Rutin and Hesperidin Revoke the Hepatotoxicity Induced by Paclitaxel in Male Wistar Rats via Their Antioxidant, Anti-Inflammatory, and Antiapoptotic Activities

Paclitaxel, one of the most effective chemotherapeutic drugs, is used to treat various cancers but it is exceedingly toxic when used long-term and can harm the liver. This study aimed to see if rutin, hesperidin, and their combination could protect male Wistar rats against paclitaxel (Taxol)-induced hepatotoxicity. Adult male Wistar rats were subdivided into 5 groups (each of six rats). The normal group was orally given the equivalent volume of vehicles for 6 weeks. The paclitaxel-administered control group received intraperitoneal injection of paclitaxel at a dose of 2 mg/Kg body weight twice a week for 6 weeks. Treated paclitaxel-administered groups were given paclitaxel similar to the paclitaxel-administered control group together with oral supplementation of rutin, hesperidin, and their combination at a dose of 10 mg/Kg body weight every other day for 6 weeks. The treatment of paclitaxel-administered rats with rutin and hesperidin significantly reduced paclitaxel-induced increases in serum alanine transaminase, aspartate transaminase, lactate dehydrogenase, alkaline phosphatase, and gamma-glutamyl transferase activities as well as total bilirubin level and liver lipid peroxidation. However, the levels of serum albumin, liver glutathione content, and the activities of liver superoxide dismutase and glutathione peroxidase increased. Furthermore, paclitaxel-induced harmful hepatic histological changes (central vein and portal area blood vessel congestion, fatty changes, and moderate necrotic changes with focal nuclear pyknosis, focal mononuclear infiltration, and Kupffer cell proliferation) were remarkably enhanced by rutin and hesperidin treatments. Moreover, the elevated hepatic proapoptotic mediator (caspase-3) and pro-inflammatory cytokine (tumor necrosis factor-α) expressions were decreased by the three treatments in paclitaxel-administered rats. The cotreatment with rutin and hesperidin was the most effective in restoring the majority of liver function and histological integrity. Therefore, rutin, hesperidin, and their combination may exert hepatic protective effects in paclitaxel-administered rats by improving antioxidant defenses and inhibiting inflammation and apoptosis.


Introduction
Paclitaxel, which stabilizes microtubules and inhibits their depolymerization during cell division, is one of the most widely used chemotherapy drugs [1][2][3][4]. Te active compound selection program founded by the National Cancer Institute in 1981 proved that paclitaxel was the only active biological ingredient that falls within this category and meets the standard that could be efectively used to manage cancer, mainly from clinical trials [5,6]. Paclitaxel is used to treat various cancers, including breast, prostate, bladder, cervical, and brain cancer [7][8][9][10]. Many diferent cancers are also treated with paclitaxel, such as aggressive and metastatic breast cancer, ovarian cancer, lung cancer, pancreatic cancer, and others [11]. However, its administration causes numerous adverse efects, including neuropathy, cardiotoxicity, and hepatotoxicity, as well cancer cells' resistance to paclitaxel chemotherapy [12][13][14]. Paclitaxel has been widely known to stimulate apoptosis. Moreover, it has been recognized to produce reactive oxygen species (ROS) that trigger mitochondrial dysfunction to release cytochrome C into the cytoplasm and activate the caspase cascade and apoptosis stimulation [15,16]. Paclitaxel promotes oxidative stress, decreases antioxidants, increases liver enzymes, and impairs renal function, which may be due to its mechanism of action and the oxidative stress that it caused [17]. Paclitaxel exacerbates liver damage during treatment and causes severe liver necrosis that may lead to mortality [18][19][20]. Paclitaxel has been reported to exert infammatory actions. It also revealed a signifcant increase in proinfammatory cytokines, such as interleukin (IL)-17A, tumor necrosis factor-alpha (TNF-α), interferon-c (IFNc), and keratinocyte, in paclitaxel-treated mice [21].
To reduce the toxicity of various organs from chemotherapeutic drugs, several studies have investigated the use of natural compounds that have antioxidant and antiapoptotic efects [22][23][24][25][26][27][28]. Citrus species are considered to be among the most economically signifcant biological resources, as they contain a variety of plant nutrients and phytochemicals with promising therapeutic properties [29]. Flavonoids have various biological efects and may confer health benefts via diferent mechanisms through antiinfammatory, antioxidant, antimicrobial, and antiproliferative regulatory activities [30][31][32]. Several natural antioxidants have been experimentally tested for their potential to protect the liver, such as rutin [33] and hesperidin [34]. Combining rutin with other drugs can reduce drug resistance and side efects of chemotherapy [35]. Rutin has tremendous medicinal potential to regulate several cell signaling and apoptotic pathways implicated in cancer progression [36]. Additionally, it induces an important mechanism in inhibiting cell proliferation in neoplastic cells in the liver tissue by hepatocellular marker enzyme and tumor incursion suppression [37]. Rutin has shown remarkable protection against acrylamide-induced oxidative deoxyribonucleic acid (DNA) damage, which may be due to its antioxidant potential [38]. Hesperidin possesses chemopreventive potential against paclitaxel-induced hepatotoxicity probably by reducing oxidative stress, infammation, apoptosis, and autophagy [39]. Furthermore, the pretreatment of hesperidin ofers powerful protective efects against cisplatin-induced hepatic damage, which is achieved by its antioxidant, anti-infammatory, and antiapoptotic activities [40]. Hesperidin's anticancer potential is controlled by ROS-dependent apoptotic pathways in certain cancer cells, despite the fact that it can be an excellent ROS scavenger and could operate as a powerful antioxidant defense mechanism [41].
Chemotherapeutic drugs such as paclitaxel have several deleterious side efects including liver injury and we aim to minimize these efects by using plant constituents with antioxidant and anti-infammatory activities. Terefore, this research aimed to scrutinize the preventative efcacy of rutin, hesperidin, and their combination on paclitaxel (Taxol)-induced liver toxicity, as well as to investigate the roles of infammation, oxidative stress, and apoptosis modulations in preventive action.

