{'fraction_non_alphanumeric': 0.03637982378043165, 'fraction_numerical': 0.021797193995026557, 'mean_word_length': 4.992273730684326, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Adenosine-rich extract of Ganoderma lucidum: A safe and effective lipid-lowering substance

Summary Ganoderma lucidum is a traditional Chinese medicine with a variety of active compounds and possesses adequate lipid-lowering and anti-atherosclerotic effects. However, its main active components and potential mechanisms still remain unclear. Here, we evaluated the anti-hyperlipidemic effect of the adenosine extract from Ganoderma lucidum (AEGL) in high-fat-diet (HFD)-induced hyperlipidemic ApoE−/− mice and explored the underlying biological mechanism by multi-omics analysis. Treatment with AEGL for 8 weeks significantly decreased the serum levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-c) by 45.59%, 41.22%, and 39.02%, respectively, as well as reduced liver TC and TG by 44.15% and 76.23%, compared with the HFD-only group. We also observed significant amelioration of hepatic steatosis without liver and kidney damage after AEGL treatment. Regulating the expression and acetylation/crotonylation of proteins involved in the PPAR signaling pathway may be one of the potential mechanisms involved in the observed lipid-lowering effects of AEGL.


INTRODUCTION
Hyperlipidemia is characterized by abnormal high levels of low-density lipoprotein cholesterol (LDL-c) and/or triglycerides (TG) in blood, and as a key risk factor for global cardiovascular and metabolic diseases, it has been responsible for the increased incidence of acute cardiovascular and cerebrovascular events in recent years (Nicholls et al., 2018;Klimchak et al., 2020;Bai et al., 2021). Deaths from cardiovascular diseases have steadily increased from 12.1 million (95% UI:11.4 to 12.6 million) in 1990, reaching 18.6 million (95% UI: 17.1 to 19.7 million) in 2019 (Du et al., 2016), and it is also estimated that there are currently more than 400 million patients with hyperlipidemia in China ([2016 Chinese guideline for the management of dyslipidemia in adults], 2016). A clinical study of 170,000 individuals has reported that the average annual incidence of major cardiovascular disease events was decreased by about 20% when a decrease of 1.0 mmol/L in LDL-c level existed in a patient individual (Baigent et al., 2010). Hence, relieving hyperlipidemia-related symptoms is a crucial strategy to hamper the progress of cardiovascular and cerebrovascular complications. However, the existing lipid-lowering drugs are plagued by various factors, including adverse reactions, drug resistance, individual differences in treatment responses, or poor efficacy (Hirota et al., 2020). Therefore, it is urgently needed to discover novel hypolipidemic agents with more efficiency and less adverse effects.
Ganoderma lucidum is a Basidiomycete fungus with high medicinal value in traditional Chinese medicine (TCM). It contains a variety of essential bioactive compounds and thus has unique advantages in treating or preventing many diseases. Previous studies have found that polysaccharide and ganoderic acid extract from Ganoderma lucidum can help to reverse obesity and effectively alleviate insulin resistance (Chang et al., 2015;Yang et al., 2018). In addition, the ethanol extract of Ganoderma lucidum spore has been found to possess lipid-lowering and anti-atherosclerotic effects that occur owing to the extract's increasing expression of LXRa and downstream genes related to cholesterol transport and metabolism (Lai et al., 2020). Adenosines are an important bioactive component in Ganoderma lucidum, which have several derivatives (Khan et al., 2015). Previous researches have shown that adenosine can ameliorate metabolic disorders by activating brown adipose tissues (Cypess et al., 2015;Gnad et al.,2014Gnad et al., , 2020. However, the side effects of pure adenosine limit its clinical application. Therefore, as an adenosine-rich extract from a Protein post-translational modifications (PTMs) are crucial biological processes that add polypeptides or chemical groups to protein amino acid residues in order to control various cellular processes by altering protein stability, localization, activity, or interactions (Keenan et al., 2021). Several kinds of physiological activities and diseases are not only associated with the abundance of certain proteins but are essentially regulated by PTMs of various other proteins as well (Yang et al., 2017). The metabolism of lipids involves an accurate and complex regulation network, such as gene expression, transcription and translation, and protein PTMs (Harney et al., 2021), and as one of the most common PTMs, protein acetylation modification, in particular, plays an important role in lipid metabolism. Previous studies have shown that the acetylation of several potentially important proteins, such as Hadha, Acat1, and Ehhadh, may be crucial regulatory factors in the development of fatty liver disease . Lysine crotonylation (Kcr) is another type of PTM that has been newly identified on histones and has been shown to be related to transcription activation and cell signaling pathways . However, the biological functions related to lipid metabolism of histone crotonylation have not yet been reported.
In this work, we first evaluated the lipid-lowering effects of adenosine-rich AEGL in HFD-induced ApoE À/À mice and then explored its underlying mechanisms based on proteomics, protein PTMs, and lipidomic analysis.
