Emodin alleviated hyperlipidemia and hepatic lipid metabolism in zebrash larvae fed a high-cholesterol diet via AMPK/SREBP-2/PCSK9/LDLR signaling pathway

Emodin (EM) is one of bioactive components extracted from Rheum palmatum L. (Dahuang), which possesses numerous pharmacological activities including hypolipidemic effect. However, the potential action of EM on hyperlipidemia (HLP) remains unclear. Here, the theraputic effect of EM against HLP were investigated. In this study, the hypolipidemic properties of EM were evaluated using high-cholesterol diet (HCD)-stimulated zebrash larvae model. The body weight, body length and body mass index (BMI) was measured. The total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by corresponding assay kits. Tg (il: eGFP) zebrash were utilized to observe vascular cholesterol accumulation and Tg (mpx: eGFP) zebrash to visualize and quantify neutrophil inammation. The hepatic lipid deposition and hepatic histopathology were analyzed by Oil red O staining and H&E staining, respectively. Finally, the underlying mechanism of EM were investigated using real-time quantitative PCR (RT-qPCR) analysis to assess the gene levels of adenosine monophosphate-activated protein kinase alpha (AMPKα), sterol regulatory element binding protein (SREBP-2), convertase subtilisin kexin 9 (PCSK9), low-density lipoprotein receptor (LDLR), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), adenosine triphosphate binding cassette transporter A1 and binding cassette transporter G1 transporter G1; 3-hydroxy-3-methyl-glutaryl reductase.

With the improvement of life quality and dietary standards, hyperlipidemia (HLP) and other metabolic syndrome are becoming more prevalent (1). HLP is a pathological condition in which there are abnormalities in the levels of lipids in the blood as a result of lipid metabolism dysfunction, which represents the predictive risk factor for cardiovascular and hepatic diseases (2). Substantial evidence has demonstrated that HLP plays a crucial role in driving the development of atherosclerosis, because excessive lipids accumulate in the arterial intima, of which cholesterol exerts the greatest impact (3)(4)(5).
The liver constitutes the central organ of lipid metabolism involved in lipid digestion, absorption, transportation, decomposition and synthesis. It was been reported that the deposition of lipids in the liver may lead to hepatotoxicity and in ammatory response, thereby exacerbating lipid metabolism disorder (6, 7). Currently, the hypocholesterolemic drug statins are the mainstay for treating HLP, but long-term administration of statins may cause adverse effects, such as gastrointestinal events and musculoskeletal pain (3,8). Therefore, seeking safe, economical and alternative agents is necessary for the treatment of HLP.
Rheum palmatum L. (Dahuang), a common Chinese herb, is skilled in promoting blood circulation and removing blood stasis achieving signi cant therapeutic effects on atherosclerotic cardiovascular disease and chronic liver diseases (9,10). Emodin (EM, Fig. 1a), a major bioactive component in Dahuang, exhibits numerous biological activities including anti-in ammatory, antioxidant, lipid-lowering and hepatoprotective effects (11)(12)(13). EM attenuated atherosclerotic lesions through enhancing antioxidant capacity and modulating sphingomyelin synthesis in rabbits (14). Another study showed that EM reversed the abnormal total cholesterol (TC), triglyceride (TG) and hepatic steatosis in zebra sh with nonalcoholic fatty liver disease (NAFLD) induced by egg powder (15). Moreover, EM administration inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), a key enzyme in TC synthesis, in vitro experiment (16). However, the effect and underlying mechanism of EM on HLP in vivo experiments are still limited.
Compared with mammalian models, the zebra sh (Danio rerio), a small freshwater sh, is considered as an emerging and promising tool for drug screening and development due to its unique strengths, including small sizes, transparent embryo, low feeding cost and short experimental period (17). Intriguingly, zebra sh and mammals share similarities in hepatocellular composition, function as well as genetics (18). Moreover, zebra sh is also resemble to human in lipid metabolism, such as lipid absorption in intestine and cholesterol transport mediated by lipoproteins (19,20). These strengths make them an excellent model utilized to study lipid metabolism-related diseases, including HLP, NAFLD and atherosclerosis (15,21). Therefore, this study aimed to explore the effect of EM on lipid metabolism in HLP zebra sh fed with high-cholesterol diet (HCD), which provided a theoretical basis for the research on the therapeutic mechanism of HLP and the development and utilization of EM. Preparation of high cholesterol diet (HCD) and drug solutions Ordinary larval zebra sh feed (Azoo 9 in 1 Arti cial Rotifera) was purchased from Taikong Corp. (Taiwan, China) as the normal feed. The feed mainly contained 41.6% crude protein and 5.6% crude fat. The HCD was prepared by mixing a diethyl ether solution of cholesterol with normal feed to get 4% (w/w) cholesterol in the diet after diethyl ether evaporation (22,23). For the aim of analyzing vascular cholesterol accumulation in zebra sh larvae, both HCD and normal feed were additionally added with 10 µg/g of Cholesteryl BODIPY® 542/563-C11 in the dark (22). Due to the low solubility of EM in water, the drug was solved with DMSO to 1mg/mL and diluted with cultured zebra sh water (less than 0.1% DMSO v/v).