Experimental Design.
Adult male Wistar rats were subdivided into 5 groups in this study (6 rats per group).
(i) Normal group: rats in this group were orally administered with 5 mL 1% carboxymethylcellulose (CMC) (vehicle in which rutin and hesperidin are dissolved)/Kg body weight (b. wt) every other day and 2 mL isotonic saline (0.9% NaCl) (vehicle in which paclitaxel is dissolved)/Kg b. wt twice per week via the intraperitoneal (i.p.) route for 6 weeks. (ii) Paclitaxel-administered control group: this group of rats received paclitaxel at a dose of 2 mg/Kg b. wt (in 2 mL 0.9% NaCl) by i.p. injection [42] twice a week on the 2 nd and 5 th days of each week for 6 weeks, an equivalent dose of 1% CMC (5 mL/Kg b. wt) was also given orally every other day. (iii) Paclitaxel-administered group treated with rutin: this group of rats received paclitaxel as in the paclitaxel-administered control group, as well as rutin orally every other day at a dose of 10 mg/Kg b. wt [43] (dissolved in 5 mL of 1% CMC) for 6 weeks. (iv) Paclitaxel-administered group treated with hesperidin: this group of rats received paclitaxel as in the paclitaxel-administered control group, as well as hesperidin orally every other day at a dose of 10 mg/ Kg b. wt [44] (dissolved in 5 mL of 1% CMC) for 6 weeks. (v) Paclitaxel-administered group treated with rutin and hesperidin combination: this group of rats received paclitaxel as in the paclitaxel-administered control group, as well as rutin and hesperidin combination orally every other day at a dose of 10 mg/Kg b. wt (dissolved in 5 mL of 1% CMC) for 6 weeks.

Blood and Liver
Sampling. Under inhalation anesthesia [45], blood samples were collected from the jugular vein into gel and clot activator tubes after a 6-week treatment with the prescribed dosages. Blood samples were allowed to clot at room temperature and then centrifuged for 15 minutes at 3,000 rounds per minute (rpm). For various biochemical experiments, sera were quickly separated, split into four portions for each animal, and kept at −30°C. Following decapitation and dissection, livers were dissected for biochemical testing and histopathological examination, with each rat's liver tissue being quickly weighed and washed with isotonic saline (0.9% NaCl). A part of the liver was preserved in bufered formalin for 24 hours, then cut and placed in 70% alcohol for histopathologic analysis. Te Tefon homogenizer (Glas-Col, Terre Haute, IND, USA) was used to homogenize approximately 0.5 g of each liver tissue into 5 mL 0.9% NaCl. Te homogenates were then centrifuged for 15 minutes at 3,000 rpm, and the supernatants were aspirated and frozen at −30°C until employed in the assessment of oxidative stress marker-related biochemical and antioxidant parameters.

