Targeting ROS/NF‑κB Signaling Pathway By The Seedless Black Vitis Vinifera Polyphenols in ­CCl4‑intoxicated Kidney, Lung, Brain, And Spleen in Rats

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Oxidative stress is defined as an imbalance between the reactive oxygen species (ROS) toxicity and the physiological antioxidant defense system that scavenges ROS or ameliorates the resulted damage, The generated ROS that destroys all the cell aspects can yield lethal effects,2 and plays a vital role in the pathogenesis of numerous human organs, including the brain, lung, liver, spleen, and kidney-5. Consequently, decreasing the production of ROS is critical for preventing more oxidative damage and; for cell survival. Nevertheless, massive cellular damage causes removal of the damaged cell to maintain the surrounding healthy cells6, Various toxic chemicals such as methanol, carbon tetrachloride (CC1,). aromatic hydrocarbons and bromobenzene can induce systemic organ injury by producing ROS8. The CCl was extensively used as a cleaner, solvent, and degreaser, both for home and industrial use. In addition, it is widely used as an experimental model to study the hepatotoxic effects of plant extracts and drugs, since the liver is the main organ exposed to CCl, toxicity. Other organs can also be affected by its toxicity such as the kidney, lung, and spleen.

Numerous traditional remedies have been studied in laboratory animal models for their protective capabilities against different chronic diseases, toxins, and toxic chemicals-4. Vitis vinifera(VV) is one of the folk remedies plants and functional foods, belongings to the Vitaceae family. It is a non-climacteric fruit and the most cultivated and consumed edible fruit in the world. VV has various health-benefiting effects like its immune-enhancing, anti-inflammatory, neuroprotective, antioxidant, anti-apoptotic, and anti-carcinogenic properties. These promoting effects are related to its functional constituents, which include minerals, amino acids, proteins, sugars, organic acids, vitamins, and phytochemicals(flavonoids, carotenoids, tannins, anthocyanins, and proanthocyanins).

The protective power of different types and parts of VV such as stems, leaves, seeds, and juice against diverse hepatotoxic agents, including CCl, has been examined-5,17.In addition, our recently published work investigated, for the first time, the hypnotherapeutic potential and synergistic activity of the extracted seedless(both pulp and skin）black VV polyphenols（VVP） against CCl-induced hepatotoxicity². Fortunately， no previous study has attempted to investigate the therapeutic efficacy of VVP against CClA-induced systemic toxicity (kidney, lung, brain, and spleen toxicity) in rats, to compare their therapeutic values in these target organs. Therefore, the current study was conducted to investigate this point in addition to comparing the therapeutic potential of these isolated polyphenols in the four studied organs using the heatmap clusters plots. The effects of the black VVP on the oxidative damage and necroinflammation by which CCl induced systemic destruction, were also evaluated here. For an explanation of the results, the phytochemical content and the in vitro antioxidant activities of VVP were examined.

Results

Phenolic composition and antioxidant potential of VVPF. The present study found that the total phenolic content of VVPF was 58.387±0.755 mg gallic acid eq. g-'VV PF. In addition, this study reported the presence of 16.718±1.858 mg catechin eq of flavonoids,1.098±0.342 mg Quercetin(QR) eq of flavanols and 3.467±0.466 mg cyanidin-3-glucoside (Cy-3-GLC) eq of anthocyanins in each 1 g of VVPF.

The VVPF showed a potent in vitro antioxidant activity with a total antioxidant capacity (TAC) value of 709.589±25.939 mg Butylated hydroxytoluene (BHT)eg. g-1VVPE. Moreover, it had potent ferric reducing power and was able to scavenge 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid(ABTS) radical (Fig. 1A, B). The results also showed that the ferric reducing power of VVPF was significantly(p<0.05)higher(lower IC50 value "50% inhibitory concentration") than that of ascorbic acid (Asc), while its ABTS scavenging power was lower (higher IC50 value) than that of BHT.

The influence of VVPF on CCl-induced systemic toxicity. The VVPF exhibited a potent ameliorating impact on the CCl-induced toxicity in kidney, lung, brain, and spleen tissues. This was demonstrated by a substantial improvement in systemic oxidative stress and neuroinflammation.

