Neuroprotective Effect of Baicalein Against Oxaliplatin-Induced Peripheral Neuropathy: Impact on Oxidative Stress, Neuro-inflammation and WNT/β-Catenin Signaling

Oxaliplatin, an effective anti-cancer agent used in the treatment of colorectal cancer, is associated with severe dose-limiting side effects like peripheral neuropathy, which currently remains a major unmet clinical need. This study was designed to investigate the possible neuroprotective potential of a bioflavonoid, baicalein in an experimental model of oxaliplatin-induced peripheral neuropathy. Rats were administered with a dose of 4 mg/kg oxaliplatin i.p. twice per week for four weeks, and were evaluated for behavioral and functional nerve parameters, followed by biochemical, immunohistochemical and western blot analysis. This study shows that baicalein reversed oxaliplatin-induced behavioral deficits and significantly prevented oxaliplatin-induced sensory nerve conduction deficits in rats. Molecular analysis revealed baicalein significantly strengthened the antioxidant defense system by enhancing the expression of MnSOD, HO-1, and GSH levels. Baicalein treatment neutralized the oxaliplatin-induced neuroinflammation, which was evident from the significant loss of inflammatory mediators like TNF-α, IL-6 and a shunted NF-κB nuclear translocation. Additionally, baicalein treatment resulted in a significant downregulation of active β-catenin, Wnt5b and Wnt3a proteins. In line with the in vivo evidences, baicalein treatment in Neuro2a cells attenuated oxaliplatin-induced ROS, mitochondrial superoxide levels and improved neuritogenesis. Additionally, baicalein did not alter the cell viability of oxaliplatin in HCT-116 cell line. Collectively, these results suggest that baicalein may be useful for management of peripheral neuropathy associated with oxaliplatin.


Introduction
Oxaliplatin, a third-generation platinum-based antineoplastic agent and a component of FOLFOX therapy, is used as a first-line treatment in advanced or metastatic colorectal cancer [1]. However, platinum compounds are long associated with neurotoxicity, manifested as either a brief acute syndrome or a dose limiting cumulative sensory neuropathy [2]. Acute neurotoxic syndrome is typically exacerbated by the cold exposure and is characterized by transient paresthesia and/or dysesthesia and muscular spasms in the distal extremities. Chronic neurotoxicity mainly produces sensory dysfunction with distal paresthesia leading to sensory ataxia and functional impairment [3,4]. The incidence of peripheral neuropathy due to oxaliplatin ranges from 81.5-98% and increases when the cumulative dose reaches 540 mg/m 2 or more [5]. Clinical approaches for treating peripheral neuropathy are limited to drugs that provide only symptomatic relief from neuropathic pain and none of the pharmacological interventions have been found to impact the underlying pathomechanism of chemotherapy-induced peripheral neuropathy [6]. Hence, it is worth exploring novel, effective therapeutic strategies that may tackle oxaliplatin-induced neuropathic pain without interfering with its anti-cancer properties. The major hurdle in designing a treatment strategy targeting OIPN is that the mechanism of induction of anticancer activity and mechanism of induction of neurotoxicity is linked, making it difficult to reduce the neurotoxicity without compromising the anti-cancer activity [7]. Though the underlying pathomechanisms of OIPN remain elusive, a number of contributing factors have been identified, which include ion channel dysfunction [8,9], central glial cell activation [10], organelle failure [11][12][13], oxidative stress [14], neuronal apoptosis [15], and activation of various stress signaling mechanisms like NFκB, MAPK, JNK pathways [16]. In addition to these, Wnt signaling which has a massive impact on the functioning of the nervous system is gaining attention in recent times [17]. The involvement of Wnt signaling in pain pathology through the β-catenin-dependent pathway (canonical Wnt signaling) in the spinal cord is well documented [18][19][20]. Hence, a pharmacological strategy with a well-established safety profile, targeting multiple sites and without significantly altering the anti-cancer property of oxaliplatin will be a useful one in the management of OIPN.
Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is a conventional Chinese herbal medicine, purified from the roots of Scutellaria baicalensis Georgi, widely known for its usage in the treatment of bacterial and viral infections. It exerts a wide range of properties including antineoplastic [21,22], neuroprotective [23][24][25] cardioprotective [26,27] and renoprotective [28,29] effects. Baicalein has shown its potent anti-oxidant and anti-inflammatory activity in various in vitro and in vivo models [28]. Recent studies also suggest baicalein's role in the negative regulation of the WNT/β-catenin signaling pathway which may be used [30]. Though the efficacy of baicalein in diabetic neuropathy is established [23], the role of baicalein in oxaliplatin-induced peripheral neuropathy has never been investigated. The current study was aimed to explore the neuroprotective mechanisms of baicalein against oxaliplatin-evoked neurotoxicity in an experimental model of OIPN.

