Alleviation Effects of Microbial Metabolites from Resveratrol on Non-Alcoholic Fatty Liver Disease

Resveratrol (RSV), a polyphenolic stilbene, has been widely studied for its protective effects against non-alcoholic fatty liver disease (NAFLD) by modulating intestinal microbiota. The microbial metabolites after RSV supplement would contribute to the bioeffects of RSV, while their impacts on NAFLD were unclear. Therefore, this study aimed to investigate the beneficial effects of the main microbial metabolites from RSV on lipid metabolism by combining in vitro and in vivo models. The mice were fed a high-fat diet and injected with RSV, 3-hydroxyphenyl propionic acid (3-HPP), and 4-HPP for 13 weeks (n = 6). Body weight, serum parameters, histological analysis, and gene expression involved in lipid metabolism were quantified. Our results suggested that 100 μM of 3-HPP and 4-HPP inhibited lipid accumulation more significantly than parent RSV in an oleic acid-induced HepG2 cell line. Furthermore, 3-HPP, 4-HPP, and RSV effectively reduced liver weight and body weight, improved hepatic steatosis, and alleviated systemic inflammation in NAFLD mice. In addition, the results of quantitative real-time PCR showed that 3-HPP and 4-HPP altered the expression of cholesterol influx and efflux genes to a stronger extent than RSV. These results indicate that 3-HPP and 4-HPP are effective in regulating hepatic lipid metabolism.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease affecting 25% of adults and 3-10% of children globally [1,2]. NAFLD is manifested by hepatic steatosis, inflammation, gut microbiota dysbiosis, and insulin resistance without an alcoholic abuse history and can even progress to steatohepatitis, fibrosis, and hepatocellular carcinoma [3]. The imbalance between neutral lipid acquisition (biosynthesis and import) and neutral lipid disposal (conversion, degradation, and export) plays a vital role in hepatic steatosis [4]. Moreover, NAFLD is strongly associated with multiple epidemics [5][6][7], such as obesity and cardiovascular disease, which pose a heavy economic burden on patients [8]. Until now, there is no drug for treating NAFLD clinically.
Therefore, this work aimed to assess the efficacy of microbial phenolic acids after RSV supplementation on NAFLD using an oleic acid (OA)-induced cell model and an HFD-induced mice model. Our findings suggest that 3-HPP and 4-HPP can attenuate hepatic steatosis and systemic inflammation, and their effects were the same as those of RSV. Interestingly, 3-HPP and 4-HPP mainly influenced cholesterol transport, while RSV primarily affected FA oxidation and transport.

Cell Culture
The human hepatocellular carcinoma cell line HepG2 was gifted by Professor Hongbo Hu (China Agricultural University, Beijing, China). The cells were cultured in DMEM containing a 1% supplement of antibiotic solution (10,000 IU/mL penicillin and 10 mg/mL streptomycin) and 10% fetal bovine serum in an incubator with constant H 2 O vapor pressure (5% carbon dioxide, 37 • C). When the cell confluence approached 70%, the cells could be passaged. Cultures between the 4th and 10th passages were used for different experiments.
OA mother solution (100 mM) was prepared by dissolving sodium salt of OA powder in double distilled water at 70-75 • C. It was then combined with 15% (w/v) BSA solution to obtain the OA stock (10 mM/10% BSA). The stock was filtered and diluted with DMEM to obtain the OA working solution (1 mM OA/1% BSA). The NAFLD cell model was induced using the working solution, which was treated with HepG2 cells for 24 h. As a control, 1% BSA solution was used to treat cell cultures. HepG2 cells were then treated with RSV and phenolic acids and the OA working solution for 24 h.

CCK8 Assay
HepG2 cells were seeded in 96-well plates with a proper density (100,000 cells/mL). After 12-14 h, the cells were treated with various concentrations (25-200 µM) of RSV and phenolic acids in the absence and presence of the OA working solution for 24 h. The cells were then treated with 10% CCK8 reagent solution for 1 h in a dark environment. Following this, the absorbance was measured at 450 nm wavelength.

