The Synergistic Protective Effect of γ-Oryzanol (OZ) and N-Acetylcysteine (NAC) against Experimentally Induced NAFLD in Rats Entails Hypoglycemic, Antioxidant, and PPARα Stimulatory Effects

This study estimated that the combined effect of γ-Oryzanol and N-acetylcysteine (NAC) against high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats also estimated some of their mechanisms of action. Adult male rats were divided into seven groups (n = 8 each) as control, control + NAC, control + γ-Oryzanol, HFD, HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanol. NAC was administered orally at a final concentration of 200 mg/kg, whereas γ-Oryzanol was added to diets at a concentration of 0.16. All treatments were conducted for 17 weeks and daily. Both NAC and γ-Oryzanol were able to reduce final body weights, fat weights, fasting glucose, fasting insulin, serum, and serum levels of liver function enzymes as well as the inflammatory markers such as tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6), and leptin in HFD-fed rats. They also improved hepatic structure and glucose tolerance, increased adiponectin levels, and reduced serum and hepatic levels of triglycerides (TGs) and cholesterol (CHOL) in these rats. These effects were concomitant with a reduction in the hepatic levels of lipid peroxides (MDA) and serum levels of LDL-C, but also with an increment in the hepatic levels of superoxide dismutase (SOD) and glutathione (GSH). Interestingly, only treatment with γ-Oryzanol stimulated the mRNA levels of proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) in the liver and white adipose tissue (WAT) of rats. Of note, the combination therapy of both drugs resulted in maximum effects and restored almost normal liver structure and basal levels of all the above-mentioned metabolic parameters. In conclusion, a combination therapy of γ-Oryzanol and NAC is an effective therapy to treat NAFLD, which can act via several mechanisms on the liver and adipose tissue.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is a common liver disease that is associated with obesity, insulin resistance (IR), and diabetes mellitus (DM) and is characterized by excessive lipid accumulation in the liver [1]. The severity of the diseases ranges from simple steatosis, which rapidly progresses into more advanced stages, including nonalcoholic steatohepatitis (NASH), fibrosis, apoptosis, and hepatic carcinoma [2]. To date, the precise mechanism responsible for the development of NAFLD and NASH is still not completely understood, but was identified as a crosstalk mechanism that includes numerous organs-including the liver, adipose tissue, and muscle-and involves hormonal disturbances, IR, and impaired adipose [3].
Hepatic steatosis and insulin resistance, hyperlipidemia, fasting hyperglycemia, and hepatocyte damage are clinical features in patients and animals with NAFLD. However, increased de novo lipogenesis and gluconeogenesis, overproduction of reactive oxygen

Experimental Design
The animals were adapted for 1 week and then randomly segregated into 7 groups (n = 8 each) (1) STD group: Fed STD and orally treated 5% CMC as a vehicle; (2) STD + NAC-treated rats: fed STD and orally administered NAC (200 mg/kg) dissolved in 5% CMC. (3) STD-ORL-fed rats: fed STD containing 0.16% γ-Oryzanol; (4) HFD-fed rats: fed HFD and orally administered 5% CMC, (5) HFD + NAC-treated rats: fed HFD and orally administered NAC solution (200 mg/kg); (6) HFD + OZ-fed rats: fed HFD containing 0.1% γ-Oryzanol; and (7) HFD + NAC + OZ-fed rats: fed HFD containing 0.1% γ-Oryzanol and co-treated with NAC (200 mg/kg). All experiments were continued for 17 weeks. NAC and vehicle were given by gavage. Changes in body weight and food/calorie intake were calculated every 2 weeks. In our preliminary data (not shown), treating the control rats with a combination of OZ and NAC for 4 weeks resulted in significant hypoglycemia in rats which prevented us from giving both treatments to the control rats.

Dose Selection
The regimen and dose of γ-Oryzanol (0.16%) were based on a recent study by Wang et al. [40], who reported a potent ability of this compound to attenuate liver weights, hepatic steatosis, and hyperglycemia in HFD-fed rats. In addition, the dose of NAC (200 mg/kg) was adopted from another study in rats which showed protective potential against HFD-mediated NAFLD [41].