Drugs and Chemicals
Oxaliplatin was a generous gift from Astron pharmaceuticals (Ahmedabad, India). All chemicals including baicalein were obtained from Sigma-Aldrich, USA, unless stated otherwise. FBS was procured from Gibco (life technologies). TNF-α and IL-6 ELISA kits were purchased from BioLegend, San Diego, USA. IHC detection kit was purchased from Pathn-Situ Biotechnologies Pvt Ltd. India.

Cell Lines
Mouse neuroblastoma cell line, Neuro2a, was procured from National Centre for Cell Science, Pune, India, and HCT-116 cell line was obtained from the ATCC, Rockville, USA. These were cultured in MEM and McCoy's 5a media, respectively, containing 10% FBS, 1% streptomycin/penicillin, L-glutamine (2 mM) and were maintained in a CO 2 incubator.

Measurement of Cell viability by MTT Assay
HCT-116 cell line was seeded at an appropriate cell density in a 96-well plate and allowed to attach overnight. Later on cells were treated with different concentrations of oxaliplatin (12.5 μM to 100 μM) and also in combination of baicalein (01 to 50 μM) then again incubated for 24 h. Thereafter, MTT solution was added and the cells were incubated for 4 h. Then, the media containing MTT was discarded and the insoluble formazan crystals were dissolved using 100 μl DMSO and the absorbance was measured spectrophotometrically at 570 nm (Spectramax M4, USA) [31].

Measurement of Intracellular ROS by DCFDA
Neuro2a cells were sub-cultured in a 24-well plate and were allowed to attach overnight; the cells were then dealt with oxaliplatin (50 μM) and baicalein (1 and 3 μM) drug solutions for 24 h, and were stained with DCFDA dye at a concentration of 10 μm for 30 min to detect the levels of intracellular ROS. The fluorescent signal at an excitation wavelength of 485 nm and an emission wavelength of 535 nm was measured by multiplate reader (Spectramax M4, USA. Images were visualized using a fluorescence microscope (Nikon, Japan) [32].

Measurement of Mitochondrial Superoxide Anion (O 2 − ) by MitoSox Staining
Neuro2a cells were seeded in a 24-well plate and were allowed to attach overnight; the cells were dealt with oxaliplatin and baicalein with designated concentrations for 24 h. Then, cells were stained with MitoSox Red (5 μM) for 30 min. The fluorescent signal at an excitation wavelength of 530 nm and emission wavelength of 580 nm was measured by Spectramax M4, USA. Images were visualized using a fluorescence microscope (Nikon, Japan) [33].

Measurement of Mitochondrial Membrane Potential by JC-1 Staining
Neuro2a cells were plated in a 24-well plate and were allowed to attach overnight. Later, cells were treated with oxaliplatin and baicalein drug solutions with designated concentrations and incubated for 24 h. Later on cells were exposed to JC-1 at a concentration of 1 μg/ml for 20 min. Afterward, images were visualized using a fluorescence microscope (Nikon, Japan) [31].

Evaluation of Neurite Outgrowth
Neuro2a cells were used as neurodegeneration model to demonstrate oxaliplatin-induced neurite degeneration.
Cells were seeded at 1 × 104 cells/cm2 onto a 12-well plate and used for experiments on the following day. Cells were induced for neurite growth by adding nerve growth factor (NGF) (50 ng/ mL). Then, they were exposed to drug solutions with indicated concentrations and neurite outgrowth was measured after 24 h of treatment. Morphometric analysis was performed on digitalized images of live cells taken under phase-contrast illumination using an inverted microscope (NIKON, USA). The neurite length was measured by software (Image J 1.36; Wayne Rasband, National Institutes of Health, MD, USA) tracing the length of the distance between the cell periphery and the tip of the longest neurite for each cell in a field. Data from the 6 wells with 10 fields in each were pooled. Neurite outgrowth is expressed as average neurite length (μm) and the percentage of cells bearing neurites (%) [34].

RT-PCR Studies
Trizol method was used for total cellular RNA extraction, and the cDNA was generated with the help of Verso cDNA synthesis kit. Real-time PCR was performed on SYBR Green Master Mix. Triplicate PCR reactions were performed on 1 μl of cDNA, and each reaction underwent 35 cycles of annealing at 48 °C for 20 s, extension at 72 °C for 25 s and denaturation at 94 °C for 15 s. Relative gene expression was calculated using the comparative ΔCt method and normalized to β-actin. RT-PCR primers were designed using Primer Express software (version 3.0.17). The following primers were used: β-actin: forward 5 1 -AGA CCT CTA TGC CAA CAC AG-3 1 and reverse 5 1 -ACT CAT CGT ACT CCT GCT TTG-3 1 ; β-catenin: forward 5 1 -CTT GGT AGG GTG GGA ATG -3 1 and reverse 5 1 -GCC CTC TCA GCA ACT CTA -3 1 . [35].

