The Comprehensive Effects of Carassius auratus Complex Formula against Lipid Accumulation, Hepatocarcinogenesis, and COVID-19 Pathogenesis via Stabilized G-Quadruplex and Reduced Cell Senescence

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Overexpression of Cluster of Differentiation 36 (CD36) and Telomerase Reverse Transcriptase (TERT) in Hepatocellular Carcinoma (HCC)
CD36, also called fatty acid translocase, is a cell membrane glycoprotein that uptakes long-chain fatty acids into cells and widely expressed in multiple cell types. [15] Hepatic CD36 expression normally is low, but is increased in the fatty liver in rodents. [16] In addition, CD36 transcript levels were elevated in the liver of mice with steatohepatitis by high-fat diet and in genetic obesity models. [16,17] CD36 has been regarded as a prognostic biomarker of several cancer types, including breast cancer, prostate cancer, ovary cancer, and colon cancer. [18] CD36 overexpression promotes fatty acid uptake and reduces fatty acid oxidation to facilitate the progression of NAFLD [19] and even HCC. [20] Telomere is a region of repetitive nucleotide sequences at each end of a chromosome, and telomerase adds guanine-rich repetitive sequences to maintain the length of telomeres and overcomes telomere shortening during DNA replication. [21] Telomerase has two core subunits: the catalytic component telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). [22] TERC is expressed in most human cells, but TERT is strictly suppressed. Consequently, the telomerase activity depends on TERT activation in human cells. [23] In contrast to human normal cells, cancer cells have a higher telomerase activity to prevent telomere shortening. [24] Integrations of HBV in the TERT promoter are not random for HCC, [25] and TERT reactivation is observed in 60% of HCC. [5]

Using CD36 and tert Transgenic Zebrafish Models for the Anti-HCC Effects of CACF
Zebrafish (Danio rerio) is an excellent model for biomedical research with many advantages including fast sexual maturation, many offspring, transparent embryos for observation, and low husbandry cost. In addition, it has up to 87% genomic conservation with humans. [26] With these advantages, zebrafish has been used in various biomedical research fields related to human diseases like cancer research and drug screening. [27] Studies in zebrafish model have revealed molecules with similar activities and pharmacokinetic properties to those in mice and humans. [28] Zebrafish is widely used for investigating carcinogenesis and developing efficient drug screening platforms for anticancer therapeutics, [29] is applied to study liver cancer. [30] Activated β-catenin expression in the zebrafish fed with cholesterol-enriched diets for 8 days developed HCC at 13 daypost-fertilization (dpf), representing an NASH-driven HCC. [31] Xenograft is a rapid screening system to examine tumor cell proliferation and migration. Thus, the xenograft of human cancer cells into zebrafish embryos becomes a good model to validate the potential agents for anticancer effect in vivo. [27b] In this study, we reported that CACF possesses antihepatoma cell proliferation in vitro and in vivo. In addition, CACF exhibits antilipid accumulation effect by reducing the elevated gene expression of lipogenesis, cholesterol synthesis, and inflammation markers in CD36 transgenic fish under high-fat diet. CACF can attenuate the enhanced cell proliferation marker gene expression in two distinct HCC conditions: CD36 transgenic fish under high-fat diet (representing NASH-driven HCC) and tert transgenic fish (representing 80% of HCC overexpressing TERT). Furthermore, the transcriptomic analysis discloses the comprehensive protective effects of CACF, including the possibility of lowering the chance of coronavirus infection. Moreover, CACF can decrease the elevated expression of interferonbeta (INF-β) and CXCL10 in hepatoma cell and reduce the cell senescence in the liver of the tert transgenic fish. Interestingly, we found CACF contains antimicrobial activity against some Gram-positive and Gram-negative bacteria. Importantly, we found CACF may serve as a G-quadruplex ligand which may help to explain its multifaceted functions. Together, our results

The Preparations of Different Forms of CACF
The CACF and NRICM101 were provided by Everprofit Biotech Inc. in Taipei, Taiwan. The extraction process is as follows. Fresh Carassius auratus was washed and simmered in wine at 50 °C. The organ was removed, and stewed with Rhizoma dioscoreae, Lycium chinense, and Rehmannia glutinosa Libosch, and then filtered and condensed the mixture. The paste was dried at 60-70 °C, and then ground into fine powder. Different solvents were used to dissolve CACF because it was tried to improve the solubility. In the beginning, 100% ethanol was used to dissolve the powder and centrifuge at 3000 rpm for 1 min to obtain the soluble supernatant (EtOH-CACF). The final concentration of ethanol for immersing zebrafish larvae is 1%. Because 1% of ethanol caused some abnormality to the zebrafish, water was then used to dissolve the powder, and there were some insoluble particles (Whole-CACF), and centrifuge at 3000 rpm for 1 min to obtain the soluble supernatant (Soluble-CACF). In cell culture experiment water is used to dissolve the powder and to get the supernatant by centrifugation and to be sterilized by filter. To improve the solubility, the company provided new formula CACF, which can be completely dissolved in water and were used for experiments, which was labeled as New-CACF. All the names have been kept consistent in the main text and figure legends as EtOH-CACF, Whole-CACF, Soluble-CACF, and New-CACF. The amount of CACF for Human is 2 g per day. Human adult dose: 2 g per day, and the average body weight for human is ≈50 kg in Taiwan, that equal to 2 g/50 kg = 0.04 g kg −1 = 0.04 mg g −1 .
Because zebrafish larvae were immersed with CACF solution, so the concentration was estimated as 0.04 mg mL −1 .

MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay or WST-1 assay (Takara, Japan) was used in accordance with the manufacturer's protocol to determine cell viability. [32] Normal liver cells or hepatoma cells were seeded in a 96-well plate with 1 × 10 4 cells in each well, the cells with or without CACF treatment with four repeats were performed to measure cell viability in a time-dependent manner.

