Mitigative Effect of Graphene Oxide Nanoparticle in Maintaining Gut-Liver Homeostasis against Alcohol Injury

1. Introduction

2. Materials and Methods

2.2. Methods:

2.2.1. Synthesis and Characterization of Graphene Oxide Nanoparticle (GO):

The mixture of H₂SO₄ and H₃PO₄ were prepared in 9:1 ratio. In cooling condition this mixture was added on 1g of graphite powder and 9g of KMnO₄. This reaction is exothermic. After the mixture is ready, it was kept on stirring condition for 12 h and 60°C temperature maintained. After 12h of stirring the oxidation reaction was ceased using of ice cold distilled water. To see the oxidation, 10 mL of 30% H₂O₂ was added to the mixture. As H₂O₂ was added and it created bubble in solution. Then the mixture is first filtered through Whatman filter paper 1. Then collected filtrate was washed with distilled water, 30% HCl solution. HCl wash was given till the sulphate and manganese impurities were removed. After washing with HCl again water wash was given. To get raw material the collected pellet was air dried for 12 h at 50°C. then the material was exfoliated using sonication method [10,11].

After exfoliation sample was again air dried for 12 h at 50°C then characterization of the material was done using UV visible spectrophotometer, Scanning Electron Microscopy, and Fourier Transform Infrared Resonance. For FTIR, KBr palate were prepared and transmission were analyzed.

2.2.2. Cell Culture Studies:

HepG2 and Caco2 cell line were procured from NCCS, Pune. HepG2 cell line was maintained using 10% FBS and MEM media with 1X non-essential amino acid while for Caco2, 20% FBS and DMEM low glucose was used. For the experimentation purpose cells were treated with low serum media. Both the cell line 1X antibiotic and antimycotic mixture was used which contained penicillin, streptomycin and amphotericin.

(A)

Toxicity study:

In brief, cells were seeded in adequate amount to 96 well plate. As the confluency reach to 60-70% they were treated with different amount of GO (10 to 100 μg/mL). The concentration was in increasing amount. Next day, GO media was removed and cells were washed with 1X PBS thoroughly as unremoved GO can interact with MTT and might give false positive result [12]. As the cells were washed, they were treated with final concentration of 0.5 mg/mL MTT. After 60-90 mins MTT containing media removed and formazan crystals were dissolved in 100% DMSO. Then results were observed in multimode reader at 570 nm.

(B)

Cell damage analysis:

DAPI staining:

For DAPI, cells were seeded in 96 well plate (HepG2 25,000 cell/well and Caco2 10,000 cells/well) and next day cells were treated with 10 and 50 μg/mL GO and 0.1M ethanol. After treatment, cells were incubated and their growth characteristics were observed after 24h. Next day, cells were washed with 1X PBS and fixed with 4% neutral buffered saline (NBS). After fixation cells were washed with distilled water till the traces of fixative agent was removed. Further they were incubated for 15min with 100 µM DAPI stain in dark condition for 15 min. After incubation, extra DAPI solution was removed and cells were further washed with PBS. After wash cells were observed under fluorescent microscopy in DAPI filter. Their excitation and emission was also noted via multimode reader [13].

AO-EtBr staining:

Acridine orange and ethidium bromide were used to check cell death. For which live cells were observed under fluorescent microscope. Initially in 96 well plate, cells were seeded (HepG2 25,000 cell/well) and following day they were treated with of 10 and 50 μg/mL GO and 0.1M ethanol. On the next day, cells were washed with PBS and encountered with 100 μg/mL AO/EtBr solution and immediately observed under FITC (485 excitation /595nm emission) and TRITC (540 excitation /590 nm emission) filter. In merged channel, image cells were observed and up to 300 cells were counted to screen live and dead cells [14,15].

Lipid Accumulation study:

Due to ethanol, cells produces more amount of lipid and transferred near to cell membrane [16]. Cells were seeded in 96 well plate and next day treated with 0.1M ethanol and 10 and 50 μg/mL GO. After 24 h of treatment they were washed with PBS and incubated for fixation in NBS for 15-30 mins. Then, cells were washed with Distilled Water and incubated for 15 min with working Oil red O (ORO) solution. After incubation, extra ORO was removed by washing cells with Phosphate Buffered Saline (PBS) and observed under light microscope. Further to take absorbance, PBS was removed and 100% IPA was added in each well and shaken for few minutes to extract stain from cells and solution was taken in empty well. Absorbance was taken at 570 nm and relative fold change over basal was measured [17,18].

