Preparation and characterization of β-alanine–modified gadofullerene nanoparticles
The β-alanine–modified gadofullerene (GF-Ala) nanoparticles were prepared in the alkaline environment via nucleophilic attack of amino groups and hydroxyl ions (25) (Fig. 1B). After purification and freeze-drying, the structural compositions of GF-Ala were thoroughly characterized by Fourier transform infrared (FTIR) spectroscopy, elemental analysis (EA), and thermogravimetric analysis (TGA). First, the stretching vibration bands of N (O)─H (ca. 3400 to 3300 cm−1), C─H (ca. 2900 to 2800 cm−1), and C═O (1740 cm−1) in the FTIR could be ascribed to the hydroxyl and β-alanine groups of GF-Ala. The bands of C═C (1570 cm−1), C─C (1410 cm−1), and C─N (O) (ca. 1320 cm−1) could be assigned to [email protected]82 carbon cages and their bonded groups (Fig. 1C). Then, the weight loss before 115°C was assigned to 10.96 weight % (wt %) bound water in GF-Ala based on the TGA result (fig. S1A). As shown in the EA results, there were 53.99 wt % C, 3.42 wt % H, and 3.04 wt % N in GF-Ala, suggesting that each carbon cage was composed of ca. 5 β-alanine groups. Last, the average structure formula of GF-Ala could be described as [email protected]82(OH)~20(NHCH2CH2COOH)~5•13H2O (calculated values: 54.01 wt % C, 3.53 wt % H, 3.25 wt % N, and 31.92 wt % O). The size and zeta potential of GF-Ala were investigated by dynamic light scattering (DLS). Hydrodynamic diameters were 89.3 nm [polydispersity index (PDI), 0.227] in water as well as 86.6 nm (PDI: 0.307) in normal saline (NS) (Fig. 1D), and zeta potential (pH 7.0) was −44.0 ± 2.0 mV. In addition, GF-Ala was also highly stable without showing any aggregation in water, saline, and fetal bovine serum (FBS) even after centrifugation at 10,000 rpm for 10 min (Fig. 1E).
Hydroxyl radicals (•OH) are known as the most robust ROS induced by lipid accumulation in hepatic steatosis (26). As the carbon cage has an extended π-bond system with high electron affinity, GF-Ala could quench •OH via directly adding with •OH by ethylenic bonds or donating hydrogen atoms to form water. Here, we tested the ability of GF-Ala to eliminate •OH in vitro by electron spin resonance (ESR). The •OH were derived from H2O2 under ultraviolet (UV) light and captured by 5, 5-dimethyl-1-pyrroline N-oxide (DMPO). More than 90% of •OH were eliminated by 50 μM GF-Ala (Fig. 1F). Given both  fullerene and gadofullerene derivatives acting as excellent radical scavengers, we also prepared β-alanine–modified  fullerene, C60-Ala, via a similar method. Comparatively, GF-Ala exhibited a more excellent elimination of •OH than that of C60-Ala, which might be more carbon atoms and embedded gadolinium ion in GF-Ala (fig. S1B). All these results indicate that GF-Ala has narrow distribution, excellent stability, and superior antioxidant property in physiological media.
In vivo anti-hepatic steatosis treatment of GF-Ala
To investigate whether GF-Ala nanoparticles could treat hepatic steatosis in vivo, we established two animal models. One is caused by feeding with fructose in WT mice. Briefly, C57BL/6J mice (male, 6 weeks old; n = 12) were fed with drinking water containing 30% fructose and standard normal chow diet ad libitum. After 8 weeks, mice were divided into two groups randomly and treated with GF-Ala [6 μmol/kg, intraperitoneally (ip)] or NS for 15 days. Another six C57BL/6J mice (male, 6 weeks old) were fed with sterile water and a normal diet for 8 weeks and treated with NS for 15 days (fig. S2A). There was no significant difference in body weight among each group (fig. S2B). The liver-to-body ratio and oil red O staining show that excessive lipids in the mice fed with fructose are remarkably reduced after GF-Ala treatment (fig. S2, C and D).
Given that it takes a long time to obtain this model and there are large intragroup differences in mice, we chose another leptin-deficient mouse model, ob/ob obese (OB) mice, commonly used for hepatic steatosis study. First, we treated OB mice with GF-Ala (6 μmol/kg) for 10, 15, and 30 days (Fig. 2A). The liver of OB mice had obvious hepatomegaly and is pale and bloodless. We found that the liver’s hepatomegaly and abnormal appearances in OB mice were prominently improved after 10 days with GF-Ala administration (Fig. 2B). However, according to the hepatic hematoxylin and eosin (H&E) staining and oil red O staining, there were still a few lipid droplets in the liver after 10 days of treatment. When the period was prolonged to 15 days, we detected better anti-hepatic steatosis effects of GF-Ala. Pathology results also showed that the curative effects of hepatic steatosis in 15-day treatment are similar to those in 30-day treatment (fig. S2, E and F). Therefore, we chose 15 days in the following research, and three concentrations of GF-Ala were used: low dosage (L): 1.5 μmol/kg, medial dosage (M): 3 μmol/kg, and high dosage (H): 6 μmol/kg (Fig. 2C).
We recorded the body weights of both WT mice and OB mice every 3 days during the 15-day treatment (fig. S2G). The average weight of WT mice treated with saline was increased about 2 g, and the OB mice gained approximately 7 g at the end of the treatment. The body weights of OB mice went up slowly after GF-Ala treatment. Notably, the body weights of OB mice only increased ~4 g in the GF-Ala H group, in which the weight growth rate had fallen nearly by half compared with the OB mice treated by saline (Fig. 2D). Notably, the average feed consumption in both WT mice and OB mice declined after GF-Ala treatment with concentration dependence (fig. S2H). Besides, the total weights of primary adipose tissues (including subcutaneous, perinephric, and gonadal fat) and adipose tissue–to–body weight ratio had been significantly declined by GF-Ala treatment in a dose-dependent manner (Fig. 2, E and F).