Transgenic Zebrafish Lines
The transgenic zebrafish lines Tg(fabp10a:CD36:cmlc2:GFP) and Tg(fabp10a:tert:cmlc2:GFP) were established as described previously. [33] Tg(fabp10a:tert:cmlc2:GFP) was generated via Tol2 gateway recombination. [34] Then, tert was amplified from zebrafish tert cDNA and cloned by rapid amplification of cDNA ends (RACE). The middle-entry clone:pME-tert was produced and used to generate the expression construct, together with p5E-fapb10a, p3E-pA, and pDEST-Tol2-CG2, via LR reaction. The expressing plasmid pTol2-fabp10a-tert-pA/CG2 was purified. Afterward, the sequence was confirmed and microinjected into AB wild-type zebrafish. The embryos carrying the transgene were selected by screening the green fluorescent protein expression in the heart of 3 dpf injected embryos. The independent transgenic fish lines were raised to sexual maturity (around 3 months), crossed with AB wild-type fish to generate F1 fish, and self-crossed to obtain F2 transgenic fish. Fin-clip method was used to ensure all the transgenic fish containing tert transgenic DNA insertion and overexpression of tert mRNA through quantitative polymerase chain reaction (Q-PCR). All experiments were performed by using F2 homozygous fish.

Zebrafish Maintenance
Zebrafish were maintained at the Zebrafish Core Facility at NTHU-NHRI (ZeTH) in accordance with previously described procedures. [32,33,35] All zebrafish experiments were carried out under the approval of the Institutional Animal Care and Use Committee (IACUC) at NHRI under the number NHRI-IACUC-110114-A.

Embryo Collection and Embryonic Toxicity Test
Zebrafish embryos were collected in accordance with the standard protocol. [32,33] The embryos were then placed in a 100 mm dish with E3 solution (5 mm NaCl, 0.17 mm KCl, 0.33 mm CaCl 2 , and 0.33 mm MgSO 4 , pH 7.0) [36] and incubated at 28 °C. At 16-22 h post-fertilization (hpf), they were washed with 0.0016% bleach solution to clean and improve their survival rate. Unfertilized and dead embryos were removed at 6 hpf, and the embryos were kept in a 100 mm dish with fresh E3 solution.
The collected embryos were placed in a 12-well polystyrene plate, with 10 embryos per well and incubated in 28 °C. Different concentrations of CACF (0.01 to 4 mg mL −1 ) were added into the wells, and the buffer was renewed every day throughout the experiment. The mortality and morphological characteristics were recorded.

Examination of Cell Proliferation and Migration in Zebrafish via Xenotransplantation Assay
Zebrafish xenotransplantation assay was performed as previously described. [35b,c,37] For dechorionation, 1 dpf zebrafish eggs were placed in 0.003% PTU/E3 medium at ≈20 hpf. Then, 20 µg mL −1 pronase (ChemCruz, Santa Cruz Biotechnology, Texas, USA) was added to remove the membrane. The embryos were under stirring, and transferred to another glass beaker until they were completely dechorionated, and then maintained in the fresh PTU/E3 medium. For cell injection, ≈9 × 10 5 HepG2 cells were collected in PBS added with 5 µL of CFSE (Life Technologies, Invitrogen, MA, USA) and then incubated at 37 °C for 15 min. After centrifugation, the cells were washed with PBS, the cell pellet was resuspended with 20 µL of PBS and kept at 37 °C. Next, 0.016% tricaine in the PTU/E3 medium was used to anaesthetize 2 dpf fish. The prepared CFSE-labeled HepG2 cells were injected into zebrafish embryo yolks via microinjection. The fish were maintained in the PTU/E3 medium and placed in a incubator with temperature slowly adjusted from 28 to 37 °C. The injected and healthy 3 dpf embryos were selected and transferred into a 96-well plate under a fluorescent microscope. Fluorescent signals were taken using an ImageXpress Micro device (Molecular Device, CA, USA) at 3 dpf (1 dpi) and 5 dpf (3 dpi). The change in fluorescent signal area was calculated using MetaXpress 2.3 (Molecular Device, CA, USA).

Feeding Protocol and CACF Treatment
Wild-type and CD36 transgenic zebrafish were used in this experiment. After the zebrafishes were cross-mated, their embryos were collected and incubated in E3 solution at 28 °C the next day (0 dpf). Then, each embryo was washed with 6% and 8% bleach solution to clean the surface of the embryo envelope at 16-22 hpf. The larvae were fed starting from 5 dpf (day-post-fertilization) four times daily with either a normal diet (12% fat) or a high-fat diet (24% fat) supplemented with 20 mL of Paramecium. For each treatment, the transgenic embryos were raised in tanks with 800 mL of water and separated into two groups. Each group was composed of 50 larvae for the control and treatment experiments.
For the experiments with CD36 transgenic fish, the larvae were divided into two groups: experiments performed on 15-day (liver lipid accumulation inhibition) and 30-day (liver cancer inhibition). For the 15-day experiment, the larvae were fed from 5 to 15 dpf and fasting for 2 days until 17 dpf to remove food and feces in the gastrointestinal tract for clearer staining observation. Then, they were sacrificed at 18 dpf. For the 30-day experiment, the larvae were fed from 5 to 30 dpf and fasting for 2 days until 32 dpf. At 33 dpf, they were sacrificed.
For the experiments with tert transgenic fish, the larvae were fed with normal larvae food for 20 mL of Paramecium four times a day with an interval of 2 h between meals. The zebrafish larvae were placed in a petri dish containing 25 mL of clean E3 solution (control group) or CACF in E3 solution, soaked in the specific drug overnight until 9:00 the next morning, and moved to a fish tank with 800 mL water. The fish were fasting for 1 day before the samples were collected for RNA extraction and hematoxylin-eosin staining.

