The Amelioration Role of Acacia Senegal on the Systemic Oxidative Stress and Necroinflammation Induced by Carbon Tetrachloride in Rats


 Acacia Senegal (AS, Gum Arabic) is a polysaccharide with several potential health benefits that is recognized by the Food and Drug Administration (FDA) agency as a secure dietary fibre. The present research evaluated the systemic oxidative and necroinflammatory stress induced by CCl4 administration and the alleviating effect of AS aqueous extract (ASE, 7.5 g/Kg b.w.). The results demonstrated the presence of certain phenolic compounds in ASE as well as its in vitro potent scavenging ability to ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), NO, and lipid peroxide radicals. Also, the outcomes observed an improvement in the CCl4-induced liver, lung, brain, and spleen toxicity by reducing ROS, lipid peroxidation, NO, and the pro-inflammatory mediators. Moreover, the TAC (total antioxidant capacity) as well as the enzymatic and non-enzymatic antioxidants were significantly upregulated in these organs after the treatment with ASE. These results were confirmed by enhancing the morphological features of each organ. Therefore, ASE can ameliorate the systemic toxicity induced in rats by CCl4 due to its antioxidant and anti-inflammatory potential, which is related to its phytochemical composition.


Introduction
Oxidative stress exhibits an asymmetry between the exposure to the reactive oxygen species (ROS) and the ability of the physiological system to directly scavenge the reactive intermediates or adjust the outcome's damage 1 . Disturbances in the normal redox state of cells can produce lethal results by building ROS that damage all cell parts, involving lipids, proteins, and DNA 1,2 . ROS performs a crucial role in the pathogenesis of various human organs, including liver, heart, lung, spleen, and kidney 3,4 . Various toxic chemicals, such as carbon tetrachloride (CC1 4 ), bromobenzene, aromatic hydrocarbons, and methanol 5 , are implicated in the development of organ damage by generating ROS 5,6 . CCl 4 is a prevalent hepatotoxin used to provoke liver complications in various experimental studies 7,8 . Its hepatotoxic effect is demonstrated histologically to induce brosis, hepatocellular death, and cirrhosis 9,10 . CCl 4 is incorporated with its quick break by cytochrome P450 (CYP2E1) in liver cells, providing trichloromethyl radicals (CCl 3 *) driving lipid peroxidation and subsequent membrane impairment 4,9 . In addition to this, CCl 4 caused the activation of hepatic Kupffer cells that generate in ammatory mediators and ROS, yielding an ending in hepatic parenchymal cells' distress 2,10 . CCl 4 can also induce similar damage to other organs such as kidney, lung, and spleen 11 .
Nowadays, numerous functional foods and their ingredients were involved in the amelioration of the chemical's toxicity due to their antioxidant and anti-in ammatory roles 12 Acacia Senegal (AS, Gum Arabic) is one of these functional foods that have numerous medicinal effects. It is a neutral or slightly acidic dietary bre that is present naturally as mixed magnesium, calcium, and potassium salt polysaccharidic acid 13,14 . Gum Arabic has anti-in ammatory and antioxidant activities and protects from kidney, liver, and heart injury. It also ameliorates some biochemical, physiological, and behavioral effects in rats 15,16 besides its ability to relieve the disadvantageous effects of chronic kidney malfunction in humans 14,16 . Few previous studies have assessed the ameliorative effect of AS on the drug and chemical-induced toxicity 17,18 , but no study has evaluated its effect on CCl 4 -induced systemic damage.
Thus, the present work was conducted to evaluate the therapeutic effect of AS aqueous extract (ASE) on the CCl 4 -induced toxicity in rat liver, lung, brain, and spleen tissues. The necroin ammatory and oxidative stress mediators have been examined due to their critical role in systemic destruction. Moreover, the phytochemistry and the in vitro antioxidant activity of ASE was assessed to provide a more persuasive proof of its therapeutic opportunities.

The ameliorating effect of ASE on CCl 4 -induced systemic toxicity
The results of the present study demonstrated that ASE was able to ameliorate the CCl 4 -induced toxicity in liver, lung, brain, and spleen. This was indicated by a signi cant improvement in the oxidative stress and necroin ammation in these tissues.

