30H48O2	Roburic acid	Prenol lipids	Yes
76	POS	19.44	[M + H]+	375.1062	375.1075	-3.26	375.1074, 317.0654, 299.0548, 345.0603, 360.083, 342.0728, 359.0756	C19H18O8	Casticin	Flavonoids	Yes
77	POS	19.59	[M + H]+	403.1375	403.1388	-3.11	403.1385, 373.0914, 388.1153, 183.0288, 211.0234, 330.0726, 327.0858, 358.0663	C21H22O8	Nobiletin	Flavonoids	Yes
78	POS-NEG	19.61	[M + H]+	285.0748	285.0758	-3.57	285.0753, 229.0858, 167.0338	C16H12O5	Biochanin a	Isoflavonoids	Yes
79	POS	19.83	[M + H]+	315.0852	315.0863	-3.73	315.0858, 300.0623, 167.0339, 283.0598, 243.065, 255.065	C17H14O6	Pectolinarigenin	Flavonoids	Yes
80	POS	19.96	[M + H]+	369.1323	369.1333	-2.59	369.1329, 151.0752, 384.3342, 81.071, 135.0443, 95.0863, 67.0555, 109.1017, 93.0706, 247.0956	C21H20O6	Suchilactone	Furanoid lignans	NO
81	POS	20.25	[M + H]+	373.1271	373.1282	-3.00	373.1278, 343.0809, 358.1041, 297.0749, 300.0618, 183.029, 211.0234, 328.0575	C20H20O7	Tangeretin	Flavonoids	Yes
82	NEG	20.61	[M-H]−	763.4290	763.4274	2.09	763.4391, 59.0113, 71.0113, 85.0269, 113.022, 75.0062, 99.0064, 89.0219	C41H64O13	Momordin ic	Prenol lipids	Yes
83	NEG	20.70	[M-H]−	269.0456	269.0455	0.42	269.0457, 225.0551	C15H10O5	Emodin	Anthracenes	Yes
84	POS	20.81	[M + H]+	389.1219	389.1231	-3.15	389.123, 359.0761, 149.0235, 110.0607, 341.0638, 374.1005, 55.0556, 57.0713, 388.3064, 67.0556	C20H20O8	Artemetin	Flavonoids	Yes
85	POS	21.25	[M + H]+	245.1166	245.1172	-2.80	189.0546, 131.0493, 245.1169	C15H16O3	Osthole	Coumarins and derivatives	Yes
86	POS-NEG	21.64	[M-H]−	617.4060	617.4059	0.19	617.4064, 75.0061, 85.027, 113.0221	C36H58O8	Oleanolic acid-28-O-beta-d-glucopyranoside	Prenol lipids	NO
87	POS	22.08	[M + NH4]+	554.2375	554.2384	-1.70	415.1749, 340.1304, 371.148, 325.1055, 342.1064, 299.0914, 327.0862, 297.1118, 373.1281, 295.0959	C30H32O9	Schisantherin a	Tannins	Yes
88	POS	22.21	[M + NH4]+	532.2529	532.2541	-2.16	415.175, 340.1301, 371.1485, 342.1079, 325.1074, 327.086, 373.1279, 295.0958, 313.1051, 299.0905	C28H34O9	Schizantherin b	Tannins	Yes
89	POS	22.57	[M + H-H2O]+	301.2151	301.2162	-3.67	301.2155, 255.2104, 119.0859, 109.1019, 147.1171, 105.0705, 133.1014, 258.2786, 95.0863, 145.1012	C20H30O3	Isosteviol	Prenol lipids	Yes
90	POS	22.88	[M + H-H2O]+	219.1739	219.1743	-2.15	219.1743, 205.1585, 203.1427, 81.0711, 200.1281, 57.0713, 121.1016, 153.937, 119.086, 93.0706	C15H24O2	Dihydroartemisinic acid	Prenol lipids	Yes
91	NEG	23.11	[M + FA-H]−	269.2123	269.2122	0.39	269.2124, 83.0476, 269.0457, 225.2216	C15H28O	Β-eudesmol	Prenol lipids	Yes
92	NEG	23.19	[M-H]−	469.3324	469.3323	0.10	469.3324	C30H46O4	Glycyrrhetinic acid	Prenol lipids	Yes
93	POS	23.51	[M + H]+	417.2262	417.2272	-2.39	417.2268, 316.1305, 301.107, 285.1119, 402.2032, 242.0936, 347.1484, 119.0858, 258.0876	C24H32O6	Schizandrin a	Tannins	Yes
94	POS	24.96	[M + H-H2O]+	439.3562	439.3570	-1.99	439.3566, 203.1793, 191.1794, 189.1637, 95.0862, 109.1019, 107.0861, 119.086, 121.1015, 133.1013	C30H48O3	Oleanolic acid	Prenol lipids	Yes