Except for the reduction of body weight and adipose tissues, the liver-to-body weight ratio in OB mice decreased after GF-Ala treatment (Fig. 2G). Furthermore, the alanine transaminase (ALT) and aspartate transaminase (AST), two primary biomarkers of hepatic function, were returned to normal levels in medial and high dosage groups (Fig. 2, H and I). The hepatic pathology was used to precisely evaluate the anti-hepatic steatosis effects of GF-Ala in different dosages. H&E staining exhibited extensive hepatocellular vacuoles, lipid droplets, and hepatocyte damage in the liver of OB mice. Those typically pathological changes in hepatic steatosis were highly reversed by GF-Ala treatment (Fig. 2J). In oil red O staining, there was a ~90% reduction of hepatic neutral lipid content in OB GF-Ala H group compared with OB NS group (Fig. 2K). Also, the hepatic free fatty acid (FFA) and total TG were also reduced after the 15-day treatment (Fig. 2, L and M). Together, we strongly confirm that GF-Ala has a superior anti-hepatic steatosis effect on both fructose-induced mice and OB mice.
Because of the decline of weight gain after GF-Ala treatment, we further studied the influence of weight loss on hepatic steatosis using orlistat, an approved anti-obesity agent inhibiting lipid absorption in intestine. We treated OB mice with orlistat for 15 days by oral administration (50 mg/kg, every day). The body weight gain slowed down by orlistat (fig. S3A), but no improvement on hepatic steatosis was obtained in such a short period (fig. S3B).
Quantitative proteomics study
To gain insight into the protein interaction in GF-Ala treatment, we performed a tandem mass tag (TMT) based on the quantitative proteomics profiling technique. We detected 4233 proteins, among which 4222 proteins coexisted within four experiment groups, and 3945 proteins were identified and quantified in the Venn diagram (Fig. 3A). The WT NS, WT GF-Ala H, OB NS, and OB GF-Ala H groups were coupled pairwise to form four pairs, and the proteins with a fold change >1.2 and P < 0.05 were considered as differential proteins. There were 777 differential proteins in WT NS versus OB NS group, 314 differential proteins in OB NS versus OB GF-Ala H group, and 196 differential proteins in both two. Subsequently, we subjected these 196 differential proteins with three categories (cellular component, molecular functions, and biological process) based on the Gene Ontology (GO) annotation (Fig. 3, B to D). It was found that the differential proteins associated with ER and mitochondria might be the major executors, which mainly involve protein binding and lipid metabolism processes.
According to the pathogenesis of hepatic steatosis (27), we divided the differential proteins into three categories in heat map based on lipid homeostasis, including lipid biosynthesis, lipid catabolism, and lipid transport (Fig. 3E). We found that GF-Ala had positive improvements on these differential protein expressions in OB mice, all of which tended to be reversed to normal levels compared with the nonhepatic steatosis controls. Moreover, GF-Ala had little impact on these differential proteins in normal mice. With the assistance of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, we further illustrated the changes of key differential proteins in GF-Ala treatment.
(i) In the process of lipid biosynthesis, two vital differential proteins, including acetyl–coenzyme A (CoA) carboxylase 1 (ACACA) and fatty acid synthase (FASN) in fatty acid biosynthesis, were notably up-regulated in OB mice, which were markedly down-regulated by GF-Ala treatment. Other differential proteins related with lipogenesis, such as adenosine triphosphate (ATP)–citrate synthase (ACYL), acetyl-CoA synthetase, cytoplasmic (ACSS2), thyroid hormone-inducible hepatic protein (THRSP), and elongation of very long chain fatty acids protein 5 (ELOVL5), were also increased in OB mice and decreased by GF-Ala treatment (fig. S4).
(ii) For lipid catabolism, cytochrome P450 4A10 (CYP4A10), ATP-binding cassette subfamily member 2 (ABCD2), carnitine palmitoyltransferase IA (CPT1A), acyl-CoA dehydrogenase for medium-chain fatty acids (ACADM), and peroxisomal bifunctional enzyme (EHHADH) were all notably up-regulated in OB mice as well as down-regulated by GF-Ala treatment (fig. S5).
(iii) For lipid transport, NPC intracellular cholesterol transporter 2 (NPC2), prolow-density lipoprotein receptor–related protein 1 (LRP1), and ApoB100 were decreased in hepatic steatosis mice, and all of them were elevated after GF-Ala treatment. Perilipin 2 (Plin2), a protein associated with fat storage, was with a high expression in OB mice, which was distinctively reduced after GF-Ala treatment (fig. S6).
To highlight key proteins, STRING tool was used to detect the functional relations and networks between these proteins. Here, we obtained a network of above 37 differential proteins from the STRING database using the default setting (Fig. 3F). The gray lines between nodes represent functional associations between proteins. The darker the line is, the more interactions the proteins are. There are several highly interconnected nodes among the proteins of FASN, ACACA, ACADM, CYP4A10, EHHADH, Plin2, ApoB, LRP1, and NPC2. All these proteins and relevant proteins were verified by Western blot (WB) in the following research.
Accelerative lipid transport in GF-Ala hepatic steatosis treatment
We sought to ascertain differential proteins selected from the above analysis. First, the protein expressions of ACACA and FASN, related to fatty acid biosynthesis, exhibited no significant change before and after treatment in OB mice (fig. S7, A and B). CPT1A and ACADM were involved in the mitochondrial fatty acid β-oxidation pathway (28), and the protein expressions of both two had no visible change after the treatment in OB mice (fig. S7, C and D). Nevertheless, EHHADH, the protein of peroxisomal fatty acid β-oxidation pathway, was up-regulated in OB mice and significantly reduced by GF-Ala treatment (Fig. 4A). CYP4A10, participating in microsomal fatty acid ε-oxidation, was also increased in OB mice and decreased by GF-Ala treatment (Fig. 4B). All these changes indicate that GF-Ala effectively regulates the lipid catabolism by reducing fatty acid peroxisomal β-oxidation and microsomal ε-oxidation. However, these differential proteins have positive regulations in lipid catabolic process, and the decline of them after treatment suggests that they are subsequent consequences instead of driving factors during the treatment of hepatic steatosis.
