Pharmacologic Inhibition of Porcupine, Disheveled, and β-Catenin in Wnt Signaling Pathway Ameliorates Diabetic Peripheral Neuropathy in Rats

ARTICLE IN PRESS The Journal of Pain, Vol 00, No 00 (), 2019: pp 1−15 Available online at www.jpain.org and www.sciencedirect.com

Pharmacologic Inhibition of Porcupine, Disheveled, and b-Catenin in Wnt Signaling Pathway Ameliorates Diabetic Peripheral Neuropathy in Rats Kahkashan Resham, and Shyam S. Sharma Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Punjab, India

Abstract: Wnt signaling pathway has been investigated extensively for its diverse metabolic and pain-modulating mechanisms; recently its involvement has been postulated in the development of neuropathic pain. However, there are no reports as yet on the involvement of Wnt signaling pathway in one of the most debilitating neurovascular complication of diabetes, namely, diabetic peripheral neuropathy (DPN). Thus, in the present study, involvement of Wnt signaling was investigated in DPN using Wnt signaling inhibitors namely LGK974 (porcupine inhibitor), NSC668036 (disheveled inhibitor), and PNU74654 (b-catenin inhibitor). Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg) to male Sprague−Dawley rats. Diabetic rats after 6 weeks of diabetes induction showed increased expression of Wnt signaling proteins in the spinal cord (L4−L6 lumbar segment), dorsal root ganglions and sciatic nerves. Subsequent increase in inflammation, endoplasmic reticulum stress and loss of intraepidermal nerve fiber density was also observed, leading to neurobehavioral and nerve functional deficits in diabetic rats. Intrathecal administration of Wnt signaling inhibitors (each at doses of 10 and 30 mmol/L) in diabetic rats showed improvement in painassociated behaviors (heat, cold, and mechanical hyperalgesia) and nerve functions (motor, sensory nerve conduction velocities, and nerve blood flow) by decreasing the expression of Wnt pathway proteins, inflammatory marker, matrix metalloproteinase 2, endoplasmic reticulum stress marker, glucose-regulated protein 78, and improving intraepidermal nerve fiber density. All these results signify the neuroprotective potential of Wnt signaling inhibitors in DPN. Perspective: This study emphasizes the involvement of Wnt signaling pathway in DPN. Blockade of this pathway using Wnt inhibitors provided neuroprotection in experimental DPN in rats. This study may provide a basis for exploring the therapeutic potential of Wnt inhibitors in DPN patients. © 2019 by the American Pain Society Key words: Diabetic peripheral neuropathy, porcupine, disheveled, b-catenin, Wnt. ype 1 diabetes is often associated with many comorbid complications, out of which, diabetic peripheral neuropathy (DPN), stands out to be one of the most common one, across the globe, affecting almost 50 to 60% of diabetic patients worldwide.8,53 DPN manifests as sensory symptoms in a distal “gloveand-stocking” distribution, thereby causing a debilitating pain in the form of parasthesias, hyperalgesia, and allodynia.15,45 Hyperglycemia, the characteristic feature

T

of type 1 diabetes, has long been thought to instigate DPN pathology, either through direct neurotoxicity, or from the activation of secondary pathways. A plethora of mechanisms including hyperglycemia-induced cytokines, advanced glycation end products (AGE) production, chemokines, protein kinase C activation, mitochondrial dysfunction, nuclear factor-kB activation, endoplasmic reticulum (ER) stress, and oxidative stress have been proposed to underlie the pathophysiology of DPN.63,7

Received November 28, 2018; Revised February 22, 2019; Accepted March 15, 2019. This study was funded by the Department of Pharmaceuticals, Ministry of Chemicals & Fertilizers, Government of India to carry out this research work in National Institute of Pharmaceutical Education and Research, S.A.S. Nagar. There is no conflict of interest. Address reprint requests to Prof. Shyam S. Sharma, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical

Education and Research (NIPER), Sector 67, SAS Nagar, Mohali, Punjab 160062, India. E-mail: [email protected] 1526-5900/$36.00 © 2019 by the American Pain Society https://doi.org/10.1016/j.jpain.2019.04.010