Measurement Triglyceride (TG) Level of HepG2 Cells
HepG2 cells were seeded in 60 mm dishes. The cells were treated with 1% BSA solutions (C group), the OA working solution (OA group), the OA working solution with RSV (RSV group), and the OA working solution with phenolic acids (phenolic acids group) for 24 h. After these treatments, the cells were collected and then crushed with PBS by using an ultrasonic cell crusher (Scientz, Ningbo, China). Then, the protein concentration of the crushed liquid was measured with the BCA Assay Kit to normalize the number of cells and the TG level in the cells was measured using the TG Assay Kit (Jiancheng, Nanjing, China).

ORO Staining of HepG2 Cells
HepG2 cells were seeded in 35 mm dishes with slides. The cells were treated with 1% BSA solutions (C group), the OA working solution (OA group), the OA working solution with RSV (RSV group), and the OA working solution with phenolic acids (phenolic acids group) for 24 h. After these treatments, the cells were washed and fixed for 25-40 min. Following this, these were washed and stained with 60% (v/v) ORO solution in the dark for 1 h. Lastly, the cells were sealed with pure water to observe and photograph under an inverted microscope (Nikon, Tokyo, Japan) at 400× magnification. The results of ORO staining were semi-quantified using Image-Pro Plus 6.0.

Feeding and Grouping of Mice
The study protocol was approved by the China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethics Committee (Beijing, China) (No. AW70702202-4-1). A total of 30 4-week-old male C57BL/6J mice (16-18 g) were bought from Beijing Vital River Laboratory Animal Technology Co Ltd., China. After 7 days of adaptive feeding in a barrier environment (10-14 h light/dark cycle, 22 ± 2 • C, 55 ± 5% humidity), mice were kept in cages (three mice per cage) and classified into 5 groups with 6 mice in each group: the normal diet (ND), HFD, RSV, 3-HPP, and 4-HPP group.
For 13 consecutive weeks, the ND group was supported with a control diet (Research Diets, New Brunswick, NJ, USA; D12450J) and the other groups were fed HFD (Research Diets, New Brunswick, NJ, USA; D12492). The macronutrient composition of the two diets is shown in the Supplementary Materials, Table S2. For intraperitoneal injection (i.p.), 3-HPP, 4-HPP, and RSV were dissolved in PEG400 and then diluted with saline to obtain 20% (v/v) PEG solutions. The mice in the RSV, 3-HPP, and 4-HPP groups were intraperitoneally injected with the PEG solutions at a dose of 24 mg/kg BW twice a week. The mice of the ND and HFD groups were intraperitoneally injected with 20% PEG with saline.
On the 89th d, the mice were fasted for 16 h, weighed, and sacrificed. Following this, we collected their blood, white adipose tissue (epididymal fat, inguinal fat, and perirenal fat), and liver. The serum samples were acquired by centrifuging blood for 25 min at 3300 rpm at 4 • C. After weighing, a piece of epididymal fat and a piece of liver tissue that were approximately 1 cm 3 in size were fixed with 4% paraformaldehyde at a ratio of 10:1 for 24 h. The remaining samples were loaded into cryovials, placed in dry ice, and subsequently stored at −80 • C.

Intraperitoneal Glucose Tolerance Test (IPGTT)
On the 82nd d, the mice were fasted for 16 h. Following this, the mice were intraperitoneally injected with 20% (w/v) glucose solution (2.0 g/kg BW). Blood samples were collected from the tail vein at several time points, and blood glucose levels were measured with glucometers (Active, Roche, Switzerland).

Histopathological Examination
Fixed epididymal fat and liver were embedded with optimal cutting temperature compound and quickly frozen. When the specimen became white and hard, it was sectioned (8-10 µm) and dyed with hematoxylin and eosin (H&E) or ORO and hematoxylin. Staining results were visualized under an inverted microscope at 200× magnification and photographed using a digital camera. The results of H&E staining of liver tissues were scored on the basis of the NAFLD Activity Score (NAS) [43]. Histological features were scored on a scale of 0-3 for steatosis, lobular inflammation, and ballooning. NAFLD was considered to be present when the total score was above 4 and absent when the total score was less than 3. The graphs of ORO staining of liver tissues and H&E staining of epididymal fat samples were semi-quantified using Image-Pro Plus 6.0 and Image J.