Adenosine extract of Ganoderma lucidum alleviates hyperlipidemia in ApoE À/À mice Currently, several mouse models including high-fat-diet (HFD)-fed wildtype mice, ob/ob obese mice, LDLR À/À mice and ApoE À/À mice, are popularly used to investigate the anti-hyperlipidemic effects of various drugs. As the response of wildtype mice to HFD-induced hypertriglyceridemia is unstable (Gao et al., 2010) and previous studies have shown that Ganoderma lucidum and its related components can improve hyperlipidemia through modulating LDLR and leptin (Meneses et al., 2016), so we chose ApoE À/À mice to investigate the anti-hyperlipidemic effects and potential mechanisms of AEGL on HFD-induced hyperlipidemia. We fed ApoE À/À mice with HFD for 4 weeks then exposed them to low and high doses of AEGL (7.63 mg/kg/d and 15.26 mg/kg/d) and distilled water for 8 weeks. Serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c) in both the low-and high-dose AEGL groups were significantly lower than those in the HFD group, while the serum levels of high-density lipoprotein cholesterol (HDL-c) of three groups had no significant differences (Figure 2A). Statistically, TC, TG, and LDL-c were decreased by 30. 24%, 43.42%, 35.96% and 28.67%, 29.14%, 38.35% in the low-and high-dose AEGL groups, respectively, as compared to the HFD group ( Figure 2B). Liver function, as assessed by serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) did not appear to be affected by AEGL ( Figure 2C). In addition, neither dosage of AEGL To explore the lipid-lowering effects of AEGL, we designed a study where 6-week-old male ApoE À/À mice were fed with a HFD for 4 weeks and subsequently treated with a 7.63 mg/kg/d dosage of AEGL for 8 weeks.
As expected from the above results, both bodyweight and bodyweight gain ( Figures 3A and 3B) were significantly lower after AEGL treatment, and the serum TC, TG, and LDL-c were decreased by 45.59%, 41.22%, and 39.02%, respectively, in the AEGL group as compared to the HFD group ( Figures 3C and 3D).
Subsequently, we focused on the adipose tissues, one of the major sites of lipid metabolism, and on the activation of uncoupling protein 1 (UCP1), a protein involved in the anti-obesity effects of adenosine (Cypess et al., 2015;Gnad et al.,2014Gnad et al., , 2020. . Hematoxylin/eosin (H&E) staining of scapular and inguinal adipose tissue and Western blot analysis of UCP1 both showed that AEGL had no significant effect on adipose histopathology but reduced UCP1 expression in scapular and inguinal adipose tissue compared to the HFD group (Figures S1A-S1C), suggesting that AEGL did not stimulate adipose browning. Contrastively, the liver TC and TG in the AEGL group were significantly lower, by 44.15% and 76.23%, respectively, compared to the HFD group ( Figures 3E and 3F). Oil red O staining also showed that AEGL dramatically ameliorate hepatic steatosis ( Figure 3G), indicating that AEGL may alleviate hyperlipidemia via the modulation of liver lipid metabolism.
Adenosine extract of Ganoderma lucidum alters hepatic protein expression and posttranslational modifications involved in fatty acid metabolism Regulations on protein expression and PTMs (e.g. phosphorylation, acetylation, and lysine crotonylation) have been widely indicated to participate in various physiological and pathological processes (Radamaker et al., 2021). We, therefore, performed proteomics on liver protein, aiming to uncover the underlying mechanism of AEGL's lipid-lowering effects. Principal component analysis (PCA) showed that the protein profiles in both the HFD and AEGL groups had marked separation. Similarly, levels of the acetylation and lysine crotonylation modification were also evidently altered by AEGL treatment ( Figure 4A). Moreover, we performed KEGG gene sets and GO biological process analysis to explore certain signaling pathways associated with AEGL's lipid-lowering effects. The KEGG gene sets showed that the differential proteins were mostly enriched in the lysosome, peroxisome proliferator-activated receptors (PPAR) signaling pathways, as well as peroxisome and steroid biosynthesis ( Figure 4B). GO biological process analysis showed that both the small-molecule metabolic process and lipid metabolic process were affected by AEGL ( Figure 4C), suggesting that AEGL can regulate lipid metabolism, especially fatty acid metabolism.
To improve the accuracy of the analysis, we further narrowed down candidate proteins with a score greater than 60 in the modification-specific proteomics study. KEGG gene sets analysis showed that the acetylated iScience Article modified proteins were mostly enriched in the PPAR signaling pathway and fatty acid metabolism (Figure 4D), and the small-molecule metabolic process, organic acid metabolic process, lipid metabolic process, fatty acid metabolic process, lipid catabolic process, lipid oxidation, and fatty acid beta-oxidation were all affected by AEGL to some degree ( Figure 4E). Additionally, the differential crotonylated modification proteins were enriched in fatty acid metabolism and the PPAR signaling pathway as well ( Figure 4F). Finally, our GO biological process once again showed that the lipid metabolic process was affected by AEGL ( Figure 4G). Together, our proteomics and PTMs data suggest that the activation of the PPAR signaling pathway plays a key role in AEGL's lipid-lowering effects.
To explore further how AEGL affects protein expression or PTMs related to fatty acid metabolism, we combined proteomic and PTMs in HFD-induced ApoE À/À mice with or without AEGL treatment in order to understand the anti-hyperlipidemia effects of AEGL better. Based on a fold change (AEGL/HFD) greater than 1.5 and a p value less than 0.05,  iScience Article primarily detected the protein expression and modification levels in fatty acid metabolism, and the results showed that the proteins in the PPAR signaling pathway, such as Fabp1, Acsl5, Abcd3, Ech1, Acox1, Cpt2, and Hadh were mostly down-regulated. However, the acetylation modifications of those proteins were mostly up-regulated, while crotonylation modification levels were regulated to varying degrees ( Figure 5D and Table 2). We then assessed the expression levels of several proteins in the table by Western blot analysis and Parallel reaction monitoring (PRM) ( Figure S2 and Table S1), and evaluated modifications of these proteins by Co-immunoprecipitation and Western blot ( Figure S3). Our results showed that the profiles of protein expression and modification were basically consistent with proteomics.