Oil Red O Staining and Quantification Analysis
Oil Red O staining was performed in accordance with previous procedures. [32,33,35b,c] The larvae were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C, washed twice with 1× PBS for 5 min at each time, and immersed in 80% and 100% 1,2-propylene glycol (Cat#, W294004, Sigma) at room temperature each for 20 min. The larvae were stained with 0.5% Oil Red O (Cat# O0625, Sigma) dissolved in 100% 1,2-propylene glycol in the dark while shaking very gently at room temperature overnight. The stained larvae were washed twice with 1× PBS for 5 min each time. Then, 80% and 100% 1,2-propylene glycol were used to wash the stained background color for 20 min each and kept in 80% 1,2-propylene glycol. Oil droplet accumulation was observed under a microscope, and photos were taken (Olympus SZX10). Pooled larvae were placed in one tube, washed with 1× PBS, added with 300 µL of 4% NP40 in 100% isopropanol, and incubated at room temperature overnight. Absorbance at 490 nm with 570 nm for one well with 90 µL each (triplicate) was measured as a reference.

Total RNA Isolation
Total RNA was isolated in accordance with a previously described protocol [32,35b,c] and the manufacturer's instructions (NucleoSpin RNA kit, Macherey-Nagel, USA). The RNA samples were suspended in 40 µL of RNase-free H 2 O and stored at −80 °C.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
cDNA was synthesized in accordance with a previously described protocol [32,35b,c] and the manual of the iScript cDNA synthesis kit (BIO-RAD, USA). It was then stored in a freezer at −20 °C.

Q-PCR
Q-PCR was performed in triplicate by using an SYBR Green Q-PCR Master Mix kit (Applied Biosystems) and an ABI PRISM 7900 System in accordance with a previously described protocol. [32,35b,c] Fold change was calculated based on ΔΔCt by normalizing with internal control actin expression inside the group, and compared with that of the control group by using the relative Ct method. The fold change is calculated by 1.94 −ΔΔCt considering the Q-PCR reaction efficiency. The nucleotide sequences of the primers are listed in Table 1.

Detection of Senescence-Associated β-Galactosidase (SA-β-Gal) Accumulation
The Cellular Senescence Detection Kit -SPiDER-βGal (DOJINDO, Kumamoto, Japan, SG03) was used for detecting the SA-β-gal-positive senescent cells as described previously. [39] Specifically, five larvae were placed in each well of a 24-well plate, washed once with 500 µL of PBS, and then added with 500 µL of PBS containing 4% PFA for 10 min at room temperature for fixation. The supernatant was removed and the larvae were washed three times with 500 µL of PBS. 500 µL of SPiDER-βGal working solution in Mcllvaine buffer (pH 6.0, 1/1000 dilution) was added to the wells for 30 min at 37 °C. After removing the supernatant, the larvae were washed twice with 500 µL of PBS, then observed and photographed with a confocal microscope. Cellular Senescence Plate Assay Kit -SPiDER-βGal (DOJINDO, Kumamoto, Japan, SG05) was used for quantifying the SA-β-gal accumulation as described previously. [39] Specifically, ten larvae per eppendorf were washed once with 500 µL PBS, then RNase free beads with autoclaved and 400 µL lysis buffer were added to the eppendorf. Next, put the eppendorf into the homogenizer for 1 min to break up the larvae, and incubated them at room temperature for 9 min to allow the lysis buffer to fully lyse the larvae. After centrifuging for 2 min at 13 000 × g, 50 µL of supernatant was transferred to each well of a 96-well black plate, and 50 µL of SPiDER-βGal working solution was added to each well and incubated at 37 °C for 30 min. Then 100 µL of stop solution was added to each well and fluorescence was measured in a fluorometer (excitation: 500-540 nm, emission: 540-580 nm). Triplicate experiments were performed for each sample, normalized to no larvae control (only added reaction reagents) to remove background signal, and fold changes were calculated using WT as 1.

Detection of the G-Quadruplex Binding Activity of CACF In Vitro and In Vivo
To detect the G-quadruplex binding activity of CACF in vitro, nondenaturing polyacrylamide gel electrophoresis was applied Adv. Biology 2023, 7, 2200310 Table 1. Primer sequences for Q-PCR.

Species
Target and silver was stained as described before. [40] The CACF and PDP were incubated with the annealed RNA samples (5′-GGCUGGCAAUGGCGG-3′) at 4 °C for 16 h, and performed gel electrophoresis by using 15% acrylamide native gel with 1× TB buffer containing 10 mm KCl at 4 °C, and the gel was silver stained by using the Pierce Silver Stain Kit (Thermo Scientific cat: 24612) following the manufacturer's protocol. After gel electrophoresis, the gel was washed twice using ultrapure water, fixed with 30% ethanol:10% acetic acid solution overnight, then washed twice with sensitizer working solution for 1 min. Then, the gel was washed twice with UltraPure DNase/ RNase-Free Distilled Water (Invitrogen, cat: 10977015), stained with stain working solution for 30 min. Following, the gel was washed twice with ultrapure water, and developed for 2-3 min until bands appear, then stop with 5% acetic acid for 10 min.
The image of the G4-complex was quantified by ImageJ. The positive control PDP, Pyridostatin Trifluoroacetate Salt (RR82) is a G-quadruplex stabilizer with Kd of 490 nm in a cell-free assay, was order from Selleck Chemicals (Catalog No. S7444).
To detect the G-quadruplex binding activity of CACF in vivo, BG4 antibody was used to detect the G-quadruplex as described previously. [41] The anti-DNA G-quadruplex structures antibody, clone BG4, from Escherichia coli DNA G-quadruplex (BG4) was purchased (Sigma-Aldrich: MABE917-25UL). HepG2 cells (2.4 × 10 5 ) were seeded in Nunc Lab-Tek II CC2 Chamber Slide System (Thermo Scientific, cat: 154917) for 24 h, and prefixed with a solution of 50% DMEM and 50% methanol/acetic acid (3:1), and washed with methanol/acetic acid (3:1), fixed with methanol/acetic acid (3:1) at RT for 10 min, and then permeabilized with 0.1% Triton X-100 (Merck) in PBS at RT for 3 min. HepG2 cells were exposed to blocking solution (2% milk in PBS, pH 7.4) for 1 h at RT and then incubated with 1:100 of BG4 antibody in blocking solution (O/N, in cold room). HepG2 cells were then incubated with 1:800 of a rabbit antibody against the DYKDDDDK epitope (Cell Signaling, cat #2368) in blocking solution for 1 h at room temperature, and then were incubated at RT with 1:1000 Alexa Fluor 488 goat anti-rabbit IgG (Life technologies, cat: A11008) in blocking solution for 1 h at room temperature, and then cells were washed three times for 5 min each with 0.1% Tween-20 in PBS under gentle rocking. For nuclear staining, the cells were added with three drops of prolong gold antifade with DAPI and mounted with cover glass. The images were taken under fluorescent microscope.

