Modulatory Effects of Cilostazol; an Nrf2/HO-1 activator against NAFLD in Rats Con rmed by Molecular Docking and FTIR Studies

Nonalcoholic fatty liver disease (NAFLD) is a complex hepato-metabolic syndrome with multi-etiological pathways. No effective drugs have been settled for the effective therapy of NAFLD. The purpose of this study was to investigate the modulatory effects of cilostazol (CILO, 50 and 100 mg/kg.p.o.) against NAFLD induced by high fat diet rich in cholesterol (HFDCH) for 10 weeks. Thirty male Sprague dawely rats were divided into 4 groups (8 rat / group). Normal control group supplied with normal chow diet. Control positive group received high fat diet rich in cholesterol for 10 weeks. In addition, two CILO groups received (CILO, 50 and 100 mg/kg.p.o.). Oral administration of (CILO; 100 mg/kg) showed promising results in reducing fasting glucose and insulin levels. Moreover, CILO could reduce the elevated hepatic lipids, oxidative stress biomarkers and inflammatory cytokines. In addition, CILO succeeded to restore the total protein levels and activate nuclear factor erythroid-related factor 2/ heme oxygenase-1 (Nrf2/HO-1) activity. Furthermore, administration of CILO for NAFLD rats succeeded to show corrected and normalized FTIR spectra. We also investigated the plausible binding interactions of CILO with various biological targets using a molecular docking approach, and the results showed that CILO had an excellent docking energy score and significant binding interactions with the core amino acids involved in the active pocket for the enzymes studied. This study depicts that CILO exerted new intervention for NAFLD due to its complementary antihyperlipdemic, anti-inflammatory effects and antioxidant potential, via Nrf2/HO-1 activation.


Introduction
Nonalcoholic fatty liver disease (NAFLD) has a dramatically rising incidence in the western world and its prevalence continues to increase as a health crisis in society because hepatic lipid accumulation can trigger hepatocyte damage, inflammation and fibrogenesis (1).
NAFLD is strongly associated with the risk of more severe conditions such as atherosclerosis and cardiovascular diseases (2). Liver is the chief modulator of the biological pathways associated with the mechanism of cholesterol metabolism, cholesterol synthesis, uptake from chylomicron remnants, re-uptake from high-density lipoproteins (HDL) as well as the release of cholesterol under very-low density lipoproteins (VLDL), and biliary acids production. All of these pathways have a dominant effect on the regulation of the concentrations of plasma cholesterol and subsequently increasing the lipoprotein triglyceride (TGS) -particles in the blood, leading to fatty disposition in the peripheral tissues and liver. Inflammation and oxidative stress are most crucial precipitating factors associated with liver injury (3).
NAFLD induced by high fat diet rich in cholesterol (HFD-CH) was related primarily to an oxidative injury and inflammation (4). Under normal and oxidative conditions, regulation of redox hemostasis occurs chiefly at the nucleus, and the nuclear factor erythroid-related factor2 (Nrf2) / hemoxygenase-1 (HO-1) signaling pathway is an important mediator for modulation of such responses (5). The impairment in the cellular redox homeostasis causes deviation in the level of inflammatory mediators such as nuclear factor kappa-b (NF-κB) and tumor necrosis factor (TNF-α) which are required for repairing and regeneration of hepatocytes (6). Therefore, to avoid harmful oxidative conditions and to restore the redox homeostasis, activation of (Nrf2/HO-1) must being achieved (7). Hepatic injury is a detrimental factor for the severity of NAFLD. Several experimental and clinical attempts have been postulated for the therapy of the NAFLD, but none have shown prominent results on liver biomarkers and hepatic regeneration (8). Thus, urgent necessities for discovering safe therapeutic agents that can alleviate the precipitating factors and could reduce the hepatic accumulation of fats surrounding the liver are being mandatory.
Cilostazol (CILO) represents an attractive area in research due to its significant results in reducing reactive oxygen species (ROS) by acting as a non-enzymatic antioxidant (9). CILO is a quinolone derivative approved from FDA to treat intermittent claudication due to peripheral vascular disease (10). It was also used principally for the management of thrombotic diseases due to its anti-platelet activates. It also inhibits apoptosis and inflammatory changes in different models via decreasing the production of intracellular ROS (11).
Molecular Docking is a theoretical technique for studying protein-ligand interactions and recognition. Small molecule ligands are analysed in their interactions with receptor bio-macromolecules to find out how well they bind and how strong their affinity is, and then these interactions are used to develop structure-based drug design, which can be used to study molecular mechanisms of pharmacological effects, predict the structure of protein or ligandligand complexes, and allow targeted drug discovery to occur (12). The Increasing of computing and improving insights from computational chemistry approaches, the transformational influence of application of machine learning models to chemical sciences (13) . Therefore, this study was conducted to evaluate the possible modulatory effects of cilostazol against metabolic, biochemical and molecular alterations induced by high fat diet rich in cholesterol (HFD-CH) that mimics the pathophysiological features of NAFLD in human depending on Nrf2/HO-1 activation and its anti-inflammatory properties. Molecular docking studies help to predict the best active potential compounds against NAFLD proteins.