RNA Isolation and qPCR Analysis
Liver samples were pre-treated by liquid nitrogen milling and then total RNA was isolated. The concentration and purity of RNA were then assessed by detecting OD230, OD260, and OD280 using NanoDrop 2000 (Thermo Fisher, Darmstadt, Germany). High-purity RNA samples were converted into cDNA. Quantitative real-time polymerase chain reaction (qPCR) analysis was performed using the BioRad CFX96 System. The primer sequences (Tsingke Biotechnology, Beijing, China) are listed in Table 1 and their amplification efficiency is shown in the Supplementary Materials, Table S1. Finally, the expression of target genes was standardized to the gapdh expression and estimated with the 2 −∆∆Ct method. Table 1. Primers of quantitative real-time polymerase chain reaction (qPCR).

Statistical Analysis
Data are expressed as the means ± standard errors (SEMs). SPSS 20.0 was selected to compare statistically and GraphPad Prism 8 was used to show the results. All experiments were repeated 3 times in this work. One-way analysis of variance (ANOVA) was used to assess the significant difference among the 5 groups. p < 0.05 was considered markedly different in accordance with Duncan's test.

Phenolic Acids Reduced Lipid Contents in OA-Induced NAFLD Cell Model
The molecular structures of phenolic acids and RSV are shown in Figure 1A; their effects on the accumulation of hepatic lipids were assessed. HepG2 cells were incubated with increasing concentrations of RSV and phenolic acids (25-200 µM) with and without OA for 24 h. Following this, cell viability was assessed. RSV treatment caused no discernible toxicity in cells even at 175 µM (Supplementary Materials, Figure S1). The effects of RSV and phenolic acids on lipid accumulation induced by OA were assessed based on TG levels and ORO staining results. Treatment with RSV, 3-HBA, 3-HPP, and 4-HPP at 100 µM reduced the TG levels by 8.55%, 6.94%, 16.87%, and 18.52%, respectively ( Figure 1C,D); this was accompanied by a decrease in lipid droplets ( Figure 1B,E). Moreover, 3-HPP and 4-HPP were more efficacious than RSV in inhibiting the accumulation of lipids. Therefore, the effect of 3-HPP, 4-HPP, and RSV on NAFLD was further assessed using an in vivo model.

3-HPP and 4-HPP Had Little Influence on Lipid Anabolism in Adipocytes of HFD Mice
Five-week-old C57BL/6J mice were supported with an ND or HFD (Figure 2A). The mice were intraperitoneally injected with vehicle, 3-HPP, 4-HPP, or RSV. During this 13week period, HFD significantly induced body weight gain in the mice, while 3-HPP, 4-HPP, and RSV reduced it without any change in the energy intake ( Figure 2B,G,H). HFD

3-HPP and 4-HPP Had Little Influence on Lipid Anabolism in Adipocytes of HFD Mice
Five-week-old C57BL/6J mice were supported with an ND or HFD (Figure 2A). The mice were intraperitoneally injected with vehicle, 3-HPP, 4-HPP, or RSV. During this 13week period, HFD significantly induced body weight gain in the mice, while 3-HPP, 4-HPP, and RSV reduced it without any change in the energy intake ( Figure 2B,G,H). HFD mice had more white fat and a higher white fat index. Compared with RSV supplementation, 3-HPP and 4-HPP supplementation failed to reduce white fat weight and white fat index; this result was consistent with that of histological analysis of epididymal fat by H&E staining (Figure 2C-F). These results suggest that 3-HPP and 4-HPP were unable to reduce lipid anabolism in white adipose tissue.

3-HPP and 4-HPP Alleviated Liver Damage and Hepatic Steatosis in HFD Mice
After 13 weeks, compared with ND, HFD significantly increased liver weight and liver index; however, 3-HPP, 4-HPP, and RSV supplementation reduced liver weight and liver index ( Figure 3A,B). Then, serum and hepatic levels of parameters indicative of lipid accumulation and liver injury were assessed. Compared with HFD, supplementation with 3-HPP, 4-HPP, and RSV significantly reduced hepatic TC and TG levels and ALT, AST, TG, TC, and LDL-c contents in serum ( Figure 3G-N). Moreover, histological analysis revealed that HFD induced histological features of NAFLD, such as lipid droplet accumulation, lipid vacuole formation, ballooning, and lobular inflammation. Interestingly, 3-HPP, 4-HPP, and RSV supplementation markedly ameliorated these histological features to a similar level to ND mice ( Figure 3C-F).