Network pharmacology suggests the possible mechanisms of the flavonoid compounds against diabetic inflammation

PTF exerts anti-diabetic inflammation action. Oral administration and external application are the traditional use of PT in clinical practice. To reveal the anti-inflammatory mechanisms of PTF and its active constituents, 47 flavonoid compounds from PT were used to perform network pharmacology analysis. As shown in Fig. 2A, 165 targets (Table S3) were screened by intersecting 281 flavonoid compound-targets and 9019 disease-targets for type 2 diabetes. Figure 2B indicated the interaction between the intersecting targets through straight line. The key candidate targets were identified by the node degree value which was evaluated by the node size and color. The top 10 key candidate targets were tumor protein P53 (TP53), BCL2 apoptosis regulator (BCL2), estrogen receptor 1 (ESR1), heat shock protein 90 alpha family class A member 1 (HSP90AA1), AKT Serine/Threonine kinase 1 (AKT1), HSP90AB1, tumor necrosis factor (TNF), matrix metallopeptidase 9 (MMP9), PPARγ, and prostaglandin-endoperoxide synthase 2 (PTGS2). Flavonoid compound-target-disease network (Fig. 2C) displayed that multiple flavonoid compounds, including pectolinarigenin, kaempferol, isorhamnetin, naringenin, quercetin, TYP, and I3ON, might exert anti-diabetic effects. Furthermore, in addition to the top 20 KEGG molecular pathways (Fig. 2D) and the top 30 GO terms (Fig. 2E), which suggested the potential mechanisms of the flavonoid compounds against diabetes and inflammation, the intersecting targets were related to multiple inflammation- and lipid metabolism-related pathways and biological processes (Fig. 3). In especial, both the key candidate target TNF and the candidate target IKBKB were enriched to the inflammation-related pathways, including the ‘Lipid and atherosclerosis’, ‘IL-17 signaling pathway’, ‘Toll-like receptor signaling pathway’, ‘NOD-like receptor signaling pathway’, ‘NF-κB signaling pathway’, et al. (Fig. 3A). The key candidate target PPARγ was closely associated with the ‘Lipid and atherosclerosis’, ‘AMPK signaling pathway’, and multiple lipid metabolism- and inflammation-related biological processes such as ‘fatty acid metabolic process’, ‘fatty acid oxidation’, ‘negative regulation of sequestering of triglyceride’, ‘negative regulation of inflammatory response’ and so on (Fig. 3B). Besides, the intersecting targets including PPARγ were correlated monocyte/macrophage biological processes such as ‘macrophage differentiation’, ‘monocyte differentiation’, ‘positive regulation of macrophage proliferation’, ‘macrophage derived foam cell differentiation’ and ‘negative regulation of macrophage chemotaxis’ (Fig. 3C). These results implied the possible mechanisms of PT flavonoids against diabetic inflammation.

Fig. 2.

Effects of the flavonoid compounds on type 2 diabetes based on network pharmacological analysis. (A) A Venn diagram indicates the intersecting targets between the flavonoid compound targets and the type 2 diabetes targets. (B) Protein-protein interaction (PPI) network based on the intersecting targets. A straight line represents the interaction between two target proteins. The degree value, evaluated by the node size and color, positively correlated with node importance. (C) Flavonoid compound-target-disease network. (D) The top 20 KEGG enrichment pathways. (E) The top 30 GO terms referring to biological process (BP), cellular component (CC), and molecular function (MF)

Fig. 3.

The main KEGG pathways and biological process involving key candidate target-related inflammation and lipid metabolism, and the biological process involving monocyte/macrophage functions. Left: KEGG pathway terms (A) or GO terms (B, C); Middle: statistical evidence; Right: the enriched genes

PTF and TYP reverse PA-induced activation of NLRP3 inflammasome in RAW264.7 macrophages, respectively

NLRP3 is a NOD-like receptor protein. PA was applied to induce NLRP3 inflammasome activation. Herein, PA was administered in conjunction with 1 ng/ml LPS pretreatment for 3 h, a dose-dependent increase in the secretion of IL-1β and IL-18 was noted in macrophages (Fig. 4A). Moreover, the presence of 0.5 mM PA led to a significant enhancement in the protein expression of NLRP3, Caspase-1, and Caspase-1 cleavage (Caspase-1 p10) (Fig. 4B). Hence, the concentration of 0.5 mM PA was selected as the optimal dose to stimulate NLRP3 inflammasome activation in LPS-primed macrophages.