Oral Glucose Tolerance Test (OGTT)
On the last day of the feeding protocol, each rat of every group was fasted for 12 h and then exposed to the OGTT procedure [42,43]. In brief, the rats were orally treated with glucose solution (2 g/kg glucose), and then 0.25 mL EDTA-blood samples were collected from the tail at baseline (0.0 min) and after different time intervals (30,60,90, and 120 min). All blood samples were centrifuged at 1100× g, and supernatants were directly used to measure plasma glucose (Cat No. 10009582, Cayman chemicals, Ann Arbor, MI, USA) and insulin levels (Cat. No. 589501, Ann Arbor, MI, USA). Furthermore, the IR homeostasis model assessment (HOMA) was calculated according to the following equation: HOMA-IR = ([glucose (mg/dL) × insulin (ng/mL)]/405). All analyses were conducted for n = 8 samples per group.

Blood and Tissue Sampling
Two days after the OGTT, the rats were fasted again for 12 h and then anesthetized with a ketamine/xylazine solution at a ratio of 80:10 mg/mg. One ml of blood was collected from each rat using the cardiac puncture into plain tubes and used to separate the serum (1100× g/10 min/room temperature). Euthanasia was achieved by cervical dislocation. In addition, livers were dissected, weighed, and cut into small pieces. White adipose tissue (WAT) pads-including the inguinal, epididymal, peritoneal, and mesenteric-were identified, separated, and weighed. All tissues were then stored at −80 • C until further use. Parts of the liver of each rat were fixed directly in 10% formalin and sent to the pathology lab for further histological analysis.

Biochemical Analysis in the Serum
Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transpeptidase (GGT) were measured in the serum of each rat using the rats' special ELISA kit and as per instructions (Cat. No. MBS264975; Cat. No MBS269614; Cat. No. MBS9343646, and MyBioSorces, San Diego, CA, USA; respectively). All analyses were conducted for n = 8 rats/group.

Biochemical Analysis of Lipids of All Fractions
Stool from each rat was collected during the last week using metabolic cages. Lipids were extracted from the livers and stools using the methanol/chloroform method established by Folch et al. [44]. Serum, hepatic, fecal TGs, and CHOL were measured using assay kits (Cat. No. ECCH-100, BioAssay Systems, Hayward, CA, USA and Cat. No. 10009582, Cayman Chemicals, Ann Arbor, MI, USA). Serum levels of FFAs, high-density lipoproteincholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were measured using the following assay kits (Cat. No. MBS014345, MyBioSource, San Diego, CA, USA, Cat. No. STA-394, cell Biolabs, San Diego, CA, USA Cat. No. 79960; Crystal Chemicals, Houston, TX, USA). All procedures were conducted for n = 8 rats/group.

Biochemical Analysis in the Liver Homogenates
Frozen liver samples were homogenized in isotonic solution and centrifuged at 1200× g/10 min/4 • C. The supernatant of each sample was frozen at −80 •  . This protocol extracted the total RNA using the Qiagen extraction (Cat. No. 74004). The purity of the RNA was determined using the absorbance 260/280. The first-strand cDNA was synthesized using the supplied commercial kit (Cat. No. K1621 ThromoFisher Waltham, MA, USA, respectively). Amplification of mRNA was conducted using the Ssofast Evergreen Supermix kit (Cat. No. 172-5200, BioRad, Hercules, CA, USA) and Bio-Rad qPCR amplification (model CFX96) as instructed by the kit. The following steps were followed for each target: (1) heating (1 cycle/98 • C/30 s), (2) denaturation (40 cycles/98 • C/5 s), (3) annealing (40 cycles/60 • C/5 s), and (4) melting (1 cycle/95 • C/5 s/step). The relative mRNA expression of PPARα was presented after the normalization of GAPDH using the 2∆∆CT method. All procedures were performed as instructed by the kit manufacturer's instructions.

Histopathological Evaluation
The livers were dehydrated in xylene and alcohol of decreasing concentrations-i.e., 100%, 90%, and 70%. The tissue was then placed in wax and cut with a microtome into slices of 3-5 µM thickness. All tissue slices were stained with Harris haematoxylin (H)/glacial acetic acid solution, de-stained with 1:400 v/v HCL/ethanol (70%) solution, and then stained with eosin (E). Further, the tissue slices were then dehydrated with ethanol and xylene. A mounting media was added, and the tissue slice was covered with a coverslip. The next day, all tissue was examined under a light microscope and photographed at 200×.

Statistical Analysis
All data were analyzed using the GraphPad Prism analysis software (version 8, San Diego, CA, USA). The normality of the data will be tested using the Kolmogorov-Smirnov test. The one-way ANOVA test was used for the analysis using Tukey's test as post hoc (p < 0.05). All data were expressed in the results as means ± standard deviation (SD).