Organ/body weight ratio and liver function markers
The organ/b.w ratio results showed a non-signi cant difference between all the studied groups for the liver and brain tissues. However, a signi cant (p < 0.05) elevation in these ratios was observed in the lung and spleen tissues after CCl 4 administration only, by 276.431 and 86.067%, respectively (Fig. 1C).
The serum analysis for the liver function markers (Fig. 1D) showed signi cant (p < 0.05) decline in the ALT activity (75.146%) and albumin level (36.707%) in the rats that were injected with CCl 4 compared to the control rats. The remaining studied serum markers (AST and total proteins) revealed slight changes (7.323% and 3.287%, respectively) than the control. Concerning rats in the CCl 4 -ASE group, the ALT activity and albumin level were signi cantly (p < 0.05) increased by 604.471% and 21.081%, respectively, but there was no change in the AST activity (21.254%) and total protein level (6.134%) compared to those in the CCl 4 group. The V group displayed a non-signi cant difference in the ALT activity (13.255%), AST activity (2.834%), albumin level (3.584%), and total protein level (7.658%), relative to the control. Similarly, the serum of rats that administered ASE alone showed slight differences in the AST activity (5.883%), albumin level (12.640%), and total protein level (9.550%) relative to control. In contrast, the activity of ALT was signi cantly (p < 0.05) elevated (195.865%) in the rats of this group compared to that in C group.
Regarding rats that were injected with olive oil alone (V group), they showed non-signi cant changes in all the studied oxidative stress parameters. The changes in ROS, TAC, TBARS, NO, GPX, GSH, SOD and MPO were 7.520%, 21.979%, 21.823%, 34.118%, 3.536%, 3.339%, 12.207%, and 4.219%, respectively, compared to those in the control rats. On the other hand, the rats that were administered ASE alone for 10 days (ASE group) showed a non-signi cant change in the levels of ROS, TAC, TBARS, and NO and the activity of SOD and MPO than the control (7.779%, 6.143%, 5.492%, 1.859%, 3.550%, and 5.953%, respectively). While the activity of GPX and the level of GSH were signi cantly (p < 0.05) elevated by 12.783% and 20.611%, respectively when compared to those in the C group (Fig. 2C).
Improving the CCl 4 -induced brain redox state disturbance by ASE Figure 3A and B shows the toxic effect of CCl 4 on brain tissue through the induction of oxidative stress, which is indicated by the signi cant (p < 0.05) rise in the levels of intracellular ROS (185.182 %), NO (140.579%), and TBARS (480.468%) as compared to the C group. Otherwise, there was a non-signi cant (36.179%) difference in the MPO activity in the brain tissue of rats injected with CCl 4 and the control ones ( Fig. 3D). Moreover, the cellular redox state (TAC), the activity of GPX and SOD, and the level of GSH were extremely reduced by 63.778%, 42.000%, 37.463 %, and 58.677%, respectively related to the C group ( Fig. 3A, C, D). Treatment of CCl 4 -injected rats with ASE (CCl 4 -ASE group) signi cantly incremented the cellular redox state of the brain tissue. This was shown by a signi cant (p < 0.05) decrease in the levels of accumulated intracellular ROS (28.951%), TBARS (61.184%), and NO (46.038%), respectively relative to that of CCl 4 group (Fig. 3A, B). In contrast, the data analysis found a signi cant increase (p < 0.05) in the TAC (48.006%) and GSH (59.799%) levels and the GPX (51.519%) and SOD (72.019%) activities after ASE treatment compared to those in the CCl 4 group (Fig. 3A, C, D). While a non-signi cant change (41.511%) was observed in the activity of MPO in this group compared to those in the CCl 4 group (Fig. 3D).
The results also detected a signi cant (p < 0.05) elevation in the ROS, TBARS, and NO levels in the brain of rats injected with olive oil only (V group) compared to those in the control group (Fig. 3B). However, the antioxidant indices (Fig. 3A, C, D) in the brain of these rats, such as the TAC (44.444%), GSH (29.822%), GPX (17.578%), and SOD (30.741%) were signi cantly (p < 0.05) diminished relative to control. But the MPO activity decreased insigni cantly (17.903%) in the brain tissue of these rats, relative to the control group (Fig. 3D). On the other hand, administration of ASE alone (ASE group) insigni cantly affected the levels of ROS (11.584%), TAC (0.500%), TBARS (11.519%), NO (0%), and GSH (8.239%) or the activities of GPX (3.209%), SOD (7.587%), and MPO (30.220%) compared to those in the control group.
Improving the CCl 4 -induced lung redox state disturbance by ASE Figure 4A, B shows that CCl 4 caused harm to the lung redox state with a massive rise in the levels of ROS (591.297%) and NO (483.908%), compared to those in the C group, which led to an incredible formation of TBARS (361.120%). Also, compared to that of the control group, there was a signi cant increase in the MPO activity by 57.318%. Conversely, the lung enzymatic (GPX and SOD) and non-enzymatic (GSH) antioxidants enormously reduced by 69.426%, 27.635%, and 82.387%, respectively, compared to those in the C group (Fig. 4C, D). Consequently, TAC of lung tissue signi cantly dropped in the CCl 4 -injected rats by 65.780%, related to the control group (Fig. 4A). The treatment with ASE after CCl 4 -injection (CCl 4 -ASE group) improved the redox state in lung tissue extremely. This occurred due to the signi cant (p < 0.05) decrease in the studied oxidative stress parameters, ROS, NO, TBARS, and MPO levels, by 43.448%, 50.098%, 72.211%, 20.594%, respectively, compared to those in the CCl 4 group (Fig. 4A, B, D). Besides, ASE signi cantly (p < 0.05) enhanced the lung antioxidant system (SOD, GPX, and GSH) by 48.505%, 160.157%, and 386.431%, relative to the CCl 4 group (Fig. 5C, D). These results concomitantly led to an increase in the lung tissue TAC (64.683%) related to that in the CCl 4 group (Fig. 5A).
On the other hand, the lung tissue of rats in the vehicle (V) group showed an alteration in the redox state parameters. Hence ROS, NO, and TBARS signi cantly (p < 0.05) increased by 43.619%, 158.818%, and 24.490%, compared to the C group. Also, the antioxidant parameters, including GPX (22.390%), GSH (24.611%), and SOD (3.469%) depleted signi cantly (p < 0.05) and as a result, the TAC (27.389%) decreased compared to the control group (Fig. 4A, C, D), while the activity of MPO in the lung of the rats in this group insigni cantly increased (0.456%), compared to that in the C group. Nevertheless, the administration of ASE alone for 10 days (ASE group) displayed non-signi cant changes in the studied oxidative stress parameters in lung tissue compared to the C group. These parameters include ROS (12.146 %), TBARS (2.306%), NO (10.344%), MPO (11.409%), GPX (18.502 %), GSH (5.813%), SOD (10.135%), and TAC (4.137%).

