Part2: Geraniol Ameliorates Doxorubicin-Mediated Kidney Injury Through Alteration Of Antioxidant Status, Inflammation, And Apoptosis: Potential Roles Of NF-kB And Nrf2/Ho-1
Jun 07, 2022
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3. Results
3.1. Geraniol Protects Kidneys against Doxorubicin-Mediated Injury
This study examined blood creatinine, albumin, and BUN levels to determine whether Dox administration caused the renal injury. As expected, Dox treatment caused significant increases in BUNand creatinine, alongside a significant reduction in albumin (Figure 1A-C). Geraniol, pre-treatment, ameliorated the observed abnormalities in creatinine, albumin, and BUN levels, indicating that it may protect against Dox-mediated renal injury.
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3.2. Geraniol Protects against Doxorubicin-Mediated Oxidative Stress
To validate if geraniol supplementation may reduce Dox-associated oxidative stress and improve antioxidant capacity, we quantified the amount of MDA and GSH, the activity of CAT, and the gene and protein expression of Nrf-2, Ho-1, SOD-2, and GPx-1 in kidney tissue. Compared with the control group, a single Dox injection (20 mg/kg)resulted in a substantial rise in MDA along with significant reductions in GSH content and CAT activity (Figure 2A-C). It also significantly decreased the gene and protein expression of Nrf-2, Ho-1, GPx-1, and SOD-2(Figure 3A-G). However, prophylactic supplementation with geraniol substantially recovered the modifications of these parameters in a dose-dependent manner. These findings reveal the potential antioxidant action of geraniol.
3.3.Geraniol Protects against Doxorubicin-Mediated Kidney Inflammation
Geraniol's anti-inflammatory activities have been demonstrated elsewhere. To further confirm the potential anti-inflammatory action of geraniol in the context of Dox treatment, we used Western blots to evaluate the expression of proteins important in inflammatory regulation (NfkB-p65, TNF-a, and IL-6). The results showed that expression changes driven by Dox were restored in the rats treated with geraniol (Figure 4A-D), and indicate that geraniol has anti-inflammatory effects.
3.4. Geraniol Protects against Doxorubicin-Mediated Apoptosis
To examine whether geraniol suppresses apoptosis induced by Dox, we measured the expression of pro-apoptotic and anti-apoptotic protein markers. When compared with the control group, a single dosage of Dox led to a significant increase in the renal tissue expression of pro-apoptotic proteins (Bax and cleaved caspase-3) and a substantial decline in the levels of the anti-apoptotic protein Bcl-2(Figure 5A-D). Meanwhile, supplementation with geraniol rectified those abnormalities. These findings suggest that pre-treatment with geraniol reduces Dox-mediated apoptosis in the rat kidney.
3.5. Geraniol Protects against Doxorubicin-Mediated Alteration in Kidney Architecture
Finally, to further confirm our results, we examined the histopathology of renal tissue. Rats in the control group had normal tubules and glomeruli (Figure6A), while those with administered Dox exhibited glomerular congestion and tubular destruction (Figure 6B). We found that supplementation with geraniol restores the Dox-mediated damage in a dose-dependent manner (Figure 6C, D).
4. Discussion
Chemotherapeutic medicines are frequently employed to treat different types of cancer; however, these drugs often also destroy physiological homeostasis in numerous organs and can lead to physiological adverse effects in non-tumor cells, mostly due to free radical formation and oxidative stress toxicity42]. The goal of this investigation was to determine whether geraniol could protect Wistar rats against Dox-mediated kidney damage. Accordingly, we investigated Dox-mediated inflammation, apoptosis, and oxidative stress, and evaluated whether geraniol can act as a preventive agent.
Albumin, creatinine, and blood urea nitrogen(BUN)are reliable indicators of kidney damage[11]. Normal renal tissue does not permit albumin to flow out from the bloodstream to the urine, while also filtering creatinine and BUN from the bloodstream into the urine. However, in the event of renal dysfunction, these processes are disturbed: albumin is excreted in the urine, resulting in reduced serum concentrations, and creatinine and BUN are not filtered correctly, resulting in elevated serum values[11]. In the current work, Dox treatment caused increases in serum creatinine and BUN alongside a significant decrease in albumin. The results are in fair agreement with the published reports [11. We also found prophylactic supplementation with geraniol to attenuate these changes, suggesting that geraniol may prevent renal damage caused by Dox.