Lipidomics analysis of fatty acid metabolism
We further performed lipidomics analysis to examine the readouts of lipid metabolism regulated by AEGL. As shown in Figure 6A, glycerides, free fatty acids, and carnitines in the AEGL group were markedly separated from the HFD group. A volcano plot of glyceride profile revealed that among 230 glycerides, 62 were decreased in AEGL-treated mice (AEGL/HFD ratios > 2-fold; p value < 0.05) ( Figure 6B). Among these, TAG56: Carnitine is a key substance in mediating fatty acid oxidation, and we found that 16:1-carnitine was significantly increased in the AEGL group, while 20:0-carnitine, 11:0-carnitine, and 10:0-carnitine were markedly decreased after AEGL treatment ( Figures 6E and 6F). Given that acetyl CoA generated by fatty acid oxidation can enter the tricarboxylic acid (TCA) cycle, we then measured the levels of organic acids in order to investigate the effects of AEGL on the TCA cycle. Malate and aconitate were found to be increased in the AEGL group ( Figure 6G), supporting that AEGL, indeed, affected the TCA cycle. These results are in line with GO biological process analysis regarding acetylation and crotonylation modification.

DISCUSSION
As a major cause of cardiovascular and cerebrovascular complications, hyperlipidemia is threatening global public health with increasing incidence (Klimchak et al., 2020). Among the current lipidlowering drugs, statins can effectively reduce cholesterol levels, but their effect on triglyceride levels is not significant (Nicholls et al., 2018). Bates can promote the degradation of triglycerides, yet exhibiting no obvious effect on reducing cholesterol levels (Nicholls et al., 2018). Consequently, there is still room to exploit lipid-lowering drugs with multi-targets and more effectiveness. Ganoderma lucidum, a widely used TCM to treat metabolic diseases in China, possesses various active compounds, such as polysaccharides, organic acids, and nucleosides. Compared with polysaccharides and organic acids, little is known about the lipid-lowering effects of the nucleoside compounds in Ganoderma lucidum.
Our previous work reported that AEGL was favored to reduce cholesterol and triglycerides. ApoE À/À mice fed with high-fat diet usually accompany increased serum TG, TC, LDL-c, and decreased HDL-c, and are commonly used hyperlipidemia model (Wu et al., 2014). We hypothesized that the efficacy of AEGL may be related to UCP1 which partially depends on the signaling pathway of leptin. Therefore, ApoE À/À mice are superior to ob/ob (leptin À/À ) and Ldlr À/À mice in investigating the lipid-lowering effect of AEGL and exploring its potential mechanism. Hence, in the present study, we established hyperlipidemia in ApoE À/À mice with HFD-feeding for 4 weeks and evaluated AEGL's lipid-lowering activity by pharmacodynamics, and then preliminarily explored its mechanism based on multi-omics. The results showed that AEGL could significantly reduce lipid levels in blood and liver, as well as significantly ameliorate hepatic steatosis without causing significant damage to liver and kidney. By integrating analysis of proteomics, modification-omics, and lipidomics, we found that AEGL mainly exerted its lipid-lowering effect by promoting fatty acid metabolism.
The liver and adipose tissue are the most critical organs to regulate lipid metabolism, and studies have shown that synthetic adenosine plays a lipid-lowering role primarily by activating adipose tissue (Gnad et al.,2014(Gnad et al., , 2020. However, the pathological examination of mouse adipose tissue and the expression of UCP1 in our study indicated that AEGL did not exert lipid-lowering effects by acting on adipose tissue. In particular, liver oil red O staining showed that AEGL was able to marked reduce the accumulation of lipid droplets induced by HFD feeding. Consistently, liver TC and TG were also reduced upon AEGL treatment. Therefore, we conclude that AEGL has a lipid-lowering effect by affecting liver fat metabolism. Fatty acids (FAs) are important raw materials for the synthesis