Animal and experimental groups
The wild-type AB-line zebra sh, Tg ( il: eGFP) zebra sh with vascular endothelial cells expressing green uorescent protein (GFP) and Tg (mpx: eGFP) zebra sh with neutrophils expressing GFP were provided by zebra sh laboratory of Pharmacy College, Chengdu University of TCM. All adult zebra sh were raised and maintained in the zebra sh breeding system (circulated water temperature 27.5 ± 1°C, pH 7.2-7.6, electrical conductivity 500-550 microns/cm and a 14 h light/10 h dark cycle). Zebra sh embryos were obtained by naturally mating and cultured at 28°C in a constant temperature incubator. wild-type AB-line or transgenic zebra sh larvae at 5 days post fertilization (dpf) were randomly divided into ve groups in 6-well plate. The ve groups were prepared as follows: Normal feed as the control group, 4% HCD as the HCD group, 4% HCD plus 0.125 µg/mL EM as the EM-L group, 4% HCD plus 0.25 µg/mL EM as the EM-M group, 4% HCD plus 0.5 µg/mL EM as the EM-H group. All groups were fed equal mass feed 30 mg/day (twice daily for 10 days) followed the schedule (Fig. 1b)

Measurement of growth parameters
After EM administration for 10 days, body weight and length of anesthetized larval zebra sh with tricaine were analyzed. Body length of zebra sh larvae from the front end of the mouth to the end of tail showing in (Fig. 2a) was photographed under leica M165-FC microscope and measured by Image Pro Plus software. Body mass index (BMI) was calculated as body weight (g) divided by the square of body length (cm). Measurement of biochemical parameters 30 euthanized zebra sh larvae were washed 3 times by precooled phosphate buffered saline (PBS) and then homogenized with 270 ul normal saline. After centrifugation at 4°C, 2500 rpm for 10 min, the separated supernatant was used to determine the levels of TC, TG, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by speci c commercial assay kits purchased from Nanjing Jiancheng Bioengineering Insititute (Nanjing, China).

Confocal image analysis
In order to observe vascular cholesterol accumulation, Tg ( il: eGFP) zebra sh larvae were fed with normal fed or 4% HCD for 10 days. The uorescent images were captured under Olympus FV-OSR confocal microscope. Vascular vessels in the green channel and uorescent cholesteryl ester in the red channel were excited at 559 nm and 488 nm respectively. Mean uorescence intensity (MFI) of caudal arteries were analyzed by OLYMPUS Stream software.

Oil red O staining
Before staining, zebra sh larvae were euthanized with high concentration of tricaine and then xed in 4% paraformaldehyde (PFA) at 4°C for 12 h. Brie y for oil red O staining, larvae were washed 3 times with PBS and orderly submerged in 25, 50, 75 and 100% 1, 2-propanediol at room temperature for 15 min, this was followed by 6 h incubation in freshly prepared 0.5% Oil red O dye solution in the dark. Subsequently, the samples were sequentially rinsed in 100, 75, 50 and 25% 1,2-propanediol for approximately 30 min and washed twice with PBS. Eventually, the images were photographed under leica M165-FC microscope and analyzed for lipid deposition in the liver. Integrated optical density (IOD) value of liver, a relative quantitative index of lipid deposition in liver, was measured by Image Pro Plus software.