For lipid transport, LRP1, NPC2, MTTP, and LPL had almost no change after GF-Ala treatment (fig. S7, E to H). However, we found that Plin2, involved in the formation of lipid droplets, was highly expressed in OB mice but observably reduced after GF-Ala treatment (Fig. 4C). It might be the consequence of the reduction of hepatic lipid droplets. Besides, ApoB100 and ApoB48 in liver were highly reduced in OB mice, but both of them were enhanced by GF-Ala treatment (Fig. 4D). It is known that ApoB100 takes part in removing hepatic TG from liver to serum, and conversely, ApoB48 is used to transport TG from the diet into the liver. Thus, our results reveal that GF-Ala boosts hepatic TG transport and maintains the hepatic lipid metabolic homeostasis. However, the mRNA expression of Apob in OB mice was lower than that in normal mice, which was not changed by GF-Ala treatment (Fig. 4E). It suggests that GF-Ala only regulates the protein expression of ApoB100 by inhibiting its posttranslational degradation instead of promoting its production at the source.
To uncover the above anti-hepatic steatosis mechanism in detail, we established a model of hepatocellular steatosis by incubating L02 hepatocytes with oleic acid (OA) (29). First, we tracked the subcellular localizations of GF-Ala by cryopreservation transmission electron microscope (cryo-TEM). GF-Ala nanoparticles (black arrows) are mainly distributed in endosomes (E) (fig. S8A). However, the membrane structure is missing in some of the endosomes (red arrows), indicating that GF-Ala could escape from endosomes to the cytoplasm. Some of GF-Ala nanoparticles are also distributed around ER. Then, to investigate the optimum concentration of OA, L02 cells were incubated with 0.5, 1, 1.5, and 2 mM OA for 24 hours, and the proportion of living cells was identified by measuring the Hoechst 33342 fluorescence intensity through microplate reader. It shows that the quantity of L02 cells has no change within 1.5 mM OA (fig. S8B). The relative lipid content in L02 cells was determined by Nile Red staining. We found that OA treatment significantly induced lipid accumulation in L02 cells, and the lipid content in L02 cells treated with 1.5 mM OA increased almost sixfold higher than that in normal L02 cells (fig. S8C). Our study reveals that GF-Ala reverses the lipid accumulation in L02 cells, and the lipid content is decreased ca. 31.3% by 0.2 mM GF-Ala (Fig. 5A). TG content in the extracellular medium was determined at different times. GF-Ala significantly increased TG content in the medium after 24 hours (Fig. 5B). It indicates that the decrease of intracellular lipid accumulation results in the increase of extracellular TG after GF-Ala treatment. In addition, we measured the protein expressions of ApoB100 in OA-induced L02 cells at different times. We found that OA could enhance intracellular ApoB100 quickly (6 hours), which was degraded over time (24 hours). GF-Ala could inhibit the degradation of ApoB100 in OA-induced L02 cells, consequently promoting TG transport (Fig. 5C). Considering that the degradation of ApoB100 is known to be closely associated with ROS and lipid peroxidation, we determined the amount of intracellular ROS and malondialdehyde (MDA) after GF-Ala treatment. The lipid accumulation in OA-induced L02 cells increases ROS, appearing as stronger fluorescent signals of 2′-7′-dichlorodihydrofluorescein (DCF) in flow cytometry. Notably, GF-Ala could reduce excessive ROS in L02 cells (Fig. 5D). The similar decline of DCF and Nile Red signals after GF-Ala treatment was also observed by confocal microscopy (Fig. 5D), which positively supported the above results. In addition, our data also show that the content of MDA, one of the typical biomarkers of lipid peroxidation, is increased in OA-induced L02 cells but observably decreased by GF-Ala (Fig. 5E). Together, we reveal that GF-Ala could effectively improve hepatocellular steatosis and inhibit the degradation of ApoB100 by reducing excessive ROS and lipid peroxidation in OA-induced L02 cells.
Improvements of lipid peroxidation and abnormal mitochondria by GF-Ala
The hepatic lipid accumulation induces overloaded lipid catabolism, causing lipid peroxidation and mitochondrial dysfunction, finally leading to further hepatocyte damages. We measured the levels of serum MDA and hepatic MDA in OB mice. Both of them were increased in OB NS group (serum: 5.62 ± 1.00 nmol/ml and liver: 3.55 ± 0.30 nmol/mgprot) and decreased after GF-Ala treatment in different levels (serum: 2.52 ± 0.88 nmol/ml and liver: 3.01 ± 0.24 nmol/mgprot) (Fig. 6, A and B), showing similar results to OA-induced L02 cells. We used cryo-TEM to observe the microstructure of hepatocellular mitochondria. The images displayed that hepatic mitochondria abnormally appeared with ruptured membranes, messy cristae, lost matrix, and internal vacuoles in the OB NS group (Fig. 6C). Inversely, seldom ruptured mitochondria were observed in OB GF-Ala group, and the structural integrity of mitochondria had been improved.