Changes in Calorie Intake, Fat Deposits, and Body Weights
Food consumption and calorie intake were measured over the 12 weeks of the study. HFD-fed rats showed a significant and progressive increase in food intake from week 3 to week 12, as compared to control rats, NAC, or γ-Oryzanol ( Figure 1A,B). They also had significantly higher weights of mesenteric, peritoneal, subcutaneous, and epidydimal fats, as well as final body weights (Figure 2A-D and Table 1). HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanol showed a significant reduction in the calorie intake during the whole weeks of the study ( Figure 1A,B) and all their fat pads, as well as final body weights, were significantly lower than those HFD-fed rats ( Table 1). However, the maximum reduction in all these measures was seen in the HFD + NAC + γ-Oryzanol-treated rats, with levels that were not significantly varied with the control rats ( Figures 1A,B and 2A-D, and Table 1).

Changes in Fasting Glucose, Insulin, and Glucose Tolerance
HFD-fed rats showed a significant increase in fasting glucose levels and the concentration of glucose measured at 15, 30, 60, 90, and 120 min after the OGTT, as compared to control rats ( Figure 1C,D, Table 1). They also had significantly higher levels of insulin and HOMA-IR (Table 1). Serum glucose levels measured during the OGGT, fasting glucose and insulin levels intervals and levels of HOMA-IR were not significantly varied between the control and control + NAC-treated rats but showed a significant reduction in control + γ-Oryzanol-treated rats ( Figure 1C,D, Table 1). A significant decrease was seen in HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanol-treated rats ( Figure 1C,D, Table 1). Of note, only standard and non-significant glucose curves-as well as normal levels of fasting glucose, fasting insulin, and HOMA-IR, like those seen in the control rats-were seen in HFD + NAC + γ-Oryzanol-treated rats ( Figure 1C,D, Table 1).

Changes in SBP and Serum Parameters
Systolic blood pressure (SBP)-as well as serum levels of leptin, TNF-α, IL-6, FFAs, AST, ALT, and GTT-were significantly increased, but serum levels of adiponectin were significantly decreased in the HFD-fed rats as compared to all control groups (Tables 1 and 2). The levels of all these parameters were significantly reversed in HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanol-treated rats with normal basal levels to be seen in the latter group (Tables 1 and 2). Interestingly, no significant variations in the majority of all these markers were seen between the control, NAC, and γ-Oryzanol-treated control rats. Only adiponectin showed a significant increase, and FFAs showed a significant decrease in the NAC-treated control rats as compared to the control group (Tables 1 and 2).  The value of significance was determined at p < 0.05. a: significantly different as compared to the control group; b: significantly different as compared to the NAC-treated control rats; c: significantly different as compared to the γ-oryzanol-treated rats; and d: significantly different as compared to the HFD-fed group; e: significantly different as compared to the HFD + NAC-treated rats; and f: significantly different as compared to the HFD + γ-oryzanol-treated rats. The value of significance was determined at p < 0.05. a: significantly different as compared to the control group; b: significantly different as compared to the NAC-treated control rats; c: significantly different as compared to the γ-Oryzanol-treated rats; and d: significantly different as compared to the HFD-fed group; e: significantly different as compared to the HFD + NAC-treated rats; and f: significantly different as compared to the HFD + γ-Oryzanol-treated rats. Data are presented as means ± SD (n = 8/group). The value of significance was determined at p < 0.05. a: significantly different as compared to the control group; b: significantly different as compared to the NAC-treated control rats; c: significantly different as compared to the γ-Oryzanol-treated rats; and d: significantly different as compared to the HFD-fed group; e: significantly different as compared to the HFD + NAC-treated rats; and f: significantly different as compared to the HFD + γ-Oryzanol-treated rats. Data are presented as means ± SD (n = 8/group). The value of significance was determined at p < 0.05. a: significantly different as compared to the control group; b: significantly different as compared to the NAC-treated control rats; c: significantly different as compared to the γ-Oryzanol-treated rats; and d: significantly different as compared to the HFD-fed group; e: significantly different as compared to the HFD + NAC-treated rats; and f: significantly different as compared to the HFD + γ-Oryzanol-treated rats. AST: aspartate aminotransferase (AST); ALT: alanine aminotransferase; and GTT: gamma-glutamyl transpeptidase.