Hematoxylin and eosin (H&E) staining
After xed with 4 % PFA overnight, larvae was processed in accordance with standard procedures of H&E staining. The whole sh was embedded in para n, sectioned and the histopathological changes of zebra sh liver was observed under leica M165-FC microscope.

Determination of in ammatory level
Studies have shown that uorescent neutrophils in Tg (mpx: eGFP) zebra sh larvae can be used to track the in ammatory response (24,25). Therefore, we use Tg (mpx: eGFP) zebra sh to visualize and quantify neutrophil in ammation. Images were collected by microscope and the average number of neutrophils in the blood vessels of tail were counted by Image Pro Plus software.

RNA Extraction and Real-time quantitative PCR (RT-qPCR) analysis
Total RNA of 40 larvae in each group was extracted using Animal Total RNA Isolation kit and then dissolved in 65 µL RNase-free ddH 2 O. The total RNA purity was analyzed by measuring the OD260/280 value using a nucleic acid/protein analyzer. The RNA was reverse-transcribed by 5×All-In-One RT MasterMix with AccuRT for the synthesis of cDNA. The resultant cDNA was applied as template for qPCR analyses with EvaGreen 2×qPCR MasterMix-No Dye on the StepOnePlus Real-time uorescent quantitative PCR system. The reaction conditions were as follows: 95°C for 10 min, 95°C for 15 s and 60°C for 60 s (40 cycles). The calculated mRNA expression data were expressed as relative expression ratio normalized to GAPDH by 2 −ΔΔCt method. The designed sets of gene primer sequences for RT-qPCR were synthesized by TSINGKE Biological Technology (Chengdu, China) listed in Table 1.

Statistical analysis
Statistical analysis was performed using SPSS version 20.0 software. All experimental data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare differences among multiple groups and Least signi cant difference (LSD) was used for comparison between the two groups. When p < 0.05 level, the results were considered to be statistically signi cant. Graphpad Prism 8.0 software was used to generate graphs.

Results
Effect of EM on the growth of zebra sh larvae with HLP After 10 days of feeding, the growth parameters including body weight and length of each group were determined. Our data showed that HCD increased zebra sh BMI (p < 0.05) and promoted obesity (Fig. 2bd). However, compared with HCD group, Body weight, length and BMI were decreased signi cantly in EM-M and EM-H group (p < 0.05), but not EM-L group.

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EM improved lipid pro les and liver function The levels of TC, TG, LDL-C and HDL-C were measured to con rm the lipid-lowering of EM. The results indicated that the levels of TC, TG and LDL-C in HCD group were markedly increased and the HDL-C level was low as compared to control group (p < 0.01) (Fig. 3a-d). Interestingly, EM reduced the concentration of TC and LDL-C and increased HDL-C level in a dose-dependent manner. However, TG had no signi cant effect at all doses of EM except EM-H group (p < 0.01). The biochemical indexes ALT and AST were measured to investigate liver function (Fig. 3e, f). There was a increase in ALT and AST activities in HCD group compared to control group, whereas the intervention of EM signi cantly decreased the activities of ALT and ALT (p < 0.01).

EM reduced vascular cholesterol accumulation
Fluorescence images were shown in Fig. 4a, b. Cholesterol tagged with red uorescent cholestery ester accumulated in the green blood vessel including caudal artery (arrows in Fig. 4a). The extent of vascular cholesterol accumulation of larvae from trunk to tail, including caudal artery in each group were observed (Fig. 4b). The uorescence level of vascular red cholesterol was signi cantly higher (p < 0.001) in larvae fed with HCD than in larvae fed with normal feed, but this increase was suppressed signi cantly when EM administrated (Fig. 4c). Among which, EM-M and EM-H groups obviously reduced blood lipid in zebra sh fed with HCD.

EM alleviated hepatic lipid accumulation and liver histological damage
The results of Oil red O staining were shown in Fig. 5a, there was no obvious lipid deposition in the liver of control group, whereas the HCD group showed darker staining. Compared with HCD group, the lipid droplets dyed red in the EM treatment groups reduced markedly, which consistent with IOD (p < 0.001) (Fig. 5b). Among which, EM-M and EM-H treatment groups showed obvious attenuation of hepatic lipid staining. Subsequently, liver histopathological observation was performed. H&E staining results (Fig. 5c, d) suggested that the liver tissues were intact and the hepatocytes were closely arranged in control group. However, it was found that there were a large quantity of vacuoles in the liver, and deformed and irregularly arranged hepatocytes in HCD group. In contrast, the vacuoles in the liver tissue reduced and the arrangement of hepatocytes was tended to be regular after treating with EM, especially in EM-H group.