Fig. 4.

Pollen Typhae flavonoids attenuate PA-induced NLRP3 inflammasome activation in macrophages. (A) After pretreatment with or without LPS (1 ng/ml) for 3 h, RAW264.7 macrophages were stimulated with PA (0.125 ~ 0.5 mM) for 16 h, IL-1β and IL-18 in culture supernatants were analyzed by ELISA. (B) The macrophages were pretreated with LPS (1 ng/ml) for 3 h, followed by PA (0.5 mM) treatment for 16 h, the protein expression of NLRP3, Caspase-1, and Caspase-1 p10 was analyzed by western blotting. The uncropped blots were presented in Supplementary Fig. S4. (C) RAW264.7 macrophages were treated with PTF (0 ~ 0.5 mg/ml), TYP (0 ~ 100 µM) or I3ON (0 ~ 100 µM) for 16 and 20 h, the cell viability was detected by CCK-8 assay. ^*P < 0.05, ^**P < 0.01 versus 0.0 mg/ml PTF (0.0 µM TYP or I3ON). (D) The macrophages were pretreated with LPS (1 ng/ml) or plus different concentrations of PTF (0.03125 ~ 0.5 mg/ml), TYP (10 ~ 100 µM), I3ON (10 ~ 100 µM) for 3 h before exposure to PA (0.5 mM) for 16 h, IL-1β and IL-18 in culture supernatants were checked by ELISA. (E and F) The macrophages were pretreated with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), AICAR (2 mM), MCC950 (10 µM) for 3 h followed by exposure to PA (0.5 mM) for 16 h, the protein (E) and gene expression (F) of NLRP3, Caspase-1 and IL-1β were checked by western blotting and Real-time PCR, respectively. The uncropped/unedited blots were presented in Supplementary Fig. S5. Each column shows the mean with SD (n = 3–7). ^*P < 0.05, ^**P < 0.01 versus LPS (1 ng/ml); ^#P < 0.05, ^##P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM)

Next, PT flavonoids were evaluated for their effects on cell viability. TYP and I3ON are quality markers of PT according to Chinese pharmacopoeia (2020). As shown in Fig. 4C, treatment with PT flavonoids for 16 and 20 h, including PTF, TYP, and I3ON, exhibited little toxicity in RAW264.7 macrophages. Importantly, PTF and TYP decreased PA-induced secretion of IL-1β and IL-18 in a dose-dependent manner in LPS-primed macrophages (Fig. 4D), respectively. But I3ON did not exhibit any significant impact on the secretion. Furthermore, treatment with 0.5 mg/ml PTF and 100 µM TYP reversed the PA-induced protein expression of NLRP3, Caspase-1, Caspase-1 p10, and IL-1β p17 in the macrophages, respectively. Similarly, the inhibitor of NLRP3, MCC950, also reversed the expression of these proteins, implying the inactivation of NLRP3 inflammasome (Fig. 4E). INF39, another NLRP3 inhibitor, also canceled NLRP3 inflammasome activation in type 2 diabetic rats [26]. It is postulated that PT flavonoids likely affect the LPS-induced priming step for activation of NLRP3 inflammasome. While PTF reduced the mRNA levels of IL-1β, it did not affect the gene expression of NLRP3 or Caspase-1, suggesting a weaker impact on the priming step compared to the activation step induced by PA. On the other hand, TYP unchanged the gene expression of NLRP3, Caspase-1, and IL-1β (Fig. 4F), indicating little effects on the LPS-induced priming step. The results showed that PTF and TYP effectively reverse the PA-induced activation of NLRP3 inflammasome in RAW264.7 macrophages, respectively.

Molecular docking implies the potential target proteins for TYP regulating inflammation

Network pharmacology indicated that the intersecting targets regulated inflammatory response, fatty acid metabolism, and the NOD-like receptor, AMPK, and NF-κB pathways, suggesting the potential mechanisms of the flavonoid compounds against inflammation. TYP, a key quality marker of PT according to the Chinese Pharmacopoeia (2020), exerts anti-inflammation effects. PTF was reported to increase AKT protein expression in vitro [21], and fail to significantly decrease blood TNF-α in vivo [24]. Accordingly, molecular docking analysis was used to reveal the interaction between TYP (Fig. 5A) and the inflammation- and lipid metabolism-related proteins. As shown in Fig. 5B, all binding energies were less than or equal to -7 kcal/mol between TYP (ligand) and the proteins (receptors), including NLRP3 inflammasome, fatty acid transport protein CD36, lipid synthesis-related proteins (FAS, PPARγ), and AMPK. Among them, the binding energy between TYP and NLRP3 was the smallest, as low as -10.5 kcal/mol. In general, there is a negative correlation between affinity and binding energy. The binding energy which does not exceed − 7 kcal/mol implies the strong affinity between the ligand and the receptor. Figure 5C-H showed 3D binding structures of TYP and the proteins, exhibiting the active sites, including the residues and the hydrogen bonds.