3-HPP and 4-HPP Alleviated Liver Damage and Hepatic Steatosis in HFD Mice
After 13 weeks, compared with ND, HFD significantly increased liver weight and liver index; however, 3-HPP, 4-HPP, and RSV supplementation reduced liver weight and liver index ( Figure 3A,B). Then, serum and hepatic levels of parameters indicative of lipid accumulation and liver injury were assessed. Compared with HFD, supplementation with 3-HPP, 4-HPP, and RSV significantly reduced hepatic TC and TG levels and ALT, AST, TG, TC, and LDL-c contents in serum ( Figure 3G-N). Moreover, histological analysis revealed that HFD induced histological features of NAFLD, such as lipid droplet accumulation, lipid vacuole formation, ballooning, and lobular inflammation. Interestingly, 3-HPP, 4-HPP, and RSV supplementation markedly ameliorated these histological features to a similar level to ND mice ( Figure 3C-F).

3-HPP and 4-HPP Improved Systemic Inflammation and Insulin Resistance in HFD Mice
Glucose homeostasis and inflammation have been reported to induce NAFLD [44]. Compared with ND mice, HFD mice exhibited increased blood glucose levels, HOMA-IR index, and insulin levels after fasting. The results revealed that 3-HPP, 4-HPP, and RSV supplementation ameliorated HOMA-IR index and insulin levels ( Figure 4C-E). However, areas under the blood glucose curve after a glucose bolus in the 3-HPP and 4-HPP groups were parallel to that in the HFD group, indicating that 3-HPP and 4-HPP were unable to improve glucose tolerance ( Figure 4A,B). Furthermore, the levels of proinflammatory cytokines (TNF-α, IL-8, and IL-6) were markedly higher in mice serum

3-HPP and 4-HPP Improved Systemic Inflammation and Insulin Resistance in HFD Mice
Glucose homeostasis and inflammation have been reported to induce NAFLD [44]. Compared with ND mice, HFD mice exhibited increased blood glucose levels, HOMA-IR index, and insulin levels after fasting. The results revealed that 3-HPP, 4-HPP, and RSV supplementation ameliorated HOMA-IR index and insulin levels ( Figure 4C-E). However, areas under the blood glucose curve after a glucose bolus in the 3-HPP and 4-HPP groups were parallel to that in the HFD group, indicating that 3-HPP and 4-HPP were unable to improve glucose tolerance ( Figure 4A,B). Furthermore, the levels of proinflammatory cytokines (TNF-α, IL-8, and IL-6) were markedly higher in mice serum of the HFD group than the ND group. 3-HPP, 4-HPP, and RSV supplementation reduced the levels of proinflammatory cytokines, suggesting that RSV, 3-HPP, and 4-HPP can effectively ameliorate systemic inflammation ( Figure 4F-H).

3-HPP and 4-HPP Regulated the Gene Expression of Lipid Metabolism in HFD Mice
The expression levels of genes in β oxidation of FA, FA transport, and cholesterol transport were assessed. In Figure 5B, the HFD group exhibited obviously upregulated mRNA expression of genes in FA transport (cd36, fabp1, and slc27a5). Interestingly, only RSV supplementation downregulated the expression of cd36, illustrating that 3-HPP and 4-HPP had little influence on FA transport. Moreover, compared with ND, long-term HFD reduced the gene expressions of FA β oxidation (acadm, cpt1a, and pdk4). RSV, 3-HPP, and 4-HPP upregulated the expression of acadm, while only RSV upregulated the expression of cpt1a ( Figure 5A). The expression levels of abca1, abcg5, and abcg8, markers of cholesterol export, were downregulated in HFD mice. Whereas, the gene expression of cholesterol export, such as ldlr and scarb1, was not significantly influenced by HFD. RSV supplementation upregulated the expression of abca1 and abcg8, 3-HPP supplementation influenced the expression of abcg8 and scarb1, and 4-HPP administration upregulated the