Immunofluorescence Studies
Neuro2a cells at a suitable cell density were cultured on coverslips in a multi-well plate and were allowed to settle. Once after the attachment of the cells, they were treated with the indicated concentrations of drug solutions for a day. Thereafter, cells were washed, fixed using 4% paraformaldehyde solution and permeabilized with 0.2% Triton-X for 15 min each at room temperature, and then blocked with 3% BSA in PBS for 30 min. Later, cells were incubated with NF-κB primary antibody at 1:400 dilution (Cell Signaling Technology, Beverly, MA) at 4 °C for 12 h. After washing with PBS-T, secondary antibody conjugated with Rhodamine (Santa Cruz Biotechnology, Inc., Texas) at a dilution of 1:200 was added and incubated for 2 h at room temperature in a dark place. Thereafter cells were washed with PBS-T. Then, the coverslips were mounted with antifade mounting medium with DAPI (Vector laboratories) on a glass slide. Visualization of the fluorescent images was done using confocal laser scanning microscope (Leica TCS SP8) [34].

Animals
Healthy male SD rats weighing 250-300 g were used in this study. They were fed on standard diet and water ad libitum. Optimal laboratory conditions like constant temperature of 23 °C ± 1 °C and relative humidity 55% ± 10% with 12:12-h light and dark cycle were maintained during the entire study. The animal study protocols were duly approved by the Institutional Animal Ethics Committee (IAEC)-NIPER Hyderabad, and all the experiments were conducted in accordance to prevailing guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Study Design
SD Rats were randomized into five groups consisting of 8 animals each. The groups included normal control (NC), oxaliplatin control (OC), two baicalein treated (5 and 10 mg/ kg, once daily for 4 weeks) and the perse group. Oxaliplatin at a dose of 4 mg/kg (i.p in 5% dextrose solution) twice every week for 4 weeks was injected. The treatment groups consist of baicalein at 5 mg/kg (OC + BA1) and 10 mg/kg (OC + BA2), i.p. in DMSO and baicalein alone treated group consists of normal rats treated with high dose of baicalein (BA2). Baicalein dose range used in the studies were chosen based on existing literature. At the end of the study, animals were euthanized with CO 2 anesthesia, and sciatic nerves and L4-L6 region of spinal cord were collected on the spot. The collected tissue samples were stored at -80˚C to carry out biochemical and protein expression studies and in 10% NBF for immuno-histochemical studies.

Functional Assessment
Power lab system was employed to assess motor and sensory nerve conduction velocity. The receiving electrodes were placed on the muscle of foot and by utilizing bipolar needle anode; 3 V stimulus was applied at sciatic nerve (proximal to sciatic notch) and tibial nerve (proximally to ankle). The latencies of muscle action potential were recorded by Lab chart software. H-reflex latencies and negative M-Wave deflection were measured to calculate the related SNCV and MNCV, utilizing the distance between the stimulation points and latencies. The results being expressed in m/sec.

Behavioral Assessment
Cold Allodynia Cold allodynia was assessed via acetone spray method by spraying 100 µl of acetone onto the plantar surface of hind paw. The animals were kept under observation for the next 20 s and responses were scored. Score 0 represents, no response; Score 1 represents, quick withdrawal or flicking of the paw; Score 2 represents, prolonged withdrawal or repeated flicking; and Score 3 represents, repeated flicking with licking of the paw. The minimum score obtained was 0 and the maximum score was 9.
Mechanical Allodynia Calibrated Von Frey hairs were used to explore the dynamic responses to mechanical stimulus. After acclimatizing animals in a transparent chamber on a perforated surface, the filaments were applied eventually from lower to higher pressure, perpendicular to plantar surface of rat's feet. While applying sufficient pressure to bend the filaments partially for 2-3 s, responses were noted. The response was considered as positive, when there was a quick withdrawal of the paw, with its licking. The test was repeated for three times for each animal and an interval of 5 min was provided in between each reading.
Mechanical Hyperalgesia Randall-Selitto apparatus was utilized to assess mechanical hyperalgesia. Pressure was applied on the surface of the hind paws. The paw withdrawal responses to the corresponding pressure (g) were recorded. In order to avoid the damage to the paw, a cutoff pressure of 250 g was used [36].
Thermal Hyperalgesia Thermal hyperalgesia of the paw toward radiant heat (thermal stimulus) was performed by Plantar Test (Hargreaves Method) using IITC Plantar Analgesia Meter (IITC, Life Sciences, USA). The pain threshold index was taken as latency of the foremost escaping sign, i.e., paw licking or paw flicking, while keeping a cutoff time of 20 s [37].