The results revealed that the organ wt/b.w ratio for the kidney, lung, and spleen tissues had a non-significant change between all groups. However, a significant(p<0.05)decrease was detected in the brain wt/b.w.ratios of rats in CCl(39.891%)and V(45.355%) groups as compared to the control "C" group. However, there was an anon-significant change in these ratios in rats of the CCl,+VVPF group relative to the CCl group (Fig. 1D).

The ameliorating impact of the VVPF on the CCl-induced renal oxidative stress. The data of this study revealed that the CCl induced oxidative stress in the kidney that was manifested by a signifcant (p<0.05)elevation in the level of ROS(203.169%)and NO(545.833%).In addition, the thiobarbituric acid reactive substances(TBARS,321.790%)level and the activity of myeloperoxidase (MPO, 321.579%) were increased in the CCl group,as compared to the C group (Fig,2A-C). These results were accompanied by a massive decrease in the level of TAC(53.121%) and reduced glutathione (GSH,80.647%) and the activity of the enzymatic antioxidants(superoxide dismutase"SOD,45.727%" and glutathione peroxidase"GPX, 50.909%") as shown in Fig.2A, C, D. However, there was a non-significant change in all of these oxidative stress markers in the kidney of rats in the V "vehicle" group (Fig.2A-D).

The present study reported a good improvement in the oxidative stress parameters after administration of the VVPF for ten days(CCl+VVPF group).Hence the levels of ROS, NO,and TBARS and the activity of MPO were significantly(p<0.05)reduced relative to that of the CCl,group by 44.747%,81.297%,67.439%,and 35.362%, respectively(Fig.2A-C).In contrast, the level of TAC(47.429%) and GSH(81.899%)and the activity of SOD (57.936%) and GPX(91.769%)were significantly(p<0.05) upregulated in the kidney of the rats in this group (Fig.2A, C, D). While the intake of VVPF alone for ten days(VVPFgroup)had an anon-significant effect on all of the previous parameters, except for the MPO(decrease by 31.513%)and SOD(elevated by 17.413%) activities, which were significantly (p<0.05) changed as related to the control group.

The ameliorating impact of the VVPF on the CCl-induced oxidative stress in the lung. In the lung, CCl, also induced oxidative stress by elevating the levels of ROS, NO. T-BARS. and the activity of MPO significantly (p<0.05),by 131.883%,516.092%,332.458%,and 57.319%,respectively as compared to the control rats(Fig.3A-C). Moreover, the level of TAC(65.779%), GSH(81.081%), as well as the activity of both SOD (53.195%)and GPX (66.797%)were greatly depleted in the lung after CCl, injection(Fig.3A, C, D). The results also found that the injection of rats with olive oil for 3 weeks(V group)led to a significant (p<0.05)increase in the ROS and TBARS levels. Moreover, it substantially diminished the TAC and GSH contents in addition to the SOD and GPX activities, relative to the control (Fig. 3A-D). The percentages of these changes were 38.146%, 23.430%,34.877%,19.018%,41.998%, and 15.715%, respectively.

Administration of VVPF for ten days(CCl,+VVPF group)greatly ameliorated the disturbance in the pulmonary redox state that was induced by CCl.This was achieved by a significant(p<0.05) decrease in the ROS, NO,and TBARS levels, and MPO activity by 65.510%,58.955%,75.435%,and 55.496%,respectively, as related to the CCl, group (Fig.3A-C). However, the levels of TAC (136.084%) and GSH(445.627%),as well as the activity of SOD(137.817%)and GPX(166.154%)were significantly (p<0.05)upregulated (Fig.3A,C,D).While the administration of VVPF alone for ten days(VVPF group) not only had no damaging effect on the lung but also enhanced the pulmonary redox state. This has clearly appeared from its ability to enormously decrease the MPO activity(53.951%) and increase the GSH level(5.854%)and GPX activity(54.323%), as compared to the control untreated rats (Fig. 3C, D).