of triglycerides and cholesterol (Donnelly et al., 2005;Zhang et al., 2013). In hepatocytes, fatty acids are esterified to glycerol 3-phosphate (G3p) and cholesterol to produce TG and cholesterol esters, respectively. The lipidomics analysis in our study showed that the level of hepatic triacylglycerol (TAG) was significantly decreased by AEGL, and the oil iScience Article red O staining showed that hepatic steatosis was alleviated by AEGL, indicating that AEGL can either reduce the synthesis or accelerate the decomposition of hepatic TAG. However, in the results from our multi-omics analysis, the key enzymes of liver TG degradation had no obvious change, but many proteins related to FA metabolism changed significantly.
In our present results, the crotonylation of ACSL1 at k387 was significantly down-regulated, and the acetylation of FASN at k287 was significantly up-regulated. Previous studies have shown that post-translational modification can affect the function of these proteins. For instance, histone acetylation can affect the function of FASN by impairing the binding of srebp-1csre and chrebpchore (Du et al., 2017), and the modification levels of myristoylation, acetylation, and phosphorylation of ACSL1 protein have been found to change under enhanced fatty acid oxidation (Zhang et al., 2021). However, the question of whether the elevation of acetylation modification level at the k287 site of FASN and the down-regulation of crotonylation modification level at the k387 site of ACSL1 affect their functions remains unanswered. Therefore, whether the lipid-lowering effect of AEGL is related to these modifications will be a question to be future studied.
The results of lipidomics showed that 62 kinds of TAGs in mouse liver decreased significantly after treatment with AEGL. Among the 62 TAGs significantly down-regulated by AEGL, TAG54:2 (18:0), TAG51:0 Decreased synthesis or increased the decomposition of these TAGs can lead to the accumulation of corresponding free FAs (FFAs) in the liver, and FFAs can then be used to synthesize TAG, thus increasing the TG storage of the liver and the over-production of VLDL, resulting in hepatic steatosis and the increase of serum TG, TC, and LDL (Donnelly et al., 2005). Interestingly, the amount of these FAs in the present study did not change significantly in the liver, and some FAs even showed a downward trend, suggesting that the rate of FA decomposition in the liver was greatly accelerated.
As presented in our results, many proteins related to FA metabolism changed significantly, and most of them were enriched in the peroxidase proliferators activated receptors (PPAR) signaling pathway ( Figure  7). In particular, FA binding proteins (FABPs) were expressed in various tissues including adipocytes and liver. These proteins mediate lipid transport and inflammation by preventing excessive accumulation of long-chain FAs (Du et al., 2017). Researchers have reported that increasing the expression of FABP in the liver can enhance the uptake of medium or long-chain FAs and promote the process of intracellular targeting to the nucleus (Huang et al., 2002;Baran et al., 2019), and our study showed that FABP5 protein was highly expressed in the AEGL group, suggesting that AEGL may promote the uptake of unesterified longchain FAs and then interact with PPAR to start transcription in the nucleus.  iScience Article Our Functional enrichment analysis showed that the acetylation and crotonylation of proteins related to FA metabolism were significantly changed, especially the related proteins in the PPAR signal pathway. Acetylation and lysine crotonylation are one of the most common epigenetic modifications and have been shown to be the primary factors that regulate the oxidative metabolism of FAs. The acetylation modification levels of these proteins were significantly up-regulated, while the crotonylation modification levels of these proteins were significantly up-regulated or down-regulated. Among the proteins with significant modification changes, liver carnitine