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Despite these numerous pathways being elucidated in the pathogenesis of DPN, current treatment options remain limited owing to the complexity of the pathophysiology of DPN, thus posing a challenge to the development of new pharmacologic agents for its treatment. Wnt signaling pathway, an evolutionary conserved pathway in mammals, has multifarious roles in cell proliferation and differentiation,42 stem cell maintenance,58 neuronal development, and carcinogenesis,54,66 to name a few. Wnt ligands are synthesized in the ER and secreted as cysteine-rich glycoproteins after being palmitoylated by membrane bound o-acyl transferase enzyme named porcupine (PORCN), present on the ER. These ligands then bind to the frizzled receptors on the plasma membrane, leading to inhibition of the b-catenin destruction complex comprising of glycogen synthase kinase-3b, casein kinase 1, Axin2, adenomatous polyposis coli, by a scaffolding protein, disheveled (Dvl). This further promotes b-catenin accumulation in the cytoplasm and its subsequent translocation into the nucleus to interact with T-cell/lymphoid enhancer factor transcription factors and transcription of Wnt target genes like c-myc, CyclinD1, matrix metalloproteinase 2 (MMP2), and so on.29,6 Recently, Wnt signaling pathway has been investigated in neuropathic pain models like chronic constriction injury, tumor cell-induced, and other nerve injury models.14,28,51,67,68 Multiple studies have suggested the hyperglycemia-induced aberrant activation of Wnt signaling pathway in diabetes-associated complications like retinopathy,3,13,49 cardiomyopathy,61,27 and nephropathy,44,69 signifying a positive correlation between the activation of Wnt signaling and the hyperglycemiainduced damages in the respective tissues. However, the role of Wnt signaling in DPN has not been investigated yet. In this study, we hypothesized that hyperglycemia in diabetes could lead to Wnt signaling activation in the spinal cord, dorsal root ganglions (DRGs), and sciatic nerves of diabetic animals, which leads to DPN. Furthermore, we also assessed the therapeutic potential of pharmacologic interventions targeting Wnt signaling pathway in DPN.

Methods Animals Animal experiments were performed after approval was provided by the Institutional Animal Ethics Committee of National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Punjab. Adult male Sprague− Dawley rats (250−270 g) were fed on standard diet and water ad libitum. The animals were housed in a room maintained at approximately 24 § 1°C temperature and humidity of 55 § 5% with a 12-hour light/dark cycle. The animals were acclimatized for at least 2 to 3 days before the initiation of the experiment and were observed for any signs of disease throughout the study period.

Diabetic Peripheral Neuropathy Model Diabetes was induced by a single dose of streptozotocin (50 mg/kg, intraperitoneally) injection in the animals. All

the animals having blood glucose above 300 mg/dL were considered diabetic and included in the study.20 The pain-associated behavioral and functional parameters were recorded after 6 weeks of diabetes, after which, the animals were humanely killed and spinal cord (lumbar L4−L6 segment), lumbar DRGs, and sciatic nerves as well as hind paw skin (plantar surface) were collected for protein expression studies.

Experimental Design and Treatment Schedule LGK974, NSC668036, and PNU74654 (Wnt signaling inhibitors) were purchased from Santa Cruz Biotechnology (Santa Crus, California) and the doses (10 and 30 mmol/L) were selected based on previous literature.25,36,57 Wnt signaling inhibitors were dissolved in DMSO (10% v/v) and injected intrathecally (using Hamilton syringe with a 30G needle) to anesthetized animals (anesthesia used was halothane) between L5 and L6 lumbar vertebrae in a volume of 20 mL/rat on 3 consecutive days to 6-week diabetic rats (Fig 1). No adverse events were reported with the treatment of inhibitors in diabetic and control group animals injected with the inhibitors. The experimental groups were composed of control group (C) (age-matched na€ıve rats), diabetic (D), diabetic rats treated with 10% v/v DMSO (D + vehicle), diabetic rats treated with LGK 10 mmol/L (D + LGK10), diabetic rats treated with LGK 30 mmol/L (D + LGK30), control (C) rats treated with LGK 30 mmol/L (C + LGK30), diabetic rats treated with NSC 10 mmol/L (D + NSC10), diabetic rats treated with NSC 30 mmol/L (D + NSC30), control rats treated with NSC 30 mmol/L (C + NSC30), diabetic rats treated with PNU 10 mmol/L (D + PNU10), diabetic rats treated with PNU 30 mmol/L (D + PNU30), and control rats treated with PNU 30 mmol/L (C + PNU30) group. Each group consisted of 6 to 8 animals. Parameters like pain-associated behavioral and nerve functional parameters were measured after 30 minutes of the last of injection of the drug. The animals were humanely killed and spinal cords (lumbar L4−L6 segment), lumbar DRGs and sciatic nerves as well as hind paw skin (plantar surface) were collected for protein expression studies.