Determination of Liver Function Biomarkers in Serum.
ALT and AST activities were assessed according to the method of Gella et al. [46]. Te activities of GGT and ALP were assayed using the methods of Schumann et al. [47] and Schumann et al. [48], respectively. Te activity of LDH was measured as previously described by Pesce [49]. Te levels of serum albumin and total bilirubin were measured according to the procedures of Doumas et al. [50] and Jendrassik [51], respectively.

Liver Oxidative Stress and Antioxidant Biomarkers'
Analysis. Chemical reagents prepared in the laboratory were used to evaluate liver oxidative stress and antioxidant biomarkers. Te method provided by Preuss et al. [52] was used to estimate liver lipid peroxidation (LPO). Briefy, 0.15 mL 76% TCA was added to 1 mL liver homogenate to precipitate the protein. Te isolated supernatant was then colorenhanced with 0.35 mL TBA. At 532 nm, the produced pale pink color was identifed after 30 minutes in an 80°C water bath. Te standard was MDA. On the other hand, GSH concentration in the liver was evaluated by adding 0.5 mL DTNB or Ellman's reagent (as a color-developing agent), and phosphate bufer solution (pH, 7) to homogenate supernatant after protein precipitation by centrifugation, as described by Beutler et al. [53]. At 412 nm, the generated yellow colors in the samples and GSH standard were measured and compared to a blank. Te activity of liver GPx was determined using a modifed version of the procedure described by Matkovics et al. [54]. Te remaining GSH after it has been converted by the enzyme to GSSG (oxidized glutathione) and deducting the residual from the total is the basis of this approach. Briefy, 50  Following the discovery of residual GSH in the sample, the enzyme activity was measured by converting GSH to GSSG. Te activity of the liver SOD was measured using the method of Marklund and Marklund [55]. SOD inhibits pyrogallol autoxidation, which is the basis for the reaction. Superoxide ions are necessary for the process to take place. One unit of enzyme is equivalent to the quantity of enzyme required to reduce extinction changes by 50% in one minute as compared to the control.

Histological Investigations.
After the fast decapitation and dissection of each rat, 3 mm 3 pieces of liver from all groups were preserved in 10% neutral phosphate-bufered formalin (pH 7.2) for 24 hours. Te fxed livers were transferred to the Pathology Department of Beni-Suef University's Faculty of Veterinary Medicine in Egypt for additional processing, wax blocking, sectioning, and hematoxylin and eosin (H&E) staining [56]. Histological scores were determined by examining the stained liver sections. Six random felds were estimated for each section. Te number of sections in each group is six. Degenerative change, fatty change, infammatory cell infltration, necrosis, vascular congestion, and Kupfer cell proliferation were among the graded lesions. Scoring of these hepatic lesions was calculated based on Khafaga et al. [57] and Wasef et al. [58] and graded as follows 0 � none; 1 ≤ 25%; 2 � 26-50%; 3 � 51-75%; and 4 � 76-100%.