Experimental Animals
Male Sprague dewily rats weighing 120-150 g were used throughout the study. Rats were acclimatized to optimal environmental laboratory conditions provided by the animal research colony (National Research Centre, Cairo, Egypt). Rats were provided with standard laboratory diet ad libitum with free access to water. The study was conducted in accordance with the ethical standards approved by the Medical Research Ethics Committee (MREC) of the National Research Centre, Dokki, Egypt (No.13111).

Induction of NAFLD
Induction of NAFLD in rats by daily administration of high fat diet rich in cholesterol (HFD-CH) composed of (55% fat, 4% cholesterol powder, 20% carbohydrate and 21% protein, with equal quantities of minerals, vitamins and fibers (14). Cilostazol and cholesterol powder were purchased from sigma-Aldrich (USA).In addition, all other chemicals and kits were supplied from Randox.co, UK.

Experimental design
Thirty male sprague dawely rats, were divided into 4 groups (8 rat / group). Normal control group supplied with normal chow diet. Control positive group received high fat diet rich in cholesterol for 10 weeks. In addition to, two CILO groups received (CILO, 50 and 100 mg/kg.p.o.) for 2 weeks and continued for 8 weeks concurrent with high fat diet rich in cholesterol (15). At the end of the experiment, rats were fasted overnight and blood samples were collected from the retro-orbital plexus of ether-anesthetized animals and centrifuged (800×g, 4°C, 20 min) to separate serum that was analyzed for estimation of glucose and insulin levels.
Rats were sacrificed and liver tissues were excised, homogenized and divided into two parts. Part one is centrifuged at 4°C (4000 rpm/min, 5 min), and the supernatants were analyzed for determination of hepatic concentration of further biochemical indices. Part two is freeze dried and prepared for investigation of molecular alterations by the aid of fourier transform infrared (FTIR) spectroscopy.

Assessment of GSH and MDA levels in hepatic homogenate
The hepatic concentration of reduced glutathione (GSH) was assessed depending on a colorimetric reaction where reduction of 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB) by SH group of glutathione occurs forming 2-nitro-S-mercaptobenzoic acid. The forming product was measured spectrophotometrically at 412 nm (21). In addition, the hepatic content of lipid peroxidation product, expressed as malondialdehyde (MDA) was determined through a reduction reaction occurs between one molecule of malondialdehyde with two molecules of 2thiobarbituricacid under optimal conditions at pH, 3.5) forming pink coloured product that can be detected spectrophotometrically at 532 nm (22).

Assessment of NO, SOD and HO-1 levels in hepatic homogenate
Nitric oxide (NO) concentration was measured in liver homogenate via griess reaction , that involved in reduction of nitrate to nitrite, where with a mixture of naphthyl ethylene diamine and sulfanilamide was estimated spectrophotometrically at 540 nm (23).
Superoxide dismutase (SOD) activity was measured in liver homogenate at 420 nm by aspectrophotometer using colorimetric kit (24). Hepatic concentration of heme oxygenase-1 (HO-1) was measured spectrophotometry at 450 nm by using Eliza kit and HO-1 concentration was expressed as ng/mg tissue (25).