Behavioral Pain Parameters Heat Hyperalgesia A Hargreaves plantar apparatus was used to assess heat hyperalgesia in rats. All the animals were acclimatized for 10 to 15 minutes before starting the experiment. The infrared heat source was placed beneath the glass surface where the rat hind paw was placed. The paw withdrawal latency was recorded for each animal with a cutoff time of 15 seconds. Three readings per animal were taken randomly with an interval of 15 minutes between 2 consecutive applications for a single animal, and the mean was calculated.37,20

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Figure 1. Experimental design and treatment schedule. Diabetes was induced in male Sprague−Dawley rats by administering streptozotocin intraperitoneally at a dose of 50 mg/kg. After 6 weeks of diabetes induction, treatment with pharmacologic agents targeting Wnt signaling pathway (LGK974, NSC668036, and PNU74654) was given intrathecally for 3 consecutive days each at doses 10 and 30 mmol/L to diabetic rats. Behavioral (heat, cold, and mechanical hyperalgesia) and functional pain parameters (MNCV, SNCV, and NBF) were assessed after the treatment and then the rats were humanely killed for protein expression studies (Western blotting, immunohistochemistry, and IENFD).

Cold Hyperalgesia This parameter was assessed using tail immersion test. The rats were acclimatized for 10 to 15 minutes in the hands of the experimenter before performing the experiment. The tail of the animal was immersed in cold water (10°C § 1°C) and the latency for withdrawal of the tail was recorded with a cutoff time of 15 seconds. Three readings were taken for each animal, and the mean was calculated.37

Mechanical Hyperalgesia A Randall Selitto apparatus (IITC Life Science, Woodland Hills, California) was used to assess the latency of paw withdrawal upon application of pressure on the plantar surface of the animal. The rats were acclimatized in the hands of experimenter for 15 minutes. The minimum pressure required to withdraw the paw was recorded as the threshold for mechanical hyperalgesia in the animal. The cutoff pressure was kept as 300 g. The test was performed for 3 times randomly on each hind paw with an interval of 10 minutes between 2 consecutive applications, and the mean was calculated.35,38

Functional Studies Nerve Conduction Velocity Power Lab 8sp system (AD Instruments, Sydney, Australia) was used to measure both motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) as previously described. Briefly, rats were anesthetized and body temperature was maintained with homeothermic blanket. Sciatic nerve was stimulated using bipolar electrodes (3 V) at the sciatic notch (proximal) as well as the ankle (distal; for MNCV) and the Achilles tendon (distal; for SNCV), respectively, and receiving electrodes were placed on the foot muscles. The latencies were recorded via bipolar electrodes: negative M-wave deflection for MNCV and H-wave deflection for SNCV. Nerve conduction velocities were

calculated by dividing the distance between the stimulating and recording electrode with the difference between proximal and distal latencies and expressed in meters per second.38,62

Nerve Blood Flow Nerve blood flow (NBF) was measured using Laser €rfa €lla, Sweden). Rats were Doppler system (Perimed, Ja anesthetized and body temperature was maintained using a homeothermic blanket. The sciatic nerve was exposed by putting incision on the left flank and the laser Doppler probe (tip diameter, 0.85 mm) was applied just in contact with the sciatic nerve from top. Flux measurement was obtained for period of 5 to 10 minutes after stabilizing the reading for 10 minutes for all animals. The blood flow was recorded in arbitrary perfusion units.62