Immunohistochemical Investigations of Caspase-3 and TNF-α.
Te liver samples, secured with 10% neutral bufered formalin, were processed, blocked, and divided into 5-μm-thick sections that were fxed on positive-loaded slides (Fisher Scientifc, Pittsburgh, PA, USA) at the National Cancer Institute's Pathology Department. Te immunohistochemical reactions in the liver sections were investigated according to the method described by previous publications [59][60][61][62][63]. Briefy, after antigen retrieval, liver sections were incubated for 1 hour with diluted primary antibodies (dilution: 1-100 in phosphate bufer saline) for caspase-3 or TNF-α (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Diluted biotinylated secondary antibodies (dilution: 1-200 in phosphate bufer saline) of DakoCytomation Kit were added and incubation was carried out for 15 minutes at 37°C. Ten, using a DakoCytomation Kit, horseradish peroxidase conjugated with streptavidin was added and incubated for another 15 minutes. A reaction of 3,3′-diaminobenzidine (DAB) substrate was used to visualize the bound antibody complex, which was counterstained with hematoxylin. Immunostaining was comparable across all research groups since all liver slices were incubated under the same conditions with the same antibody dilutions and for the same period. A light microscope was used to examine the immunostained liver sections and determine the degree of cell immunopositivity. A digital camera was used to capture photos of the liver section (Leica, DM2500M Leica, Wetzlar, Germany). ImageJ (1.51d), a free software program, was used to measure the area percentage of immune positivity for caspase-3 and TNF-α reactions according to Khafaga

Efects on Serum Parameters Related to Liver Function.
Te serum AST, ALT, GGT, LDH, and ALP activities, as well as the total bilirubin level, increased signifcantly (p < 0.05) after rats were given paclitaxel intraperitoneally for 6 weeks. When compared to the corresponding normal controls, paclitaxel administration resulted in a signifcant decrease in serum albumin level, with a documented percentage change of −37.37%. Te treatment of paclitaxel-administered rats with rutin and/ or hesperidin resulted in substantial decreases in increased serum AST, ALT, LDH, ALP, GGT, and total bilirubin levels when compared to the paclitaxeladministered control group. Te treatment with rutin and its combination with hesperidin, on the other hand, resulted in a signifcant change in albumin levels, with recorded percentage changes of +31.72 and + 34.41%, respectively, whereas the treatment with hesperidin produced a nonsignifcant improvement (p > 0.05). Moreover, compared with the paclitaxel-administered control group, the treatment of paclitaxel-administered rats with rutin and hesperidin combination was the most efcacious in improving the elevated serum AST, ALT, LDH, ALP, and total bilirubin levels, as well as the decreased albumin levels. Hesperidin treatment was the most efective in lowering GGT activity, with a recorded percentage change of −33.33% (Table 1).

Efects on Liver Oxidative Stress and Antioxidant Defense
Parameters. Paclitaxel was given intraperitoneally to rats for six weeks, resulting in a highly signifcant rise in liver LPO and a highly signifcant decrease in liver GSH content, as well as SOD and GPx activities. Te treatment of paclitaxeladministered rats with rutin, hesperidin, and their combination signifcantly decreased liver LPO. Hesperidin seemed to be the most efective in lowering the increased LPO product in the liver. In contrast to the paclitaxel-   Evidence-Based Complementary and Alternative Medicine 5 administered control group, paclitaxel-administered rats treated with rutin, hesperidin, or their combination showed a signifcant improvement in lowered liver SOD and GPx activities. Te treatment of paclitaxel-administered rats with rutin and hesperidin caused a signifcant increase in the GSH content (Table 2).