Changes in Serum, Hepatic, and Stool Lipid Profile
Various lipid fractions were measured in the stools, livers, and blood of all groups of rats (Table 3). There was a significant increment in the serum, hepatic, and fecal levels of TGs and GHOL in HFD-fed rats as compared to control rats (Table 3). They also had significantly higher serum levels of LDL-C and hepatic levels of FFAs but low levels of HDL-c (Table 3). A reversal of these lipid levels in the liver and serum was confirmed in the HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanol-treated rats (Table 3). Among all these groups, normal basal hepatic and serum levels of TGs and CHOL, as well as normal serum levels of LDL-C and HDL-c, were seen only in the HFD + NAC + γ-Oryzanol-treated rats (Table 3). However, fecal TGs and CHOL were not significantly varied between the control HFD rats and HFD-fed groups of all treatments, as well as between the STD-fed, NAC, and γ-Oryzanol-treated control rats (Table 3). It remains worth mentioning that serum and hepatic levels of TGs and CHOL, as well as serum levels of LDL-C, were significantly lower, but serum levels of HDL-C were significantly higher in both the NAC and γ-Oryzanol-treated control rats, as compared to control rat-fed the STD (Table 3).

Changes in Hepatic Antioxidant and Inflammatory Markers
Hepatic levels of TNF-α, IL-6, and MDA were not significantly different, but levels of SOD and GSH were significantly higher in NAC and γ-Oryzanol-treated control when compared to the control group of STD-fed rats ( Figure 3). However, HFD-fed rats showed a significant increment in the hepatic levels of TNF-α, IL-6, and MDA and showed a significant decline in the levels of GSH and SOD, as compared to all control groups (Figure 3). A significant reduction in the levels of TNF-α, IL-6, and MDA that is parallel with a significant increase in the levels of SOD and GSH was seen in the livers of HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanol-treated rats, as compared to HFD-fed ( Figure 3). In line with all other results, normal basal hepatic levels of all these biochemical endpoints were only seen in the HFD + NAC + γ-Oryzanol-treated rats ( Figure 3). Table 3. γ-Oryzanol (OZ) and N-acetylcysteine (NAC) and their combination improve lipid profile in the serum and livers of high-fat-diet (HFD)-fed rats. Hepatic levels of TNF-α, IL-6, and MDA were not significantly different, but levels of SOD and GSH were significantly higher in NAC and γ-oryzanol-treated control when compared to the control group of STD-fed rats (Figure 3). However, HFD-fed rats showed a significant increment in the hepatic levels of TNF-α, IL-6, and MDA and showed a significant decline in the levels of GSH and SOD, as compared to all control groups ( Figure  3). A significant reduction in the levels of TNF-α, IL-6, and MDA that is parallel with a significant increase in the levels of SOD and GSH was seen in the livers of HFD + NAC, HFD + γ-oryzanol, and HFD + NAC + γ-oryzanol-treated rats, as compared to HFD-fed ( Figure 3). In line with all other results, normal basal hepatic levels of all these biochemical endpoints were only seen in the HFD + NAC + γ-oryzanol-treated rats ( Figure 3).

Changes in mRNA Levels of PPARα and CPT-1
A significant increase in the mRNA levels of PPARα and CPT-1 was seen in γ-Oryzanoltreated control rats as compared to control (STD) rats ( Figure 4A-D). No significant variations in the mRNA of these genes were seen between the STD-fed and NAC-treated control rats ( Figure 4A-D). Hepatic mRNA levels of PPARα and CPT-1 ( Figure 4A,C) and WAT mRNA levels of PPARα and CPT-1 ( Figure 4B,D) were significantly decreased in the livers and WAT ( Figure 4B,D) of HFD-fed rats as compared to control, γ-Oryzanol, and NAC-treated control rats. On the contrary, hepatic mRNA levels of PPARα and CPT-1 ( Figure 4A,C) and WAT mRNA levels of PPARα and CPT-1 ( Figure 4B,D) were significantly increased in the livers HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanoltreated rats as compared to HFD-fed rats. The maximum reduction in the expression of both genes was seen in the livers of HFD + NAC + γ-Oryzanol, as compared to HFD + NAC and HFD + γ-Oryzanol-treated rats ( Figure 4A-D). cantly increased in the livers HFD + NAC, HFD + γ-oryzanol, and HFD + NAC + γ-oryzanol-treated rats as compared to HFD-fed rats. The maximum reduction in the expression of both genes was seen in the livers of HFD + NAC + γ-oryzanol, as compared to HFD + NAC and HFD + γ-oryzanol-treated rats ( Figure 4A-D).