The ameliorating impact of the VVPF on the CCl-induced oxidative stress in the brain. Figure 4 demonstrates the ability of the CCl, to induce imbalance in the cellular redox state of the rat brain. This was explained by the massive elevation in the cellular contents of ROS(198.719%), NO(140.580%), and TBARS (461.917%), and the activity of MPO (98.253%), as linked to the C group (Fig. 4A-C). Conversely, the TAC (66.333%)and the amounts of GSH(63.233%), along with the activity of SOD(37.463%)and GPX(38.788%), were drastically reduced when compared to the untreated control rats(Fig. 4A, C, D). The data also revealed the toxic effect of olive oil when it was administered for 3 weeks(V group). This toxicity was dictated by its influence on rising ROS(49.829%) and TBARS(15.612%)levels while declining TAC(44.444%) and GSH(29.822%)levels, as well as SOD(30.741%) and GPX(13.015%)activities when compared to the C group (Fig.4A-D).

The administration of VVPF for ten days(CCl,+VVPF group) was sufficient to improve the brain oxidative stress condition caused by CCl, infection. After VVPFintake, the levels of ROS(37.175%). NO(47.879%).TBARS (69.551%), as well as the activity of MPO (66.516%), were dramatically diminished when related to the CCl group (Fig.4A-C).In addition, the TAC and GSH levels, as well as the activities of SOD and GPX were significantly (p<0.05)elevated when compared to the rats in the CCl, group (Fig.4A, C, D).The percentages of increase were 95.544%,105.136%,71.815%,and 41.951%,respectively.On the other hand, the intake of VVPF alone (VVPF group) had no toxic effects on the brain. In addition, VVPF was able to significantly(p<0.05)increase the GSH level(22.809%) and the GPX activity(14.292%), relative to the control group as shown in Fig.4D.

The ameliorating impact of the VVPF on the CCl-induced splenic oxidative stress. Figure 5 elucidates the effect of the CCl4 injection on the cellular redox state of the spleen. In comparison to the control group, the levels of ROS(270.080%), NO(182.979%), and TBARS(395.516%) and the activity of MPO (165.852%) were markedly increased in the spleen of rats after 3 weeks of injection with CCl (Fig.5A-C). Furthermore, the splenic TAC and the level of the non-enzymatic antioxidant (GSH), as well as the activities of the enzymatic antioxidant(SOD and GPX)were significantly(p<0.05) decreased as compared to the C group (Fig. 5A, C, D).The percentages of this decrease were 64.035%,74.867%,35.894%,and 77.092%,respectively. However, no dramatic changes were observed in the spleens of rats after injection with olive oil (V group).

Our data found that the treatment with VVPF for ten days after CCl injection (CCl,+VVPF group)showed great effects on the splenic oxidative stress. This was clearly inferred from the ability of VVPF to normalize most of the cellular redox state parameters. In this group, the levels of ROS,NO,TBARS, and the activity of MPO were significantly (p<0.05)reduced relative to the CCl (Fig.5A-C)by 58.276%,52.519%,68.816%,and 62.098%, respectively. In contrast,the levels of TAC and GSH and the activities of SOD and GPX were significantly (p<0.05)increased(Fig.5A,C,D)by 185.366%,278.584%,48.936%,and 332.962%,respectively.Concerning the VVPF group, the TAC and GSHlevels, along with the SOD and GPX activities, were substantially enhanced in the spleens of rats that were gavage-fed VVPF for ten days (Fig.5A, C, D)by 78.947%,14.803%.32.784%.and 14.872%, respectively. However, all other examined oxidative stress parameters(ROS, NO, TBARS, and MPO)were non-significantly (p'0.05)affected in the spleen of these rats by 6.274%,6.808%,15.306%, and 21.574%, respectively.