Fig. 5.

Molecular docking results. (A) The two-dimensional chemical structure of typhaneoside (TYP). (B) The binding energy between the ligand (TYP) and the receptor proteins. (C-H) 3D binding structures of TYP and the proteins including NLRP3 (C), Caspase-1 (D), CD36 (E), PPARγ (F), FAS (G), and AMPK (H). The proteins and the ligand are marked in white and bright blue, respectively. The residues involving interaction between ligand and receptor are shown as green sticks. Yellow dashed lines represent hydrogen bonds, with the number meaning the hydrogen bond distances

PTF and TYP promote activation of the AMPK-ROS pathway in PA-induced macrophages, respectively

To demonstrate the potential mechanisms by which PT flavonoids regulated NLRP3 inflammasome, we investigated the effects of PTF and TYP on the AMPK and the NF-κB pathways. AICAR, an agonist of AMPK, deactivated PA-induced NLRP3 inflammasome in macrophages, consistent with PTF and TYP results (Fig. 4E), suggesting the close relationship between the AMPK pathway and NLRP3 inflammasome. Here, PA markedly reduced p-AMPK/AMPK ratio, and decreased the upstream p-LKB1/LKB ratio to some extent in LPS-primed macrophages. Conversely, PTF, TYP, and the positive control AICAR significantly increased the p-AMPK/AMPK ratio and p-LKB1/LKB ratio (Fig. 6A), respectively, suggesting activation of the AMPK pathway. Moreover, the inhibition of IL-1β and IL-18 secretion by PTF or TYP was either abolished or attenuated by Compound C, an inhibitor of AMPK (Fig. 6B). Interestingly, N-Acetyl-L-cysteine (NAC), an antioxidant, also significantly decreased IL-1β and IL-18 secretion, and reduced PA-induced ROS overproduction. NLRP3 inflammasome is directly activated by ROS. Inhibiting ROS production suppresses its activation [27]. But specific inhibition of NLRP3 does not affect upstream generation of ROS [28]. Similarly, PTF, TYP, and AICAR also inhibited ROS overproduction, respectively. And Compound C attenuated or abolished the inhibitory effects of PTF or TYP on ROS overproduction (Fig. 6C). Additionally, although PTF decreased p-IκBα/IκBα ratio to some extent, and TYP obviously reduced p-IκBα/IκBα ratio, neither PTF nor TYP significantly affected the downstream p-NF-κB p65/NF-κB p65 ratio (Fig. 6D). The data showed that PTF and TYP promote the activation of the AMPK-ROS pathway in PA-induced macrophages, thereby suppressing NLRP3 inflammasome activation.

Fig. 6.

Effects of Pollen Typhae flavonoids on the AMPK-ROS and NF-κB pathways in PA-induced macrophages. (A) RAW264.7 macrophages were pretreated with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), AICAR (2 mM) for 3 h before treatment with PA (0.5 mM) for 16 h, the protein expression of AMPK and LKB1, as well as their phosphorylation were analyzed by western blotting. The uncropped blots were presented in Supplementary Fig. S6. (B) The macrophages were pretreated with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), Compound C (10 µM), NAC (10 mM) for 3 h prior to treatment with PA (0.5 mM) for 16 h, IL-1β and IL-18 in culture supernatants were checked by ELISA. (C) After preincubation with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), Compound C (10 µM), NAC (10 mM), AICAR (2 mM) for 3 h followed by incubation with PA (0.5 mM) for 16 h, the macrophages were used to detect intracellular ROS by DCFH-DA method. (D) After pretreatment with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM) for 3 h followed by PA (0.5 mM) treatment with for 16 h, the protein expression was analyzed by western blotting. The uncropped blots were presented in Supplementary Fig. S7. Each column shows the mean with SD (n = 3–4).^*P < 0.05, ^**P < 0.01 versus LPS (1 ng/ml); ^#P < 0.05, ^##P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM); ^§P < 0.05 versus LPS (1 ng/ml) plus PA (0.5 mM) and PTF (0.5 mg/ml); ^&P < 0.05, ^&&P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM) and TYP (100 µM)

PTF and TYP ameliorate PA-induced lipid metabolism dysfunction in RAW264.7 macrophages, respectively