Histopathological Studies
Livers from control, NAC, and γ-OZ showed normal liver features, including central veins, sinusoids, and hepatocytes ( Figure 5A-C). On the other hand, increased cytoplasmic lipid vacuolations with an increased number of cells with karyolysis, pyknosis, and karyorrhexis were seen in the livers of HFD-fed animals ( Figures 5D and 6A). HFD co-treated with NAC or γ-OZ, as well as those co-treated with a combined dose of NAC ( Figure 6B) and γ-OZ ( Figure 6C), as well as those co-treated with both NAC and γ-OZ ( Figure 6D) showed improvement in the structure of the hepatocytes with a significant reduction in the number of and size of cytoplasmic vacuoles. In addition, these tissues showed an increased number of normal hepatocytes. Almost normal tissue features were seen in the combined treatment group.

Histopathological Studies
Livers from control, NAC, and γ-OZ showed normal liver features, including central veins, sinusoids, and hepatocytes ( Figure 5A-C). On the other hand, increased cytoplasmic lipid vacuolations with an increased number of cells with karyolysis, pyknosis, and karyorrhexis were seen in the livers of HFD-fed animals (Figures 5D and 6A). HFD cotreated with NAC or γ-OZ, as well as those co-treated with a combined dose of NAC ( Figure 6B) and γ-OZ ( Figure 6C), as well as those co-treated with both NAC and γ-OZ ( Figure 6D) showed improvement in the structure of the hepatocytes with a significant reduction in the number of and size of cytoplasmic vacuoles. In addition, these tissues showed an increased number of normal hepatocytes. Almost normal tissue features were seen in the combined treatment group.  were taken from control, NAC, and + γ-Oryzanol-treated animals, respectively, and showed normal liver pathology, including normal central vein (CV) in which normally appearing hepatocytes are radiating (long white arrow). In addition, these cells had normally sized sinusoids (short white arrow). (D): was taken from an HFD-fed animal and showed a typical image of liver steatosis. The CV was greatly enlarged with evidence of some cell necrosis around it (short white arrow). The majority of the cell cytoplasm was filled with fat droplets (long black arrow). In addition, karyolysis (black arrowhead), pyknosis (short arrow), and karyorrhexis (short black arrow) in a majority of the hepatocytes (curved black arrow) were dominant and in large numbers. Immune cell infiltration was also seen (large white arrow). cytoplasm was filled with fat droplets (long black arrow). In addition, karyolysis (black arrowhead), pyknosis (short arrow), and karyorrhexis (short black arrow) in a majority of the hepatocytes (curved black arrow) were dominant and in large numbers. Immune cell infiltration was also seen (large white arrow).

Figure 6.
Liver sections from HFD model rats and other HFD treated groups: (A): was taken from HFD-fed animals and showed increased fat accumulation in the cell cytoplasm and the appearance of large fat vacuoles (long black arrow). Note the increased number of cells with karyolysis (black arrowhead), pyknosis (short arrow), and karyorrhexis (short black arrow). In addition, immune cell infiltration was evident (white arrow). (B,C): were taken from HFD + NAC and HFD + γ-oryzanoltreated animals, respectively, and showed an obvious improvement in liver structure with an increased number of normal cells (Blue arrow) and sinusoids (small curved arrow). However, some pathological findings, including swollen cells (long black arrowhead), cells filled with fat (long large arrow), dilated sinusoids (large, curved arrow), and necrotic cells (very long black arrow), are still seen. (D): was taken from an HFD + γ-oryzanol-treated rat and showed almost normal liver features like the control group. The majority of the cells in the field (long black arrow), as well as the sinusoids (short curve arrow), appeared normal. However, some necrotic cells (black arrowhead) and dilated sinusoids (long curved arrow) are still present but were few. Figure 6. Liver sections from HFD model rats and other HFD treated groups: (A): was taken from HFD-fed animals and showed increased fat accumulation in the cell cytoplasm and the appearance of large fat vacuoles (long black arrow). Note the increased number of cells with karyolysis (black arrowhead), pyknosis (short arrow), and karyorrhexis (short black arrow). In addition, immune cell infiltration was evident (white arrow). (B,C): were taken from HFD + NAC and HFD + γ-Oryzanol-treated animals, respectively, and showed an obvious improvement in liver structure with an increased number of normal cells (Blue arrow) and sinusoids (small curved arrow). However, some pathological findings, including swollen cells (long black arrowhead), cells filled with fat (long large arrow), dilated sinusoids (large, curved arrow), and necrotic cells (very long black arrow), are still seen. (D): was taken from an HFD + γ-Oryzanol-treated rat and showed almost normal liver features like the control group. The majority of the cells in the field (long black arrow), as well as the sinusoids (short curve arrow), appeared normal. However, some necrotic cells (black arrowhead) and dilated sinusoids (long curved arrow) are still present but were few.