Improving the CCl 4 -induced spleen redox state disturbance by ASE
The results showed that CCl 4 injection can also in uence spleen oxidative stress and cause a massive elevation in ROS (292.700%) and NO (182.978%) levels, which leads to an excessive generation of lipid peroxide (TBARS, 395.516%), compared to that of the C group (Fig. 5A, B). Likewise, there was a signi cant (p < 0.05) elevation in MPO activity (165.852%) compared to the control group (Fig. 5D). In contrast, there was a signi cant (p < 0.05) falling in the spleen antioxidant content, GPX (77.092%), GSH (75.735%), and SOD (50.388%), related to the control group (Fig. 5C, D). Therefore, the TAC (63.063%) of spleen tissue signi cantly (p < 0.05) diminished in the CCl 4 -injected rats, compared to the C group On the other hand, the spleen of rats that were injected with olive oil only for 3 weeks (V group) showed a slight increase in the levels of ROS (10.537%), TAC (0.677%), and TBARS (16.592%), compared to the C group ( Fig. 5A, B). Similarly, there was a slight change in the activities of SOD (5.972%) and MPO (36.980%) (Fig. 5D). However, the level of NO signi cantly (p < 0.05) increased by 36.504% and in contrast, the activity of GPX and the level of GSH signi cantly (p < 0.05) reduced in the spleen of rats in this group by 15.281% and 25.714%, respectively, relative to the control rats ( Fig. 5B, C). Further, the administration of ASE alone for 10 days (ASE group) showed a non-signi cant difference in the levels of splenic ROS (0.692%), TBARS (0.605%), NO (0.426%), and GSH (0.186%), compared to the C group. Likewise, the activities of MPO (5.071%), GPX (2.398%), and SOD (7.924%) changed slightly. Nevertheless, the level of TAC (64.865%) signi cantly (p < 0.05) increased in the spleen tissue after ASE intake, compared to that in the C group.
The present study reported that the administration of olive oil only (V group) did not greatly in uence the fold expression of the pro-in ammatory and pro brotic cytokines in the liver compared to the those in the control group (Fig. 6A). Hence, the percentage change of these mediators, including NF-κB, iNOS, COX-2, TNF-α, COL1A1, and TGF-β1, compared to those in the control group was 7.250%, 15.535%, 0.686%, 8.699%, 7.412, and 15.794%, respectively. Similarly, in the brain tissue, there was a small change in the fold expression of the studied in ammatory mediators (9.327%, 16.204%, and 10.658%, respectively), except for the COX-2, which showed a signi cant upregulation in its fold expression by 79.0641%, compared to that in the C group (Fig. 6B). While in the lung, the fold expression of the pro-in ammatory cytokines, comprising of NF-κB, iNOS, COX-2, TNF-α, IL-1β, and IL-8 were signi cantly (p < 0.05) upregulated compared to those in the C group (Fig. 6C). The percentage change for these mediators were 290.183%, 229.548%, 286.581%, 48.410%, 234.688%, and 117.364%, respectively. Regarding the spleen tissue, there was a signi cant (p < 0.05) elevation in the fold expression of some pro-in ammatory mediators (NF-κB "177.005%", iNOS "68.012%"), and no changes in the others (COX-2 "71.528%", TNF-α "6.648 %") after the injection with the olive oil, compared to those in the C group (Fig. 6D).
Comparison of the ASE therapeutic e ciency in the four studied organs Supplementary Fig. 1 represents the heatmap plot, which clusters the oxidative stress and necroin ammation tested parameters that were observed after the treatment with ASE (CCl 4 -ASE) in the different studied organs. The color in the heatmap elucidates the quantity (% increase relative to CCl 4 group) of these different studied parameters, the higher the quantity, the darker the color. Figure (1 A) showed two clusters, one for the liver and spleen, and the other for the lung and brain. This gure illustrates the high capability of ASE in elevating the level of TAC and the activity of GPX and SOD in the liver and spleen, more than the lung and brain. However, it increased the GSH level in the lung more than in the other organs. While the heatmap in Supplementary Fig. 1 (B) demonstrates two clusters, the liver with the lung and the brain with the spleen. The gure shows the ability of ASE to decrease the levels of ROS, NO, TBARS, NF-κB, iNOS, and TNF-α, and the activity of MPO in liver and lung more than the brain and the spleen. Therefore, these heatmaps revealed that the liver was the most in uenced tissue, and the therapeutic values of ASE and the brain were the lowest.