Kirby-Bauer Disk Diffusion Susceptibility Test for CACF
Disk diffusion method by the Kirby-Bauer is a standardized technique for testing rapidly growing pathogens in clinical. Direct suspension of colonies to a 0.5 McFarland standard and swabbed onto the agar. Filter disks of CACF (16 µg mL −1 ) were put on the agar, the zone size of inhibition around the disk was measured after 16-18 h incubation. [42]

Statistical Analysis
All of the data were preprocessing by normalization to the control and evaluation and removing of outliers. The data presentation were shown as mean ± SD. 10 or five larvae were included for each batch as specified in the figure legends, and more than three batches were used for statistical analysis. All statistical analysis was conducted by using one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups, unpaired Student's t-test (two-tailed) for two groups (Figure 1G and Figure 3), or Kaplan-Meier survival analysis (Figure 2). All figures were drawn by using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The P values are presented as GraphPad (GP) formatting: ns: P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001.

CACF Decreases Hepatoma Cell Viability but Benefits Normal Liver Cell Viability
We investigated the effect of CACF on the cell viability by using two hepatoma cell lines (Huh7 and Hep3B), and two normal liver cell lines (Clone9 and L02). We used different solvent to dissolve the CACF powders: ethanol extracted CACF (EtOH-CACF), the water-dissolved CACF (Whole-CACF), the water-soluble CACF (Soluble-CACF), and new formula of CACF (NEW-CACF) which has been improved the water-solubility. All of the CACF treatment significantly reduce the cell viability in both hepatoma cells, suggesting the antihepatoma effect by CACF ( Figure 1A-D). To examine the effects of CACF on normal liver cell viability, similar dosages of the Whole-CACF and the Soluble CACF were used to treat normal liver cells. Interestingly, the treatments of the Soluble-CACF could significantly enhance the cell viability in both Clone9 and L02 normal liver cells ( Figure 1E,F), the treatments of the Whole-CACF could increase the cell viability in Clone9 but not in L02 cell ( Figure 1E,F), suggesting the Soluble-CACF may have better hepatoprotective effect in vitro. The New-CACF showed significant antihepatoma activity in Hep3B cells and increased the cell viability in L02 normal liver cell ( Figure 1G). Thus, the data indicated that CACF possesses both antihepatoma activity and hepatoprotective effects to normal liver cells in vitro. This finding is similar to previous studies that Lycium chinense exhibits hepatic protection effects, [10,11] and in accordance to our previous finding that oligo-fucoidan could reduce hepatoma cell viability and protects normal hepatocytes. [43]

The Optimal CACF Dosage Was Determined by Zebrafish Embryonic Toxicity Assay
We next examined CACF functions in vivo using zebrafish model. To avoid toxicity and find the optimal dosage of CACF in zebrafish, we tested the different concentrations of the Whole-CACF and the Soluble CACF on the zebrafish embryos to examine the survival rates for 5 days (Figure 2A,B). The results showed that all the embryos survived at the concentrations of less and equal to 0.16 mg mL −1 for both Whole-CACF and Soluble-CACF, and increasing the CACF concentration up to 0.4 mg mL −1 started to show the toxicity. The concentration of 4 mg mL −1 produced the highest mortality rate. Since the dosage for human is 2 g per day, which is equal to 0.04 mg mL −1 to immerse the zebrafish embryos, thus we chose 0.04 mg mL −1 for treating the zebrafish, which did not elicit embryonic toxicity in the following experiments.

CACF Displayed an Antihepatoma Cell Activity in Xenotransplantation Assay
To verify the antihepatoma cell activity of CACF in vivo, we conducted a xenotransplantation assay by injecting hepatoma cells to 2-day-old zebrafish embryos and treated them with CACF. The EtOH-CACF exhibited significant antihepatoma activity against Hep3B and Huh7 cells (Figure 3A,B). The Whole-CACF had strong antihepatoma activity against Hep3B cell, while the Soluble-CACF significantly reduced Huh7 cell proliferation ( Figure 3C,D). Therefore, the CACF treatments displayed antihepatoma cell activities in the xenotransplantation model in vivo. However, there seems to be slightly differences between different solvent preparations.

CACF Exhibited an Antilipid Accumulation Effect on CD36 Transgenic Fish under High-Fat Diet
Lipid accumulation derived from high-fat diet is one of the early progressions leading to liver cancer. Previously, we found CD36 transgenic fish develops fatty liver at 15 days under high-fat diet (24% fat). [33] To examine whether the CACF treatments can reduce lipid accumulation in vivo, we fed the CD36 transgenic zebrafish with high-fat diet with or without CACF for 15 days and analyzed lipid accumulation via Oil Red O staining. The lipid accumulation of CD36 transgenic zebrafish with the high-fat diet was significantly increased compared to that of the control (Figure 4A,C). The treatment of New-CACF significantly reduced the lipid accumulation in the CD36 transgenic zebrafish under the high-fat diet ( Figure 4A,B). The treatment of EtOH-CACF had the similar effect to decrease lipid accumulation in CD36 transgenic zebrafish model ( Figure 4C,D). Thus, CACF treatments reduce lipid accumulation in zebrafish model.