Biochemical Parameters
Estimation of MDA and GSH Levels Oxidative stress in tissues was assessed by using Lipid peroxidation assay and GSH assay [38,39]. Homogenization of the spinal tissue was carried out with phosphate buffer. The supernatant was collected after centrifuging the spinal cord samples for 15 min (at 10,000 rpm and 4 °C). The resulting supernatant was utilized to determine protein content, MDA and GSH levels. TBARS was measured to assess the level of lipid peroxidation. One hundred μl of the protein supernatant was mixed with thiobarbituric acid 0.8%, SDS 8.1%, acetic acid 20% and incubated at 95˚C on water bath for 120 min. After centrifuging the samples, the absorbance of supernatant (MDA levels) was measured at 532 nm spectrophotometrically. For GSH estimation, to a 50 μl of supernatant (TCA added), 120 μl of DTNB reagent was added and allowed to incubate for 10 min at room temperature, protected from light. The absorbance of the formed yellow color dianion was measured spectrophotometrically at 412 nm.
Estimation of Inflammatory Markers ELISA estimations were performed using commercially available IL-6 and TNF-α assay kits. It was carried out according to the manufacturer's instructions.

Immunohistochemical Analysis
Rehydrated spinal cord and sciatic nerve sections were incubated with primary antibodies WNT5b (Novus biological, USA, at a dilution of 1:200), non-phospho β-catenin, GFAP (Cell Signaling Technology, Beverly, MA, US, at a dilution of 1:200), 8-OHdG (Abcam, Cambridge, UK, at a dilution of 1:200) for overnight at 4 0 c with constant shaking, followed by incubation with secondary antibody for 1 h at room temperature. Further, these microsections after washing were stained (using DAB) and counterstained (using hematoxylin) to observe under light OPTIKA microscope [40].

Western blotting Analysis
Spinal tissue was homogenized using T-PER lysis buffer with 1% phosphatase inhibitor and protease inhibitor each. Protein samples containing equal protein content were loaded into the well to separate them on SDS polyacrylamide gel; thereafter transferred on PVDF membrane. Membrane was blocked by using 3% BSA solution for 2 h at room temperature, and incubated overnight with primary antibodies of β-catenin, non-phospho β-catenin, WNT3a, WNT5b, NF-κB, phospho NF-κB, p38, phospho p38, SOD2, β-actin (at a dilution of 1:1000, Cell Signaling Technology, Beverly, MA, USA) and COX-2, iNOS (at a dilution of 1:200, Abcam, UK) at 4 0 c. After washing the membranes, they were incubated with a horseradish peroxidase-conjugated secondary antibody (at a dilution of 1:20,000), and visualized using Fusion-FX chemiluminescence imager (Vilber Lourmat, France). The relative band densities were quantified by densitometry using Image J software, NIH, USA. β-actin expression were checked to confirm equal loading of proteins [33].

Statistical Analysis
Results were demonstrated as the mean ± SEM. Behavioral results data were analyzed statistically using two-way ANOVA and end-point data were analyzed statistically using one-way analysis of variance with the help of GraphPad Prism version-5.0 (GraphPad software, San Diego, CA). "Bonferroni's Multiple Comparison Test" was utilized for follow-up analysis. Results with probability level of p values < 0.05 reflected statistical significance.

Baicalein did not Interfere with the Anti-Cancer Efficacy of Oxaliplatin in HCT-116 Colorectal Carcinoma Cell Line
It is of critical importance to determine the interaction of oxaliplatin with baicalein, if any. Cell viability assay was performed in human colorectal carcinoma cell line, HCT-116. The IC 50 of oxaliplatin alone and that co-treated with baicalein at a concentration of 3 μM were found to be 12.70 ± 0.78 μm and 11.83 ± 1.34 μm, respectively. This indicates that co-treatment of baicalein with oxaliplatin did not alter its cytotoxicity.

Baicalein Mitigates Oxaliplatin-Induced Cellular and Mitochondrial ROS in N2a Cells
To evaluate the redox status in Neuro2a cells upon treatment with oxaliplatin at a concentration of 50 μM, we performed DCFDA and MitoSox staining, to measure the levels of intracellular ROS and mitochondrial superoxide (O 2 − ), respectively. Augmented intracellular ROS was indicated by increased green intensity and elevated mitochondrial superoxide levels were shown by enhanced red fluorescence and both of them were increased significantly (p < 0.001, p < 0.01, respectively) in oxaliplatin treated cells when compared to the untreated control cells as shown in Fig. 1. However, baicalein treatment at 3 μM significantly (p < 0.001, p < 0.01, respectively) reduced intracellular ROS and mitochondrial superoxide levels, suggesting a potential free radical scavenging property of baicalein.