palmitoyltransferase 1a (CPT1A) and peroxisome ABC transporter ABCD3 are essential in b-oxidation and peroxisome oxidation, respectively (Cacciola et al., 2021;Violante et al., 2019). Existing studies have shown that ABCD3, acyl CoA oxidase 2 (ACOX2), Acaa1a, and Ehhadh proteins are the key regulatory enzymes of peroxisome oxidation (Kersten and Stienstra, 2017;Nenicu et al., 2007), and the expression levels of CPT2, ACADVL, ACAA2, HADHA, ACADSB, and ACOX1 contribute to FAs b-Oxidation. We found that the acetylation modification levels of the above proteins, as well as the crotonylation modification levels of ACOX1 and ACSL5 were up-regulated after AEGL, while the crotonylation modification levels of FABP2, ACSL1, ACSM3, and ehhadh were down-regulated, In addition, from our lipidomics results, we found that the key substances 20:0-carnitine, 11:0-carnitine, and 10:0-carnitine required for FA oxidation were significantly reduced in the AEGL treatment group, which also indicated that FA oxidation was promoted. Combined with the results of proteomics and modification omics, we can see that the changes in acetylation and crotonylation modification in the FA oxidation pathway can, eventually, accelerate FA b-oxidation.
Furthermore, based on lipidomics, we found that AEGL increased the content of polyunsaturated FAs (PUFAs), but decreased the content of saturated and monounsaturated FAs. Studies have shown that increased PUFAs can inhibit the occurrence of hyperlipidemia. However, the sharp decrease of malonyl CoA and polyunsaturated FA (PUFAs) caused by ACC inhibitor, a drug for the treatment of nonalcoholic fatty liver, is one of the primary causes of side effects of hyperlipidemia treatment. Reduced PUFAs lead to an increase in the expression of SREBP and its downstream adipogenesis and VLDL secretion genes, and lower PUFAs also significantly inhibit FA oxidation, thus preventing lipid degradation in plasma (Zhang et al., 2022). Other studies have shown that 2 u-3PUFA can reduce plasma triglycerides by 20% to 30% (Zhang et al., 2022). In animal models, dietary intake rich in linoleic acid (LA) can increase the expression of LDL receptors in the liver and effectively reduce LDL (Dekker et al., 2000). These results suggest that the increase in liver PUFAs by AEGL may be AEGL's potential mechanism in reducing cholesterol and improving hyperlipidemia.
Whether acetyl coenzyme A, the prestigious metabolite of FA b-oxidation, can promote the TCA cycle merits its own discussion. To begin, aconitate is an intermediate from citric acid to isocitrate, and malic acid is also an important metabolite in the TCA cycle. Our lipidomics results showed that these substances were significantly increased, demonstrating that AEGL promoted the process of the TCA cycle. The enhancement of TCA also indicates an increase in fatty acid oxidation. Excess lipids, eventually, can be decomposed into CO 2 and H 2 O to produce ATP (Choi et al., 2021), which improves hyperlipidemia and hepatic steatosis.
Our results indicate that AEGL is a lipid-lowering drug worthy of in-depth research and exploration. Because the action mechanism of AEGL is different from current mainstream lipid-lowering agents, AEGL provides possibilities for therapeutic combinations with other medications to manage blood lipids more effectively and safely. If AEGL can be deeply studied and developed, it may provide new therapeutic drugs for hypertriglyceridemia, hypercholesterolemia, and even nonalcoholic fatty liver. This study showed that AEGL effectively reduced the levels of lipid in both serum and liver in HFD-fed ApoE À/À mice. The possible biological mechanism of AEGL's lipid-lowering was found to occur through regulating the expression of FA-metabolism-related proteins, as well as the up-regulating of acetylation and regulation of crotonylation modification levels of these proteins. Our study not only presents a new candidate for the treatment of hyperlipidemia but also provides ideas for mechanistic studies of multitarget drugs based on a multi-omics research model.

