[8] and [10]-Gingerol reduces urothelial damage in ifosfamide-induced hemorrhagic cystitis via JAK/STAT/FOXO signaling pathway via IL-10

Acrolein is the main toxic metabolite of ifosfamide (IFO) that causes urothelial damage by oxidative stress and inflammation. Here, we investigate the molecular mechanism of action of gingerols, Zingiber officinale bioactive molecules, as an alternative treatment for ifosfamide-induced hemorrhagic cystitis. Female Swiss mice were randomly divided into 5 groups: control; IFO; IFO + Mesna; and IFO + [8]- or [10]-gingerol. Mesna (80 mg/kg, i.p.) was given 5 min before, 4 and 8 h after IFO (400mg/kg, i.p.). Gingerols (25 mg/kg, p.o.) were given 1 h before and 4 and 8 h after IFO. Animals were euthanized 12 h after IFO injection. Bladders were submitted to macroscopic and histological evaluation. Oxidative stress and inflammation were assessed by malondialdehyde (MDA) or myeloperoxidase assays, respectively. mRNA gene expression was performed to evaluate mesna and gingerols mechanisms of action. Mesna was able to protect bladder tissue by activating NF-κB and NrF2 pathways. However, we demonstrated that gingerols acted as an antioxidant and anti-inflammatory agent stimulating the expression of IL-10, which intracellularly activates JAK/STAT/FOXO signaling pathway.


Introduction
Ifosfamide (IFO) is an alkylating antineoplastic agent, belonging to the oxazaphosphorine group (Wang and Wang 2012;Ali et al. 2014). A side effect of IFO treatment is the occurrence of hemorrhagic cystitis (HC). HC is an inflammatory process in the bladder mucosa and is characterized by the presence of hematuria, pain in the pubic region, dysuria, urinary incontinence, and nocturia (Martins et al. 2012;Dornelas-Filho et al. 2018). Acrolein is the main toxic metabolite formed during IFO hepatic metabolism. Acrolein enters uroepithelial cells and activate an intense production of reactive oxygen or nitrogen species, leading to peroxynitrite production. Thereafter, peroxynitrate causes lipid peroxidation, protein oxidation, DNA strand breaks and, consequently, necrotic cell death (Korkmaz et al. 2007). Clinical features of acrolein-bladder-toxicity leads to urothelial damage causing edema, ulceration, neovascularization, hemorrhage, and the expression of many pro-inflammatory and anti-inflammatory mediators (Mbanefo et al. 2019;Matz and Hsieh 2017;Smaldone et al. 2009).
The incidence of HC complications is between 10 and 40% of patients treated with IFO even with the use of an uroprotective agent, such as mesna (sodium 2-mercaptoethane sulfonate). Clinical and experimental studies have shown that prophylactic measures are unable to completely block inflammatory events in HC. Studies have shown that 66.7% of patients had histological changes in the bladder wall and all patients had microscopic lesions such as edema, hemorrhage, 1 3 mucosal hematoma, telangiectasia, even when following mesna standard clinical protocol (Lima et al. 2007).
Synthetic and natural compounds had been tested and recently reviewed as an alternative to mesna (Matz and Hsieh 2017), since mesna treatment does not completely treats or prevents acrolein-induced cystitis. Among the natural compounds presented, Zingiber officinale bioactive molecules like gingerols have become promising alternatives for the treatment of several diseases, as they present antiinflammatory, antioxidant, and anti-carcinogenic properties, showing cardioprotective and hepatoprotective effects (Ajayi et al. 2015;Halawany et al. 2017). Gingerol compounds also Fig. 1 Gingerol reduced IFO-induced bladder damage as demonstrated by macroscopic scores of hemorrhage, edema, and BWW. A Macroscopic scores of hemorrhage and edema. B Bladder wet weight, results are expressed as mean ± SEM (N = 6-7). * P < 0.05 of ANOVA test compared to control group, while # P < 0.05 of ANOVA test compared to IFO-treated group. Panels C and D show edema scores, and hemorrhage scores are expressed as * P < 0.05 of ANOVA test compared to control group, while # P < 0.05 of ANOVA test compared to IFO-treated group showed promising therapeutic effects against gentamicin and cisplatin nephrotoxicity (Rodrigues et al. 2014;Kuhad et al. 2006), and presented beneficial effects in cerebral damage after ischemia-reperfusion (Luo et al. 2021) or sepsis (Rodrigues et al. 2018). However, the mechanisms involved in the bladder tissue protection of these compounds in CH induced by IFO is unknown. Thus, the present study aims to investigate the potential protective effects of [8]-and [10]-gingerol against IFO-induced-cystitis.