Liver Histological Changes.
Histopathological fndings of the liver specimens from diferent experimental groups are presented in Figure 1 and Table 3. Te normal group's liver sections revealed normal histological structures in the form of a thin-walled central vein and normal hepatocytes forming the hepatic cords radiating from the central vein toward the periphery and alternating with narrow blood spaces, the sinusoids, which are lined with single-layered Kupfer cells on histopathological analysis (Figure 1(a)). Conversely, the livers of the paclitaxel-administered group showed marked pathological changes in the form of central vein and portal area blood vessel congestion, marked degenerative changes, including fatty changes and moderate necrotic changes with focal nuclear pyknosis in certain areas, focal leukocytic infltration (mainly mononuclear cells), and Kupfer cell proliferation (Figure 1(b)). Tese changes were altered to some extent in diferent paclitaxel-treated groups. Tese changes were amended to some extent by treatments of paclitaxel-administered groups. First, rats treated with paclitaxel/rutin showed severe degenerative and fatty changes associated with moderate necrotic changes and focal leukocytic infltration associated with moderate proliferation of Kupfer cell activation (Figure 1(c)). Second, pathologic changes in the paclitaxel/hesperidin-treated group were relatively similar to those in the paclitaxel/ rutin-treated group (Figure 1(d)). Finally, the treatment of paclitaxel/rutin/hesperidin produced a good improvement in liver histological changes compared with other treated rats. Moderate degenerative changes and mild necrotic changes accompanied by the mild Kupfer cell proliferation were noted (Figure 1(e)). Te signifcantly elevated histological lesion scores of degenerative changes, fatty changes, necrosis, infammatory cells, congestion, and activated Kupfer cell proliferation in the paclitaxel-injected group were signifcantly decreased by treatments with rutin, hesperidin, and their combination. Te combinatory treatment was the most efective in improving the degenerative and fatty changes (Table 3). Figures 2 and 3, immunohistochemical detection of expressed caspase-3 and TNF-α in the liver was performed. Caspases-3 and TNF-α immunohistochemistry reactivity was very feeble in the liver sections of normal control rats, indicating that their expression levels are very low. Caspase-3 and TNF-α staining in the livers of paclitaxel-administered rats was highly positive, as shown by a dense cytoplasmic brownish-yellow color that suggested their high expression, with percentage changes of +549.29% and +309.55%, respectively, in comparison to the control group. Rutin, hesperidin, and their combination signifcantly reduced the enhanced caspase-3 activity and TNF-α concentration in paclitaxel-administered rats. Te treatment of paclitaxeladministered rats with rutin and hesperidin combination was the most successful in lowering caspase-3 and TNF-α expressions.