3-HPP and 4-HPP Regulated the Gene Expression of Lipid Metabolism in HFD Mice
The expression levels of genes in β oxidation of FA, FA transport, and cholesterol transport were assessed. In Figure 5B, the HFD group exhibited obviously upregulated mRNA expression of genes in FA transport (cd36, fabp1, and slc27a5). Interestingly, only RSV supplementation downregulated the expression of cd36, illustrating that 3-HPP and 4-HPP had little influence on FA transport. Moreover, compared with ND, long-term HFD reduced the gene expressions of FA β oxidation (acadm, cpt1a, and pdk4). RSV, 3-HPP, and 4-HPP upregulated the expression of acadm, while only RSV upregulated the expression of cpt1a ( Figure 5A). The expression levels of abca1, abcg5, and abcg8, markers of cholesterol export, were downregulated in HFD mice. Whereas, the gene expression of cholesterol export, such as ldlr and scarb1, was not significantly influenced by HFD. RSV supplementation upregulated the expression of abca1 and abcg8, 3-HPP supplementation influenced the expression of abcg8 and scarb1, and 4-HPP administration upregulated the expression of abca1, abcg5, abcg8, and ldlr ( Figure 5C). These results suggest that 4-HPP had the maximum influence on cholesterol export and import in our study.  abca1, abcg5, abcg8, and ldlr (Figure 5C). These results suggest that 4-HPP had the maximum influence on cholesterol export and import in our study.

Discussion
Increasing evidence has shown that RSV treatment has protective effects on NAFLD by modulating gut microbiota [45]. Intriguingly, RSV treatment influenced gut microbiota structure and stimulated gut microbiota to produce phenolic acids [26]. These small metabolites can enter systemic circulation and influence host health [27,36,46]. Hence, we assessed whether the microbiota-derived phenolic acids of RSV could alleviate NAFLD. In this study, we assessed the effects of 3-HPP, 4-HPP, and RSV by combining in vitro and in vivo models to understand their bioactivity and molecular mechanism.
In the OA-induced HepG2 cell line, 3-HPP and 4-HPP were more effective in reducing lipid accumulation than RSV. By contrast, 3-HBA and 4-HBA did not affect lipid contents and 4-HPA had a weak lipid-lowering activity in HepG2 cells. This result suggests that the molecular structure of metabolites might be related to their effects on lipid accumulation and for the same benzene ring substitution, the length of the carboxyl chain had greater impacts on lipid-lowering activity than the position of the hydroxyl group. As a previous study indicated, the chemical structure of phenolic acids influences their antimicrobial potential [47].