Analysis of fourier transform infrared spectroscopy (FTIR) spectra in hepatic tissue
FTIR spectroscopy is a reliable and non-invasive tool capable of providing strong vision on the functional and structural alterations of macromolecules within tissue cell (26).
Hepatic tissue samples were lyophilized for 24 h. An equal and small amount of dried liver tissue samples were gently mixed with potassium bromide (KBr) crystalls under a suitable pressure (1200 psi) for eight minutes to produce KBr pellets. In order to gain the same thickness of each pellet, samples were weighted and subjected to the same pressure. Pellets were scanned at 4 cm-1 within the mid-IR spectra (3200-400 cm-1) at room temperature and the spectra was recorded using a Perkin Elmer Spectrum (Perkin Elmer Inc, USA) equipped with a DTGS detector (27).

Statistical analysis
All statistical comparisons were done using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, and the findings are expressed as mean SEM (8) rats. Graph Pad Prism v. 8.0 was used to examine the data (GraphPad Software, Inc., CA, USA).
When the p value is less than 0.05, the difference is considered significant.

Computational analysis
The crystal structure of target proteins were retrieved from the protein data bank at http://www.rscb.org./pdb using codes as illustrated in Table 1

Effect of CILO on Blood Hg, Hct and MCHC concentrations in NAFLD rats
NAFLD in rats was induced by continuous daily consumption of HFD-CH for 10 weeks revealed no significant difference in the concentration of blood Hg, Hct and MCHC compared with normal control rats. oral treatment of rats with CILO (50 and 100 mg/kg) for 2 weeks and continued for 8 weeks concurrent with HFD-CH revealed also no significant difference in the concentration of blood Hg, Hct and MCHC compared with control positive group ( Table 2).

Effect of CILO on the serum levels of glucose, insulin, total lipids and total proteins in NAFLD rats
Adding high fat diet rich in cholesterol to rats for 10 consecutive weeks was associated with an increase in the levels of fasting serum glucose, insulin and total lipids reaching about 171%, 147% and149%, respectively, compared with normal healthy rats. oral treatment of rats with CILO (50 mg/kg) for 2 weeks and continued for 8 weeks concurrent with HFD-CH was associated with a decrease in the levels of fasting serum glucose, insulin and total lipids reaching about 73% , 89%, respectively. Administration of CILO (100 mg/kg) for NAFLD rats succeeded to restore the normal fasting glucose, insulin and total lipids levels ( Figure 1).
Induction of NAFLD in rats by continuous daily consumption of HFD-CH for 10 weeks showed a decrease in total protein levels reaching about 75%, compared with normal control group. oral treatment of rats with CILO (50 mg/kg) for 2 weeks and continued for 8 weeks concurrent with HFD-CH was associated with an increase in the levels of fasting serum total protein levels reaching about 121%, respectively, compared with HFD-CH received rats.
Administration of CILO (100 mg/kg) for NAFLD rats succeeded to restore the fasting normal total protein levels ( Figure 2).

Effect of CILO on serum AST, ALT, ALP levels and hepatic concentration of TGS and TC in NAFLD rats
Adding HFD-CH for 10 consecutive weeks was associated with an increase in the levels of serum AST, ALT, ALP levels, hepatic concentration of TGS and TC reaching about 6 folds, 3 folds, 6 folds, 153 % and 235% respectively. oral treatment of rats with CILO (50 mg/kg) for 2 weeks and continued for 8 weeks concurrent with HFD-CH was associated with a decrease in the levels of serum AST, ALT, ALP levels, hepatic concentration of TGS reaching about 35%, 50%, 32%, 72% , respectively and normalizing the levels of hepatic TC. Administration of CILO (100 mg/kg) for NAFLD rats succeeded to reduce the levels of serum AST, ALT, ALP levels, hepatic concentration of TGS reaching about 18%, 25%, 25%, respectively and normalizing the levels of hepatic TGS and TC (Table 3).