Baicalein Prevents Loss of Mitochondrial Membrane Potential (Ψm) and Improved Neuritogenesis in N2a cells
In comparison with the control group, the cells treated with oxaliplatin showed very less fluorescence intensity reflecting loss of Ψm. However, baicalein treatment significantly (p < 0.001 at 3 μM) prevented Ψm loss, showing its ability to maintain the integrity of Ψm as shown in Fig. 2. There was no significant alteration in baicalein alone treated cells at 3 μM when compared with untreated control cells. Further, NGF treated N2a cells, upon exposure to oxaliplatin reduced the number of cells bearing neurites (p < 0.01) and average neurite length significantly (p < 0.001) as represented in

Baicalein Attenuates Oxaliplatin-associated Behavioral and Functional Alterations and Blocks Neuropathic Pain
Repeated administration of oxaliplatin resulted in the development of OIPN as evident by a marked reduction in the paw withdrawal latencies to thermal stimuli, paw withdrawal threshold to Von Frey fibers and Randall-Selitto analgesiometer and reduced mean score of allodynia to acetone spray test as compared to normal control group. Baicalein treatment significantly restored the paw withdrawal latencies (p < 0.001) to Von Frey fibers, paw withdrawal threshold (p < 0.001) to Randall-Selitto analgesiometer ( Fig. 3b and Fig. 3d, respectively). The mean score of allodynia in response to acetone spray significantly (p < 0.001) decreased with baicalein compared to oxaliplatin rats by the 28th day as represented in Fig. 3. In oxaliplatin control group, there was a significant reduction in SNCV (p < 0.001), while MNCV remained unchanged. However, treatment with baicalein showed a significant (p < 0.05) prevention of oxaliplatin-induced lowering of SNCV as shown in Table 1. Indeed, baicalein alone treated group did not show any significant functional and behavioral changes compared to normal control rats.

Baicalein Attenuated Oxaliplatin-mediated Oxidative Stress
MDA is an end product of lipid peroxidation and is a reliable indicator of oxidative stress. Intra-peritoneal administration of oxaliplatin significantly elevated the levels of MDA (p < 0.001) in comparison with normal control group, whereas the group treated concurrently with oxaliplatin and baicalein at 5 mg/kg and 10 mg/kg significantly (p < 0.05 and p < 0.001, respectively) showed reduction in the MDA levels in the spinal cord in comparison with oxaliplatin control group as shown in Table 1. Marked reduction in the levels of GSH, an anti-oxidant enzyme was observed upon treatment with oxaliplatin in comparison with the normal control group (p < 0.001). Baicalein treatment at 5 mg/kg and 10 mg/kg significantly replenished the diminished GSH levels in spinal cord (p < 0.05 and p < 0.001, respectively) in comparison with the oxaliplatin control group as shown in Table 1. The IHC analysis of dorsal part of lumbar part of spinal cord showed a significant rise (p < 0.001) in the expression of 8-OHdG in oxaliplatin control group compared to the normal control group. However, baicalein treatment at higher doses significantly inhibited the elevated levels of 8-OHdG in spinal cord (p < 0.001) as shown in Fig. 4a. To investigate the redox profile in the spinal cord upon treatment with oxaliplatin, western blot analysis was carried out. It revealed a significant decrease in the expression of MnSOD (p < 0.01) and HO-1 (p < 0.05) in oxaliplatin control group in comparison with the normal control group. However, oxaliplatin control animals when treated with 10 mg/ kg of baicalein showed a significant increase in MnSOD (p < 0.001) and HO-1 (p < 0.001) levels as shown in Fig. 4b.

Neuro-protective Activity of Baicalein on Oxaliplatin-induced Neuro-inflammation
Oxaliplatin control group demonstrated a marked increase in the levels of TNF-α and IL-6 (p < 0.01 and p < 0.001, respectively) in the spinal cord, confirming the induction of neuroinflammation upon oxaliplatin administration. However, baicalein treatment at 5 mg/kg (p < 0.01) and 10 mg/ kg (p < 0.001) significantly halted the escalation of TNF-α and IL-6 in a dose-dependent manner in comparison with the oxaliplatin control animals as shown in Fig. 5c. Additionally, western blot analysis revealed an increase in the iNOS (p < 0.001), COX-2 (p < 0.001), NF-κB (p < 0.01), phospho NF-κB (p < 0.001), p-38 activation (p < 0.001) and phospho p-38 activation (p < 0.001) expression in oxaliplatin group when compared to normal control group. However, oxaliplatin control group, when treated with baicalein, showed a decrease in the levels of iNOS, COX-2, phospho NF-κB and phospho p-38 as shown in Fig. 6. IHC analysis showed a significant increase (p < 0.001) in GFAP positivity in the oxaliplatin group compared to the control group; this indicates that oxaliplatin significantly augmented the activation of astrocytes. However, treatment with baicalein at high doses significantly inhibited this elevated level of GFAP in spinal-cord (p < 0.01) as represented in Fig. 5a. Given the anti-inflammatory role of baicalein in in vivo oxaliplatin control (4 mg/kg, ip), OC + BA1 and OC + BA2: oxaliplatin control rats treated with baicalein at 5 mg/kg and 10 mg/kg, ip, respectively, and BA2: normal control rats treated with baicalein (10 mg/kg, ip) Table 1 Effect of oxaliplatin and baicalein on functional and biochemical characteristics after 28th day: Results were expressed as mean ± SEM (n = 6 for Nerve conduction studies and n = 3 for biochemical studies).^^^ p < 0.001 vs. NC, * p < 0.05, ** p < 0.01 vs.