Discussion
Paclitaxel is a drug that is commonly used to treat a variety of cancers. Its use may have a variety of adverse efects on several organs, including the liver, kidneys, and heart [67][68][69][70]. Despite remarkable progress in cancer research, compounds derived from natural resources are powerful candidates for cancer treatment [71]. Flavonoids and other reported phenolic components were discovered to have impressive antioxidative, cardioprotective, anticancer, antibacterial, antidiabetic, hypertensive, anti-infammatory, and immune response enhancing efects as well as to protect skin from harmful ultraviolet radiation, making them outstanding drugs for pharmaceutical and medical use [72][73][74].
Tis study showed that the intraperitoneal injection of paclitaxel in the form of Taxol at a dose of 2 mg/Kg b. wt twice a week for 6 weeks caused hepatotoxicity, which was manifested biochemically by a signifcant increase in serum activities of cytosolic enzymes (ALT, AST, and LDH) due to their leakage into the bloodstream from injured hepatocytes [75]. Elevated serum ALT and AST levels in hepatocellular damage have been previously reported in paclitaxel-induced hepatotoxicity models [76][77][78][79][80][81]. Furthermore, the activity of LDH increased in paclitaxel-administered rats [82]. Te LDH activity is elevated in patients with cancer and as a result of tissue damage; it is a common marker of toxicity. Additionally, we found a signifcant elevation in serum activities of membrane-bound enzymes (ALP and GGT) as a result of the increased rate of bile duct production and/or regurgitation in the blood after bile duct blockage [83]. Tese fndings are similar to those reported by Ortega-Alonso et al. [84] who stated that the alteration of membrane permeability of liver cells and bile ducts triggers the release of their specifc enzymes, notably GGT and ALP. Moreover, paclitaxel administration led to a signifcant increase in the total bilirubin content [85,86], and this increase may be indicative of a specifc liver injury and loss of function [87]. Te serum albumin level was signifcantly reduced in paclitaxel-administered rats, which agrees with Wang et al. [88], who found that serum albumin concentration decreased signifcantly following chemotherapy. A decrease in albumin concentration, as observed in paclitaxeladministered rats, indicated insufciency of albumin synthesis by the liver due to hepatopathy [89]. Tese biochemical parameter alterations strongly correlate with hepatic histopathological changes in the form of central vein and portal area blood vessel congestion, marked degenerative changes, including fatty changes and moderate necrotic changes with focal nuclear pyknosis in certain areas, focal leucocytic infltration, and Kupfer cell proliferation. Te current fndings are congruent with those of Salahshoor et al. [80] who showed obvious changes and damage in the liver following paclitaxel treatment. Additionally, 6 Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 7 hepatotoxic efects following paclitaxel therapy were observed [90,91]. It has been also found a distinctive hepatocellular carcinoma in hepatic histological sections in all groups following paclitaxel treatment was observed [85].
Rutin and/or hesperidin treatment of paclitaxeladministered rats successfully reduced increased blood ALT, AST, LDH, ALP, and GGT activities, as well as serum total bilirubin levels, by stopping further paclitaxel-induced  Data are expressed as Mean ± SEM (n � 6). a p < 0.05: signifcant compared with the normal group. b p < 0.05: signifcant compared with the paclitaxel-injected group. c p < 0.05: signifcant compared with the paclitaxel-injected group treated with both rutin and hesperidin. Scoring of hepatic histological lesions was calculated and graded as follows 0 � none; 1 ≤ 25%; 2 � 26-50%; 3 � 51-75%; and 4 � 76-100%. 8 Evidence-Based Complementary and Alternative Medicine hepatocellular damage and stabilizing membrane activity, thereby decreasing the leakage of these enzymes into the general circulation. Te treatments potentially increased the reduced serum albumin level. Moreover, most hepatic histopathological changes were efectively improved by these treatments. Similar observations have been reported by Hozayen et al. [92] who stated that the pretreatment with rutin, hesperidin, and their combination can protect the liver against the hepatotoxic efect of doxorubicin by ameliorating the elevated AST, ALT, ALP, and c-GT activities. Tis is attributed to the hepatoprotective potential of rutin [33] and hesperidin [34]. It was found that hesperidin reduces the severity of sodium arsenate (SA)-induced liver damage [93]. Rutin administration restored the elevated ALT, LDH, AST, and ALP levels in 5-fuorouracil (FU)-treated rats and improved the hepatic structure to normal [24]. Furthermore, rutin treatment improved carflzomib-induced elevated levels of direct bilirubin in rats [94].
Enzymatic and nonenzymatic antioxidant substances are components of antioxidant defense systems. GSH has a tripeptide structure and is a potent nonenzymatic antioxidant. SOD, catalase, and GPx are additional enzymatic antioxidants for ROS defense [95,96]. Paclitaxel administration increases the formation of oxygen-free radicals, decreases antioxidants (SOD and GPx) and GSH content, and increases LPO, which results in liver damage. Tese results are consistent with those of Harisa [97] who reported that paclitaxel induces oxidative stress through decreased GSH content and increased MDA levels. In addition, it was reported that paclitaxel increases ROS and MDA concentrations and decreases SOD activity [82], indicating that paclitaxel induces changes in protein expression associated with apoptosis and ROS generation (Figure 4). ROS activates several mechanisms by damaging cell membranes and macromolecules in cells, resulting in infammation and cell death [98]. Terefore, oxidative stress, which is caused by paclitaxel administration, may cause the production of active oxygen species, including pure oxygen, H 2 O 2 and superoxide radicals, which destroy