EM ameliorated in ammatory response
To detect the in ammatory response in the process of hyperlipidemia, Tg (mpx: eGFP) zebra sh larvae were selected to be examined under a microscope. The uorescent images of in ammatory response in larvae fed with normal feed, HCD and HCD added with EM were shown in Fig. 6a. Compared with control group, the green uorescent-labeled neutrophils at the vascular site obviously increased in HCD group larvae. No difference in neutrophil number was found between HCD group and EM-L group (p > 0.05). However, neutrophil number diminished signi cantly at the vascular site in EM-M and EM-H group (p < 0.001) compared with HCD group (Fig. 6b). These results demonstrated that EM effectively inhibited the migration and recruitment of neutrophil in vascular site, thereby ameliorating vascular in ammatory response in HLP zebra sh.

Effect of EM on mRNA expression of lipid metabolism relevant molecules
To determine the under mechanism of EM on alleviating hyperlipidemia in HCD-induced zebra sh larvae, the genes expression of lipid metabolism relevant molecules including AMPKα, SREBP-2, PCSK9, LDLR, HMGCR, ABCA1 and ABCG1 were detected by RT-qPCR. The results (Fig. 7a) showed that, compared to control group, the genes expression of AMPKα, LDLR, ABCA1 and ABCG1 in HCD-induced zebra sh were signi cantly decreased, while SREBP-2, PCSK9 and HMGCR were increased (p < 0.001). After 10 days treatment with EM, the mRNA expression levels of AMPKα, LDLR, ABCA1 and ABCG1 were markedly increased, while SREBP-2, PCSK9 and HMGCR were lower in a dose-dependent manner.