Baicalein Attenuates Oxaliplatin-induced Activation of Wnt Signaling
Wnt ligands, namely Wnt5b and Wnt3a, are a well-known activator of canonical β-catenin signaling. As Wnt signaling plays a critical role in the development of nervous system, we investigated the expression of its key mediators in the spinal cord by western blot analysis. It showed elevated levels of Wnt5b (p < 0.001) and Wnt3a (p < 0.001) in oxaliplatin group in comparison with normal control. However, oxaliplatin control group, when treated with low dose of baicalein at 5 mg/ kg, showed an attenuated expression of Wnt5b (p < 0.01) and Wnt3a (p < 0.05). Significant reduction in the levels of Wnt5b (p < 0.001) and Wnt3a (p < 0.001) was seen in oxaliplatin control rats treated with higher dose of baicalein at 10 mg/kg as shown in Fig. 8a. The immunohistochemical analysis too disclosed a marked increase in the expression of Wnt5b characterized by brown-colored cells in the spinal cord (p < 0.001) and sciatic nerve (p < 0.001) in rats with oxaliplatin-induced neuropathy as represented in Fig. 7b, while baicalein treatment at higher doses significantly attenuated these changes in spinal cord (p < 0.01) by abrogating the levels of Wnt5b. However, no prominent changes in the expression of Wnt5b were observed in sciatic nerve. As β-catenin is a substrate of the destruction complex consisting of GSK3β, Axin, APC, binding of Wnt ligands to the destruction complex frees β-catenin and the levels of active non-phosphorylated β-catenin rises in the cell. In context to this we studied the expression of active β-catenin in spinal cord and sciatic nerve. Expression levels of active β-catenin were also increased (p < 0.001) in oxaliplatin group in comparison with the normal control. Baicalein at 10 mg/kg dose significantly inhibited this effect in spinal cord (p < 0.05) and sciatic nerve (p < 0.001) by abrogating the levels of active β-catenin (Fig. 7a). At the mRNA level too, oxaliplatin treated group showed a significant increase in the mRNA levels of the β-catenin (p < 0.05) but the group given baicalein treatment showed significant downregulation of β-catenin (p < 0.01) in comparison with the oxaliplatin control animals as shown in Fig. 8b.