Except for the morphology change of mitochondria, more indexes of mitochondrial function were analyzed in OA-induced L02 cells. The stabilization of mitochondrial membrane potential (MMP) is the prerequisite for oxidative phosphorylation, ATP production, and other mitochondrial functions. The decline of MMP indicates the mitochondrial abnormality and early cellular apoptosis. We evaluated the MMP of L02 cells by JC-1, which exhibited higher green and lower red fluorescence intensity in decreased MMP. The OA model had a higher green/red ratio than control, and GF-Ala reversed this ratio (Fig. 6D), indicating that GF-Ala could inhibit the decline of MMP in OA-induced L02 cells. The components of NADH [reduced form of nicotinamide adenine dinucleotide (NAD+)] oxidative respiratory chain, including the activities of mitochondrial complexes I, III, and IV, were analyzed at the same time. The activities of complexes III (fig. S9A) and IV (fig. S9B) remain unchanged at normal levels in both OA model and GF-Ala treatment groups. However, OA model had higher complex I activity, which catalyzed NADH to NAD+ and generated ROS in hepatocytes. Notably, GF-Ala reinstated the complex I activity of OA-induced L02 cells (Fig. 6E). Then, we analyzed the content of NADH, which is produced in cellular respiration and participates in the respiratory chain. Both NADH and its oxidation state, NAD+, increased in the OA model and decreased by GF-Ala treatment (Fig. 6F). It indicates that excessive lipids induce hepatocellular mitochondria to produce more NADH and NAD+. However, GF-Ala could bring NADH content back to normal and prevent mitochondria from metabolic stress effectively. Together, we reveal that GF-Ala could improve mitochondrial abnormality in hepatocytes suffering from hepatic steatosis.
In vivo biodistribution and toxicity of GF-Ala
At last, we studied the biodistribution of GF-Ala (intraperitoneal) by labeling it with Cyanine5.5 (Cy5.5) fluorescent dye and tracked GF-Ala with an in vivo fluorescent imaging system (fig. S10A). It was observed that GF-Ala mainly distributed into the pancreas, liver, spleen, lung, and kidney. The clear majority of GF-Ala could be excreted from organs, except for the pancreas, 30 days after injection. After the treatment, the pathology results of main organs (heart, spleen, lung, kidney, and pancreas) show no palpable pathological lesions or changes in GF-Ala–treated mice (fig. S10B), suggesting the low toxicity of GF-Ala in vivo.
The defining feature of hepatic steatosis is the abnormal accumulation of hepatic TG, which induces to generate excessive ROS and lipid peroxidation. It will lead to the dysfunctions of hepatic lipogenesis, lipolysis, and lipid transport, aggravating hepatic lipid accumulation. Given the pathogenesis of hepatic steatosis, researchers explored numerous strategies for treatment. For example, Aramchol (the inhibitor of stearoyl CoA desaturase), ND-630, and NDI-010976 (the inhibitors of aceyl-CoA carboxylase) were tested in fatty liver patients to inhibit de novo lipogenesis and stimulate fatty acid oxidation (30, 31). At the same time, some other medications target peroxisome proliferator–activated receptors (PPARs) to regulate gluconeogenesis, β-oxidation, and the hormone fibroblast growth factor (FGF)–21 (7, 32). In addition, several antioxidants have been used for fatty liver treatment. Vitamin E, a fat-soluble antioxidant, has been applied in multiple clinical trials for hepatic steatosis therapy, but it needs a long continuous course and high dose to take effect (33).
Fullerene  and gadofullerene ([email protected]82) with conjugated carbon cage structure and unique properties have been widely applied in the biomedicine field. The previous studies had reported that both fullerene and gadofullerene derivatives have superior antioxidant properties (20, 34, 35). Recently, we found that gadofullerene nanoparticles could prominently reduce blood glucose by regulating hepatic insulin resistance and reversing pancreatic abnormalities for type 2 diabetes mellitus treatment (24). Because both diabetes and hepatic steatosis belong to metabolic disorders, here, we attempted to exploit gadofullerene nanoparticles for hepatic steatosis therapy. We used one kind of dietary mouse model and another genetic mouse model, which could reflect hepatic histopathology and pathophysiology of human hepatic steatosis with different features. The WT mice were fed with fructose for 8 weeks, showing the feature of hepatic steatosis with massive lipid accumulation observed by oil red O staining. Similarly, the OB mice, having a mutation in the leptin gene, could also develop into the hepatic steatosis. GF-Ala observably reversed hepatic steatosis in diet-induced mice and OB mice (Fig. 2 and fig. S2). The effects were strongly confirmed by the improvements of hepatomegaly, hepatic lipid accumulation, and aminotransferase abnormalities.
Inspired by their anti-hepatic steatosis, we performed a proteomic profiling study to identify the protein targets in GF-Ala treatment. We found that GF-Ala could up-regulate lipid transport and down-regulate hepatic lipid catabolism. Boosting hepatic lipid transport is known as an available strategy for anti-hepatic steatosis. Nevertheless, few studies have been reported to treat hepatic steatosis regarding this solution. Some researches focus far more on cholesterol transport, as cholesterol is closely associated with cardiovascular diseases (36, 37). The lipid or lipoid transports rely on kinds of apolipoprotein, such as apoA-II for cholesterol transport, ApoB48 for dietary TG transport, and ApoB100 for hepatic TG transport. In our study, the protein expression of ApoB100 was decreased in OB mice and up-regulated by GF-Ala (Fig. 4D). However, the mRNA expression of hepatic Apob had no change in GF-Ala treatment, suggesting that GF-Ala did not influence the source of ApoB100. There are three posttranslational degradations of ApoB100 protein: ER association degradation (ERAD), post-ER presecretory proteolysis (PERPP), and receptor-mediated degradation (12). The severe scarcity of lipids stimulates ERAD during the translocation of ApoB100 across the ER, but hepatic lipids are overmuch in hepatic steatosis. For receptor-mediated degradation, our result shows no difference in hepatic LDL receptors before and after GF-Ala treatment (fig. S7H). PERPP is a nonproteasomal pathway caused by diverse metabolic factors, including excessive intracellular ROS. We established a hepatocellular model of hepatic steatosis to investigate the degradation of ApoB100 during GF-Ala treatment. The data show that GF-Ala could decrease the excessive ROS resulting from lipid accumulation and inhibit the degradation of ApoB100 in hepatocytes, most possibly through the PERPP pathway.