Effect of CILO on hepatic oxidative stress biomarkers in NAFLD rats
NAFLD in rats was induced by continuous daily consumption of HFD-CH for 10 weeks revealed an increase in the concentration of hepatic MDA and NO reaching about 250% and 5 folds . In addition, a decrease in hepatic GSH reaching about 36%, compared with normal control rats. oral treatment of rats with CILO (50 and 100 mg/kg) for 2 weeks and continued for 8 weeks concurrent with HFD-CH revealed a decrease in the concentration of hepatic MDA and NO reaching about 43% and 32%. In addition, an increase in hepatic GSH reaching about 32%, compared with control positive rats. Oral administration of CILO (100 mg/kg) for NAFLD rats succeeded to normalizing the levels of hepatic GSH, MDA and NO (Table 4).

Effect of CILO on hepatic levels of pro-inflammatory cytokines in NAFLD rats
Adding HFD-CH for 10 consecutive weeks was associated with an increase in the hepatic levels of TNF-α and NF-κB reaching about 364% and 453%respectively, compared with NAFLD group. oral treatment of rats with CILO (50 mg/kg) for 2 weeks and continued for 8 weeks concurrent with HFD-CH was associated with a decrease in the hepatic levels of TNF-α and NF-κB reaching about 50% and 29% , respectively, compared with control positive group.

Effect of CILO on hepatic levels of Nrf-2 expression and HO-1 levels in NAFLD rats
Administration of HFD-CH for 10 consecutive weeks for was associated with a decrease in the hepatic levels of Nrf-2 expression and HO-1 reaching about 21% and 40% respectively,

Effect of CILO on hepatic molecular alterations in NAFLD rats
Significant macromolecular bonding frequency regions are the characteristic features of the FT-IR spectra of hepatic specimens of HFD-CH. The most critical infrared absorbance bands are established between 1450 and 650 cm1, amide I and II region between 1700 and 1500 cm1, and C-H stretching region between3070 and 2800 cm1. The C-H stretching region exhibits infrared bands of lipids with a small contribution from carbohydrates and proteins. The spectral bands 3012, 2957, 2923, and 2853 cm1 are attributed to C¼CH, CH3 asymmetric, CH2 asymmetric, and CH2 symmetric stretch, respectively. Administration of CILO (100 mg/kg) for NAFLD rats succeeded to show corrected and normalized FTIR spectra (Figure 4).

Molecular docking study using MOE
Due to the current problems and intricate challenges faced by medicinal chemists, docking is a highly demanding and efficient discipline in order to rationally develop novel therapeutic compounds for treating human disease. In our study the potential of CILO as inhibitors for several biological targets including, peroxisome proliferator-activated receptor gamma (PPARγ), Peroxisome proliferator-activated receptor alpha (PPARα), heme oxygenase-1 (HO1), transcription factor NF-E2-related factor 2 (Nrf2) tumor necrosis factor-alpha (TNF-α), glutathione reductase, inducible nitric oxide synthase (iNOS) and catalase, was investigated using molecular docking technology. The binding affinity and interaction manners of the cilostazol with the selected enzymes were depicted in Table 1 Figure 5a,b).
In the interaction with PPARα (PDB ID: 3ET1, Table 1) , cilostazol formed two H-bonds acceptor with the amino acid TYR 334 (Figure 6a,b) and exhibited strong docking score of - acceptor with the core amino acid ARG 483 (Table 1, Figure 8a,b).
In the instance of tumor necrosis factor-alpha (TNF-α; PDB ID: 2AZ5), CILO displayed good interaction with the enzyme active pocket by forming four H-bonds donor and two pi-H interaction with the key amino acids GLY 121 and LEU 55 (Table 1, Figure 9a,b).