Increased free fatty acid (FFA) causes lipid accumulation and subsequent ROS overproduction in liver cells [29]. Herein, elevated PA evidently increased intracellular FFA and triglyceride (TG) contents, and raised diglyceride (DAG) content to some extent in LPS-primed macrophages (Fig. 7A-C). But PA did not change monoglyceride (MAG) or lecithin (LEC) contents (Fig. 7D and E). PTF at the concentrations of 0.125 and 0.5 mg/ml as well as TYP at the concentrations of 40, 80 and 100 µM substantially reduced intracellular FFA content, respectively. And PTF at the concentrations of 0.125, 0.125 and 0.5 mg/ml as well as TYP at the concentrations of 40 and 100 µM markedly decreased TG content, respectively. Moreover, treatment with 0.5 mg/ml PTF dramatically declined LEC content, and slightly reduced DAG and MAG. Similarly, treatment with 40 µM TYP decreased DAG to some extent, but TYP at the concentrations of 40, 80 and 100 µM did not significantly affect MAG or LEC contents. Additionally, AICAR also remarkably decreased FFA and TG contents, and downregulated DAG content to some extent. PF-05175157, an inhibitor of Acetyl-CoA Carboxylase (ACC) which is responsible for fatty acid synthesis through promoting generation of malonyl CoA, obviously reduced TG content, and slightly diminished FFA and DAG contents.

Fig. 7.

Effects of Pollen Typhae flavonoids on intracellular lipid contents in PA-induced macrophages. RAW264.7 macrophages were preincubated with LPS (1 ng/ml) or plus PTF (0.125, 0.25, 0.5 mg/ml), TYP (40, 80, 100 µM), PF-05175157 (10 µM), AICAR (2 mM) for 3 h before exposure to PA (0.5 mM) for 16 h, intracellular lipids including FFA (A), TG (B), DAG (C), MAG (D) and LEC (E) were analyzed by ELISA and Enzyme assay. Each column shows the mean with SD (n = 3–4). ^*P < 0.05, ^**P < 0.01 versus LPS (1 ng/ml); ^#P < 0.05, ^##P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM)

To ascertain the potential mechanisms, we examined the protein expression involving intracellular lipid metabolism. Figure 8 showed that continuously elevated PA increased CD36 protein expression, and enhanced the protein expression of PPARγ, FAS, and DGAT1. Furthermore, the elevated PA levels promoted the protein expression of CPT-1 and Atp5a1 (Fig. 8A), leading to an increase in ATP (Fig. 8B) and ROS production (Fig. 6C) in LPS-primed macrophages. Importantly, 0.5 mg/ml PTF and 100 µM TYP substantially decreased PA-induced protein expression of CD36, PPARγ, FAS, and CPT-1. Moreover, PTF reduced DGAT1 protein expression to some extent, and TYP significantly diminished DGAT1 protein expression. Although PTF and TYP did not change the increased expression of Atp5a1, they further increased intracellular ATP production and reduced ROS production. Interestingly, the positive control PF-05175157 and AICAR abolished above proteins expression. AICAR, but not PF-05175157, still markedly decreased ATP production. Additionally, PTF decreased CD36 mRNA levels, but failed to affect gene expression of DGAT1, CPT-1 A, Fas and Atp5a1. TYP also unchanged mRNA levels of CD36, DGAT1 and CPT-1 A (Fig. 8C). Collectively, PTF and TYP improved PA-induced lipid metabolism dysfunction in macrophages, respectively.

Fig. 8.

Effects of Pollen Typhae flavonoids on the expression of lipid metabolism-related proteins and ATP content in macrophages. The macrophages were pretreated with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), PF-05175157 (10 µM), AICAR (2 mM) for 3 h before treatment with PA (0.5 mM) for 16 h, the protein expression (A) was analyzed by western blotting, intracellular ATP content (B) was checked using an Enhanced ATP Assay Kit, and gene expression (C) was determined by Real-time PCR. The uncropped blots were presented in Supplementary Fig. S8. Each column shows the mean with SD (n = 3–4). ^*P < 0.05, ^**P < 0.01 versus LPS (1 ng/ml); ^#P < 0.05, ^##P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM); ^&P < 0.05, ^&&P < 0.01 versus LPS (1 ng/ml) + PA (0.5 mM) + AICAR (2 mM)

Improving intracellular lipid metabolism affected PA-triggered Caspase-1 activity in RAW264.7 macrophages