Lipid peroxidation is a hallmark of oxidative stress, and many investigations have shown that Dox administration leads to increased levels of malonaldehyde (MDA), a product of lipid peroxidation [7,43]. We confirmed that Dox caused a significant rise in MDA levels within kidney tissue and, moreover, that pre-supplementation with geraniol significantly and dose-dependently reduced this effect. Younis and colleagues, likewise, demonstrated that geraniol substantially reduces MDA levels in methotrexate-mediated kidney injury. These results suggest that geraniol may be effective in treating Dox-mediated kidney damage due to its potent inhibition of lipid peroxidation.
Living organisms utilize various enzymatic and non-enzymatic antioxidants to remove free radicals, thereby offering an effective defense against ROS. Essential anti-oxidative agents include the non-enzymatic antioxidant GSH and its oxidized analogs; GSH interacts directly with free radicals via the its-SH group. In the current study, a single Dox injection demonstrably reduced the GSH reservoir, and geraniol pre-treatment was able to reverse this effect. These findings are supported by previously published studies.
ROS production within cells is also linked to the depletion of anti-oxidative enzymes. In particular, the enzymes CAT and glutathione reductase (GR) accelerate the conversion of H2O2, and other ROS to H2O2 and O, while SOD assists in converting superoxide anion free radicals to H2O2, which are subsequently removed by CAT or GPx [45]. We found that expression of all examined antioxidant enzymes, namely SOD, CAT, and GPx-1, was statistically reduced in the Dox group compared with the control group. However, geraniol pre-administration rescued the Dox-induced reduction of SOD, GPx-1, and CAT levels, probably by scavenging ROS through its own antioxidant ability.
Various enzymes that protect cells from harmful oxidative stress have been linked to the Nrf2 pathway, and stimulating the Nrf2/Ho-1 pathway has been shown to considerably improve kidney function [21,48,49]. To mechanistically validate the protective effect of geraniol, we examined Nrf2 and Ho-1 levels in relation to Dox administration. We found levels of these proteins to be significantly lower in the Dox group than in the controls, consistent with prior studies. Meanwhile, geraniol supplementation prevented Dox-associated kidney damage by boosting mRNA and protein expression of members of the Nrf2/Ho-1 signaling pathway, which is in line with the findings of Younis et al.
Ultimately, inflammation is one of the most likely causes of Dox-mediated kidney injury. Induction of the nuclear factor kappa B(NF-kB) pathway is known to play a very important role in the pathophysiology of Dox-mediated kidney inflammation [50], with NF-kB being a transcription factor that regulates the expression of several genes associated with inflammation [51], such as those encoding TNF-α, IL-1β, and IL-6 [52,53]. Here, Dox-treated animals were found to exhibit increased levels of TNF-α, IL-6, and NfkB-p65 [54], while geraniol pre-supplementation reduced these inflammatory mediators in a dose-dependent manner. These data suggest that geraniol's anti-inflammatory action may be due to an inhibitory effect against the NF-kB pathway.
Another critical player in the etiology of Dox-mediated nephrotoxicity is apoptosis, which is regulated by the balance of pro-apoptotic and anti-apoptotic proteins. Caspase-3 and Bax are pro-apoptotic proteins that increase the porosity of the mitochondrial membrane and enable cytochrome c to leak from the intermembrane gap, triggering apoptosis through the intrinsic apoptotic pathway. In contrast, Bcl-2 is an anti-apoptotic protein located in the outer mitochondrial membrane that helps maintain the mitochondrial structure and inhibits cytochrome C leakage into the cytoplasm, preventing apoptosis. The ratio of Bax and Bcl-2 thus affects cell survival [11,24-26,55].In the current work, Dox treatment resulted in significant elevations of cleaved-caspase-3 and Bax alongside a dramatic reduction in expression of Bcl-2, a pattern reported in the published literature [11]. Geraniol pre-supplementation significantly mitigated this Dox-associated alteration in the balance of apoptotic proteins.
Finally, we performed a histopathological examination to investigate whether Dox administration causes renal damage. This examination confirmed that Dox administration leads to the congestion of the renal blood vessels, interstitial inflammation involving lymphocytes, and hemorrhage between the tubules. Meanwhile, geraniol pre-supplementation clearly alleviated the tissue damage caused by Dox.
In conclusion, the data obtained from the present investigation demonstrate, for the first time, the beneficial effects of geraniol against Dox-mediated kidney toxicity, likely realized by lowering oxidative stress, inflammation, and apoptotic tissue damage through the modulation of the NF-kB, Bax/Bcl-2, and Nrf2/Ho-1pathways. However, the exact protective mechanism of geraniol is vet to be elucidated; further study remains required to pinpoint the details of its action.