Animals and experimental drug
Female Swiss mice, weighting between 20 and 25 g were maintained on a 12-h light-dark cycle, with free access to water and standard chow (Biotec, Agua Fria, Santa Catarina, Brazil). All experimental protocols and procedures were carried out in accordance with the norms of the Brazilian National Council for the Control of Animal Experiments (CONCEA) and approved by the Ethics Committee on Animal Experimentation of the Federal University of Ceará, Fortaleza, Brazil (protocol 34/2016).

Experimental design
Animals were randomly divided into 5 distinct treatment groups (n= 6-8 animals per group). The control group was treated with Tween 80 2% p.o., 1h before, 4h and 8h after 0.9% saline, i.p.. The IFO group was treated with Tween 80 2% p.o., 1h before, 4h and 8h after the injection of IFO 400mg/kg, i.p. (Ribeiro et al. 2002). The mesna group was treated with 80mg/Kg, i.p., 5 min before, 4h and 8h after the same IFO dose described above. The mesna group was used as a positive control. Finally, the last two groups were treated with the gingerol compounds [8G] or [10G] at 25mg/ kg p.o., administered 1 h before, 4h and 8h after the IFO injection. The mice were euthanized 12 h after experimental procedures. Their bladders were collected and submitted to further analyzes as described below. The dosage and route of administration we used in this manuscript for mesna and gingerols followed the protocols described by others (Dornelas-Filho et al. 2018;Rodrigues et al. 2014;Batista et al. 2007).

Macroscopic evaluation and bladder wet weight assessment
Bladders were desiccated and emptied of their urinary content and examined for edema and hemorrhage, according to the criteria of Gray et al. (1986). Then, they were weighed, and the wet bladder weight was expressed in mg/20 g of animal weight.

Histological analysis
Bladder tissues were fixed in paraformaldehyde. They were dehydrated with 70% ethanol and processed in paraffin. The resulting blocks were cut into 5-μm-thick sections, stained with hematoxylin and eosin (H&E), and observed under an optical microscope (×500). Slides were observed blindly by a pathologist, who analyzed at least 10 different regions of the same slide in order to provide accurate histological analysis.

Assessment of myeloperoxidase activity
MPO activity was measured as an indirect biochemical marker of neutrophilic infiltration. The bladders were homogenized with 0.5% hexadecyltrimethylammonium bromide (HTAB; pH 6.0) in potassium phosphate buffer and centrifuged at 5000 rpm for 7 min at 4°C. The supernatant absorbance was measured in a spectrophotometer (Synergy H1®, BioTek, Winooski, VT, USA) at 450 nm using o-dianisidine hydrochloride and 1% hydrogen peroxide (Bradley et al. 1982). Results were expressed in units per milligram of tissue (U/mg tissue).

Oxidative stress analysis
Oxidative damage was investigated by the degree of lipoperoxidation through the concentration of malondialdehyde (MDA), which was measured through thiobarbituric acid reactive substances (TBARS). The reaction consisted of mixing the 10% bladder homogenate with 1% phosphoric acid and 0.6% thiobarbituric acid solutions. The mixture of these reagents was kept at 99°C for 45 min and then cooled for 20 min before n-butanol addition. The tube was vortexed for 1 min and centrifuged at 5000 rpm for 15 min. After centrifugation, the organic phase was collected, and its absorbance was read at 535 nm using the spectrophotometer Synergy H1® (BioTek, Winooski, VT, USA). MDA concentrations were expressed in nanomoles per milligram of tissue (nmoles/mg tissue) (Ohkawa et al. 1979).