Increased Caspase-1 activity is a hallmark of NLRP3 inflammasome activation. PTF, TYP, AICAR, and PF-05175157 were shown to ameliorate PA-induced lipid metabolism dysfunction. Herein, PF-05175157 and PTF reduced Caspase-1 activity in culture supernatants from PA-induced macrophages (Fig. 9A), respectively. And Compound C abolished the inhibitory effect of PTF. Similar to NLRP3 inhibitor MCC950, AICAR and PTF also decreased Caspase-1 activity in the cells (Fig. 9B), respectively. Lipid accumulation caused fatty acid oxidation and oxidative phosphorylation overload in mitochondrion, thus leading to excessive ROS production. Reducing ROS by NAC inhibited Caspase-1 activity (Fig. 9B) and the secretion of IL-1β and IL-18 (Fig. 6B), meaning the inactivation of NLRP3 inflammasome. The data implied that improving lipid metabolism inhibited PA-induced NLRP3 inflammasome activation.

Fig. 9.

Effects of PTF, AMPK agonist, ACC inhibitor and antioxidant on PA-induced Caspase-1 activity in macrophages. RAW264.7 macrophages were pretreated with LPS (1 ng/ml) or plus PTF (0.5 mg/ml), TYP (100 µM), PF-05175157 (10 µM), AICAR (2 mM), Compound C (10 µM), NAC (10 mM), MCC950 (10 µM) for 3 h before treatment with PA (0.5 mM) for 16 h, the Caspase-1 activity in culture supernatants (A) and the macrophages (B) was analyzed by bioluminescent method. Each column shows the mean with SD (n = 3). ^*P < 0.05, ^**P < 0.01 versus LPS (1 ng/ml); ^#P < 0.05, ^##P < 0.01 versus LPS (1 ng/ml) plus PA (0.5 mM); ^§P < 0.05 versus LPS (1 ng/ml) plus PA (0.5 mM) and PTF (0.5 mg/ml)

Discussion

Type 2 diabetes is often suffered from dyslipidemia, as evidenced by elevated serum FFA and other lipids [30]. High fat diet (HFD) promotes macrophage infiltration and increases NLRP3 inflammasome activity [11, 31]. And saturated fatty acids trigger NLRP3 inflammasome activation in macrophages in vitro [32]. Actually, NLRP3 inflammasome is increased in monocytes/macrophages, adipose tissue and liver of obese and type 2 diabetic patients [5, 11]. This is supported by the observation that NLRP3 inflammasome was activated by PA in LPS-primed macrophages. The present study revealed that PTF was rich in flavonoids containing 47 flavonoid compounds, including TYP and I3ON. Network pharmacology indicated the potential regulation of inflammatory response by the flavonoid compounds, and molecular docking implied the potential interaction between TYP and NLRP3 inflammasome. PTF and the compound TYP, but not I3ON, inhibited the secretion of IL-1β and IL-18, and reversed protein expression of NLRP3 inflammasome and IL-1β p17 in PA-induced macrophages, respectively. Moreover, PTF reduced Caspase-1 activity. These results were similar to a selective NLRP3 inhibitor MCC950. Emerging evidences showed that NLRP3 deletion or blockade abolishes the cleavage and activity of Caspase-1 in macrophages, leading to reduced secretion of IL-1β, IL-18, IL-6, and TNF-α [33, 34]. Likely, deactivation of NLRP3 inflammasome downregulates PA-induced protein expression of IL-1β, IL-18, IL-6 and TNF-α in pancreatic β cells [35]. Notably, inhibition of NLRP3 inflammasome in macrophages improves systemic inflammation and blood glucose, protects pancreatic islets, therefore protecting against adverse development of type 2 diabetes [36]. It was reported that ethanolic extract of PT reduces LPS-triggered gene expression of IL-1β and TNF-α [13], and that TYP improves inflammatory station in a rat with heart failure followed by myocardial infarction [19]. Our previous studies demonstrated that PTF improves systemic inflammatory state, promotes insulin sensitivity, and protects insulin secretion function in type 2 diabetic rats [22–24]. These findings collectively highlight the inhibitory effects of PTF and TYP on PA-induced NLRP3 inflammasome activation.