Histopathological results
The microscopic examination of the processed formalin-xed sections of the studied organs revealed the systemic toxicity of CCl 4 and the ameliorating impact of ASE on the different tested organs (Fig. 7). The control sections of the liver revealed the hepatic central vein (CV) with normal lobular architecture and radiating liver cell cords. CCl 4 injection showed severe hepatotoxicity, which indicated severe steatohepatitis with brous bands. In addition, the features of necroin ammation such as congested blood vessels with in ltration of mononuclear leucocytes and necrotic and binucleated hepatocytes were observed. The treatment with ASE (CCl 4 -ASE) restored the normal morphology of the liver with only a mild dilation of the CV. Injection with olive oil only (V group) showed severe dilation of the CV with in ltration of mononuclear leucocytes, as well as mild sinusoidal cells activation. However, no pathological changes were detected in the liver of rats that were administered ASE only for 10 days (ASE group).
Regarding the brain, the control tissues showed normal architecture with normal glial cells and neurons.
This normal morphology was changed after CCl 4 injection to severe damage with severe degeneration of neurons that was characterized by the appearance of pyknotic nuclei and vacuoles in the neuronal cells. Administration of ASE restored the architecture of the brain and relieved the CCl 4 toxicity. Injection with olive oil to rats (V group) caused mild degeneration of the neurons, which appeared with pyknotic nuclei and vacuoles in addition to the accumulation of the in ammatory cells in their vicinity. All these features re ected the induction of necroin ammation in the brain tissue of rats in the V group, while the oral administration of ASE alone (ASE group) didn't affect the normal morphology of the brain.
The lung tissue in control rats had the normal appearance of the alveolar sacs and alveolar walls. Massive disruption was observed in its morphology after injection with CCl 4 , which is characterized by in ammatory cells in ux with alveolar wall thickness and alveolar epithelium damage. Great amelioration in the lung morphology was observed following the administration of ASE (CCl 4 -ASE) compared to those in the CCl 4 group. Hence, the lung tissue seemed normal with only mild thickness in the alveolar septae.
In contrast, thickness in the alveolar septae with in ltration of the in ammatory cells and narrowing of the alveolar air spaces have appeared in the lung tissue sections of the animals in the V group, while no abnormal features were observed in the morphology of lung tissue in the rats of the ASE group.
Regarding the spleen tissue, it appears normal with well-de ned red and white pulp regions in the control rats. Severe disturbance in these regions with extreme in ltration of in ammatory cells was detected following the injection with CCl 4 . This damage was relieved after the intake of ASE (CCl 4 -ASE), except for the slight in ux of in ammatory cells. Moderate disorganized white pulp compartment with unclear regions and mild recruitment of in ammatory cells were observed in the spleen tissues of the V group. However, the spleen tissues of animals in the ASE group showed normal architecture and well distinct white and red pulp compartments like the control group.