Limitations of the study
In this study, our limitations are that we did not explore the intrinsic logic of significant changes in protein quantity and modification level in the PPAR signaling pathway, and we did not further evaluate the toxicity of AEGL through cell experiments. In addition, there are some unknown adenosine components in AEGL, which need to be further separated by HPLC and identified by NMR and other spectroscopy techniques to identify the most active monomer compounds, which is also the future research direction.

AVAILABILITY OF DATA AND MATERIAL
All data involved in this article can be presented on request.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: iScience Article Data and code availability d All data reported in this paper will be shared by the lead contact upon request. d All data obtained by mass spectrometry in this study have been submitted to the PRIDE archive with accession number PXD036478.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact on request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
The serum, adipose tissue and liver tissue involved in this study were obtained from the ApoE À/À mice. These biological samples were used to generate the data listed in Figures 2, 3, 4, 5, and 6 and Table 2. This research study met all appropriate regulatory standards.
Mouse studies were conducted using 6 week-old male ApoE À/À mice. All mice were purchased from a qualified supplier, Beijing Huafukang Biotechnology Co., Ltd. (

METHOD DETAILS Preparation of Ganoderma lucidum extract
The crude drugs Ganoderma lucidum were purchased from Dongzhimen Hospital Beijing University of Chinese Medicine, and the preparation of Ganoderma lucidum adenosine extract was 30% aqueous MeOH (v/v). The main constituents of Ganoderma lucidum adenosine extraction were analyzed and identified by using ultra-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (UPLC-Q-TOF/MS).
First, the Ganoderma lucidum was crushed, and then sieved through 40-80 meshes to obtain the Ganoderma lucidum power. The powder was soaked in 50-80% methanol aqueous solution (0.05-0.2 g/mL), and then extracted 3-6 times with ultrasonic at the power of 400-600W.
After the evaporation of the solvent under reduced pressure, the residue was fractionated by Xad-16n macroporous ( MeOH-H2O, 40% and 60%). The subfraction was further separated by preparative HPLC (MeOH-H2O, from 0% to 30%; Elution time 150-200 min). The eluents of all chromatographic peaks at the detection wavelength of 259 nm were collected, combined and concentrated under reduced pressure.
Mass spectrometry was executed on a Xevoä G2 Q/TOF (Waters Corp., Milford, MA, USA) equipped with electrospray ionization. The MS parameters were as follows. The MS scan m/z range was set to 100 to 1000 Da in either positive or negative ionization mode; the capillary voltage in positive and negative mode was set to 3.0 kV, 2.5 kV, respectively; the sampling cone voltage was set to 40; and the flow rate of desolvation gas was set to 800 L/h with a temperature of 400 C. All the data were collected in continuum mode and real-time corrected using a Lock Spray reference to ensure the accuracy. All the acquisitions were precisely controlled by Waters Masslynx v4.1 software (Waters Corporation, Milford, MA, USA).