CACF Reduced the Increased Gene Expression of Lipogenesis, Cholesterol Synthesis, and Inflammation Markers in CD36 Transgenic Fish but Had no Consistent Effect on Fatty Acid β-Oxidation
To uncover the potential molecular mechanisms for CACF inhibiting lipid accumulation, we performed Q-PCR for the lipid metabolism related genes. First, we analyzed the expression of lipogenesis markers with or without CACF treatment. The increased expression levels of acetyl-CoA carboxylase (acaca) and sterol regulatory element-binding transcription factor 1 (srebf1) under high-fat diet were significantly reduced by either the New-CACF (Figure 5A-C) or the Whole-CACF ( Figure S1A-C, Supporting Information). The results demonstrated that the CACF treatments effectively inhibited de novo lipogenesis, consequently decreasing lipid accumulation in CD36 transgenic fish.
Next, we analyzed the expression of srebf2 (which is a transcriptional factor initially embedded in ER-controlled cholesterol biosynthesis and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (hmgcra; the rate-limiting enzyme for cholesterol synthesis). The data indicated that either the New-CACF or Whole-CACF effectively inhibited the elevated expression of those key regulators of cholesterol synthesis; thus, lipid accumulation in CD36 transgenic fish was decreased by CACF treatments (Figure 5D-F; Figure S1D-F, Supporting Information). The results revealed that the CACF treatments also inhibit cholesterol biosynthesis in CD36 transgenic zebrafish model.
Liver inflammation leads to liver fibrosis, cirrhosis, and cancer. Proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), play an essential role in inflammation. Thus, we further analyzed the expression of inflammation markers, and found that their expression was significantly increased in the CD36 transgenic fish under the highfat diet and CACF treatment reduced the expression of proinflammatory cytokine (  an anti-inflammatory effect on the CD36 transgenic fish under the high-fat diet by decreasing the expression of the inflammation related genes, il1b. il6, and tnfa, implying CACF might reduce steatohepatitis in CD36 transgenic zebrafish model.

CACF Reverted the Elevated Chop Expression of CD36 Transgenic Fish under High-Fat Diet
High-fat diet can trigger ER stress. To examine whether the CACF treatment for the antilipid accumulation could reduce the expression of ER stress related genes, we analyzed the expression of ER stress target genes such as atf6, bip, grp94, and chop. The results showed that only chop expression was induced in the CD36 transgenic fish under high-fat diet and the elevated chop expression could be repressed by the CACF treatment ( Figure S2, Supporting Information). However, we did not find the similar effect on the other ER stress markers such as atf6, bip, and grp94, indicating that overall ER stress might not be affected in the CD36 transgenic fish under high-fat diet with CACF treatment. The expression of chop can also be enhanced by cellular stress or apoptosis. Therefore, the CACF treatment that reverted the upregulated chop expression in the CD36 transgenic fish under high-fat diet suggests that CACF might prevent cellular stress and apoptosis in zebrafish model.

CACF Decreased the Enhanced Expression of Cell Proliferation Markers in CD36 Transgenic Fish under High-Fat Diet and in tert Transgenic Fish
We previously reported that CD36 transgenic fish fed with a highfat diet can lead to HCC at 30 days. [33] To investigate whether CACF treatment can reduce liver cancer formation, we examined the anti-HCC effect by CACF treatment on the CD36 transgenic fish under high-fat diet by analyzing two cell cycle/proliferation markers expression. CCNE1 encodes cyclin E, which binds to CDK2 to drive cells through the G1/S cell cycle transition. CDK1 can substitute CDK2 to drive G1/S transition upon CDK2 absence. In CD36 transgenic zebrafish, the expression of CCNE1 and   proliferation markers were significantly increased under high-fat diet, and the increases can be reverted significantly with the New-CACF treatment (Figure 6A-C). The data suggest that CACF treatment can block the enhanced cell cycle/proliferationrelated gene expression in CD36 transgenic fish by high-fat diet.
TERT promoter mutation and TERT reactivation were detected in 60% of HCC. To further understand whether CACF possesses anti-HCC effect against TERT overexpression, we analyzed the expression of cell proliferation markers (ccne1 and cdk2) in wild-type and tert transgenic zebrafish with normal diet for 15 dpf and later with high-fat diet and treated with or without CACF. We found that the cell cycle/proliferation markers were increased in the tert transgenic fish and the increases can be reduced by the New-CACF ( Figure 6D-F) as well as by the Whole-CACF ( Figure S3A-C, Supporting Information). We further analyzed the expression of β-catenin downstream target genes (ccnd1 and myca) and found that they were also upregulated in the tert transgenic fish compared to the wild-type control fish. Importantly, the CACF treatment can significantly reduce the upregulated expression of ccnd1 and myca in the tert transgenic zebrafish (Figure 6G-I; Figure S3G-I, Supporting Information). Therefore, CACF may be an effective therapy to inhibit liver cancer in tert transgenic zebrafish model.