Gene transcription analysis
Total RNA was extracted following manufacturer's instructions for the PureLink® RNA Mini Kit (Thermo Fisher Scientific-Waltham, MA, USA). The quantity and quality of total RNA extracted was evaluated using a Nanodrop® (Thermo Fisher Scientific-Waltham, MA, USA). One microgram of total RNA was reverse transcribed using the GoScript® cDNA Synthesis Kit (Promega, Madison, WI, USA) using the Veriti® Thermal Cycler (Thermo Fisher Scientific-Waltham, MA, USA). The synthetized cDNA was stored at −20°C prior to qPCR. Gene transcription analysis were evaluated using the QuantStudio TM 5 Real-Time PCR System (Thermo Fisher Scientific-Waltham, MA, USA). DNA primer sequences were obtained from the National Center for Biotechnology Information (NCBI). As housekeeping gene, we used the hypoxanthine phosphoribosyltransferase 1 (HPRT-1).
For the quantitative real-time polymerase chain reaction (RT-qPCR), 7.5 μL of GoTaq® qPCR Master Mix (Promega, Madison, WI, USA), 2.4 μL of each primer (0.8 μM), and 1.0 μL of cDNA from the samples were used, supplemented by nuclease-free water to a final volume of 15μL. Negative samples were also tested, with cDNA replaced by autoclaved Milli-Q water. PCR conditions were as follows: initial denaturation period of 2 min at 95°C followed by 40 cycles of gene amplification. The cycles started with the denaturation step (15s at 95°C), followed by an annealing-extension step (1min at 60°C for most of primers) followed by the melting curve analysis (increments of 0.05°C each 5 s initiating at 60°C and ending at 95°C). Data were obtained with Design & Analysis software version 2.6.0 (Thermo Fisher Scientific-Waltham, MA, USA) based on cycle threshold values, where the observed fluorescence is 10 times greater than the baseline fluorescence for each qPCR assay. The relative expression of all genes was obtained by applying the 2 −ΔΔCT mathematical method (Livak and Schmittgen 2001).

Data analysis
Parametric data was presented as mean ± SEM and statistical analysis of significant difference were carried out by one-way analysis of variance (ANOVA) for comparisons of multiple data sets. Nonparametric data, such as gene expression data, was reported as median, minimum, and maximum. Statistical analysis of significant difference of nonparametric data were carried out by Mann-Whitney test. A p value of <0.05 was considered statistically significant for both parametric and nonparametric data.

Gingerols treatment reduced IFO-induced bladder damage
IFO administration increased BWW, edema and hemorrhage scores compared to the control group (P<0.05) ( Fig. 1A and B). Treatment with gingerol compounds and mesna were able to attenuate the increased in BWW caused by IFO (Fig. 1B). There was also a reduction in edema scores for all tested substances when compared to IFO (Fig. 1C). However, only mesna could reduce the macroscopic hemorrhage score (Fig. 1D).
Histopathological analysis of the bladders (Fig. 2) evidenced edema, mucosal erosion with ulceration, clots, hemorrhage, and neutrophilic infiltrate in animals treated with IFO (Fig. 2B). The treatment with gingerols showed a decrease in the extent of microscopic parameters, with similar results obtained by Mesna treatment (Fig. 2C, D, and E), with a reduction in urothelium desquamation (the partial or total loss of the epithelial lining), edema (accumulation of eosinophilic proteinaceous fluid in the subepithelial connective tissue), and neutrophilic infiltrate (segmented and lobulated nucleus leukocytes).
MDA levels were measured as an indicator of oxidative stress. Our results showed that MDA levels increased in animals subjected to IFO treatment (Fig. 3A), demonstrating that IFO damaged might be associated with oxidative stress induction. In addition, all tested compounds were able to decrease MDA levels, indicating their antioxidant effect (Fig. 3A). The MPO activity indicates neutrophil migration to the tissue, thus indicating inflammatory response. Our results showed that MPO was reduced by treatment with gingerol compounds and mesna indicating that they had antiinflammatory activity (P<0.05) (Fig. 3B).