ROS study:

H2-DCFDA is used to check ROS level. Cells were seeded in 96 well plate as mentioned previously and after 24hrs, they were treated with ethanol and 10 and 50 μg/mL GO. After 24h of the treatment, cells were washed with PBS and fixed. After that cells were incubated with 20μM concentration of H2-DCFDA for 1hr. Further, cells were washed to remove excessive dye. Fluorescence were observed using microplate reader, Perkin Almer at fluorescent excitation at 484/30 nm and emission at 535/30 nm. Fold over basal change graph for both the cell lines were prepared [19].

Gene Expression:

For gene expression analysis, cells were first seeded in 6 well plate and then in their exponential phase, they were treated with ethanol and 10 and 50μg/mL GO. After 24hrs of the treatment, RNA isolation was done manually by using RNAisoplus solution, TAKARA. In brief, cells were removed from plate by RNAisoplus solution and incubated with chloroform following centrifugation at 12,000g for 15min. Further, pellet was washed with 100% IPA and incubated at room temperature for 15mins. After that pellet was centrifuged at 12,000g for 10min and then collected pellet were again washed with 70% ethanol. Clear pellet was then resuspended in nuclease free water and stored at -80°C for future use. Purity of RNA was checked using Nanodrop. Further purified RNA was used for cDNA synthesis. cDNA was synthesized using kit and after that RT-qPCR was performed for gene expression study.

For gene expression, 2X SYBR green enzyme mix, 500 nM primer and 5ng concentration of cDNA were used. To activate SYBR green enzyme, first initial 95°C temp was given to final prepared solution. Further PCR was carried out at 95°C denaturation for 30 sec, 60°C for 45 sec annealing and 72°C for 1min extension.

Fold change over basal were calculated by 2^-ΔΔCt method, and mRNA expression were evaluated for HepG2 and Caco2 cells. 18s rRNA were used as reference gene.

Table 1. List of Human primers for RT-qPCR.

Table 1. List of Human primers for RT-qPCR.

Gene Name	Forward Primer	Reverse Primer
18S	GATGGTAGTCGCCGTGCC	GCCTGCTGCCTTCCTTGG
CYP2E1	AACTGTCCCCGGGACCTC	GCGCTCTGCACTGTGCTTT
EGR1	AGCCCTACGAGCACCTGAC	GGGCAGTCGAGTGGTTTG
SREBP2	CTCCATTGACTCTGAGCCAGGA	GAATCCGTGAGCGGTCTACCAT
Il6	CATCCTCGACGGCATCTCAG	GCAGAAGAGAGCCAACCAAC
TNF	CTCTTCTGCCTGCTGCACTTG	ATGGGCTACAGCTTGTCACTC
Il10	ACTGCTAACCGACTCCTTA	TAAGGAGTCGGTTAGCAGT
AMPK	AGGAAGAATCCTGTGACAAGCAC	CCGATCTCTGTGGAGTAGCAGT
NrF2	GAGAGCCCAGTCTTCATTGC	TGCTCAATGTCCTGTTGCAT
HO1	AAGCCGAGAATGCTGAGTTCA	CGGGTGTAGATATGGTACAAGGA
Occludin	CTCGAGAAAGTGCTGAGTGCCTGGAC	AAGCTTTCGGTGACCAATTCACCTGA
ZO-1	TATTATGGCACATCAGCACG	TGGGCAAACAGACCAAGC
PPAR	GGCTTCATGACAAGGGAGTTTC	AACTCAAACTTGGGCTCCATAAAG

3. Results

3.1. Characterisation of the Nanoparticles:

Graphene oxide is the material which is mainly made up with carbon and oxygen. Graphene oxide has hexagonal carbon structure which stacks with one another by π- π interaction which attributes to van der Waals interaction. C=C interaction were observed at near 224 nm. There is a bond between carbon and oxygen as well where oxygen tends to donate electron to form a stable structure which is n- π* interaction. This interaction can be observed in UV visible analysis near to 300 nm. It shows weak interaction that is attributed to C=O [20].

Fourier transform infrared spectroscopy (FTIR) is used to identify chemical bond present in the molecule. GO is mainly made up with carbon, oxygen and hydrogen. Together all three form various bonds which can be detected using FTIR. In FTIR analysis, bonds at 3334, 1734, 1627, 1230 and 1040 cm^-1 were observed. All are attributed to different chemical group stretching. Stretching near 3334 cm^-1 is attributed to carbonyl/ O-H stretching. Peak near to 1734 and 1627 cm^-1 are attributed to carboxyl and carbonyl group respectively. Band near to 1230 cm^-1 indicates the presence of C-O-C functional group. Vibration at 1040 cm^-1 indicates the activity of C-O group [21,22,23,24].