cells, DNA, proteins, and intracellular lipids, and fnally liver damage [99]. According to the fndings, rutin and hesperidin treatment remarkably reduced paclitaxel-induced oxidative stress by reducing LPO and improving GSH content along with the activities of antioxidant enzymes due to the ability of rutin to recoverfree radicals by chelating metallic iron ions [100,101] as well as the antioxidant activity and radical recovery properties of hesperidin [102,103]. Tese fndings are consistent with those of Hozayen et al. [92], who found that rutin and hesperidin signifcantly increased GSH and GPx levels in the liver and decreased the LPO level in doxorubicin-treated rats. Rutin treatment alleviated liver and kidney damage by reducing oxidative stress, endoplasmic reticulum stress, infammation, apoptosis, and autophagy caused by valproic acid [104]. Additionally, rutin has a hepatoprotective role in eliminating isoniazid-induced oxidative stress [33]. Hesperidin has been discovered to protect the brain, liver, kidneys, and oxidative damage caused by numerous toxins [105,106]. In another way, thymoquinone and costunolide are also natural products that have been shown to have an apoptotic efect to rapidly eliminate the senescent cells induced by doxorubicin and induce apoptosis of proliferative cancer cell lines [107].
Immunohistochemical investigations showed a signifcant increase in the proapoptotic protein (caspase-3) activity and pro infammatory cytokine (TNF-α) concentration in the liver of paclitaxel-administered rats. Te fndings of our investigation agree with those of Yardım et al. [108] who revealed that the mRNA levels of TNF-α and caspase-3 were higher in the paclitaxel group for the sciatic nerve and spinal cord, and the immunohistochemical expression of caspase-3 in the paclitaxel-induced bone marrow tissue was increased. Furthermore, taxanes, including paclitaxel, induced an increase in IL-1β, IL-6, and TNF-α levels in patients with cancer [109][110][111]. It was also found that circulating IL-6 and TNF-α levels were increased 3 days after a 6-dose paclitaxel regimen [112]. TNF-α is a critical mediator of infammation [113] that has been demonstrated to recruit and trigger more infammatory cells in response to increased oxidative stress [114]. TNF-α can promote hepatocyte apoptosis via binding to TNF receptors (TNFR) and death receptors, triggering the extrinsic apoptosis pathway [115][116][117] (Figure 4). Trough the permeability of the mitochondrial membrane or its transition pore apertures, paclitaxel releases apoptogenic components, including cytochrome C, into the cytosol, either directly or indirectly [118,119]. Apoptosis is facilitated by cytochrome C active caspase-9, which stimulates various caspase enzymes, including caspase-3 and caspase-7, in the presence of apoptotic protease activating factor-1 [120,121]. Te treatment of paclitaxel-administered rats with rutin and/or hesperidin suppressed the activity of caspase-3, which is a common mediator of extrinsic and intrinsic apoptotic pathways and the level of TNF-α, which is a key regulator of infammation ( Figure 4). Tese results are consistent with those of Li and Schluesener [122] who reported that hesperidin suppressed oxidative/nitrative stress, infammation, and apoptosis. Hesperidin reduced the caspase-3 activity and showed an anti-infammatory efect by decreasing the levels of TNF-α, nuclear factor kappa B (NF-κB), and IL1β in the kidney and liver tissues of rats with SA-induced toxicity [93]. It also reduced the serum level of TNF-α in arthritic rats [123]. Hesperidin decreased the elevated liver caspase-3 expression and altered serum TNFα, IL-17, and IL-4 levels in diclofenac-administered rats [124]. Additionally, rutin may have potential protective benefts against hepatotoxicity induced by doxorubicin through reducing oxidative stress, infammation, and apoptosis as well as altering the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) gene [125]. Rutin decreased the hepatic TNF-α and IL-6 levels of carbon tetrachloride-treated rats [126]. It was found that rutin signifcantly decreased caspase-3 immunopositivity in 5-FUtreated rats [24]. Te therapeutic potential of rutin can be owed to its antioxidant, anti-infammatory, antiallergic, and antiangiogenic properties [127,128]. Based on our fndings and past research studies, the intrinsic pathway, which is activated by high ROS levels, or extrinsic ligands of pathway receptors, such as TNF-α, can cause caspase-3, the apoptosis executor, to be activated in paclitaxel hepatotoxicity. Rutin and hesperidin may have reduced apoptosis by modulating both intrinsic and extrinsic apoptotic pathways by suppressing oxidative stress and signifcantly lowering increased TNF-α concentration (Figure 4). In addition, TNF-α (through canonical pathway) can activate NF-κB, which promotes NF-κB target genes involved in infammatory responses [129]. Both rutin and hesperidin may produce their antiinfammatory efects by afecting the canonical pathway of NF-κB through the suppression of TNF-α levels and in turn inhibition of TNF-α receptors (TNFR) (Figure 4).

Conclusion
Oral administration of rutin, hesperidin, and their combination could counteract paclitaxel-induced liver damage and toxicity by strengthening the antioxidant defense system and  decreasing oxidative stress and apoptosis. Additionally, it was discovered that rutin and hesperidin combined therapy was the most efective at restoring liver function and histological integrity in paclitaxel-administered rat models. However, before rutin and hesperidin be used in humans, more clinical trials are necessary to evaluate their efectiveness and safety during paclitaxel administration. Te Food and Drug Administration also needs to approve their use in human beings these evaluations. Moreover, further studies are required to scrutinize the efect on mediators of apoptosis other than caspase-3 and mediators of infammation other TNF-α to identify other targets of rutin and hesperidin in paclitaxel-administered rats.

Data Availability
All data are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.