Gingerols alters mRNA expression of inflammatory markers of hemorrhagic cystitis
IFO treatment increased expression of tumor necrosis factor-alpha (TNF-α), while mesna treatment restored it back to control levels (Fig. 4A). When compared to IFO group TNF-α expression was reduced upon mesna treatment (Fig. 4B). Like TNF-alpha, cyclooxygenase-2 (COX2) relative expression increased upon treatment with IFO, while mesna treatment restored it back to control levels (Fig. 4C). In addition, when we compared to IFO treatment group COX2 expression was reduced (Fig. 4D). Treatment with IFO, even in the presence of mesna or gingerols, increased expression of C-X-C motif chemokine ligand 1 (CXCL1) when compared to control (Fig. 4E). Furthermore, mesna treatment reduced expression of CXCL1, when compared to IFO treatment (Fig. 4F).
Expression of interleukin-1β (IL-1β) increased upon IFO treatment, but only mesna treatment reduced its expression Fig. 3 Gingerol reduced IFO-induced oxidative stress and inflammation. A MDA levels were used as an indicator of oxidative stress, results are expressed as mean ± SEM (N= 6-7). * P < 0.05 of ANOVA test compared to control group, while # P < 0.05 of ANOVA test compared to IFO-treated group. B Neutrophil migration was estimated by MPO, results are expressed as mean ± SEM (N= 6-7). * P < 0.05 of ANOVA test compared to control group, while # P < 0.05 of ANOVA test compared to IFO-treated group 1 3 when compared to IFO-treated group (Fig. 5A and B). Interleukin 6 (IL-6) expression was increased upon treatment with IFO. However, treatment with mesna and gingerols decreases expression of IL-6 when compared to IFO treatment ( Fig. 5C and D). Interleukin 10 (IL-10) expression was increased upon treatment with IFO and mesna reduced it back to control levels (Fig. 5E). Additionally, treatment with gingerols increased IL-10 expression higher then IFO and control groups (Fig. 5E and H), suggesting that gingerols could induce IL-10 expression.

Mesna and gingerols protect against ifosfamide-induced hemorrhagic cystitis via different mechanisms
IFO treatment increased expression of heme oxygenase 1 (HO-1) when compared to control (Fig. 6A). Treatment with mesna and gingerols also showed increased of HO-1 expression when compared to control (Fig. 6A); however, only mesna showed increased expression when compared to IFO treatment alone (Fig. 6B). Our results showed that mesna acts in synergy with IFO upregulating HO-1 gene.
IFO and gingerols treatment decreased expression of NAD(P)H quinone dehydrogenase 1 (NQO1) and superoxide dismutase 1 (SOD1) when compared to control, while mesna treatment restored expression of SOD1 backed to control levels and increased NQO-1 expression when compared to control ( Fig. 6E and Fig. 6C). Accordingly, mesna treatment increased both genes expression when compared to IFO treatment group ( Fig. 6D and F). This result suggests that Mesna antioxidant activity might be mediated by NQO1 and SOD1 activation.
IFO treatment decreased expression of nuclear factor erythroid 2-related factor 2 (Nrf2) when compared to control group, while treatment with mesna restored expression back to control (Fig. 6G). The expression of Nrf2 increased upon mesna treatment when compared to the IFO group (Fig. 6H). Suggesting that mesna and gingerols antioxidant activity might be mediated by different mechanisms. Considering our previous results, showing that gingerols treatment increased expression of IL-10, we investigated the FOXO signaling pathway, since IL-10 activates the FOXO signaling pathway via JAK-STAT signaling pathway.

Treatment with gingerols induce FOXO activation via JAK-STAT signaling pathway
When compared to the control group, IFO and mesna treatments did not change signal transducer and activator of transcription 3 (STAT3) expression; however, gingerol treatment increased STAT3 expression (Fig. 7A). Similarly, only gingerol treatment increased STAT3 expression when compared to IFO treatment (Fig. 7B). When compared to control group, forkhead box O 3a (FoxO3a) expression was reduced upon IFO treatment, expression was unchanged upon mesna treatment and expression was increased upon gingerols treatments (Fig. 7C). When compared to IFO treatment only gingerols treatment increased expression of FoxO3a (Fig. 7D). Sirtuin 1 (SIRT1) expression was decreased in IFO treatment group when compared to control group (Fig. 7E). SIRT1 expression was increased upon gingerol treatment when compared to IFO group (Fig. 7F). Activation of FoxO3a leads to oxidative stress resistance and DNA repair mechanisms via the induction of catalase, mitochondrial superoxide dismutase (mnSOD), growth arrest and DNA-damage-inducible alpha (Gadd45a), and ataxia telangiectasia mutated (ATM) expression. We analyzed the expression of all four genes and compared to control and IFO-treated group. Our results showed that when compared to control group catalase mRNA expression was decreased upon IFO treatment (Fig. 8A). When compared to the IFO group catalase expression was increased in mesna and gingerol treatment groups (Fig. 8B). mnSOD expression did not change ( Fig. 8C and D). Gadd45a expression decreased in all treatment groups when compared to control (Fig. 8E). When compared to the IFO group, expression of Gadd45a was increased in gingerol treatment groups (Fig. 8F). ATM expression was also reduced in all treatment groups when compared to control (Fig. 8G); however, when compared to the IFO group, there was no change in ATM expression in any treatment group (Fig. 8H).