Transcriptomic Analysis Revealed the Comprehensive Protective Effects of CACF
To further understand the comprehensive protective effects of CACF, we analyzed the transcriptomic profile of CD36 transgenic fish under high-fat diet with or without New-CACF treatment by using NGS and applying WebGestalt (WEB-based Gene SeT AnaLysis Toolkit) to analyze the enriched pathways. As shown in Figure S4   transgenic fish model fed with 24% fat diet with or without the New-CACF compared with that for the WT zebrafish fed with 12% normal fat diet. B) Representative images of Oil Red O staining of 15 dpf WT and CD36 fed with 24% fat diet with or without the New-CACF. C) The fold change of Oil Red O staining for CD36 transgenic fish fed with 24% fat diet with or without the EtOH-CACF compared with that for CD36 fed with 12% normal fat diet. D) Representative images of Oil Red O staining of 15 dpf CD36 fed with 12% and 24% fat diet with or without the EtOH-CACF. We performed three replicates, ten larvae were included in each experiment. Data were statistically analyzed with one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups. The P values are presented as GraphPad (GP) formatting: **: P ≤ 0.01; ****: P ≤ 0.0001. Figure 5. CACF reduced lipogenesis, cholesterol synthesis markers, and inflammation markers but had no consistent effect on fatty acid β-oxidation in 15 dpf CD36 transgenic fish. Fold change of lipogenesis markers: A) acaca, B) fasn, and C) srebf1; cholesterol synthesis markers: D) hmgcs1, E) srebf2, and F) hmgcra; inflammation markers: G) il1b, H) il6, and I) tnfa from CD36 transgenic fish fed with 24% fat diet with or without the New-CACF compared with that of WT fed with 12% normal fat diet. We performed three replicates, and for each Q-PCR, there were triplicates for each sample. Ten larvae were included in each experiment. Data were statistically analyzed with one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups. The P values are presented as GraphPad (GP) formatting: ns: P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001. Figure 6. CACF reduced the expression of cell proliferation markers in 15 dpf CD36 transgenic fish and tert transgenic fish. The fold change of the cell proliferation markers A) ccne1, B) cdk1, and C) cdk2 from CD36 transgenic fish fed with 24% fat diet with or without the New-CACF compared with that of WT fed with 12% normal fat diet. The fold change of D) ccne1, E) cdk1, F) cdk2, and the β-catenin downstream target genes G) ccnd1, H) myca, CACF treatment reduces the expression of lipid transport proteins, lipid metabolism, and immune response genes, which supports our findings of the antilipid accumulation and anti-inflammation effects of CACF ( Figure S4A, Supporting Information). In addition, CACF reduces DNA packaging complex ( Figure S4B, Supporting Information), PPAR signaling pathway, steroid hormones pathway ( Figure S4C, Supporting Information), and those genes are regulated by AP1 and HSF ( Figure S4D, Supporting Information). On the other hand, CACF increases defense response, positive regulation of stimulus response ( Figure S4E, Supporting Information), TGF-β signaling pathway, WNT, cadherin signaling pathway, Alzheimer's disease-presenilin pathway, integrin signaling pathway, and biological clock system ( Figure S4F, Supporting Information). Class A/1 (rhodopsin-like receptor)/ GPCR ligand binding/GPCR signal ( Figure S4G, Supporting Information), and those are regulated by NF-kB and STAT5B ( Figure S4H, Supporting Information). The heatmap of the selected differential expressed genes after CACF treatment is shown in Figure S5 of the Supporting Information. The results indicated that CACF not only inhibits NAFLD and NASH but also inhibit HCC by downregulating PPAR signaling pathway, DNA package, and epigenetic regulation in zebrafish model.

CACF Might Reduce the Chance of Infection by Coronavirus Revealed by Ingenuity Pathway Analysis
Using IPA, we further analyzed the transcriptomics of the CD36 transgenic fish under high-fat diet with or without CACF treatment. First, we found CACF treatment reduced the enhancement in organismal death, damage of liver, and cancer in the CD36 transgenic fish under high-fat diet for 15 days ( Figure  S6A,B, Supporting Information). Using canonical analysis, we found many signaling pathways were downregulated by CACF including p38 MAPK signaling, phospholipase C signaling, LPS/IL-1 mediated inhibition of RXR function, HER-2 signaling in breast cancer, T cell receptor signaling, signaling by Rho family GTPases, and androgen signaling. Interestingly, we also found that coronavirus pathogenesis pathway was reduced by CACF treatment (Table S1, Supporting Information). From the diseases and biofunctions analysis, we found organismal injury and abnormalities, organismal death was greatly reduced by CACF treatment (Table S2, Supporting Information). Lipid metabolism, transport and synthesis, and cancer formation were also decreased by CACF treatment (Table S2, Supporting Information). From the regulator effect analysis, we identified the upstream regulators of CACF that reduce transport of lipid (Table S3, Supporting Information). We also found CACF regulates genes that decrease organismal death, and the regulators for CACF to decrease lipid accumulation. The CACF effects on the regulators are to decrease lipid transport ( Figure S6C, Supporting Information), uptake of lipid ( Figure S6D, Supporting Information), and synthesis lipid ( Figure S6E, Supporting Information).
Surprisingly, we found CACF regulates the expression of genes and might reduce infection by coronavirus and pathogenesis in zebrafish model (Figure 7A). To verify the expression of genes related to coronavirus infection, we examined whether DUSP11 and CSF1 are upregulated, and FOXA2, radical S-adenosyl methionine domain containing 2 (RSAD2), and IFN-regulatory factors 7 (IRF7) are downregulated by CACF as predicted by IPA ( Figure 7A). Dual Specificity Phosphatase 11 (DUSP11) acts directly on the HCV transcripts, enables exonuclease XRNmediated restriction. [44] Colony stimulating factor 1 (CSF1) promotes a resident-type macrophage and inhibit COVID-19 infection. [45] FOXA2 is a transcription factor its expression was highly positively correlated with expression of angiotensin converting enzyme 2 (ACE2), the entry receptor for SARS-CoV-2, implying inhibiting FOXA2 can reduce the ACE2 expression further decrease the entering of COVID-19 to cells. [46] SARS-CoV-2 triggered a robust interferon response, [47] leading to a prominent induction of Interferon-stimulated genes such as RSAD2 and IRF7. [48] Our results indicated that CACF increased the expression of dusp11, csf1a, and csf1b in CD36 transgenic fish ( Figure 7B). We also found CACF could reverse the upregulation of rsad2 and irf7-1 in CD36 transgenic fish under highfat diet ( Figure 7C). To confirm these results, we used tert and tert*p53-transgenic fish and traditional Chinese medicine formula, Taiwan Chingguan Yihau (NRICM101), which is known to target COVID-19 [49] as a positive control. As figure 7D shown, CACF treatment increased the expression of dusp11, csf1a, and csf1b in tert transgenic fish as NRICM101 does, and the effect is more dramatically in tert*p53-transgenic fish. We also found CACF treatment reduced the expression of foxa2, rsad2, and irf7-1 in tert and tert*p53-transgenic fish ( Figure 7E). Whether those regulators mediate CACF effect requires further experiments to verify. Nevertheless, CACF might be able to reduce the chance of infection by coronavirus in zebrafish model is a new and exciting finding.