Notably, the weight gain of OB mice is reduced by GF-Ala treatment (fig. S2G). Hepatic steatosis is closely related to obesity in most cases. To ascertain the effect of weight loss on anti-hepatic steatosis, we used orlistat, an approved weight loss drug, to treat hepatic steatosis in OB mice. The results reveal that only weight loss alone by orlistat treatment could not effectively improve the hepatic steatosis in 15 days (fig. S3). Another anti-hepatic steatosis treatment in fructose-induced mice also shows that GF-Ala could markedly improve hepatic steatosis without apparent weight loss (fig. S2, B to D). Thus, we consider that weight loss is not a major factor during GF-Ala treatment. We would continue to study the mechanism of food intake reduction and fates of TG after removing from the liver in the following research, which might expand the applications of gadofullerene nanomaterials in obesity or other metabolic diseases.
In addition, we demonstrate that GF-Ala could not only boost hepatic TG transport but also maintain the hepatic homeostasis. Specifically, the lipid peroxidation and mitochondrial abnormality in hepatic steatosis were improved by GF-Ala (Fig. 6). In hepatocytes, the lipid accumulation accelerates cellular respiration, which generates more NADH in hepatocytes. The activity of mitochondrial complex I is also promoted in NADH oxidative respiratory chain, generating more ROS in hepatocytes. Then, the excessive ROS lead to lipid peroxidation, and the mitochondrial structure and function are damaged gradually. Meanwhile, the lipid transport would be stimulated by ROS at first, but continual damages inhibit lipid transport finally. As a result, the disorders of hepatocellular lipid metabolism strengthen the lipid accumulation in hepatocytes. GF-Ala nanoparticles break this vicious circle, eliminate the excessive ROS, improve the mitochondrial abnormality, and normalize the lipid transport.
Except for therapeutic effect, toxicity is equally important to anti-hepatic steatosis agents. We find that GF-Ala could be excreted gradually 30 days after administration without pathological lesions in the main organs (fig. S10).
In conclusion, we establish an effective solution for reversing hepatic steatosis by boosting TG transport using GF-Ala nanoparticles. GF-Ala decreases the hepatic lipid accumulation in hepatic steatosis mice within 15 days. One of the most notable findings in our study is that GF-Ala chiefly accelerates the TG outflow from hepatocytes by inhibiting the degradation of ApoB100. We also observe that GF-Ala effectively improves the mitochondrial abnormality and maintains the hepatic lipid metabolic homeostasis. Furthermore, we certify that GF-Ala could be almost all excreted from the living body and cause no severe toxicity. Our findings provide a promising anti-hepatic steatosis mechanism to activate hepatic TG transport and restore lipid metabolic homeostasis by GF-Ala. It offers a new perspective for the treatment of hepatic steatosis and obesity-associated metabolic diseases using advanced nanomaterials.
MATERIALS AND METHODS
This study aimed to develop an anti-hepatic steatosis strategy based on fullerene nanomaterials and studied the potential therapeutic mechanism. For this, we performed GF-Ala, a superior antioxidant, in both diet-induced WT mice and obesity-associated transgenic ob/ob obese mice. The anti-hepatic steatosis effects were confirmed in several aspects, such as hepatic morphology, weight, pathology, and blood biochemistry. Then, we focused on the hepatic lipid homeostasis in treatment via proteomics and molecular biology. Given the improvement in lipid transport, a relevant cellular model was built and used for mechanism study between GF-Ala and ApoB degradation.
Solid C60 and [email protected]82 (99% purity) were purchased from Xiamen Funano Co. Ltd. (Xiamen, China). Anhydrous ethanol, methanol, NaOH, H2O2, HNO3, NaH2PO4, and Na2HPO4 were purchased from Beijing Chemical Works (Beijing, China). NaCl, β-alanine, OA, glutaraldehyde, osmium tetroxide, dithiothreitol, iodoacetamide, pentobarbital sodium, urea, acetonitrile, formic acid, dithiothreitol, iodoacetamide, tetraethylammonium bromide (TEAB), Cyanine5.5 (Cy5.5), and 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nile Red, Hoechst 33342, hematoxylin, eosin, oil red O, and optimal cutting temperature (OCT) compound were purchased from Solarbio (Beijing, China). All reagents and solvents were obtained commercially and used without further purification.
Preparation of β-alanine derivative gadofullerene
Synthesis of β-alanine derivative gadofullerene (GF-Ala) or fullerene (C60-Ala) referred to the previous study in our laboratory. In short, 50 mg of [email protected]82 and 3.6 g of β-alanine were dispersed in 14% NaOH aqueous solution during ultrasonic shaking. Then, the slurry was heated to 80°C and stirred for 2 hours. After cooling to room temperature, the solution was centrifuged (8000 rpm, 10 min) to remove the insoluble substance and dialyzed against ultrapure water (Millipore, Billerica, USA) to get rid of the unreacted materials. At last, GF-Ala aqueous solution was obtained after filtering through a 220-nm pore size membrane. The concentration of GF-Ala, determined as Gd element concentration, was measured with an inductively coupled plasma source mass spectrometer (ICP-MS; NexION 300X, PerkinElmer, USA) after enough nitration. Briefly, 0.1 ml of GF-Ala solution was added into 1 ml of HNO3 and heated to 100°C for 12 hours. Then, the solution was diluted and measured by the external standard method.
Characterizations of GF-Ala
The solid GF-Ala obtained by vacuum freeze-drying was characterized by TGA and EA. GF-Ala was heated from room temperature to 600°C (5°C/min) under the nitrogen flow by using a TGA spectrometer (Shimadzu DTG-60H, Japan). EA (Flash EA 1112, Thermo Fisher Scientific, USA) was performed normally to detect carbon, hydrogen, and nitrogen contents in GF-Ala. The FTIR spectrum of GF-Ala was performed on Nicolet iN10 MX (Thermo Fisher Scientific, USA). Hydrodynamic size, zeta potential, and PDI of GF-Ala were measured using NanoZS ZEN3600 (Malvern Instruments, Enigma Business Park, Britain).