Discussion
IFO-induced hemorrhagic cystitis is directly related to the production of acrolein. We observed that IFO administration augmented animal bladder wet weight and caused edema, hemorrhage, mucosal erosion with ulceration, and neutrophilic infiltration. Bladder Inflammation and tissue oxidative stress was shown by the increase of neutrophil migration and lipoperoxidation. The experimental model to induce HC by IFO treatment as reported by other studies was corroborated by our Fig. 4 Gingerols alters mRNA expression of inflammatory markers of hemorrhagic cystitis. A Relative mRNA expression of TNFalpha compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. B Relative mRNA expression of TNF-alpha compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. C Relative mRNA expression of COX-2 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. D Relative mRNA expression of COX-2 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. E Relative mRNA expression of CxCL1 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. F Relative mRNA expression of CxCL1 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group ◂ 1 3 results (Ali et al. 2014;Dornelas-Filho et al. 2018;Dornelles et al. 2014;Leite et al. 2015). We investigated the uroprotective effects of [8]-and [10]-gingerol and compared it with mesna. Our data showed that mesna, [8]-gingerol and [10]-gingerol were all able to protect animal tissue, decreasing IFO's adverse impact in bladder wet weight, edema, microscopic alterations, neutrophil migration, and lipoperoxidation. However, only mesna was able to diminish hemorrhagic score. Overall, our results showed that mesna was better protecting the bladders than the gingerols, but the possibility of using gingerols as a treatment adjuvant of HC is also plausible, especially because they could be used upon oral administration.
The inf lammatory impact of oxazaphosphorines was well documented by Sherif (2020), in which the accumulation of acrolein could activate the NFκB pathway inducing the release of TNF-α, IL-1β, and IL-6. It was also reported that the mRNA expression level of COX2 was increased by the impact of TNF-α stimulation (Sherif 2018). Other studies have also reported the augmentation of bladder chemokines expression, such as CxCL1 (Gonzalez et al. 2014;Mbanefo et al. 2018). However, the increase of IL-10 expression observed in this study has not been reported before. IL-10 is an anti-inflammatory cytokine, which has a cytoprotective effect via diminishing macrophage activation and it was found to be reduced after cyclophosphorin injection (Keles et al. 2014). On the contrary, other studies showed increase of IL-10 expression was an acute response of cyclophosphamide-induced cystitis and probably related to a compensatory drive to counterbalance the release of inflammatory cytokines (Smaldone et al. 2009;Malley and Vizzard 2002).
The mRNA expression of HO-1, NQO-1, and SOD-1 are all inducible by the nuclear factor E2-related factor 2 (NrF-2) pathway, which is a transcription factor responsible for regulating the cellular antioxidative responses (Wilmes et al. 2011). NrF-2 diminishes bladder dysfunction through suppressing oxidative stress (Gu et al. 2018). Ni et al. (2021) showed that NrF-2 knockout mice presented severe symptoms of cystitis after cyclophosphamide administration, followed by a decrease in SOD-1 activity and increase in mRNA expression of HO-1 and NQO-1. mRNA expression of HO-1 and NQO-1 after IFO-induced cystitis was also described by Kim et al. (2014). The reduction of mRNA expression of NQO-1 presented in our work was the only data distinct to what is normally seen in the literature. However, even though NQO-1 and HO-1 are considered phase II detoxifying enzymes regulated by NrF-2 nuclear activation, it looks like HO-1 can be regulated differently, supporting the idea that one enzyme could be upregulated, but not the other (Yeligar et al. 2010). Future studies are needed to investigate the specific mechanism by which the expression of this enzymes is regulated upon IFO treatment.
Mesna and gingerols treatment diminish IFO-induced cystitis. Interestingly, the mRNA expression data showed that their mechanism of action to protect bladder dysfunction are different. Mesna blocked the oxidative and inflammatory impact of IFO by activating the NF-κB and NrF2 pathways, which was shown in our data by its impact in the mRNA expression of TNF-α, COX-2, CxCL-1, IL-6, IL-1β, HO-1, NQO-1, and SOD-1. Gingerols altered the mRNA expression of IL-6, to a lesser extent, but IL-10 and catalase at a higher degree. Based on these results, we postulated that gingerols stimulated the production of IL-10 in bladder tissue. This cytokine is known to activate the JAK-STAT signaling pathway triggering the activation of STAT3 (Mbanefo et al. 2019;Nagata and Nishiyama 2021), which consequently activates class O forkhead transcription factor (FoxO). When deacetylated by SIRT1, FoxO induces the production of antioxidative stress protein and DNA repair proteins (Oh et al. 2012;Storz 2011). Our results of mRNA expression analysis confirmed that gingerol treatment activates JAK/STAT/ FOXO signaling pathway, likely mediated by IL-10, as treatment with gingerol compounds increased expression of IL-10, STAT3, FoxO3a, and SIRT1.