Discussion
NAFLD is a hepato-metabolic syndrome that is usually implicated in sever clinical and pathological disorders and is considered the major precipitating factors for insulin resistance, diabetes mellitus and cardiovascular diseases (36). NAFLD is asymptomatic hepatic manifestation that can progress to nonalcoholic steatohepatitis (NASH), hepatic fibrosis, cirrhosis, and hepatic cancer (37).Weight loss regimen and exercise are the only accepted treatments but are often a challengeable matter for NAFLD patients. Thus, there is an urgent necessity to explore safe agents for reducing the elevated hepatic lipids. According to the authors' knowledge, this is the primary study exploring the valuable role of Cilostazol, CILO; Nrf2/HO-1 activator against pathophysiological alterations induced by HFD-CH that parallel to NAFLD in human.
Our study showed that excessive consumption of HFD-CH for 10 weeks is a reliable and FTIR analysis of the CH2, CH3 stretching region (3000-2800 cm1) and ester carbonyl band at 1740 cm1 revealed the accumulation of lipids in NAFLD group (65). To determine the CH2/CH3 ratio, a number of bands were fitted to the CH stretching region (3000-2800 cm1).
The decrease in intensity of the CH3 stretching modes along with a concomitant increase in the CH2 stretching modes was indicative of oxidative processes occurring in response to NAFLD.
The change in the CH2/CH3 ratio is consistent with apoptosis (66). Oral treatment of rats received HFD-CH with CILO protected the hepatic molecular alterations due to its antioxidant and anti-lipid peroxidative potential in the liver via Nrf2/Ho-1 induction (67).
To the best of our knowledge, the current study is the first research for utilizing CILO biochemically and computationally as inhibitor for peroxisome proliferator-activated receptor gamma (PPARγ), Peroxisome proliferator-activated receptor alpha The overall results indicated that CILO had excellent binding energy score and strong binding interactions with the core amino acids involved in the active pocket for the investigated enzymes (Table 1). It is worthy to mention that, the tetrazole ring, NH and CO groups in CILO structure played a significant role in the binding interaction with the target enzymes as shown in  Figure 5a,b). While, molecular binding between CILO and PPARα (PDB ID: 3ET1) active site have established two H-bonds acceptor between carbonyl group and TYR 334 residue with docking score of -9.39 kcal/mol (Table 1, Figure 6a, Figure 7a,b) . Interaction of cilostazol with Nrf2 (PDB ID: 5CGJ) resulted in low binding energy of -8.06 kcal/mol, with good interaction with the active pockets through formation of two H-bonds with the core amino acid ARG 483 and carbonyl group (Table 1, Figure 8a,b).
While, the complex produced by CILO and TNF-(PDB ID: 2AZ5) was stabilized by formation four H-bonds and two pi-H interactions between the NH group, tetrazole ring and the essential amino acids GLY 121 and LEU 55 (Table 1, Figure 9a,b) Figure 11a,b).  Figure 12a,b).

Conclusions
Finally, we can conclude that the modulatory effect of cilostazol against nonalcoholic fatty liver disease induced by high fat diet rich may be attributed to its anti-hyperlipedemic activity, activation of Nrf2/HO-1 pathway, amelioration of oxidative stress, reducing the expression of pro-inflammatory cytokines. Thus, CILO may be a promising supplement to be used for patients with NAFLD in the future. In addition to molecular docking proved that CILO had strong binding interactions with the core amino acids involved in the active pocket for each investigated enzymes and excellent binding energy score.                    It has not been published elsewhere and that it has not been submitted simultaneously for publication elsewhere.

Declarations
NAFLD is a hepato-metabolic syndrome that is usually implicated in sever clinical and pathological disorders and is considered the major precipitating factors for insulin resistance, diabetes mellitus and cardiovascular diseases. NAFLD is asymptomatic hepatic manifestation that can progress to nonalcoholic steatohepatitis (NASH), hepatic fibrosis, cirrhosis, and hepatic cancer. Our study revealed the modulatory effect of cilostazol against nonalcoholic fatty liver disease induced by high fat diet rich may be attributed to its anti-hyperlipedemic activity, activation of Nrf2/HO-1 pathway, amelioration of oxidative stress, reducing the expression of pro-inflammatory cytokines. Thus, CILO may be a promising supplement to be used for patients with NAFLD in the future.
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