Mechanistically, network pharmacology combined with molecular docking showed that PT flavonoids might regulate the AMPK pathway. The AMPK agonist, AICAR, another control for PTF and TYP, increased p-AMPK/AMPK ratio and suppressed PA-induced NLRP3 inflammasome activation in macrophages. Indeed, AICAR decreases NLRP3 inflammasome expression in the aorta of septic mice induced by cecal ligation and puncture [37]. AMPK activation is beneficial to improve insulin sensitivity in western diet-induced mice involving inflammation inhibition, and vice versa [38]. In the present study, PTF and TYP promoted p-AMPK/AMPK ratio and its upstream p-LKB1/LKB1 ratio in PA-induced macrophages, respectively. AMPK inhibition abolished or attenuated the inhibitory effects of PTF and TYP on the secretion of IL-1β and IL-18, and reversed the negative effect of PTF on Caspase-1 activity. It is confirmed that NLRP3 inflammasome is directly activated by a serial of biological process as the second signals such as increased endogenous ROS [27, 39]. Administration of exogenous H₂O₂ also drives its activation in hepatocytes [40]. In our study, antioxidant treatment inhibited PA-induced ROS overproduction and subsequent activation of NLRP3 inflammasome in macrophages. Similarly, antioxidant blocks D-gal-triggered NLRP3 inflammasome activation in Sertoli cells [41]. But specific inhibition of NLRP3 fails to change ROS generation [28]. Importantly, PTF and TYP diminished PA-induced ROS overproduction, respectively, which was abolished or attenuated by AMPK inhibitor. Moreover, AICAR also reversed PA-promoted ROS overproduction. According to the studies, the ethanolic extracts of PT possess strong free radical cleaning activity [13], while TYP exerts antioxidant effects in LPS-triggered HUVECs through regulating oxidases and antioxidant enzymes [17], and represses mitochondrial ROS production in ischemia-reperfusion-induced kidney cells [18]. Altogether, PTF and TYP likely regulate the AMPK-ROS pathway to inhibit PA-induced NLRP3 inflammasome activation in macrophages.

Majority of ROS production is attributed to oxidative phosphorylation in mitochondria. Continuously increased FFA promotes lipid deposition and metabolism dysfunction in tissues and cells [29]. In this study, PA addition expanded intracellular lipid accumulation. Mechanistically, PA enhanced protein expression of CD36, PPARγ, FAS and DGAT1, and elevated protein expression of CPT-1 and Atp5a1 with increased ATP and ROS production, suggesting an excessive increase in fatty acid transport, lipid synthesis and fatty acid oxidation, as well as abnormal oxidation phosphorylation in macrophages. As a fatty acid transporter, increased CD36 promotes FFA transport into tissues in a FFA-dependent fashion [42]. Moreover, activated CD36 enhances PPARγ activity, which is crucial for ATP-Binding cassette sub-family A member 1 (ABCA1)- and ABCG1-denpedent lipid efflux in macrophages [43]. Besides, PPARγ regulates adipogenesis through DGAT1 expression, with increased DGAT1 expression benefiting TG synthesis and storage capacity in macrophages [44]. PPARγ overexpression enhances CPT-1 expression and regulates lipid metabolism [45]. In line with the results, the protein expression of CPT-1, an enzyme undertaking FFA influx into mitochondrion and fatty acid oxidation, is increased by PA in vitro and by HFD in vivo [46]. Intracellular FFA overload causes enhanced fatty acid oxidation and subsequent abnormal oxidation phosphorylation in mitochondria with increased mitochondrial ROS production [46, 47]. Network pharmacology showed that PT flavonoids might regulate fatty acid metabolism. While molecular docking displayed the excellent affinity between TYP and the lipid transport- and synthesis-related proteins (CD36, PPARγ, and FAS). Experimentally, administration of PTF and TYP, respectively, decreased FFA and TG contents in the cells, and inhibited the protein expression. Moreover, TYP dramatically restrained DGAT1 expression, and PTF downregulated DGAT1 protein expression to a certain extent. CD36 deficiency was reported to inhibit the transfer of FFA to macrophages, muscle and adipose tissue [42, 48]. PPARγ inhibition decreases intracellular lipid droplets [49]. These data indicated the potential inhibition of lipid transport and synthesis by PT flavonoids. Additionally, inhibition of CPT-1-dependent pathway improves lipid overload-induced fatty acid oxidation [46]. Herein, both PTF and TYP reduced CPT-1 protein expression, maintained high expression of Atp5a1, and further increased ATP production, but decreased ROS overproduction, suggesting the improvement of fatty acid oxidation and oxidative phosphorylation. Studies have shown that PT extract decreases hepatic TG and TC contents in HFD-induced rats [50], and that PTF improves dyslipidemia in type 2 diabetic rats [22]. While TYP ameliorates liver lipid deposition and dyslipidemia through activating the FXR pathway in HFD-induced mice with non-alcoholic fatty liver disease [51].