CACF Diminished the Elevated Expression of INF-β and CXCL10 in Hepatoma Cells but not in Normal Liver Cells
HCC patients with higher CXCL10 (C-X-C motif chemokine ligand 10) expression levels had a significantly poorer overall survival rate, [50] and INF-β is the critical inducer for CXCL10. [51] To investigate the effect of CACF on INF-β and CXCL10 expression, we measured the expression of INF-β and CXCL10 with CACF treatment in two hepatoma cell lines and one normal liver cell line. We found the expression levels of INF-β and CXCL10 were significantly higher in Huh7 and in Hep3B hepatoma cells compared to those in L02 normal liver cell, and the CACF treatment can significantly reduce the expression and I) mycb from tert transgenic fish with or without the New-CACF compared with that of WT fish fed with 12% normal fat diet. We performed three replicates, and for each Q-PCR, there were triplicates for each sample. Ten larvae were included in each experiment. Data were statistically analyzed with one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups. The P values are presented as GraphPad (GP) formatting: ns: P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001. A) The predicted regulators and the effectors of New-CACF inhibiting coronavirus infection revealed by IPA analysis. We designed the Q-PCR primers to validate the IPA data, and found the expression of DUSP11 and CSF1 are upregulated and FOXA2, RSAD2, and IRF7 are downregulated by CACF as predicted. The fold change of the genes B) dusp11, csf1a, and csf1b, C) foxa2, rsad2, and irf7-1 from CD36 transgenic fish fed with 24% fat diet with or without the New-CACF (0.04 mg mL −1 ) compared with that of WT fed with 12% normal fat diet. The fold change of the genes E) dusp11, csf1a, and csf1b, E) foxa2, rsad2, and irf7-1 from tert and tertxp53 −/− transgenic fish fed with 12% fat diet with or without the New-CACF or NRICM101 compared with that of WT fed with 12% normal fat diet. For each Q-PCR, there were triplicates for each sample. Ten larvae were included in different feeding and drug groups. Data were normalized to the internal control actin, and using the control (WT) in the group as the benchmark to present the relative expression fold value. Data were statistically analyzed with one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups.
The P values are presented as GraphPad (GP) formatting: ns: P > 0.05; ***: P ≤ 0.001. levels of INF-β and CXCL10 in the two hepatoma cells (Figure 8). INF-β and CXCL10 are important cytokines for hepatoma microenvironments, those inflamatory factors might contribute to tumor progression and development. CACF can strongly reduce the levels of INF-β and CXCL10 in hepatoma cells, which might be one of the mechanisms for the antihepatoma function of CACF.

CACF Decreased the Cell Senescence-Associated β-Galactosidase Activity in the Liver of the tert Transgenic Zebrafish
Many cytokines including INF-β are markers of cellular senescence which is associated with chronic inflammation and cancer formation. To examine if CACF can decrease cellular senescence, we detected the senescence-associated β-galactosidase (SA-β-gal). which is another marker of cellular senescence [52] in the tert transgenic fish with and without CACF treatment. The data indicated that CACF can lower the elevated expression of SA-β-gal in the liver of tert transgenic fish compared to the no treatment control (Figure 9). These results support the idea that CACF may attenuate the inflammation and cell senescence for anti-HCC effect.

CACF Functions as an Antimicrobial Agent for Some Gram-Positive and Gram-Negative Bacteria In Vitro
Since we found CACF might be able to reduce the infection of coronavirus, we are curious about whether CACF can also inhibit other microbes. We performed the antibacterial susceptibility test [53] by using CACF. We found that CACF inhibits common pathogens of respiratory tract infections such as Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, and Haemophilus influenzae ( Figure S7, Supporting Information). CACF can also suppress the bacteria for foodborne diseases such as Salmonella enterica, Aeromonas hydrophila, Plesiomonas shigelloides, Vibrio parahaemolyticus, and Campylobacter jejuni. Furthermore, some bacteria with healthcare-associated infections such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa, can be curbed by CACF. However, we found that CACF cannot inhibit Acinetobacter baumannii, Enterococcus faecium, and yeast-like fungi, Candida albicans and Candida glabrata ( Figure S7, Supporting Information). With those interesting findings, we were curious about the common mechanisms for the anticancer, anti-COVID-19, and anti-microorganisms activities by CACF.