Expression Studies Western Blotting Sciatic nerves, spinal cord (lumbar L4−L6 segment), and lumbar DRGs were homogenized with lysis buffer (Tris-HCl 50 mmol/L, NaCl 150 mmol/L, SDS 0.1%, TritonX 1%, EDTA 5 mmol/L, EGTA 1 mmol/L, sodium deoxycholate 10%, protease inhibitor cocktail 1 mL/mL, PMSF 1 mmol/L, and sodium orthovanadate 1 mmol/L). Protein estimation was performed according to the Lowry method. Equal amounts of proteins were separated in SDS-PAGE (8%) and transferred to a nitrocellulose membrane (Advanced Microdevices, Haryana, India). After blocking with 5% skimmed milk, membranes were incubated with primary antibodies (Santa Cruz Biotechnology) against Wnt3a (1:1000), Dvl1 (1:500), b-catenin (1:500), c-myc (1:500), MMP2 (1:500), glucose-regulated protein 78 (GRP78; 1:500), and b-actin (1:500) for 12 hours at 4°C. After washing with TBST, the membranes were incubated with an HRP-conjugated secondary antibody (1:10,000) for 1 hour. The membranes were

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washed with TBST and bound antibody was visualized by using enhanced chemiluminescence method. The relative band intensities were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, Maryland). Protein expression was normalized with b-actin.39,2

Immunohistochemistry Sciatic nerves were fixed in 10% formaldehyde, dehydrated through graded concentrations of alcohol, embedded in paraffin, and sectioned. Sections (5 mm thick) were mounted on slides, cleared using xylene, and hydrated in graded alcohol. The immunohistochemical staining was performed according to the manufacturer’s instruction provided in the kit (Novacastra polymer based immunohistochemistry kit, Leica Microsystems, Wetzlar, Germany). Briefly, antigen retrieval was carried out for sections in citrate buffer (pH 6.0) for 10 minutes and incubated in blocking solution (3% bovine serum albumin) for 5 minutes. Then, sections were incubated with primary antibodies for b-catenin (rabbit polyclonal; Santacruz Biotechnologies) separately at a dilution of 1:50 at 4°C for 12 hours. Sections were washed with TBS 0.05 mol/L, pH 7.6, and incubated with secondary antibody at room temperature for 30 minutes. The sections were then incubated in the polymer solution for 30 minutes, washed, and then coincubated with a 3,3-o-diaminobenzidine solution in the dark, at room temperature for 5 minutes followed by washing and counterstaining with hematoxylin and observed under a light microscope.23

Immunofluorescence for Intraepidermal Nerve Fiber Density Paw tissues were cut from the plantar surface of rat hind paw skin and kept in 10% formalin for 3 to 4 hours. The tissues were dehydrated through graded alcohols and embedded in paraffin. Sections (40 mm) were cut and mounted on slides, cleared using xylene, and hydrated in graded alcohols. After antigen retrieval and blocking, sections were incubated with primary antibodies for Pgp 9.5 (mouse monoclonal; Abcam, Cambridge, UK) 1:500 at 4°C for 12 hours, washed with TBS, and incubated with antimouse DyLight-488 secondary antibody, at room temperature for 1 hour. Sections were mounted using 90% glycerol and observed under fluorescent microscope. Intraepidermal nerve fiber density (IENFD) was expressed as number of fibers per millimeter of epidermis.22

Statistical Analysis All the values were expressed as mean § standard error of the mean. Statistical significance for multiple groups was assessed by using 1-way analysis of variance followed by Tukey’s post hoc multiple comparison test using Graph Pad Prism 5 software. A P value of less than .05 was considered statistically significant.