AMPK is vital to regulate metabolism and sustain energy homeostasis. In line with PTF and TYP, the positive control AICAR improved PA-induced lipid metabolism dysfunction, leading to a reduction in ROS production. Indeed, AMPK activation has been found to decrease the synthesis of fatty acids and cholesterol in macrophages [52], as well as improve mitochondrial function and reduce ROS production in FFA-induced liver cells [53]. Conversely, AMPK deficiency increases lipid synthesis and mitochondrial ROS production [52, 54]. In the same way, ACC inhibitor PF-05175157, serving as a positive control, ameliorated PA-induced lipid metabolism dysfunction. ACC inhibition was reported to improve fatty acid oxidation and reduce ROS contents in low phosphorus-induced Zebraffsh liver cell line [55].

In addition, AMPK activation negatively regulates ACC activity, and inhibits FFA-induced NLRP3 inflammasome activation in human meibomian gland epithelial cells [56]. In the present study, similar to PTF and TYP, AICAR, antioxidant, and PF-05175157 inhibited PA-induced NLRP3 inflammasome activation, respectively. Interestingly, decreased IL-1β relieves the secretion of inflammatory cytokines such as IL-6, TNF-α and IL-17, which rescues the LKB1 and AMPK activity inhibited by inflammatory cytokines [57, 58].

Currently, there are few chemical drugs for diabetic inflammation. Aspirin, a nonsteroidal anti-inflammatory drug, is available in clinical practice. The oral anti-diabetic agents, such as thiazolidinedione insulin sensitizers and metformin, also exert anti-inflammatory effects [59]. However, they are proved to perform side effects, including gastrointestinal adverse reactions, anaphylaxis, and hepatic and renal damage [60]. Clinically, PT alone is initially used to treat diabetic fundus hemorrhage [61]. Recently, PT has been combined with other herbal medicine to treatment diabetic nephropathy and diabetic retinopathy [62, 63]. The effects are close related to the improvement of inflammation [64], providing an alternative choice for the treatment of diabetic inflammation. Moreover, few side effects were recorded or reported. The findings highlight the ability of PTF and its active component TYP to inhibit PA-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism (Fig. 10), suggesting the potential mechanisms through which PT can improve inflammation, providing evidences supporting the clinical treatment of diabetic inflammation using PT, and implying the potential use of PT flavonoid compounds as anti-diabetic inflammation lead compounds.

Fig. 10.

Proposed mechanisms by which PTF and TYP inhibit PA-induced NLRP3 inflammasome activation in macrophages

However, limitations indeed exist. The present study was not designed to confirm the findings in vivo. Due to the different extraction methods, the flavonoid composition may be varied in PT. In addition, pharmacokinetics of PT flavonoids keeps unclear. Gut metabolism specially associates with the transform, bioavailability and plasma concentration of PT flavonoids [65]. Therefore, more research on the limitations needs to conduct in future, which will promote the clinical applications of PT flavonoids.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

NLRP3

Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3

IL-1β

Interleukin-1β

LPS

Lipopolysaccharide

NF-κB

Nuclear factor κB

p-IκBα

Phospho-inhibitor of NF-κB

ROS

Reactive oxygen species

PT

Pollen Typhae

TYP

Typhaneoside

I3ON

Isorhamnetin-3-O-neohesperidoside

PTF

Pollen Typhae total flavone

PA

Palmitic acid

ox-LDL

Oxidized low-density lipoprotein

UHPLC

Ultra-high performance liquid chromatograph

MS

Mass spectrometer

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

AMPK

AMP-activated protein kinase

TLR

Toll-like receptor

FAS

Fatty acid synthase

PPARγ/PPARG

Peroxisome proliferator-activated receptor gamma

LKB1

Liver kinase B1

NAC

N-Acetyl-L-cysteine

FFA

Free fatty acid

TG

Triglyceride

DAG

Diglyceride

MAG

Monoglyceride

LEC

Lecithin

DGAT1

Diacylglycerol-O-acyltransferase 1

CPT-1

Carnitine palmitoyltransferase 1

Atp5a1

ATP synthase alpha-subunit

ACC

Acetyl-CoA Carboxylase

Author contributions

W.R. and Y.Y did the experiment and collected data. H.D., W.N., H.L., C.W., and L.L. performed index analysis. A.T. was responsible for statistical analysis. X.F. designed and did the experiments, wrote and revised the article. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant numbers 81973834 and 82260902), the Gui Style Xinglin Top Talent Funding Project of Guangxi University of Chinese Medicine (no. 2022C012) and the Cultivation Program of 1000 Young and Middle-aged Backbone Teachers in Higher Education of Guangxi (no. Gui Teacher Education [2019]81).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

In this study, permission was obtained to collect the pollen of Typha angustifolia L. from the wild habitat according to the List of National Key Protected Wild Plants in China (2021).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.