The anti-inflammatory influence of the VVPF in the kidney and lung tissues. The graphs in Figs. 2E,3E elucidate the massive elevation of the pro-inflammatory mediators in kidney and lung tissues after CCl, injection. Hence, the fold expression of nuclear factor-kappa (NF-K)B, inducible nitric oxide synthase (iNOS),cyclooxygenase(COX)-2, and tumor necrosis factor(TNF)-a was upregulated in the kidney by 505.605%,960.088%,2399.110%,and 219.150%,respectively. Similarly, the fold expression of these mediators was dramatically increased in the lung tissue by 628.041%,755.107%,4066.674%,and 351.253%, respectively. Moreover, the fold expression of the interleukin (IL)-1β and IL-8 in lung tissue was substantially elevated (Fig. 3E)by 4207.250% and 3307.650%, respectively. Regarding, the rats which were gavage-fed olive oil for 3 weeks(V group), the fold expression of NF-KB,iNOS, COX-2, and TNF-a in the kidney was non-significantly changed when compared to the control group(Fig.2E). Likewise, the fold expression of IL-1β and IL-8 in the lung tissue was non-significantly increased as related to the Cgroup(Fig.3E).In contrast, the fold expression  of NF-KB(390.183%), iNOS(329.548%),COX-2(286.581%),and TNF-α(148.410%)in the lung tissue was upregulated following the olive oil administration (Fig. 3E).

A great improving effect was observed for the VVPF(CCl,+VVPF group)on the tested pro-inflammatory mediators induced in both kidney and lung following CCl injection. The fold expression of NF-KB and TNF-α inboth tissues along with the iNOS, COX-2, IL-1β, and IL-8 were normalized(Figs.2E,3E).The % changes in the fold expression in the kidney relative to the CCl,group were 78.928%,64.111%,66.921%,69.514%,respectively. While these percentages in the lung tissue were 79.9158%,72.199%,88.049%,98.082%,88.860%,and 97.049%, respectively. Moreover, the fold expression of iNOS and COX-2 in the kidney was significantly(p<0.05)reduced when compared to that of the CCl, rats(Fig.2E)by 66.921% and 69.514%, respectively. The rats in the VVPF group showed a significant (p<0.05) ability to reduce the fold expression of the pro-inflammatory mediators in the kidney and lung tissues relative to the control group, especially for NF-KB,iNOS, and TNF-a.The percentages of reduction in the kidney were 45.702%,99.993%,and 18.134%,respectively,while these values in the lung were 90.817%,96.590%,and 96.438%,respectively. However, all other examined pro-inflammatory mediators either in the kidney(COX-2) or lung (COX-2,IL-1β,IL-8)were non-significantly(p>0.05)changed (6.109%, 13.468%,4.691%, and 9.194%, respectively).

The anti-inflammatory influence of the WVPF in the brain and spleen tissues. Like in the kidney and lung tissues, the CCl, injection caused upregulation in the pro-inflammatory mediators fold expression in the rat brain and spleen tissues(Figs.4E,5E). The fold expression of the NF-kB,iNOS, COX-2,and TNF-a was markedly upregulated in the brain by 979.924%,522.286%,355.513%, and 71.140%,respectively, and in the spleen by 1007.975%,1937.934%,549.203%,and 22.784%,respectively. However, the rats that were gavage-fed olive oil only for 3 weeks showed normal fold expression levels for all of the pro-inflammatory parameters in the spleen when compared to the control rats(Fig. 5E). The same results have been shown in the brain tissue, except for the fold expression of COX-2 that was upregulated significantly (p<0.05), relative to the C group (Fig. 4E)by 89.064%.

Regarding rats that were administered VVPF for ten days after CCl, injection (CCl+VVPF group), a dramatic decrease in the fold expression of NF-RB, iNOS, COX-2, and TNF-a in the brain and spleen, relative to the CCl-intoxicated rats were reported. The percentages of this reduction in the brain tissue were 29.574%, 64.299%,85.069%,and 34.672%,respectively.While these values in the spleen were 90.772%,94.888%,84.006%, and 24.426%,respectively. Similarly, the fold expression of NF-KB, iNOS, and TNF-a in the brain (99.376%95.678%, and 89.976%, respectively)and spleen(69.646%,92.194%,and79.936%, respectively)of rats in the VVPF group were markedly decreased in comparison to the control group. While the fold expression of COX-2 in both tissues did not change, relative to the control, after VVPF administration for ten days.