Results Effects of PORCN Inhibitor (LGK974), Dvl Inhibitor (NSC668036), and b-Catenin Inhibitor (PNU74654) on Behavioral Pain Parameters Paw withdrawal latency in the plantar test was significantly (P < .001) decreased in diabetic rats as compared with control animals. Vehicle-treated diabetic rats did not show any significant difference in the latency as compared with the diabetic rats. Intrathecal administration of LGK974 in diabetic rats attenuated heat hyperalgesia significantly at both the 10 mmol/L (P < .05) and 30 mmol/L (P < .001) doses. LGK974 treatment showed 40.91% and 76.67% reversal in heat hyperalgesia response at 10 and 30 mmol/L, respectively. Similarly, intrathecal administration of NSC668036 attenuated heat hyperalgesia significantly at both the 10 mmol/L (P < .01) and 30 mmol/L (P < .001) doses. NSC668036 showed 49.71% and 78.00% reversal of heat hyperalgesia at 10 and 30 mmol/L, respectively, in diabetic rats. PNU74654 administration attenuated heat hyperalgesia significantly at both the 10 and 30 mmol/L (P < .001) doses. PNU74654 treatment at both the doses if 10 and 30 mmol/L showed a higher reversal of heat hyperalgesia 62.71% and 90.43%, respectively, in diabetic animals as compared with LGK974 and NSC668036 groups in diabetic rats. The administration of Wnt inhibitors in control animals did not alter heat hyperalgesia as compared with the na€ıve control animals (Fig 2A). In cold hyperalgesia, diabetic animals showed a significant (P < .001) decrease in the tail flick latency as compared with the control animals. Vehicle-treated diabetic rats did not show any significant difference in the latency as compared with the diabetic rats. Intrathecal administration of LGK974 attenuated cold hyperalgesia significantly at the 30 mmol/L (P < .001) dose; however, the increase in tail flick latency at the 10 mmol/L dose was not significant. LGK974 treatment showed a 40.89% and 61.98% reversal as compared with the diabetic animals at both the doses (10 and 30 mmol/L, respectively). Similarly, intrathecal administration of NSC668036 attenuated cold hyperalgesia significantly at both the 10 and 30 mmol/L doses (P < .001). NSC668036 showed a 75.42% and 92.04% reversal of cold hyperalgesia at 10 and 30 mmol/L, respectively, in comparison with the diabetic animals. PNU74654 administration attenuated cold hyperalgesia significantly at both 10 and 30 mmol/L doses (P < .001). PNU74654 treatment at both the 10 and 30 mmol/L doses showed a reversal of 67.58% and 93.91%, respectively, in diabetic rats. The administration of Wnt inhibitors in control animals did not alter cold hyperalgesia as compared with the na€ıve control animals (Fig 2B). For mechanical hyperalgesia in the Randall Selitto test, diabetic animals showed a significant (P < .001) decrease in the paw withdrawal threshold as compared with the control animals. Vehicle-treated diabetic rats did not show any significant difference in the latency as compared with the diabetic rats. Intrathecal

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Figure 2. Effects of PORCN inhibitor, Dvl inhibitor, and b-catenin inhibitor on behavioral pain parameters. (A) PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036), and b-catenin inhibitor (PNU74654) were found to attenuate heat hyperalgesia significantly in diabetic rats. (B) PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036), and b-catenin inhibitor (PNU74654) were found to attenuate cold hyperalgesia significantly in diabetic rats. (C) PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036), and b-catenin inhibitor (PNU74654) were found to attenuate mechanical hyperalgesia significantly in diabetic rats. Vehicle-treated diabetic rats and the control rats that received treatment of inhibitors did not show any significant differences from the diabetic and naive control rats respectively. Data are expressed as mean § standard error of the mean. ***P < .001 versus control animals; ###P < .001; ##P < .01; #P < .05 versus diabetic group (n = 6−8 per group).

administration of LGK974 attenuated mechanical hyperalgesia significantly at the 10 mmol/L (P < .05) and 30 mmol/L (P < .01) doses. LGK974 treatment depicted 35.81% and 47.11% reversal as compared with the diabetic animals at both the doses (10 and 30 mmol/L, respectively). Similarly, intrathecal administration of NSC668036 attenuated mechanical hyperalgesia significantly at both the 10 mmol/L (P < .01) and 30 mmol/L (P < .001) doses. NSC668036 showed a 45.76% and 56.21% reversal of mechanical hyperalgesia at 10 and 30 mmol/L, respectively, in comparison with diabetic animals. PNU74654 administration attenuated mechanical hyperalgesia in animals significantly at both the 10 and 30 mmol/L doses (P < .001). PNU74654 treatment at both the doses 10 and 30 mmol/L showed a higher reversal of 48.47% and 57.72%, respectively, in diabetic animals as compared with LGK974 and NSC668036 groups. The administration of Wnt inhibitors in control animals did not alter mechanical hyperalgesia as compared with the na€ıve control animals (Fig 2C).