Discussion
Arabic gum has been well-known since ancient times and consists mainly of complex sugars such as galactose, rhamnose, arabinose, and glucuronic acid with about 25% proteins. In addition, the current study detected the presence of some amounts of phenolic compounds such as gallic, ellagic, benzoic, and O-coumaric acids (Fig. 1A). The presence of these compounds explains the current outcomes about the antioxidant activities of AS in vitro through scavenging the ABTS, NO, and lipid peroxide radicals. Hence, the antioxidant activities of the phenolic acids and reducing sugars were reported extensively in the previous researches 2, 19,20 .
AS is used in the traditional medicines of Arabs to improve renal function in patients with renal failure 21 .
The present study evaluated its effect on the CCl 4 -induced systemic toxicity in rats. This highly toxic chemical caused metabolomics variations for the mammalian liver, which have been widely investigated.
It is less reported in other organs 11,22,23 . To examine the systemic toxicity induced by CCl 4 , we investigated the alterations in oxidative stress, in ammation, and necrosis markers in rat liver, lung, brain, and spleen. The CCl 4 is a common hepatic toxin that induces oxidative stress, necroptosis, brosis, and in ammation in rats 2,23 . This can be due to the liver's metabolism, which is caused by CYP2E1. This catabolic process resulted in the formation of CCl 3 *, which converted to the additional reactive trichloromethyl peroxyl radical (CCl 3 OO*), following its combination with oxygen 2,24 . Formation of these radicals will augment the ROS level in the liver and induce a disruption of its redox state and in turn, cause the occurrence of the oxidative stress, leading to hepatic damage. The present study con rmed this damage in the liver after CCl 4 exposure through a signi cant (p < 0.05) depletion in the ALT activity and albumin level relative to the C group. The previous studies reported also the ability of CCl 4 to induce metabonomic changes in rat kidney, lung, and spleen 11 . In line with this study, the present work provided fundamental data on CCl 4 toxicity to various rat organs. Hence, injection with CCl 4 signi cantly (p < 0.05) induced disturbance in the oxidative stress indices and necroin ammation in the rat liver, brain, lung, and spleen tissues. This may be due to the free radical metabolites of CCl 4 inducing oxidative stress and generate in ammatory mediators in the liver, leading to an inappropriate in ammatory response and alteration in the functions of other organs 11,22,23 . The present study observed a substantially high level of ROS in the brain, lung, and spleen tissues in addition to the liver tissue. Concomitant to this, the TAC and non-enzymatic (GSH) and enzymatic (GPX and SOD) antioxidants dropped dramatically compared to those in the C group in all the studied organs after CCl 4 injection. This probably occurred due to the high cellular content of the free radicals, including the NO radicals in these tested organs that affect the cellular macromolecules, particularly the membrane lipids causing lipid peroxidation (high TBARS level) 2,10 . Peroxidation of lipids led to further formation and accumulation of ROS in the studied rat organs and resulted in the exhaustion of the cellular antioxidants such as GSH, GPX, and SOD 25,26 . The elevation in the activity of MPO after CCl 4 injection magni es the oxidative stress condition due to its vital role in the production of hypochlorous acid (HOCl) in the neutrophils, which interacted with and consumed the GSH. This reaction occurred in the presence of H 2 O 2 and halide ions and serves in the elevation of lipid peroxidation [27][28][29] . These disturbances caused severe depletion in the cellular redox environment (TAC) in all studied organs (Fig. 2-5). All these outcomes are in agreement with the previous studies of Muhammad et al. and Limiao et al. 3,16 .
The administration of ASE for 10 days to rats with systemic toxicity alleviated the CCl 4 -induced organ damage. This was clearly noticed from the signi cant (p < 0.05) decrease in the levels of NO and TBARS and the activity of MPO along with the signi cant elevation in the GSH, GPX, and SOD, compared to those in the CCl 4 group. Subsequently, the balance between the ROS level and TAC of each organ was about the achievement, indicating the reduction in the oxidative stress. These results were in accordance with the previous study of Babiker et al. 30 . The in vivo antioxidant effect of ASE was in line with our obtained in vitro results that con rm its scavenging ability to the ABTS, NO, and lipid peroxides (Fig. 1B). The ameliorating role of ASE could be linked to its composition, comprising phenolic acids, sugars, amino acids, and minerals. Hence, the free radical scavenging and antioxidant enhancement capabilities of gallic, ellagic, benzoic, and O-coumaric acids were recorded previously 31 . The AS mainly contains (97%) sugars, including L-arabinose, D-galactose, rhamnose, and glucuronic acid along with others 32 that have reducing power and potent antioxidant activities 33 . AS also contains different types of amino acids such as histidine, tyrosine, and lysine, which are generally considered antioxidant compounds 34 . In addition to this, copper and zinc are two essential minerals in the AS 35 known for their vital role in SOD antioxidant activity 36 . The current results ( Supplementary Fig. 1, heatmap plots) revealed that the improving e ciency of ASE to the CCl 4 -induced oxidative stress in the liver was more than the other organs, although the liver is the most vulnerable human organ to all aspects of injury, especially those caused by chemical toxicity, due to its essential role in xenobiotic metabolism. This may be due to the ability of this organ to regenerate itself by replacing the dead cells with new ones. Liver regeneration is a counterbalancing response to liver injury and it is de ned for some toxicants, including CCl 4 37 .
Regarding the rats in the ASE group, all the studied oxidative stress parameters were similar to the control, except for the hepatic GPX activity and GSH level, as well as the splenic TAC level, which were all signi cantly upregulated. These results not only referred to the safety of ASE on the liver, brain, lung, and spleen but also the antioxidant enhancement ability of this extract in the liver and the spleen. This capability may be attributed to the ASE phenolics such as gallic and coumaric acids, which were reported previously to upregulate the hepatic gene expression of GPX and elevate the GSH level by increasing the level of E2-related factor (the antioxidant response element regulating transcription factor) 38 . The improvement in the splenic TAC may be associated with the ability of ASE to modulate other types of the complex antioxidant network in this organ than those investigated in this study.