Further SEM and FESEM analysis of GO were done to observe structural orientation. In SEM images sheet like structure of GO was observed which was further analysed in FESEM. In FESEM, layered structured was visualised properly. Thickness was analysed using ImageJ software which showed thickness of the sheet near to 0.8 to 1nm. It showed a flaky morphology [25].

Figure 1. Characterization of GO. If there are multiple panels, they should be listed as: (a) Description of what is contained in the first panel; (b) Description of what is contained in the second panel. Figures should be placed in the main text near to the first time they are cited.

Figure 1. Characterization of GO. If there are multiple panels, they should be listed as: (a) Description of what is contained in the first panel; (b) Description of what is contained in the second panel. Figures should be placed in the main text near to the first time they are cited.

3.2. Activity in In Vitro Model:

3.2.1. Cell Viability Study:

Cells were treated with different concentration of GO ranging from 10 to 100 μg/mL. 100 mM ethanol was taken as negative control in which almost 30 to 40 % cell death observed for both the cell line while 100% viability was observed even at the highest concentration of GO treatment. To have proper results cells were washed with 1X PBS before treating with MTT.

Figure 2. A) % cell viability for HepG2 cells. B) % cell viability for Caco2 cells. Values represents the mean ± SD of six individual experiments.

Figure 2. A) % cell viability for HepG2 cells. B) % cell viability for Caco2 cells. Values represents the mean ± SD of six individual experiments.

3.2.1. Cell damage study:

A)

AO-EtBr and DAPI staining:

Cell damage was analysed using AO-EtBr staining and DAPI staining. AO EtBr analysis was done for HepG2 cells. Acridine orange and Ethidium bromide staining was used to detect live and dead cell. Dead cells were stained due to EtBr because of ruptured membrane [26]. The cells were counted in merged channel where first green fluorescence was taken and then red fluorescence was taken. In merged image, approximately 300 cells were counted. Form that 300 cells, in normal control group showed 10±5 dead cells but in ethanol control, 80±5 cells were observed red or orange in colour. It was seen that in treatment cells which were treated with both ethanol and nanoparticles showed decreased cell death.

DAPI staining is used to assess cell activity. HepG2 and Caco2 both cells were stained after 24hrs of treatment. Fluorescence images were analysed and fluorescence spectra were also measured. In both the cases, ethanol treated cells were more damaged than control cells but damage were decreased. In ethanol treated cells, chromatin condensation were increased. From 100 cells, 15±2 cells were damaged and showed higher fluorescence. Fluorescence of treatment cells were decreased and was almost near to control untreated cells. Furthermore, from approx 100 cells there were very less cells found damaged.

Figure 3. A) AO EtBr staining in HepG2 cell line. B) DAPI staining in HepG2 and Caco2 cell line. Relative fold change was analysed for both the cell lines. Statistical significance was carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. Values represents the mean ± SD of three individual experiments. The above figures are illustrative 10x objective images of three individual experiments.

Figure 3. A) AO EtBr staining in HepG2 cell line. B) DAPI staining in HepG2 and Caco2 cell line. Relative fold change was analysed for both the cell lines. Statistical significance was carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. Values represents the mean ± SD of three individual experiments. The above figures are illustrative 10x objective images of three individual experiments.

(B)

Lipid Accumulation Study:

Due to ethanol activity, lipid accumulation is increased in cells which can be observed via Oil Red O staining. Cells were analyzed microscopically by Nikon microscope and were further quantified using a spectrophotometer. Oil red O stain was extracted from cells by treating cells with 100 % isopropanol [18]. Then the extracted stain was quantified using spectrophotometer by taking absorbance at 570 nm. Due to ethanol treatment in both the cell type, lipid accumulation was increased. Increased lipogenesis was further confirmed by taking absorbance at 570nm. In treatment cells, lipid accumulation was decreased and it showed similar activity like control untreated cells. Relative fold change was done for absorbance and comparison were done in reference to ethanol treated cells.

Figure 4. Lipid accumulation. A) Relative fold change in absorbance were calculated for HepG2 cells and images was captured in phase contrast microscope. B) Relative fold change in absorbance was calculated for Caco2 cells and images were captured in phase contrast microscope. C) Images of HepG2 cell line at 10x. D) Images of Caco2 cell line at 40x. For both cell line, ethanol treated cells showed higher lipid accumulation. Statistical significance was carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. The above figures are illustrative 10x objective images of three individual experiments.