In vitro hydroxyl radical’s elimination
Forty microliters of DMPO (100 mM) and 20 μl of H2O2 (100 mM) were mixed with 20 μl of ultrapure water, 20 μl of GF-Ala aqueous solution (100 μM), and 20 μl of GF-Ala aqueous solution (200 μM). The mixtures were irradiated with a 500-W UV lamp for 4 min first, and then the X-band electron paramagnetic resonance (EPR) spectra of DMPO-OH were recorded in the dark with an EPR spectrometer (FA-200, JEOL, Japan).
Animal models and treatments
Male obese mice (OB, 5 weeks old) and their C57BL/6J lean littermates (WT, 5 weeks old) were purchased from Beijing Huafukang Bioscience Co. Inc. (Beijing, China). All mice were reared in a temperature-controlled environment with a 12-hour light-dark cycle. Besides, all mice were fed on sterile water and standard normal chow diet purchased from Beijing Huafukang Bioscience Co. Inc. (Beijing, China). All the experimental protocols involving live animals were reviewed and approved by the Animal Ethics Committee of Institute of Processes, Chinese Academy of Sciences. Different concentrations of GF-Ala were regulated to appropriate osmotic pressure via sodium chloride before treatment. Three animal experiments were performed in this work.
First, C57BL/6J mice (male, 6 weeks old; n = 12) were fed with drinking water containing 30% fructose and standard normal chow diet ad libitum. After 8 weeks, mice were divided into two groups randomly and treated with GF-Ala (6 μmol/kg, ip) or NS for 15 days. Another six C57BL/6J mice (male, 6 weeks old) were fed with sterile water and a normal diet until 8 weeks old and treated with NS for 15 days. After treatment, the mice were anesthetized and sacrificed. The livers were harvested, photographed, and fixed with 4% paraformaldehyde for histopathological examination.
Then, we preliminarily studied the treatment time of GF-Ala for hepatic steatosis on OB mice. After 1 week of feeding, the OB mice were randomly divided into four groups (n = 3, each group), and one of them was treated with NS every day by intraperitoneal injection (5 ml/kg) for 30 days. GF-Ala (1.2 mM) was performed on the other three groups every day by intraperitoneal injection (5 ml/kg) for 10, 15, and 30 days. Three WT mice were treated with NS every day by intraperitoneal injection (5 ml/kg) for 30 days in the meantime. After treatment (on the 10th, 15th, and 30th day), the mice were anesthetized and sacrificed. The livers were harvested, photographed, and fixed with 4% paraformaldehyde for histopathological examination.
Another 24 OB mice and 12 WT mice were prepared for researches on the dosage and anti-hepatic steatosis mechanism of GF-Ala. After 1 week of feeding, these OB mice and WT mice were randomly divided into four groups and two groups (n = 6, each group). (i) Blank control group (WT NS): intraperitoneal injection of NS (5 ml/kg) every day; (ii) GF-Ala control group (WT GF-Ala H): intraperitoneal injection of GF-Ala (1.2 mM, 5 ml/kg) every day; (iii) model group (OB NS): intraperitoneal injection of NS (5 ml/kg) every day; (iv) GF-Ala L group (OB GF-Ala L): intraperitoneal injection of GF-Ala (0.3 mM, 5 ml/kg) every day; (v) GF-Ala M group (OB GF-Ala M): intraperitoneal injection of GF-Ala (0.6 mM, 5 ml/kg) every day; (vi) GF-Ala H group (OB GF-Ala H): intraperitoneal injection of GF-Ala (1.2 mM, 5 ml/kg) every day. The body weights of each group were recorded every 3 days. After 15 days of treatment, all mice were anesthetized, sacrificed, and harvested with blood, liver, adipose tissue, heart, spleen, lung, kidney, and pancreas. Blood was collected and centrifuged (1200 rpm, 15 min) to gather serum for the biochemical test. Liver and adipose tissue (perirenal, subcutaneous, and gonads) were weighted, snap-frozen, and stored at −80°C. Heart, liver, spleen, lung, kidney, and pancreas of each mouse were fixed with 4% paraformaldehyde for histopathological examination. Besides, portions of liver in WT NS, OB NS, and OB GF-Ala M groups were fixed in 2.5% glutaraldehyde for TEM observation.
H&E staining: Fixed tissues, including heart, liver, spleen, lung, kidney, and pancreas, were dehydrated through a serial alcohol gradient and embedded in paraffin wax blocks. Then, 5-μm-thick sections were sliced, and standard methods were followed, including dewaxing, hydration, staining, dehydration, and sealing.
Oil red O staining: Fixed hepatic tissues were dealt with 15 and 30% sucrose solution overnight successively. Then, we embedded hepatic tissues in OCT and sliced embedded tissues into 6- to 8-μm sections by freezing microtome. Afterward, standard methods were followed, including washing, staining, differentiation, and sealing.
Both H&E staining and oil red O staining were scanned with a scanner (NanoZoomer-SQ, HAMAMATSU, Japan). Average optical density analysis of oil red O staining was performed by Image-Pro Plus 6.0 (Media Cybernetics, USA).
In cells, MDA was extracted and measured using an MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. TG was measured by using TG assay kits (Nanjing Jiancheng Bioengineering Institute, China) as described.
In serum, alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG, and cholesterol were all detected with an automatic biochemical analyzer (TBA-2000FR, Toshiba, Japan) with corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, China). According to the manufacturer’s instructions, MDA was measured using an MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China).
In liver tissues, FFA, TG, total glutathione (tGSH), and MDA were measured by using corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, China) as described. All these biochemical analyses were performed following the manufacturer’s instructions.
Proteomics experiment and bioinformatics methods
The hepatic sample (in WT NS, OB NS, and OB GF-Ala H groups in the second animal experiment) was grinded by liquid nitrogen into cell powder and then transferred to a 5-ml centrifuge tube. After that, four volumes of lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) were added to the powder, followed by sonication three times on ice using a high-intensity ultrasonic processor (Scientz, China). The remaining debris was removed by centrifugation at 12,000g at 4°C for 10 min. Last, the supernatant was collected, and the protein concentration was determined with a BCA kit (Solarbio, China) according to the manufacturer’s instructions.