The current study interestingly revealed the mild toxicity of olive oil injection on the brain, lung, and spleen tissues. These ndings were in line with the recent study of Kouka et al., who demonstrated the ability of olive oil administration to induce oxidative stress in certain tissues 39 . This may be in uenced by the multiple intakes of this antioxidant-rich oil, which has led to the consumption of the cellular antioxidant defence biomolecules, implying a prooxidant effect, which is called antioxidative stress 40 .
The differential prooxidant activities of olive oil in the different organs can be owed to the differences of the concentration, availability, time of persistence, and the distribution of the olive oil antioxidant molecules and their metabolites in different rat organs 39,41 . This assumes that these organs didn't make valuable adaptations after antioxidant ingestion, and the observed negative effect may be reversed if the olive oil was administered for a longer period 42 . Thus, the systemic toxicity in rats injected with CCl 4 is associated with CCl 4 itself, not the vehicle olive oil.
To further con rm the therapeutic potential of ASE on CCl 4 -induced systemic toxicity, we examined the level of in ammatory markers in the different tissues studied (Fig. 6). In addition, the brotic mediators were evaluated in the liver tissue. The present study found that the fold expression of NF-κB, iNOS, COX-2, and TNF-α was elevated in liver, brain, lung, and spleen tissues of the CCl 4 -injected rats. Moreover, the fold expression of IL-1β and IL-8 in the lung tissue, as well as the brotic mediators, including COL1A1 and TGF-β in the liver tissue, were signi cantly (p < 0.05) raised. The in ammatory process is the earliest healing and defence mechanism of tissue damage and there is a serious concern about the commitment of the ROS in supporting this process. Hence, ROS can activate NF-κB and, in turn, it can result in upregulating the gene expression of the relevant pro-in ammatory mediators such as iNOS, COX-2, and TNF-α 43 . Therefore, due to the increased level of ROS in the rat liver, brain, lung, and spleen after CCl 4 injection, the NF-κB with other pro-in ammatory cytokines were upregulated. Activation of the NF-κB pathway interrelated to the steatohepatitis 44 that was observed in the histopathological photomicrographs after CCl 4 injection (Fig. 7). The hepatic morphology of the rats in the CCl 4 group also showed the presence of brous bands that may be linked to the upregulation of COL1A1 and TGF-β1 gene expression (Fig. 6). These two cytokines are secreted from the activated hepatic stellate cells and serve as essential activators for the hepatic brogenesis 45 . Further, the IL-8, IL-1β, and TNF-α are the main responsible cytokines for in ammatory cells in ltration in the lung tissue. It was reported that IL-1β and TNF-α are the key regulators for IL-8 expression 46 . TNF-α and IL-1β stimulate NO production by iNOS, which induces nitrosative stress due to the formation of the highly reactive peroxynitrite radical after NO interaction with superoxide radical. As a result, the lung tissue showed thickening in the airway wall (Fig. 7), which re ected the mucosal in ammation, increase in mucus glands, muscle mass, and vessel area, as well as connective tissue deposition on the extracellular matrix 47 . The upregulation of COX-2 gene expression also can augment the CCl 4 -induced systemic damage by catalyzing the formation of prostaglandin H2 from arachidonic acid along with the production of superoxide radicals 26 . Furthermore, TNF-α and IL-1β activate cellular apoptosis and necrosis pathways by altering the balance of the receptor and the ligand 48 . On the other hand, the elevation of the MPO activity also contributed to increasing the systemic in ammatory response of the CCl 4 by activating the neutrophils 28 . Therefore, the injection of CCl 4 to rats induced systemic necroin ammation by recruitment of the in ammatory cells to the different studied organs (Fig. 7), as well as cell necrosis by upregulating the TNF-α fold expression. All of these results are in accordance with our previous recent study 2 .
The administration of ASE (CCl 4 -ASE group) relieved the systemic in ammatory state induced by CCl 4 by downregulating the gene expression of the proin ammatory mediators, including the NF-κB, iNOS, COX-2, TNF-α in all the studied organs. Also, the gene expression of IL-1β and IL-8 in lung tissue was signi cantly (p < 0.05) decreased, compared to those in the CCl 4 group. Moreover, ASE ingestion signi cantly (p < 0.05) declined the hepatic pro brotic markers (COL1A1, TGF-β) compared to those in the CCl 4 group. These results were in harmony with the histopathological data that showed an improvement in the morphology of the different studied organs (Fig. 7). The anti-in ammatory and anti brotic impacts of ASE that were con rmed previously 49,50 may be related to its potent antioxidant activity and its potential effect in reducing the main in ammatory inducer, ROS. All of these in uences may be implied in the presence of the antioxidant polyphenols in ASE such as ellagic, gallic, benzoic, and O-coumaric acids 51 . These results were in line with the previous investigations that revealed the ability of AS to block or treat the toxic signs of some drugs such as aspirin, acetaminophen, and cisplatin. Additionally, it plays a possible prophylactic role against the toxicity of some chemicals such as trichloroacetic acid and mercuric chloride [52][53][54] . The cluster heatmap plot ( Supplementary Fig. 1) revealed that the anti-in ammatory potency of ASE was higher in the liver than in the other organs. This may be due to the regeneration ability of the liver that requires certain types of cytokines such as TNF-α, NF-κB, and other factors. Hence, TNF-α is one of the hepatocytes priming factors that enhance their proliferation in addition to its stabilization role to the NF-κB for hepatic regeneration 37 . Further, the administration of ASE alone for 10 days (ASE group) demonstrated non-signi cant changes in the studied proin ammatory and pro brotic mediators in all the investigated organs. Furthermore, the architecture and morphology of the studied organs are still well preserved.
On the other hand, the injection with olive oil only (V group) had no adverse effect on the liver but increased the gene expression of certain in ammatory cytokines in the brain, lung, and spleen (Fig. 6).
These adverse effects may be owed to the inability of these organs to make adaptations after the olive oil intake as discussed above 42 . These biochemical outcomes were in agreement with the histopathological ndings that showed in ltration of in ammatory cells in the brain, lung, and spleen, along with other pathological changes. However, slight changes were observed in the liver of rats in this group (Fig. 7).