Discussion
This study demonstrates that the combination therapy of both γ-Oryzanol and NAC is an effective treatment to ameliorate NAFLD and its metabolic abnormalities (e.g., hyperlipidemia, hyperglycemia, hypertension, and IR) in rats. The novelty of this study also shows a synergistic mechanism by which NAC and γ-Oryzanol collaborate to protect such hepatic and steatosis effects by attenuating HFD-mediated oxidative stress and inflammation. In addition, γ-Oryzanol adds a powerful effect mediated by its potent hypoglycemic effect and its ability to modulate de novo lipogenesis by stimulating PPARα/CPT-1 induced FA oxidation. A full mechanism of action is shown in the graphical abstract (Figure 7).

Discussion
This study demonstrates that the combination therapy of both γ-oryzanol and NAC is an effective treatment to ameliorate NAFLD and its metabolic abnormalities (e.g., hyperlipidemia, hyperglycemia, hypertension, and IR) in rats. The novelty of this study also shows a synergistic mechanism by which NAC and γ-oryzanol collaborate to protect such hepatic and steatosis effects by attenuating HFD-mediated oxidative stress and inflammation. In addition, γ-oryzanol adds a powerful effect mediated by its potent hypoglycemic effect and its ability to modulate de novo lipogenesis by stimulating PPARα/CPT-1 induced FA oxidation. A full mechanism of action is shown in the graphical abstract ( Figure  7).