Animal experiment design
A total of 40 male ApoE À/À mice that were 6 weeks old were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). The mice were housed in a temperature-controlled (22 C) chamber with a 12-h light/dark cycle. All operations were approved by the Key Laboratory of Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine. The animals were randomly divided into 4 groups. After adaptive feeding for one week, 10 mice were fed with chow (chow group, n = 10), and 30 mice were treated with a high-fat diet(HFD) (21% lipid and 0.15% cholesterol) for 4 weeks to induce hyperlipidemia (HFD, HFD + AEGL (7.63 mg/kg/d) and HFD + AEGL (15.26 mg/kg/d) group, n = 10). The chow group and HFD group were intraperitoneally injected with distilled water, and the HFD + AEGL (7.63 mg/kg/d) and HFD + AEGL (15.26 mg/kg/d) groups were treated with AEGL at the dosages of 7.63 mg/kg/d and 15.26 mg/kg/d, respectively After 8 weeks, all mice were fasted for 12 h and then euthanized. The blood was collected and then centrifuged at 8000 rpm for 15 min at 4 C, and the serum was then stored at À80 C for further biochemical analysis.

OPEN ACCESS
iScience 25, 105214, November 18, 2022 iScience Article To find the optimal dose of the AEGL, we randomly divided the 20 male ApoE À/À mice into 2 groups (HFD and HFD + AEGL group) and fed them a HFD for 4 weeks to induce hyperlipidemia. Then the HFD group was intraperitoneally injected with distilled water, while the HFD + AEGL group was treated with AEGL at a dosage of 7.63 mg/kg/d. After 8 weeks, all mice were fasted for 12 h and then euthanized, and blood and tissue samples were collected for further examination.

Histopathological examination of liver and adipose tissue
The liver and adipose tissue were fixed in 10% formalin solution for 72 h, rinsed with distilled water, and washed once with 60% isopropyl alcohol. Paraffin-embedded liver and adipose tissue were then cut into 5 mm thick sections for hematoxylin and eosin (HE) staining, and then the sections were observed with a pathological section panoramic scanner (Leica Aperio AT2). Liver embedded in optimal cut-temperature compound (Tissue-Tek, Laborimpex) was used for oil-red O staining in order to assess hepatic steatosis.

Proteomics analysis Extraction of protein
Frozen liver tissue was pulverized in liquid nitrogen and ultrasonic cleavage with 4 times lysis buffer (8 M urea, 1% protease inhibitor, 3 mM TSA, 50 mM NAM). The resulting homogenate was then centrifuged at 12,000g for 10 min at 4 C, and the supernatant was collected for quantification using a BCA Protein Quantification Kit (Vazyme, China).

Trypsin enzymolysis
An equal amount of proteins were adjusted to the same volume with lysate, and slowly added to 20% TCA and mixed. The sample mixture was precipitated at 4 C for 2h and then centrifuged at 4,500 g for 5 min. The supernatant was discarded, and the precipitate was washed 2 to 3 times with pre-cooled acetone. After drying the precipitate, TEAB with a final concentration of 200 mM was added and then sonicated. The protein suspension was enzymolyzed with trypsin at a 1:50 ratio (protease: protein, m / m) overnight. Disulfur threitol (DTT) was added to gain a final concentration of 5 mM and reduced by 56 C for 30 min. Finally, iodiacetamide (IAA) was added to gain a final concentration of 11 mM and the suspension was incubated for 15 min at room temperature under darkness.

HPLC-MS/MS analysis
Peptides were resolved by mobile phase A, separated using an HPLC system (Thermo Fisher Scientific EASY-nLC 1000; Waltham, MA, USA) with a flow rate of 450 nL/min, and identified using a timsTOF Pro mass spectrometer (Thermo Fisher Scientific). The separation system comprised mobile phase A (0.1% formic acid and 2% acetonitrile) and mobile phase B (0.1% formic acid and 100% acetonitrile). Mobile phase B was used to separate the peptides over a 1.5 h separation time. From 0 min to 70 min, the linear gradient of mobile phase B was 6% to 24%; from 70 min to 84 min, the gradient of mobile phase B was 24% to 32%; and from 84 min to 87 min, the gradient of mobile phase B was 32% to 80%. Thereafter, mobile phase B remained at 80% from 87 min to 90 min. Our MS analysis was set to positive ion detection mode. The ion source voltage was set to 1.6 kV, and the scanning range for the secondary mass spectrometry was 100-1700 m/z. Additionally, the data acquisition used a parallel accumulation serial fragmentation (PASEF) mode. A secondary spectrogram with the number of parent ion charges in the range 0 to 5 after 10 PASEF mode acquisition after primary mass spectrometry as well as the dynamic exclusion time of tandem MS scans were set to 30 s s to avoid repeat scans of the parent ion.