Discussion
Increasing evidence has shown that RSV treatment has protective effects on NAFLD by modulating gut microbiota [45]. Intriguingly, RSV treatment influenced gut microbiota structure and stimulated gut microbiota to produce phenolic acids [26]. These small metabolites can enter systemic circulation and influence host health [27,36,46]. Hence, we assessed whether the microbiota-derived phenolic acids of RSV could alleviate NAFLD. In this study, we assessed the effects of 3-HPP, 4-HPP, and RSV by combining in vitro and in vivo models to understand their bioactivity and molecular mechanism.
In the OA-induced HepG2 cell line, 3-HPP and 4-HPP were more effective in reducing lipid accumulation than RSV. By contrast, 3-HBA and 4-HBA did not affect lipid contents and 4-HPA had a weak lipid-lowering activity in HepG2 cells. This result suggests that the molecular structure of metabolites might be related to their effects on lipid accumulation and for the same benzene ring substitution, the length of the carboxyl chain had greater impacts on lipid-lowering activity than the position of the hydroxyl group. As a previous study indicated, the chemical structure of phenolic acids influences their antimicrobial potential [47].
In NAFLD mice, intraperitoneal supplementation with 3-HPP and 4-HPP can alleviate hepatic steatosis, as indicated by liver weight, lipid contents in the liver and serum, and NAS. Surprisingly, the effects of 3-HPP and 4-HPP were the same as those of RSV. While DHR and LUN had little impact on body weight, adipose tissue mass and blood parameters in HFD-fed mice [25]. Our findings elucidated a distinct modulating effect of 3-HPP and 4-HPP on lipid accumulation, which was of significance for treating the problem of beneficial bioactivity and low bioavailability of RSV.
The liver is an essential organ for lipid metabolism. NAFLD primarily occurs as a result of gradually increased lipid accumulation. TG and TC are the major neutral lipids in the liver synthesized by FA, and hepatic steatosis results from an influx and outflux imbalance among these substances in hepatocytes [4]. 3-HPP, 4-HPP, and RSV supplementation significantly lowered lipid levels in the liver and serum, suggesting that RSV and hydroxyphenyl propionic acids can alleviate hepatic steatosis by affecting the acquisition and disposal of major hepatic lipids. The expression levels of FA transport, FA oxidation, and TC transport genes were markedly affected by 3-HPP, 4-HPP, and RSV supplementation. FA transport mainly relies on transport proteins, such as CD36, hepatic FA-binding protein 1 (FABP1), and FATP5 [48]. Interestingly, RSV reduced the upregulation of cd36 mRNA levels, while 3-HPP and 4-HPP did not influence the expression levels of marker genes of FA transport. Therefore, RSV had the ability to regulate FA transport, which was consistent with the effect of RSV supplementation with oral gavage [13]. CPT1a, medium-chain aryl-CoA dehydrogenase (MCAD), and pyruvate dehydrogenase kinase 4 (PDK4) are key enzymes that are directly involved in FA β oxidation and are regulated by peroxisome proliferator-activated receptor γ (PPARγ) [49]. Compared with HFD, 3-HPP, 4-HPP, and RSV supplementation upregulated acadm mRNA levels and downregulated pparg mRNA levels. Moreover, RSV supplementation stimulated the expression of cpt1a, which was inhibited by HFD; this finding is consistent with previous findings [50]. Scavenger receptor class B type I (SR-BI) and low-density lipoprotein receptor (LDLR) are key mediators of TC absorption from blood and ATP-binding cassette subfamily A member 1 (ABCA1), ABCG5, and ABCG8 are mediators of TC efflux [51]. We found that RSV supplementation upregulated the expression of abca1 and abcg8, 3-HPP supplementation upregulated the expression of abcg8 and downregulated the expression of scarb1, and 4-HPP supplementation increased the expression of abca1, abcg5, abcg8, and ldlr. This finding indicates that the bioeffects of 3-HPP and 4-HPP on TC transport are stronger than RSV because RSV did not regulate genes of TG absorption while 3-HPP and 4-HPP did regulate. Importantly, RSV exhibited beneficial effects on lipid metabolism by influencing FA oxidation and transport, while 3-HPP and 4-HPP exhibited beneficial effects by influencing TC transport. After i.p, the majority of RSV and 3-HPP, 4-HPP were directly entered into the blood, while oral administration RSV was metabolized and entered systemic circulation together with its metabolites. Our findings help to illustrate the diverse biological activities of RSV oral administration.
Besides hepatic steatosis, the development of NAFLD was highly associated with insulin resistance, chronic inflammation, and oxidative stress. We also found that HFD induced insulin resistance and systemic inflammation, which were reversed by 3-HPP, 4-HPP, and RSV supplementation. However, the effects of 3-HPP and 4-HPP on insulin resistance were weaker than those of RSV, which inspired us to find the real and active form in RSV decreasing insulin resistance. Finding this form would be of importance for preventing diabetes by RSV. In addition, 3-HPP and 4-HPP supplementation decreased systemic inflammation as well as RSV supplementation. Future studies could focus on antioxidant enzyme levels in the liver to assess the effects of 3-HPP and 4-HPP on oxidative stress. Moreover, the mechanisms by which 3-HPP and 4-HPP inhibit inflammation need to be explored in future studies.

Conclusions
In conclusion, we found that 3-HPP and 4-HPP, gut microbiota-derived metabolites of RSV, can alleviate NAFLD in OA-induced HepG2 cells and HFD-fed mice by ameliorating hepatic steatosis and their bioeffects were equivalent to RSV at the same concentration. While the molecular mechanisms between the parent compound and microbial metabolites on regulating lipid metabolism are different, the former focuses on FA transport and the latter focuses on cholesterol transport. Our finding helps to explain the contradiction between low bioavailability and beneficial effects of RSV and other polyphenols. Furthermore, our study illustrates the individual difference of RSV bioactivity and provides further support for nutritional intervention of RSV.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/foods12010094/s1. Table S1. The amplification efficiency of primers. Table S2. The chemical composition of diets. . Figure S1: Cytotoxicity assay of RSV and phenolic acids at doses from 0 to 200 µM with and without OA for 24 h.