Discussion
OIPN is a debilitating side effect of widely used anti-cancer agents oxaliplatin, limiting its clinical utility. The current study was designed to investigate the neuro-protective function of a Chinese herbal medicine baicalein and its outcomes against oxaliplatin-induced oxidative stress, neuro-inflammation and aberrant Wnt signaling in an experimental model of OIPN. To induce neuropathy in SD rats, oxaliplatin was given at a dose of 4 mg/kg twice every week for 4 weeks. Behavioral examinations depicting various sensory elements of neuropathic pain were evaluated before, during and after the administration of oxaliplatin. Treatment with oxaliplatin resulted in the induction of allodynia and hyperalgesia, the hallmark symptoms of neuropathy which may be due to hyper-responsiveness of Aβ-fibers and C-fibers. The thinly myelinated Aδ fibers mediate the touch sensation while the non-myelinated C-fibers mediate the cold sensation; the hyper-responsiveness of these fibers may contribute to the induction of neuropathic pain [41,42]. The increased pain hypersensitivity was confirmed by a marked decrease in the paw withdrawal latency (thermal hyperalgesia), decreased paw withdrawal pressure (mechanical hyperalgesia), decreased paw withdrawal threshold (mechanical allodynia) and decreased score of allodynia (cold allodynia) in oxaliplatin group compared to the normal control group. Additionally, reduced sensory nerve conduction velocities with no prominent changes in motor nerve conduction velocities showing ELISA analysis of IL-6 and TNF-a levels. Results were expressed as mean ± SEM (n = 3). ^^ p < 0.01, ^^^ p < 0.001 vs. NC, * p < 0.05, ** p < 0.01 vs. OC. NC: Normal control, OC: oxaliplatin control (4 mg/kg, ip), OC + BA1 and OC + BA2: oxaliplatin control rats treated with baicalein at 5 mg/kg and 10 mg/kg, ip, respectively, and BA2: Normal control rats treated with baicalein (10 mg/kg, ip) were observed upon the administration of oxaliplatin. This may be due to the fact that sensory neurons in the dorsal root ganglion had remarkably higher exposure to oxaliplatin and relatively due to the absence of its mitotoxic effect in motor neuron axons [43]. Notably, the treatment with baicalein at doses 5 mg/kg and 10 mg/kg significantly mitigated the oxaliplatin-induced behavioral deficits like thermal and mechanical hyperalgesia, cold and mechanical allodynia and improved SNCV, providing preliminary evidence of its possible neuroprotective role.
To shed light on the molecular mechanisms underlying the neuroprotective role of baicalein, we studied oxidative stress and inflammatory markers. Oxidative stress is a crucial player in the pathogenesis of OIPN [44,45]. Oxaliplatin-induced oxidative damage is likely to be mediated by increasing levels of superoxide anion, hydrogen peroxide, and hydroxyl radical. It is often characterized by high amounts of free radicals and a compromised antioxidant defense mechanism, leading to redox imbalance [46]. ROS directly targets lipids, and the presence of high amount of phosphospholipids in the spinal cord makes them more vulnerable to oxaliplatin-induced oxidative damage [45]. Levels of MDA, an end product of lipid of peroxidation, were markedly increased in the oxaliplatin treated rats compared to the normal control group. Compromised expression of antioxidant proteins like GSH, MnSOD, and HO-1, resulting in the inefficiency of cellular mechanisms to combat oxidative stress induced by oxaliplatin was observed.
While treatment with baicalein attenuated the ROS-induced lipid peroxidation it also re-established the levels of the glutathione in spinal cord tissue of rats belonging to the treatment group (Table 1). Treatment with baicalein restored MnSOD and HO-1 levels in spinal cord (Fig. 4b). 8-OHdG is a key marker in measuring endogenous damage to DNA. Free radicals generated by oxaliplatin are known to hydrolyze guanosine resulting in the formation of 8-OHdG [47]. A significantly high amount of 8-OHdG was observed in the oxaliplatin control group compared to the normal control. Baicalein reduced the expression of 8-OHdG in the spinal cord of treated rats (Fig. 4a). In agreement with the previous reports [45,47], redox imbalance was evident in the sciatic nerves and spinal cord of oxaliplatin-treated rats. And baicalein possibly protects the neurons from ROS-induced damage by alleviating the oxaliplatin-induced MDA levels and by preventing the depletion of antioxidant defenses and DNA oxidation. Consistent with our in vivo findings, oxaliplatin also induces ROS generation, including mitochondrial superoxide in N2a cells. We also observed oxaliplatin-mediated loss of mitochondrial potential which is a characteristic of mitochondrial dysfunction in N2a cells. However, baicalein treatment not only suppressed the levels of ROS and superoxide, but also preserved the mitochondrial membrane potential in a dose-dependent manner (Fig. 1,2). Over and above, baicalein also enhanced neurite outgrowth and the percentage of cells bearing neurites indicating a prominent neuro-protective role (Fig. 2). The development of pathophysiological changes in the spinal cord was evidenced by neuro-inflammatory processes including enhanced glial cell activation and transcriptional upregulation of inflammatory genes [10,45]. Recent findings suggest that the response of glial cells to chemotherapyinduced neuropathy is different from that of trauma-induced nerve injury. Predominantly astrocyte is activated in the maintenance of OIPN with a minor role for microglial activation [48,49]. Astrocytes in addition of maintaining neuronal health regulate the uptake and release of pro-inflammatory cytokines. The released pro-inflammatory cytokines in turn act on the neurons increasing their excitability and trigger the glial cells creating cascading effects, which leads to long lasting neuropathic pain [50]. Immunohistochemical evaluation revealed an upregulated GFAP expression in