CACF Might Serve as a G-Quadruplex Ligand
As we uncovered multiple functions of CACF, we would like to know the underlying mechanisms. Some novel compounds, which can bind to putative G4-forming sequences and inhibit translation of SARS-CoV-2 N protein, act as promising therapeutics for anti-COVID-19. [40] G-quadruplexes also appear in gene promoters and have the potential to serve as therapeutic targets. [54] Thus, we tested the possibility for New-CACF as a G4-ligand using in vitro assay by silver stain. We found that New-CACF solution in physiological concentration stabilizes G-quadruplexes with RG-1 G4 compared to the positive control PDP (Figure 10A,B). G4-specific antibody BG4 was used  ) without New-CACF, or with 2 or 4 mg mL −1 New-CACF treatment for 24 h. We performed three replicates, and for each Q-PCR, there were triplicates for each sample. Data were statistically analyzed with one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups. The P values are presented as GraphPad (GP) formatting: ns: P > 0.05; ****: P ≤ 0.0001.
to detect G4 structures in vivo. [41] We examined the possibility for CACF as a G4-ligand in HepG2 cells by using G4-specific antibody BG4. We found that New-CACF treatment indeed increased the BG4 antibody staining similar to the positive control PDP ( Figure 10C). Together, we provide one of the possible mechanisms for CACF exhibiting anticancer, anti-inflammation, and perhaps anti-COVID-19 might be through stabilizing G-quadruplexes.

Discussion
Liver cancer is highly heterogenous with many risk factors including HBV and HCV infection, NAFLD, NASH, alcohol consumption, aflatoxin intake, etc. [1] In Asia (except Japan), South America, and Africa, liver cancer has a high incidence and mortality rate mainly caused by HBV and aflatoxin. In most parts of the world, liver cancer results from NASH. [55] Currently, NASH-driven HCC only accounts for 2.6%, the majority is still HBV-induced HCC for 45%. With the advancement of anti-HBV vaccines and anti-HCV drugs, the incidence of viral liver cancer decreases gradually, NASH-driven HCC eventually becomes a major concern. The treatments for advanced HCC are limited, [4] and the first and second lines of therapies can only extend 8 to 13 months of survival life for advanced HCC. [5] Moreover, NASH-driven HCC responds poorly to the first-line treatment of combination Atezolizumab (anti-PDL1) and Bevacizumab (anti-VEGF). [6] The major unmet needs for HCC depend on discovering new therapies for liver cancer patients and formulating personalized precision medicine.
Traditional Chinese medicine (TCM) for primary liver cancer has been found to increase immunity and even slow down the tumor growth. [56] Huang-lian-jie-du-tang exhibits anti-HCC effect by inducing cell-cycle arrest and apoptosis. [57] Many other TCM also has been used in HCC treatment. [58] TCM feed additives significantly improve the microbial balance and prevent diseases in aquaculture. [59] CACF has been used since ancient China for treating many human chronic diseases. It has been demonstrated that CACF exhibits antidiabetic effects in mouse model. [14] Since T2DM linked to HCC formation, especially NASH-driven HCC. Thus, we set our goal to test whether CACF prevents HCC formation and investigate the underlying mechanism.
Our results demonstrated that CACF reduced hepatoma cell viability but has benefit to the normal hepatocyte in vitro. CACF exhibited antiliver cancer effect in zebrafish xenotransplantation model in vivo. In addition, CACF reduced lipid accumulation through inhibition of lipogenesis markers, cholesterol synthesis genes, and decreased cellular stress by reducing the elevated chop expression in CD36 transgenic fish under high-fat diet. CACF effectively inhibited the proinflammatory cytokines expression. Our data suggested that CACF intake may help to block NAFLD and NASH by inhibiting lipid accumulation at an early stage to prevent the progression of NASH-induced HCC. In tert transgenic fish model, CACF significantly diminished the increased expression of cell proliferation markers, indicating that CACF might be a Figure 9. CACF reduced the high expression of senescence-associated β-galactosidase (SA-β-gal) activity in tert transgenic fish by SA-β-Gal analysis. These fish were fed with normal diet from 5 to 14 dpf old and treated with or without New-CACF (0.04 mg mL −1 ), then were fixed and stained for fluorescence imaging (excitation: 500-540 nm, emission: 540-580 nm) or lysed for quantification on 16 dpf. Five larvae were included in each SA-β-Gal fluorescent staining group, and ten larvae were included in each quantitative experiment. For each group, three replicates were performed. The scale bar is 200 µm in length. The orange circle indicates the location of liver. Data were normalized to internal control (only reaction reagents, no larvae tissue) and using the control (WT) in the group as the benchmark to present the relative expression fold value. Data were statistically analyzed with one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups. The P values are presented as GraphPad (GP) formatting: *: P ≤ 0.05; **: P ≤ 0.01.
promising therapeutic agent against HCC induced by TERT activation.
The data from whole genomic transcriptomic analysis prove that the mechanisms of action of CACF appear to be multifaceted. It not only can inhibit lipid accumulation but also reduce the PPAR signal pathway in the CD36 transgenic fish model under high-fat diet. Therefore, fatty liver and hepatitis can be suppressed. CACF can also reduce DNA packaging and epigenetic control gene expression, so it may diminish the growth of cancer cells. Moreover, CACF can also positively regulate cell defenses, Alzheimer's premature aging, and activate integrins and the biological clock system, thus it may regulate and balance the overall physiological function. In conclusion, CACF exhibits a full range of cellular protection effect in the zebrafish animal models.
From the IPA analysis, it supports that CACF can prevent liver damage and cancer formation induced by CD36 overexpression with high-fat diet by decreasing lipid transport, synthesis, and metabolism through many regulators, including CCNC, ESRRG, FASN, FMO3, HNF1A, MAPK3, MAPK9, Figure 10. CACF could be a G4-ligand by in vitro and in vivo assays. A) Representative image of silver staining results after incubating the RG-1 oligonucleotides with three different concentrations of New-CACF or PDP (as a positive control). B) Quantitation results of silver staining from three batches of experiments. Data were statistically analyzed with one-way ANOVA analysis followed by multiple analysis of variance to identify the statistical significance between groups. The P values are presented as GraphPad (GP) formatting: ns: P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01. C) CACF could promote the G4 formation in HepG2 cells. HepG2 cells without treatment (Ctrl), treated with 0.04 mg mL −1 CACF, or 10 µm PDP for 24 h, G-quadruplex was revealed by G4-specific antibody BG4. The scale bar = 50.