Histopathological findings. The photomicrographs of the handled formalin-fixed pieces of the examined tissues showed the systemic toxicity of CCl, and the improving influence of VVPF on the different studied organs(Fig.6). The control sections of the kidney demonstrated the kidney cortex with normal renal tubules and glomeruli. However, the injection with CCl, for 3 weeks showed severe toxicity that was manifested by congested renal vein(CRV(with infiltration of leucocytes and the renal tubules became degenerated and lost their lining epithelial cells and their typical appearance. These alterations revealed the necroinflammatory characteristics of this tissue. The administration of VVPF(CCl,+VVPF)preserved the typical morphology of the kidney with only a slight degeneration of the tubules(SDT). Similarly, the olive oil injection (V group)demonstrated an SDT, however, no morphological changes were found in the kidney tissues of the VVPF group of rats.

Regarding the lung tissue, the control rats showed normal morphology of the alveolar sacs(AS) and alveolar walls. Severe damage was noticed in its architecture after CCl injection, which is characterized by loss of regular alveolar architecture, prevalent disorganized thickening of the alveolar septa, and alveolar space collapse. The administration of VVPF(CCl,+VVPF) for ten days restored the lung morphology. Thickness in the AS with the alveolar air spaces narrowing was observed in the lung tissue pieces of the V group rats, while the morphology of lung tissue in the rats of the VVPF group showed no abnormal features.

The brain tissue in the control rats demonstrated normal structure with normal neurons and glial cells. After injection with CCl, this typical architecture was altered to severe neurons degeneration with vacuoles and pyknotic nuclei in the neuronal cells. The gavage-fed VVPF for ten days relieved the CCl, toxicity and preserved the brain morphology. Moreover, the rats of the V and VVPF groups showed normal morphology of the brain.

The spleen tissue of the control rats showed normal morphology with well-distinct red and white pulp sections. However, the spleen of the CCl-intoxicated rats revealed massive damage in these regions with extreme inflammatory cells infiltration. This damage was mitigated after the administration of VVPF(CCl,+ VVPF). except for the minor influx of inflammatory cells. On the other hand, the rats injected with olive oil only(V group) had moderately unorganized white pulp regions with ambiguous regions and slight inflammatory cells recruitment. In contrast, administration of VVPF for ten days(VVPF group)had no adverse effect on animal spleen tissues, and the tissue showed well-defined white and red pulp compartments, as in the control group.

The impact of CCl, damage, and VVPF therapeutic effects in the four examined organs. The influences of the CCl, harmful effect, and VVPF therapeutic effect in the four tested organs were evaluated by the heatmap plots(Fig. 7). The graphs in Fig.7 cluster the studied oxidative stress and necroinflammation parameters in the rats treated with CCl(CCl, group) and VVPF (CCl,+VVPF group) in the different studied organs. The % increase of these parameters compared to the control(Fig. 7A, B)or CCl4(Fig.7C, D)group, respectively, was indicted by the color of the heatmap. The red color referred to a higher quantity of the parameter and the blue color referred to a lower one. Figure 7A, B demonstrates that the CCl, can induce more oxidative stress and redox state disturbance in the spleen (higher ROS and lower TAC) followed by the kidney, brain, and finally lung tissue. Furthermore, as shown in Fig, 7B, the spleen and brain tissues had the highest fold expression of NF-KB while the other mediators in the target organs had varying degrees of fold expressions.

On the other hand, Fig.7C, D shows the highest ability of VVPF to improve the level of TAC and GSH in addition to the activity of SOD and GPX in the lung followed by spleen, kidney, and brain(Fig.7C).In addition, this grape fraction was able to deplete thelevels of the pro-inflammatory mediators, as well as ROS, NO, TBARS, and MPOin the lung followed by the spleen, kidney, and then the brain (Fig.7D). Consequently, the heatmaps showed that the lung was the least affected tissue with the CCl, damage, and the most responsive tissue with the therapeutic values ofVVPF.