For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2 M. Last, trypsin (Corning, USA) was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4-hour digestion.
After trypsin digestion, the peptide was desalted by Strata X C18 Solid Phase Extraction (SPE) column (Phenomenex, USA) and vacuum-dried. The peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit (Thermo Fisher Scientific, USA). Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 hours at room temperature and pooled, desalted, and dried by vacuum centrifugation.
The tryptic peptides were fractionated into fractions by high-pH reversed-phase high-performance liquid chromatography (HPLC) using Agilent 300Extend C18 column [5 μm particles, 4.6 mm inside diameter (ID), and 250 mm length]. Briefly, peptides were first separated with a gradient of 8 to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into 18 fractions and dried by vacuum centrifugation.
The tryptic peptides were dissolved in 0.1% formic acid and directly loaded onto a homemade reversed-phase analytical column (15-cm length, 75 μm ID). The gradient was composed of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23 to 35% in 8 min and climbing to 80% in 3 min, then holding at 80% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to nanospray ion (NSI) source followed by tandem mass spectrometry (MS/MS) in Q Exactive Plus (Thermo Fisher Scientific, USA) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The mass/charge ratio (m/z) scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using normalized collision energy (NCE) setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan followed by 20 MS/MS scans with 15.0-s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set to 100 m/z.
The resulting MS/MS data were processed using MaxQuant search engine (v.126.96.36.199). Tandem mass spectra were searched against UniProt Muridae database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to two missing cleavages. The mass tolerance for precursor ions was set to 20 parts per million (ppm) in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set to 0.02 Da. Carbamidomethyl on cysteine was specified as fixed modification, and oxidation on Met was specified as variable modifications. False discovery rate (FDR) was adjusted to <1%, and the minimum score for peptides was set to >40.
GO annotation proteome was derived from the UniProt-GOA database (www.ebi.ac.uk/GOA/), first converting identified protein ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt-GOA database, the InterProScan soft would be used to annotated protein’s GO functional based on the protein sequence alignment method. Then, proteins were classified by GO annotation based on three categories: biological process, cellular component, and molecular function.
KEGG database was used to annotate the protein pathway, first using KEGG Automatic Annotation Server (KAAS) to annotated protein’s KEGG database description and then mapping the annotation result on the KEGG pathway database using KEGG online service tools KEGG mapper (www.genome.jp/kegg/tool/map_pathway2.html). STING v.10 online database platform (http://string-db.org/) was used for network interaction analysis of differential proteins.
Antibodies for WB were purchased from Abcam (Cambridge, UK), including anti-ApoB (ab20737), anti-CYP450A (ab140635), anti-EHHADH (ab93172), anti-Plin2 (ab52356), anti-ACACA (ab45174), anti-FASN (ab22759), anti-CPT1A (ab128568), anti-ACADM (ab110296), anti-LRP1 (ab92544), anti-NPC2 (ab114047), anti-MTTP (ab75316), anti-LPL (ab21356), and corresponding secondary antibodies. SDS, tris, glycine, and skim milk powder were purchased from BioDee (Beijing, China).
The proteins in livers were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) and quantified with a BCA protein assay kit (Beyotime, China) as described. Proteins were separated by electrophoresis. Briefly, polyacrylamide gel electrophoresis gels were prepared, and proteins were mixed with loading buffer with a 5-min heat at 95°C. Each sample was loaded with 20 μg of total protein per mini-gel well. The gels were submerged in running buffer [25 mM tris base, 190 mM glycine, 0.1% SDS (pH 8.3)]. Then, we ran the gel and stopped when the dye reached the bottom of the gel. Next, we transferred the proteins from gel to polyvinylidene fluoride membrane (Millipore, USA) by wet transfer. The transfer buffer is 48 mM tris, 39 mM glycine, 0.04% SDS, and 20% methanol. After transfer, we blocked the membrane with 5% nonfat milk for 1 hour at 4°C under agitation. Following the rinsing, we incubated with the primary antibodies at default dilution at 4°C overnight. On the next day, we rinsed the membrane and incubated with the secondary antibodies at default dilution at 4°C for 1 hour. After rinsing again, proteins were visualized by electrochemiluminescence (Millipore, USA) and imaging system (Tanon 4200, Tanon, China). The gray analysis was performed on Gel Image System version 4.00 (Tanon, China).
Quantitative real-time polymerase chain reaction measurements
Total RNA in livers was extracted with RNA isolator total RNA extraction reagent (Vazyme, Nanjing, China), and their complementary DNA (cDNA) was converted with HiScript Q RT SuperMix for polymerase chain reaction (PCR) (Vazyme, Nanjing, China). Then, the obtained cDNA was amplified with AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China), and relative expressions of Apob were quantified using a quantitative PCR (qPCR) detection system (CFX96, Bio-Rad, USA). Apob forward primer: 5′-TCCAGACAACCTCTTCCTAAAGAC-3′; Apob reverse primer: 5′-GGATGTCAATGTTTATTTTGTTCCT-3′.
Cell culture and OA-induced cellular model treatment
L02 cells were purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). They were cultured with RPMI 1640 supplemented with 10% (v/v) FBS, 1% (v/v) penicillin (100 μg/ml), and 1% (v/v) streptomycin (100 μg/ml) in a 5% CO2, 37°C incubator.
The OA stock solution was prepared by dissolving 0.4 mmol of OA into 40 ml of phosphate-buffered saline (PBS) solution containing 5% (w/v) fatty acid–free bovine serum albumin (BSA) (Equitech-Bio Inc., USA) and stored at −20°C. The culture medium was used to dilute OA stock solution to a certain concentration, and another 5% (w/v) BSA solution without OA was diluted in the same multiples as control.