oxaliplatin control group compared to normal control group which is interpreted as an increase in the activity of astrocytes, while treatment with baicalein significantly inhibited the GFAP positive cells as shown in Fig. 5a. Additionally, as astrocyte activation co-relates with the degree of sensitivity to mechanical stimuli, attenuation of activated astrocyte by baicalein resulted in a marked reversal of neuropathic pain symptoms which is evident from restored sensitivity to mechanical hyperalgesia and allodynia as represented in Fig. 3. These inflammatory effects displayed by oxaliplatin are in line with the published reports on OIPN [51][52][53][54]. Under physiological conditions, NF-κB exists in an inactive form in the cytoplasm bound to IκB. Upon stimulation with pro-inflammatory cytokines and/or free radicals, IκB is phosphorylated resulting in its ubiquitin mediated degradation. This leads to the translocation of free NF-κB into the nucleus and subsequent activation of inflammatory genes [10]. Oxaliplatin drastically increased levels of inflammatory markers in the spinal cord which may be due to cytokine mediated nociceptor sensitization in the oxaliplatin control group. Indeed, oxaliplatin treatment in N2a cells resulted in an enhanced nuclear translocation of NF-κB as observed by immunofluorescence microscopy. Treatment with baicalein ameliorated these pro-inflammatory cytokines, namely TNF-α and IL-1β, owing to its anti-inflammatory potential.
Next, we assessed the role of Wnt signaling in OIPN. Wnt signaling is involved in the regulation of synaptic plasticity, glial function and neuronal-development [17,55]. Its function has been found to be dysregulated in various neurological disorders and in various form of cancer [56][57][58].
Recently, Wnt signaling has gained importance for its probable role in the aetiology of \ neuropathic pain [59,60]. Although Wnt signaling remains silent in an unstimulated cell, it gets activated upon nerve injury [17]. Wnt ligands bind to N-terminal cysteine-rich domain of Frizzled receptors and co-receptors, leading to disruption of the destruction complex, the complex consisting of the proteins, namely Axin, GSK-3β, CK-1, and APC [61,62]. The disruption of destruction complex and its subsequent escape of β-catenin from the destruction complex leads to its stabilization and build-up in the cytoplasm and its subsequent translocation into the nucleus to activate the transcription of target genes. Recently, many studies have reported a pro-inflammatory role of Wnt signaling and were shown to orchestrate the chemotaxis of macrophages into the injury site. Of note, β-Catenin, a multi-functional protein is found to regulate the production of pro-inflammatory cytokines [63,64]. In this study, we found that oxaliplatin-induced nerve damage causes a rapid and an enduring activation of the Wnt signaling in the spinal cord. Repeated administration of oxaliplatin increased the spinal expression of well-known activators of Wnt/β-Catenin signaling, namely Wnt3a and Wnt5b. These elevated levels of Wnt ligands act as a trigger for the stabilization of β-Catenin, which in turn regulates the expression of pro-inflammatory genes. Upregulation of pro-inflammatory cytokines due to the dysregulation of Wnt signaling in the spinal cord leads to central sensitization and the abnormal firing of nerve impulses resulting in the persistence of neuropathic pain [65]. Wnt pathway is directly linked with the induction and progression of neuropathic pain and the treatment with Wnt inhibitors has yielded promising result [59,66]. The fact that Wnt signaling contributes to neuropathic pain by regulating proinflammatory cytokines IL-6 and TNF-α and baicalein being a negative regulator of Wnt/β-catenin signaling, posits that spinal blockade of Wnt signaling by baicalein may abrogate neuropathic pain induced by oxaliplatin. Interestingly, accumulating evidences suggests a strong association of Wnt pathway with the tumorigenesis and aggressiveness of cancer. The Wnt signaling was found to provide resistance against chemotherapy [58]. Indeed, inhibition of Wnt/βcatenin signaling by baicalein has been shown to promote anti-proliferative properties (22). Hence, attenuation of activated Wnt/β-catenin signaling has a dual function, leading to sensitization to chemotherapy and further providing neuroprotection (Fig. 9). Nevertheless, baicalein could provide neuroprotection while simultaneously preserving the anti-cancer activity of oxaliplatin.

Conclusion
The present study demonstrates the involvement of oxidative stress in the overactivation of Wnt/ β-catenin and neuroinflammation in the pathogenesis of oxaliplatin-induced peripheral neuropathy. Treatment with baicalein ameliorated the functional, behavioral and biochemical alterations associated with OIPN and reduced the expression of β-catenin and its downstream inflammatory mediators. Hence, our findings suggest baicalein as a potential therapeutic strategy against OIPN without compromising the anti-cancer effect of oxaliplatin. Funding For this project, we have utilized internal funding from NIPER Hyderabad & NIPER Kolkata.

Data Availability
In this article, we have included all the data after doing experiments and statistical analysis, the source of majority of materials included in material and methods section. The data that support the findings of this study are available on request.

Ethics Approval
The animal study protocols were duly approved by the Institutional Animal Ethics Committee (IAEC)-NIPER Hyderabad, and all the experiments were conducted in accordance to prevailing guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Conflict of Interest
The authors declare that there are no conflicts of interest.

Informed Consent Not applicable
Research Involving Human Participants and/or Animals All the procedures used for animal experimentations were approved by Institutional Animal Ethics Committee of NIPER Hyderabad.