Figure 4. Lipid accumulation. A) Relative fold change in absorbance were calculated for HepG2 cells and images was captured in phase contrast microscope. B) Relative fold change in absorbance was calculated for Caco2 cells and images were captured in phase contrast microscope. C) Images of HepG2 cell line at 10x. D) Images of Caco2 cell line at 40x. For both cell line, ethanol treated cells showed higher lipid accumulation. Statistical significance was carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. The above figures are illustrative 10x objective images of three individual experiments.

(C)

Reactive oxygen spices analysis:

Cells exposed to ethanol produce an increased amount of reactive oxygen species (ROS) through the activation of cytochrome p450E1. The 2',7'-dichlorofluorescin diacetate (H2DCFDA) reagent can be used to analyse this phenomenon. H2DCFDA is a non-fluorescent compound that easily enters cells. In the presence of esterase, it is converted into a non-fluorescent compound called 2'7'-dichlorofluorescin. Due to the presence of ROS, these non-fluorescent compounds quickly transform into a highly fluorescent compound known as 2',7'-Dichlorodihydrofluorescein. This fluorescence can be detected using fluorescent spectroscopy at an excitation wavelength of 484/20 nm and an emission wavelength of 535/20 nm. In this study, a Perkin Elmer spectrophotometer was used to detect fluorescence. The ethanol control group exhibited higher fluorescence compared to the control/untreated group for both cell types, but this trend was reversed in the treatment group. The relative fold change was measured for both cell types, demonstrating that ethanol-treated cells exhibited greater fluorescence compared to the treatment group.

Figure 5. ROS estimation. A) Relative fold change in fluorescence of H2DCFDA stain for HepG2. B) Relative fold change in fluorescence of H2DCFDA stain for Caco2. Statistical significance were carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. Values represents the mean ± SD of three individual experiments.

Figure 5. ROS estimation. A) Relative fold change in fluorescence of H2DCFDA stain for HepG2. B) Relative fold change in fluorescence of H2DCFDA stain for Caco2. Statistical significance were carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. Values represents the mean ± SD of three individual experiments.

(D)

Gene Expression Analysis:

Excess ethanol in the body system causes various changes in gene expression, leading to increased inflammation. According to figure 6, treatment with ethanol in cells increases the expression of genes responsible for ROS production, fat accumulation, and inflammation, while treatment with GO decreases their expression. Additionally, ethanol downregulates lipolytic genes such as PPAR family protein, which were upregulated in the treatment group. Furthermore, ethanol increases the antioxidant activity of cells by upregulating the expression of AMPK, NrF2, and HO-1, which were downregulated due to ethanol exposure. It also increases IL10 anti-inflammatory regulation to reduce inflammation.

Ethanol-induced damage to the gut permeability results in secondary damage to the liver [28]. GO helps to restore gut permeability by upregulating the expression of tight junction proteins, which are downregulated due to ethanol exposure. Moreover, it decreases inflammation by reducing the activity of TNFα.

Figure 6. Gene expression study was carried for both cell line. A) Proinflammatory and lipolytic gene expression fold change for HepG2 cells. B) Anti-oxidant and Anti-inflammatory gene expression fold change for HepG2 cells. C) Tight junction activity of Caco2 cells. D) proinflammatory TNFα activity in Caco2 cells. (EGO1-ethanol+10 μg/mL GO, EGO2- ethanol+50 μg/mL GO). Statistical significance were carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. Values represents the mean ± SD of three individual experiments.

Figure 6. Gene expression study was carried for both cell line. A) Proinflammatory and lipolytic gene expression fold change for HepG2 cells. B) Anti-oxidant and Anti-inflammatory gene expression fold change for HepG2 cells. C) Tight junction activity of Caco2 cells. D) proinflammatory TNFα activity in Caco2 cells. (EGO1-ethanol+10 μg/mL GO, EGO2- ethanol+50 μg/mL GO). Statistical significance were carried out by two way ANOVA followed by multiple group comparison where ^*p<0.05, ^**p<0.01, ^***p<0.001 and ^****p<0.0001. Values represents the mean ± SD of three individual experiments.

4. Discussion

5. Conclusions

Figure 7. Possible protective mechanism of GO for ALD.

Figure 7. Possible protective mechanism of GO for ALD.

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