L02 cells in 96-well plate were incubated with 0, 0.5, 1.0, 1.5, and 2.0 mM OA for 24 hours, respectively. Then, the media were removed, washed twice with PBS, and stained with Nile Red (excitation wavelength: 488 nm; emission wavelength: 550 nm) and Hoechst 33342 (excitation wavelength: 350 nm; emission wavelength: 461 nm) for 30 min. After that, the media were washed twice and measured with Infinite M1000 Pro (Tecan, Switzerland). The relative lipid content of each group was equal to the normalized ratio of fluorescence intensity between Nile Red and Hoechst. GF-Ala (0, 0.1, 0.2, and 0.4 mM) was coincubated with 1.5 mM OA–induced L02 cells for 24 hours, and lipid contents were measured using the same method.
In vivo ROS elimination
L02 cells were incubated with 1.5 mM OA, 0.2 mM GF-Ala, or together for 24 hours. Then, cells were collected, washed with PBS, and stained with Nile Red (excitation wavelength: 488 nm; emission wavelength: 550 nm) and DCF diacetate (DCF-DA) (excitation wavelength: 488 nm; emission wavelength: 525 nm) for 30 min. After that, the cells were washed twice and measured with a flow cytometer (Attune NxT, Invitrogen, USA). Single-dye samples were prepared for compensation regulation. The same samples were also prepared for imaging with a confocal laser scanning microscope (FV1000-IX81, Olympus, Japan).
Mitochondrial function study in cell
L02 cells were incubated with 1.5 mM OA, 0.2 mM GF-Ala, or together for 24 hours. Then, the media were removed, washed twice with PBS, and orderly stained with Hoechst 33342 (excitation wavelength: 350 nm; emission wavelength: 461 nm) and JC-1 (excitation wavelength: 515 nm, emission wavelength: 529 nm; excitation wavelength: 585 nm, emission wavelength: 590 nm) (Beyotime, China). Then, samples are imaged with a confocal laser scanning microscope (FV1000-IX81, Olympus, Japan). The fluorescence intensity analysis was performed by Image-Pro Plus 6.0 (Media Cybernetics, USA).
The NAD+/NADH Kit was purchased from Beyotime (Shanghai, China), and the activities of mitochondrial complex I, III, and IV kits were purchased from Solarbio (Beijing, China). L02 cells were incubated with 1.5 mM OA, 0.2 mM GF-Ala, or together for 24 hours. Then, cells were collected and washed with PBS, and the above analysis was performed following the manufacturer’s instructions.
TG transport study in cell
L02 cells were treated with 1.5 mM OA for 6, 12, or 24 hours with or without 0.2 mM GF-Ala. Then, the media were removed and washed twice with PBS, and a normal medium was added to each group. After 2 hours, the media were collected for the TG test, and cells were incubated with Hoechst 33342 to quantify the relative cell numbers.
ApoB100 degradation study in cell
L02 cells were treated with 1.5 mM OA for 6, 12, or 24 hours with or without 0.2 mM GF-Ala. Then, the media were removed and washed twice with PBS. The proteins in cells were extracted with RIPA lysis buffer (Beyotime, China) and quantified with a BCA protein assay kit (Beyotime, China) as described. Then, the expression of ApoB100 protein was analyzed by WB.
In vivo fluorescence imaging
GF-Ala was labeled with Cy5.5 fluorescent dye by condensation reaction under room temperature and stirring. After purification and quantitation, WT mice (C57BL/6J, n = 4) were injected with GF-Ala–Cy5.5 (1.2 mM, 5 ml/kg, ip) once. Mice were anesthetized, sacrificed, and harvested with heart, liver, spleen, lung, kidney, pancreas, and intestines on the 1st, 5th, 12th, and 30th day. Then, an in vivo fluorescent imaging system (Spectrum CT, PerkinElmer, USA) was used to study the biodistribution of GF-Ala–Cy5.5 (excitation wavelength: 675 nm; emission wavelength: 720 nm). Blank control was also performed in the meantime.
TEM of liver tissues and L02 cells
L02 cells were incubated with 0.4 mM GF-Ala for 3 hours. Then, we collected the cells and fixed them in 2.5% glutaraldehyde. Both cells and liver tissues fixed in 2.5% glutaraldehyde were steeped in 1% osmium tetroxide for 2 hours. Then, we used a serial alcohol gradient with increasing concentrations to dehydrate samples. After that, samples were embedded in Spi-pon812 resin, sectioned into approximately 70-nm slices, and then stained by uranyl acetate and lead citrate. Last, we observed these slices by TEM (Tecnai Spirit 120 kV, FEI, USA).
Data were provided as means ± SD. Comparisons between two groups or several groups were analyzed using unpaired Student’s t test or one-way analysis of variance (ANOVA) by using IBM SPSS Statistics 19 (IBM, USA). *P < 0.05, **P < 0.01, and ***P < 0.001 were considered the statistical significance of differences.
Acknowledgments: We thank the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Science for our cryo-TEM work, and L. Wang and C. Peng for their help of making TEM sample and taking TEM images. Funding: This work is supported by the National Major Scientific Instruments and Equipments Development Project (ZDYZ2015-2) and the Key Research Program of the Chinese Academy of Sciences (QYZDJ-SSW-SLH025). Author contributions: C.Z., M.Z., C.W., and C.B. designed research. C.Z., X.L., T.Y., and J. Li synthesized and characterized the materials. C.Z., X.L., J. Liu, W.J., S.L., L.L., Z.S., Z.Z., and X.W. performed the animal experiments. M.Y., C.Z., and M.Z. performed proteomics analyses. C.Z., M.Z., and X.Z. performed the cell experiments. C.Z., M.Z., C.W., and C.B. prepared the paper. All authors have read and approved the paper. Competing interests: C.B., X.L., M.Z., C.Z., and C.W. are authors on a patent application related to this work filed by Institute of Chemistry, Chinese Academy of Sciences (note: the patent had been transferred to Beijing Funakang Biotechnology Co. Ltd.) (no. CN201710560609.8, filed on 11 July 2017). The authors declare no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.