Discussion
HLP is a metabolic syndrome due to lipid metabolism imbalance, and usually represented as elevated TC and/or TG, as well as a reduction in HDL-C. HLP results in cardiovascular disease and liver injury, which seriously threatens human life (2). Lipid metabolism disorder may attributed to the interaction between genetics and environmental factors, such as irrational dietary habits. For instance, excessive consumption of HCD and high-fat diet (HFD) are potent risk factors for dyslipidemia and atherosclerosis (26, 27).
In the present study, we used HCD to induce HLP disease to evaluate the effect of EM on lipid metabolism disorder. Our results indicated that HLP zebra sh larvae model was successfully established by consuming HCD for 10 days, which was consistent with the previous study (21). As can be seen from the results, HCD promoted obesity of zebra sh larvae, as evidenced by elevated body weight, length and BMI ( Fig. 2b-d). However, after EM treatment, this situation were effectively alleviated. The translucency of larval zebra sh until 30 dpf permits observation of vascular lipid accumulation and deposition in real time, which is a unique advantage different from the HLP rodent model. It was reported that excessive lipid particularly cholesterol in the blood vessel may predispose to atherosclerosis (28). Recent evidence suggests that the accumulated lipid in the vascular wall of HLP zebra sh model is similar to the composition of early atherosclerotic plaque of humans (29,30). Compared with the control group, it was observed that large amounts of red cholesterol tagged with uorescent cholestery ester accumulated in the green blood vessel (including the caudal artery) in the HCD group (Fig. 4a-c). Intriguingly, EM remarkably improved cholesterol deposition in the area, showing its hypocholesterolemic effect. In addition, neutrophil-regulated in ammatory reaction is essential for early atherosclerosis, and neutrophils, an important immune cell, are recruited in large numbers to the injured sites of endothelial cell layer driving early atherosclerosis and plaque destabilization (31). Our results (Fig. 6a, b) found abundant neutrophils were recruited in the blood vessel of tail in HLP zebra sh larvae, while EM reversed this change. Together, this results suggested that EM reduced vascular cholesterol deposition and improved vascular in ammatory response to inhibit the occurrence of early atherosclerosis in zebra sh larave fed with HCD.
The liver is a center organ for lipid metabolism. When the generated lipid cannot be released into the blood, they are usually accumulated and deposited in hepatocytes in the form of lipid droplets. In the case of hypercholesterolemia, elevated hepatic lipid accumulation contributes to NAFLD and liver injury (32). Transaminases is sensitive to the damage of hepatocytes. ALT and AST are crucial indicators for evaluating liver damage. In this study, compared to the control group, HCD-induced larvae showed that severe lipid accumulation in the liver, hepatic steatosis and abnormal morphology of hepatocytes ( Fig. 5a-d). Whereas, the administration of EM prevented the HCD-induced abnormalities in TC, TG, LDL-C and HDL-C levels, as well as the increased activities of ALT and AST (Fig. 3a-f). These results demonstrated that EM could attenuate hepatic lipid accumulation and improve liver function of HLP zebra sh.
Adenosine monophosphate-activated protein kinase (AMPK) is considered as the key molecule that regulates biological energy metabolism and thus has become potential therapeutic target for metabolic diseases. Viollet et al. demonstrated that the improvement of lipid metabolism disorder may be attributed to AMPK activation and its downstream target genes sterol regulatory element binding protein 2 (SREBP-2) (33). The SREBP-2/PCSK9/LDLR signalling pathway is an important and effective pathway that regulates lipid metabolism (34). SREBP-2, a member of SREBPs transcription factors family, regulates the expression of genes required for cholesterol synthesis and uptake (35). Studies have shown that overexpression of SREBP-2 induced by HFD stimulation can dramatically increase genes expression involved in lipid synthesis and metabolism, which may be the cause of diet-induced lipid metabolism disorder (36). LDLR on the surface of hepatocytes is the mediator for uptaking LDL-C in the circulation, leading to decrease LDL-C level. PCSK9 is a serine protease and mainly synthesized in the liver, which is regulated by SREBP-2 (37). It is reported that PCSK9 can triggers LDLR intracellular degradation via attaching to LDLR surface and translocating to lysosomes, resulting in increase both circulating LDL-C and the risk of cardiovascular disease (37). In addition, HMGCR is the key enzyme regulated by the activity of SREBP-2 for cholesterol biosynthesis and an effective target of statins for HLP prevention and treatment (38). Thus, we determined whether EM attenuates lipid accumulation in HCD-induced HLP zebra sh larvae via modulating the AMPK/SREBP-2/PCSK9/LDLR signalling pathway.
In this study, our data (Fig. 7a) showed that EM signi cantly prevented the HCD-induced decrease of AMPK mRNA in zebra sh. Previous studies have shown that EM activated AMPK signaling pathway and promoted fatty acid oxidation, thereby exerting protective effect in NAFLD zebra sh fed with egg powder (15). Moreover, our results (Fig. 7a) indicated that the expression of SREBP-2 and its target genes involved in PCSK9 and HMGCR was signi cantly increased, while that of LDLR was decreased in zebra sh with HLP induced by HCD. However, EM reversed the increased expression of SREBP-2, HMGCR and PCSK9, but upregulated the expression of LDLR, which might be the reason of decrease of cholesterol endogenous synthesis and circulated LDL-C content. Reverse cholesterol transport (RCT) process is an effective method to reduce cholesterol in extrahepatic tissues and has been demonstrated to reduce HLP (39). Adenosine triphosphate binding cassette transporter A1 (ABCA1) and adenosine triphosphate binding cassette transporter G1 (ABCG1) both participate in regulating the secretion of cholesterol, which is bene cial to RCT (40). Our research found that EM effectively upregulated the expression of ABCA1 and ABCG1, and promoted the elimination of excess cholesterol.
Collectively, present study revealed that EM can improve obesity, reduce lipid deposition, alleviate in ammation and liver injury on HLP zebra sh larvae fed with HCD through regulating genes expression of lipid metabolism relevant factors and redistributing of lipid in the blood and liver.

Conclusion
In summary, EM can improved metabolic pro les, liver function and lipid accumulation caused by HCD. According to our work, the protective mechanism of EM against HLP was probably achieved by modulating the AMPK/SREBP2/PCSK9/LDLR signaling pathway (Fig. 7b). Therefore, EM could be a potential drug for the treatment of lipid metabolism related diseases.      Values are expressed as mean ± SD in each group. ##P < 0.01, ###P < 0.001 compared with control group; *P < 0.05, **P < 0.01, ***P < 0.001 compared with HCD group; n.s. indicates no signi cant.