Figure 7.
A graphical abstract showing the synergistic mechanistic effect of γ-oryzanol and N-acetylcysteine (NAC) against high-fat diet (HFD) induced non-fatty liver. In the figure, HFD promotes inflammation and insulin resistance (IR) and downregulates PPARα in the white adipose tissue (WAT). This activates lipogenesis in WAT, which increases the influx of inflammatory cytokines and free fatty acids (FFAs) to the liver. It also increases the release of leptin and lowers those of adiponectin. Fruthermore, the influx of dietary FFAs increases in the liver of rats. As a result of all these factors, the generation of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) are stimulated in the livers of rats which scavenge major antioxidants such as glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). In addition, the levels PPARα are decreased, which stimulates the hepatic de novo lipid synthesis and results in hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). In the presence of low antioxidant power and high levels of ROS and inflammatory cytokines, the disease can progress to non-alcoholic steatohepatitis (NASH) and liver failure. NAC inhibits oxidative stress and inflammation by stimulating major antioxidant proteins, CAT, SOD, and GSH. On the other hand, γ-oryzanol stimulates GSH, SOD, and CAT and stimulates the levels of PPARα and both the liver and WAT. This activates lipogenesis in WAT, which increases the influx of inflammatory cytokines and free fatty acids (FFAs) to the liver. It also increases the release of leptin and lowers those of adiponectin. Fruthermore, the influx of dietary FFAs increases in the liver of rats. As a result of all these factors, the generation of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) are stimulated in the livers of rats which scavenge major antioxidants such as glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). In addition, the levels PPARα are decreased, which stimulates the hepatic de novo lipid synthesis and results in hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). In the presence of low antioxidant power and high levels of ROS and inflammatory cytokines, the disease can progress to non-alcoholic steatohepatitis (NASH) and liver failure. NAC inhibits oxidative stress and inflammation by stimulating major antioxidant proteins, CAT, SOD, and GSH. On the other hand, γ-Oryzanol stimulates GSH, SOD, and CAT and stimulates the levels of PPARα and both the liver and WAT. Increased calorie intake and chronic feeding HFD promote metabolic syndrome (MetS), which is the most known risk factor for the development and progression of NAFLD and NASH [45][46][47]. HFD feeding of the rats of this study resulted in features of metabolic syndrome-including obesity, dyslipidemia, fasting hyperglycemia, hyperinsulinemia, IR, and hypertension-which validate our animal model and support other studies [1,48,49]. On the other hand, individual or combined treatment of both γ-Oryzanol and NAC attenuated all these metabolic disturbances induced by HFD. In addition, they also attenuated hepatic steatosis and reduced hepatic fat accumulation in the model rats. Furthermore, both drugs prevented the gain in fat deposits and significantly reduced the calorie intake and final body weights of these HFD-fed rats. Hence, we have concluded that both NAC and γ-Oryzanol are excellent adipogenic, anti-obesity, and anti-steatotic molecules that can prevent the development and progression of NAFLD. Interestingly, we have shown an exceptional ability of NAC and γ-Oryzanol to reduce glucose, insulin, hepatic and serum levels of TGs, and CHOL, even in control rats that fed the STD, thus confirming their dependent hypoglycemic and hypolipidemic effects.
These findings support others who have found similar hypoglycemic, hypolipidemic, insulin-improving, and anti-steatosis effects of γ-Oryzanol in sucrose, fructose, and HFDfed rats, possibly due to their effect to reduce fecal lipid excretion (inhibiting intestinal lipase), inhibit gluconeogenesis enzymes (e.g., G6PD), and suppress lipogenic enzymes (e.g., malic enzyme, SREBP-1c, and fatty acid synthase) [40,50,51]. In addition, γ-Oryzanol reduced the body weights of obese rodents and inhibited the differentiation and increase in the size of human cultured adipocytes [50,52]. It also lowered blood pressure and prevented hepatic cirrhosis in hypercholesterolemia-spontaneous hypertensive rats by reversing dyslipidemia [53]. In the same line, NAC has an independent hypolipidemic effect mediated by downregulating the hepatic FAs receptors (D36), SREBP1/2, and PPARγ [54,55]. Treatment with NAC attenuated fasting hyperglycemia, IR, and hypertension and improved peripheral insulin sensitivity in HFD and IR animal models [55][56][57]. It is noteworthy that fecal lipid levels of TGs and CHOL were not significantly different between HFD rats that received the vehicle or the individual or combined treatment of NAC and γ-Oryzanol, thus dissipating their effect on intestinal lipid absorption and contradicting those reported by Francisqueti et al. [39].
Leptin and adiponectin are the two major hormones released from adipose tissue. Leptin is the polyphagia-related hormone that stimulates food intake, whereas adiponectin is an anti-adipogenic hormone that improves insulin signaling and FAs oxidation and suppresses FA oxidation [58,59]. Low levels of adiponectin or adiponectin resistance that is concomitant with a sustained increase in leptin levels were seen in obese and NAFLD animals and subjects [60]. However, higher adiponectin levels protected against NAFLD in rats by modulating glucose and lipid metabolism, as well as insulin signaling, oxidative stress, and inflammation [61]. In this study, we have also seen higher leptin and low adiponectin levels in the sera of HFD-fed rats. Such an increase in leptin levels could explain the increase in food and calorie intake of these rats during the whole period of the study. On the other hand, the reduction in circulatory adiponectin could be considered an extra mechanism that worsens hepatic inflammation, oxidative stress, lipogenesis, and IR [61]. Interestingly, the partial reversal in the levels of these hormones in the HFD rats after γ-Oryzanol or NAC therapy may explain why these rats showed a reduction in food/calorie intake and could partially be attributed to their hepatoprotective effects. It is worth noting that treatments with γ-Oryzanol not only stimulated the release of adiponectin in HFD-fed rats but also in those fed the STD, indicating an interesting mechanism of action. Similarly, γ-Oryzanol also restored the expression and length of adiponectin levels in a stress-induced model of hypoadiponectinemia [62].
On the other hand, the increased WAT lipolysis due to IR is the major mechanism by which NAFLD develops in obese individuals and HFD-fed animals [3,4]. IR is the key player in the process that promotes hepatic oxidative stress and inflammation, mainly by increasing the influx of FFAs and cytokines from the impaired adipose tissue [3,4,6,7]. Antioxidants and anti-inflammatory agents protect against NAFLD and prevent its progression to nonalcoholic steatohepatitis (NASH) [63,64]. Levels of IL-6 and TNF-α, as well as markers of lipid peroxidations and reduced endogenous antioxidants, were estimated in the serum and livers of NAFLD animals and subjects [65]. This study also saw a similar mirror image in the livers of HFD-fed rats. Indeed, NAC and γ-Oryzanol reduced the contents of TNF-α, IL-6, and MDA but stimulated SOD and GSH in the livers of the control and HFD-fed rats. These data indicate individual and independent antioxidant and anti-inflammatory potentials of NAC and γ-Oryzanol. This can be supported by the numerous reports showing the dependent-antioxidant potential of both drugs [52,66].
Indeed, NAC is a precursor of GSH that can prevent tissue damage in a variety of disorders by boosting GSH, scavenging ROS, and suppressing inflammatory cytokine generation. NAC also prevented NAFLD in HFD, cholesterol-fed, methionine-cholinedeficient (MCD) animal models by its ability to reduce hepatic lipogenesis and alleviate oxidative stress and inflammation by boosting GSH and antioxidant enzymes [67]. Furthermore, NAC reduced the expression of TNF-α, IL-6, and IL-1β by suppressing NF-κB [68]. Likewise, γ-Oryzanol stimulated hepatic levels of GSH and SOD, CAT, glutathione peroxidase (GPx), and glutathione reductase (GR) and reduced lipid peroxidation in HFD and sucrose-fed animals [50]. Such antioxidant potential of γ-Oryzanol was explained by its exceptional ability to donate hydrogen from its ferulic acid constituent, as well as by its ability to inhibit iron-driven hydroxyl radical formation [50,[69][70][71]. Additionally, γ-Oryzanol prevented neuromotor defects and reduced dopamine by suppressing lipid peroxidation and oxidative stress by upregulating SOD, CAT, and glutathione-S-transferase in the Drosophila melanogaster model of Parkinson's disease [72]. Moreover, Islam et al. [73] demonstrated that this molecule could prevent inflammatory colitis in rats by suppressing the transcription of different inflammatory cytokines (i.e., IL-6, IL-1β, TNF-α) and decreasing leukocyte infiltration. A year later, the same authors have shown a potent ability of γ-Oryzanol to suppress NF-κB in LPS-stimulated RAW 264.7 [71].
Nonetheless, PPARα agonists such as fibrate are currently used clinically to treat hyperlipidemia and reduce the risk of CVDs [74]. In general, PPARα can be found in numerous tissues-including the heart, liver, and adipose tissue-where its major function is to stimulate FA oxidation by increasing the expression of many related genes such as CPT1/2 and other uncoupling protein (UCP1-3) [74,75]. PPARα can also stimulate the synthesis of HDL-c and reduce levels of LDL-c [75]. In addition, the activation of the PPARα suppressed hepatic and adipose tissue lipogenesis by inhibiting SREBP1c through the activation of Insig2a [9]. PPARα can also prevent tissue inflammation by suppressing the NF-κB and the synthesis of IL-1β, TNF-α, and IL-6 [76]. PPARα is significantly depleted in the liver, WBC, and adipose tissue of HFD-fed and obese animals or subjects [13,[77][78][79]. Pharmacological or genetic overexpressing of PPARα reduced the gain in body weights, decreased serum levels of TGs and CHOL, increased insulin sensitivity, suppressed the increase in adipocyte size, and stimulated hepatic and adipose tissue mitochondrial FAs oxidation by upregulating CTP1, CPT2, and UCP1-3 [12,[80][81][82].
Similar to this evidence, mRNA levels of PPARα and CPT1 were significantly decreased in the livers of HFD-fed rats. However, only the treatment with γ-Oryzanol stimulated these genes' levels in the liver and adipose tissue of HFD-fed rats. They also stimulated the expression of these genes in the livers and adipose tissues of the control rats too. The data suggest that γ-Oryzanol acts as an anti-obesity, antihyperlipidemic, and anti-adipogenic molecule by stimulating PPARα. This finding is novel and shows that Oryzanol may act as a PPARα agonist to treat obesity, hyperlipidemia, and IR in metabolic conditions. However, since NAC does not affect the expression of PPARα, we dissipate this mechanism from its action. The most interesting finding in this study is that co-therapy with both NAC and γ-Oryzanol was the most effective therapy, which completely abolished the above-mentioned metabolic, oxidative stress, and inflammatory markers. This could be due to their synergistic effects, each of which acts by different mechanisms, as discussed above.