Conclusions
The current study revealed the toxic effect of CCl 4 on rat liver, brain, lung, and spleen (systemic toxicity) and the therapeutic impact of ASE. The biochemical and histopathological ndings explained the mechanism of ASE in alleviating this toxicity through a decrease in systemic oxidative stress and necroin ammation induced by CCl 4 . The phenolic compounds, sugars, and other valuable constituents of ASE are the main cause for this potency. Therefore, ASE can be a promising extract that can be used for

Animals
Fifty male Albino rats were obtained from MISR University for Science and Technology with pet welfare (assurance number: A5865-01). The rats were adapted under ordinary circumstances of about 30°C temperature with a 12-hour light-dark period for two weeks. Throughout this time, the rats had free access to tap water and regular nutrition as required. All experimental protocols and methodology were carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) and approved by the Alexandria University Committee of Animal Care and Use. This study was carried out in compliance with the ARRIVE guidelines.

Preparation of ASE
The AS (NCBI:txid138043) was provided from Egypt and handled to prepare the crude extract. It was crushed using an electric grinder (Telstar, Terrassa, Spain) to receive the powder; and 50 g of it was dissolved in 200 mL of distilled water and left for 24 h in a refrigerator (4°C) to be dissolved completely.
The solution was ltered to eliminate the impurities and then lyophilized to get the powdered extract (ASE), which was stored at -20°C until used.

Phytochemical analysis of ASE
The total amount of avonoids and phenolics in ASE were assessed using the spectrophotometric and chromatographic methods. Total phenolics were measured by Folin-Ciocalteau reagent and the calibration curve of gallic acid 55 . Total avonoids were estimated using 5% sodium nitrite and 10% AlCl 3 .
The developed yellow color with ASE or the catechin standard was recorded at 510 nm 56 .
The High-performance liquid chromatography (HPLC) using 12 different types of pure phenolic standards were used to detect the phenolics in ASE. Twenty microliters of ASE was distributed on the Zorbax Eclipse plusC18 column (100 mm × 4.6 mm Agilent Technologies, Palo Alto, CA, USA). The separation was accomplished at 284 nm using a mobile phase of methanol, acetonitrile, and 0.2% H 3 PO 4 with a ternary linear elution gradient and a ow speed of 0.75 mL/min 57 . In vitroantioxidant activities of ASE The antiradical (ABTS and NO radicals) activities and β-carotene-linoleate bleaching analysis of ASE were tested. The 50% inhibitory concentration (IC50) for each test was recognized by the GraphPad Instat software version 3.
The ABTS + radical cation-decolorization assay was used to study the ability of ASE to neutralize ABTS + radical to ABTS 58 . ASE or BHT (standard antioxidant) was mixed with ABTS + radical (7 mM ABTS was incubated with 140 mM potassium persulphate for 16 h at 25 C) in dark. Then the absorbance of the blue color was recorded at 734 nm to calculate the percentage inhibition of ABTS + radical.
The NO scavenging ability of ASE was detected by measuring the generated bright-reddish-purple azo dye color from Griess reaction at 490 nm. Sodium nitroprusside and Griess reagent (0.1% naphthylethylenediamine dihydrochloride, 2% phosphoric acid, and 1% sulfanilamide) 59 was used in this reaction.
The anti-lipid peroxidation effect of ASE can be determined by examining the β-carotene-linoleate bleaching. The assay was performed using the emulsion of linoleic acid, β-carotene, and Tween-80 60 . The ability of ASE to scavenge the produced radicals from linoleic acid oxidation was indicated by the decrease in the rate of β-carotene bleaching. The absorbance of the β-carotene color was recorded at 490 nm immediately (a) and after 180 min (b). Then the value ethof the degradation rate (DR) of ASE, control Evaluation of the systemic anti-toxicity effect of ASE Experimental design Fifty male Albino rats (weighing 140-200 g, six weeks' age) were randomly divided into ve groups (ten animals in each). The handling of animals in each laboratory group is presented in Fig. 8. The systemic toxicity in rats was induced by intraperitoneal (IP) injection with 50% CCl 4 in olive oil (1 ml /kg b.w.) every Sunday and Wednesday each week for three weeks 61,62 . The animals in CCl 4 -ASE group was orally administered with ASE (7.5 g/kg b.w., dissolved in water) by gavage, every day for ten days. At day thirty, rats were anaesthetized and dissected immediately to obtain the blood by cardiac puncture and the tissues (liver, brain, lung, and spleen). The heparinized blood was centrifuged for 15 min at 6000 rpm to collect plasma for liver function parameters quanti cation. Liver, brain, lung, and spleen tissues were cleaned with cold saline (0.9% NaCl) and weighed. Then, the small parts were xed in 10% formalin for histopathological examination. The remaining tissues were stored at -80°C until utilized in the biochemical and molecular investigations.