The FoxO family of DNA transcription factors have been implicated in many cell processes including cell cycle, apoptosis, DNA repair, stress resistance, and metabolism. FoxO is also regulated by oxidative stress (Furukawa-Hibi et al. 2002). Few studies have described FoxO activation as one of the intracellular downstream genes triggered by gingerols, specially related to apoptosis of cancer cells (Kiptiyah et al. 2017). This study shows, for the first time, that oral administration of gingerols could be used as an adjuvant treatment of ifosfamide-induced cystitis. Like other studies (Sheng et al. 2020;Choi et al. 2017), our work demonstrates that gingerols anti-inflammatory activation could be related to the increase of IL-10 mRNA expression; furthermore, we found that gingerols may exerts its bladder protection by the activation of the JAK-STAT signaling pathway mediated by the expression of IL-10. Gingerols alters mRNA expression of inflammatory interleukins of hemorrhagic cystitis. A Relative mRNA expression of IL-1Beta compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. B Relative mRNA expression of IL-1Beta compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. C Relative mRNA expression of Il-6 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. D Relative mRNA expression of IL-6 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. E Relative mRNA expression of Il-10 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. F Relative mRNA expression of IL-10 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group ◂ Fig. 6 Mesna alters mRNA expression of antioxidant proteins of hemorrhagic cystitis. A Relative mRNA expression of HO-1 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. B Relative mRNA expression of HO-1 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. C Relative mRNA expression of NQO1 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. D Relative mRNA expression of NQO1 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. E Relative mRNA expression of SOD1 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. F Relative mRNA expression of SOD1 compared to IFO-treated group. Results are expressed as median, minimum, and maximum SD (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. G Relative mRNA expression of Nrf2 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. H Relative mRNA expression of Nrf2 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group Fig. 7 Gingerols alters mRNA expression of STAT3/FoxO3a in hemorrhagic cystitis. A Relative mRNA expression of STAT3 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. B Relative mRNA expression of STAT3 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. C. Relative mRNA expression of FoxO3a compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. D Relative mRNA expression of FoxO3a compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. E Relative mRNA expression of SIRT-1 compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. F Relative mRNA expression of SIRT-1 compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group Fig. 8 Gingerols alters mRNA expression of FoxO3a downstream targets in hemorrhagic cystitis. A Relative mRNA expression of Catalase compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. B Relative mRNA expression of catalase compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. C Relative mRNA expression of MnSOD compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. D Relative mRNA expression of MnSOD compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. E Relative mRNA expression of Gadd45a compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. F Relative mRNA expression of Gadd45a compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group. G Relative mRNA expression of ATM compared to control group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to control group. H Relative mRNA expression of ATM compared to IFO-treated group. Results are expressed as median, minimum, and maximum (N= 6-7). * P < 0.05 of Mann-Whitney test compared to IFO-treated group ◂