Conclusions
Chronic administration of a combination of NAC and γ-Oryzanol is an effective therapy to prevent NAFLD and other features of metabolic syndrome. This combination synergistically reduces fasting hyperglycemia, hyperlipidemia, and hypertension; improves peripheral and hepatic insulin sensitivity; alleviates hepatic oxidative stress and inflam-mation; and prevents lipid accumulation in the WAT and liver. However, while both treatments are potent hypoglycemic and antioxidant molecules, γ-Oryzanol stimulates WAT and hepatic FA oxidation by stimulating the PPARα/CPT1 axis. In addition, given the high safety and pre-clinical use of both drugs in humans, these data encourage further subclinical and clinical use of this combination which could present a novel treatment for obesity, IR, and NAFLD.

Limitations of the Study
Despite our findings, this study still has some limitations. Even our data have shown a stimulatory effect of both NAC and γ-Oryzanol on the hepatic and WAT levels of PPARα, and this finding remains observational and more experiments using PPARα-deficient cells or animals may validate this. In addition, a dose-response, as well as a time-dependent designed study are required to precisely study the metabolic effect of both treatments on liver lipid metabolism. Furthermore, targeting other signaling pathways such as SIRR1 and AMPK-which normally regulate lipid metabolisms, biogenesis, and oxidation and can regulate PPARα-were not studied in this study and should be targeted in a future study to examine the upstream mechanism of action of these drugs.

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.