Plasma investigation
Using the speci c kits, the levels of liver function parameters (ALT, AST, total proteins, and albumin) in the separated plasma specimens of all the examined groups were measured colorimetrically.

Biochemical assessment of the CCl 4 -induced systemic oxidative stress
The oxidative stress (cellular redox state disturbance) was determined in the studied organs by quanti cation of the intracellular ROS, TAC, lipid peroxidation, NO, MPO, and the enzymatic and nonenzymatic antioxidant markers. The organ homogenates were obtained by homogenizing one gram of each studied organ separately in 10 mL of cold PBS. Then the cleared homogenates were collected after centrifugation for 30 min (6000 rpm, 4°C) for the analyses 2 .
The concentration of ROS was determined by mixing an equal volume of the diluted homogenate (dilution with PBS, 2-fold) with 5 µM DCFH-DA uorescent probe (diluted 1000-fold with 10 µM dimethyl sulfoxide). The amount of the uorescence was recorded at 485 (excitation) and 520 nm (emission) after keeping the reaction mixture in dark at 37°C for 5 min. Then the ROS level in each studied organ was calculated using the H 2 O 2 calibration curve 63 .
The TAC of the tested organs was determined using the ABTS radical cation method by mixing 2 mL of the radical solution with 20 µL of each organ homogenate or standard (BHT) or PBS (control) 58 . The preparation of ABTS solution and the method proceeded as indicated above in the in vitro assay to obtain the percentage of inhibition. Then the TAC of each studied organ was calculated as BHT equivalent/g tissue using the BHT standard curve.
The level of lipid peroxidation was measured colorimetrically using TBA reactive substances (TBARS) assay using the standard curve of TMP 64 . NO was examined using Griess reaction as referred in the in vitro part 60 .
The activity of MPO as µmoL of H 2 O 2 /min was quanti ed spectrophotometrically using 16.7 mg% ODD and 1.2% H 2 O 2 65 , while the activity of the enzymatic antioxidants (GPX and SOD) were examined by Rotruck method 66 and pyrogallol autooxidation method 36 , respectively. The speci c activities of these enzymes were calculated as IU/mg protein using the value of the protein content in each organ homogenate that was measured by the biuret method using the speci c kit. Ellman's reagent (5, 5′-dithio bis2-nitrobenzoic acid) was used to determine the nonenzymatic antioxidant (GSH) level using the GSH calibration curve 67 .
Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) The necroin ammation in all the studied tissues and the brotic markers in the liver tissue were assessed at the molecular level using the qRT-PCR technique. The NF-кB, iNOS, COX-2, and TNF-α were evaluated here as good markers of necroin ammation in all the examined tissues, and IL-1β and IL-8 (essential pulmonary pro-in ammatory cytokines) were assessed in lung tissue. In addition, the COL1A1 and TGF-β1 (critical hepatic brosis cytokines) were tested in liver tissues.
Each studied tissue was homogenized in lysis buffer containing β-mercaptoethanol and then centrifuged (14,000 rpm for 5 min) and the total RNA was extracted from the obtained supernatant, using Gene JET RNA Puri cation Kit and quanti ed. Then the cDNA Synthesis Kit was used to synthesis the cDNA. The levels of target genes expression were determined via real-time PCR using SYBR green master mix kit, speci c primers (Supplementary Table 1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. The comparative Ct method (number of threshold cycle at cross-point between ampli cation plot and threshold) was used to calculate the fold expression of the studied genes 2 .

Histopathological study
The formalin-xed tissue specimens were embedded in para n wax then each sample was cut into small slices (5 µm thickness) and stained with hematoxylin and eosin following the standard histopathological examination protocol. Then, the phase-contrast microscope was used to visualize the pathological features of the examined tissues in all the studied groups, and high-resolution images (200x magni cation) were captured 2 .

Statistical analysis
The results (symmetric with skewness values from 0 to 0.941) of the present study were expressed as mean ± SE of seven rats. The parametric analysis, one-way analysis of variance (ANOVA) using Duncan's test, was used to determine the difference between the mean values in all the studied groups. The analysis was done by SPSS software version 16 and the signi cance was considered at p-value < 0.05. The GraphPad Instate software version 3 was used to calculate the IC50 values for the in vitro antioxidant analyses.