Effects of PORCN Inhibitor (LGK974), Dvl Inhibitor (NSC668036), and b-Catenin Inhibitor (PNU74654) on Nerve Functional Parameters Diabetic rats showed a significant decrease in MNCV (P < .001), as compared with control rats. Intrathecal administration of LGK974 improved MNCV significantly at the 30 mmol/L (P < .01) dose; however, at the 10 mmol/L dose, the change was not significant. LGK974 treatment showed 54.6% and 78.49% reversal in MNCV 10 and 30 mmol/L, respectively, in diabetic rats. Similarly, intrathecal administration of NSC668036 improved MNCV significantly at the 30 mmol/L dose (P < .01) with no significant changes at the lower dose (10 mmol/L). NSC668036 showed a 55.17% and 77.85% reversal of MNCV at 10 and 30 mmol/L, respectively, in comparison with the diabetic animals. PNU74654 administration showed significant improvement in MNCV at the higher dose, namely, 30 mmol/L (P < .001). PNU74654 treatment at both doses

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(10 and 30 mmol/L) showed a reversal of 70.17% and 93.35%, respectively, in diabetic rats (Fig 3A). Diabetic rats showed a marked decrease in SNCV (P < .001) as compared with control rats. Intrathecal administration of LGK974 restored SNCV significantly at the 30 mmol/L dose (P < .05); however, at the 10 mmol/L dose, the change was not significant. LGK974 treatment showed 42.77% and 58.96% reversal in SNCV as compared with the diabetic animals at both the 10 and 30 mmol/L doses, respectively. Similarly, intrathecal administration of NSC668036 improved SNCV significantly at the 30 mmol/L dose (P < .01), with no significant changes at the lower dose (10 mmol/L). NSC668036 showed 34.53% and 56.22% reversal of SNCV at 10 and 30 mmol/L, respectively, in comparison with the diabetic animals. PNU74654 administration showed significant improvement in SNCV at the higher dose (30 mmol/L; P < .001). PNU74654 treatment at both doses (10 and 30 mmol/L) showed a reversal of 52.17% and 67.29%, respectively, in diabetic animals (Fig 3B).

Diabetic rats showed significant decrease in NBF (P < .001) as compared with control rats. Intrathecal administration of LGK974 increased NBF significantly at both the 10 mmol/L (P < .05) and 30 mmol/L (P < .01) doses. LGK974 treatment showed 40.22% and 43.55% reversal in NBF as compared with the diabetic animals at both doses (10 and 30 mmol/L, respectively). Similarly, intrathecal administration of NSC668036 improved NBF significantly at both the 10 mmol/L (P < .01) and 30 mmol/L (P < .001) doses. NSC668036 showed 46.84% and 48.31% reversal of NBF at 10 and 30 mmol/L, respectively, in comparison with the diabetic animals. PNU74654 administration showed significant improvement in NBF at both 10 and 30 mmol/L doses (P < .001). PNU74654 treatment at doses of 10 and 30 mmol/L showed a reversal of 47.09% and 51.43%, respectively, in diabetic animals (Fig 3C). Vehicle-treated diabetic rats and the control rats that received treatment of inhibitors did not show any significant effects on nerve functional parameters (MNCV,

Figure 3. Effects of PORCN inhibitor, Dvl inhibitor, and b-catenin inhibitor on nerve functional parameters. (A) PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036), and b-catenin inhibitor (PNU74654) were found to improve MNCV in diabetic rats. (B) PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036), and b-catenin inhibitor (PNU74654) were found to improve SNCV in diabetic rats. (C) PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036), and b-catenin inhibitor (PNU74654) were found to restore NBF in diabetic animals. Vehicle-treated diabetic rats and the control rats which received treatment of inhibitors did not show any significant differences from the diabetic and naive control rats respectively. Values are expressed as mean § standard error of the mean. ***P < .001 versus control animals; ###P < .001; ##P < .01; #P < .05 versus diabetic group (n = 6−8 per group).

ARTICLE IN PRESS Resham and Sharma SNCV, and NBF) as compared with the diabetic and naive control animals.

Effects of PORCN Inhibitor (LGK974), Dvl Inhibitor (NSC668036), and b-Catenin Inhibitor (PNU74654) on Protein Expressions Western Blotting Wnt3a, b-catenin, c-myc, Dvl1, MMP2, and GRP78 expression was significantly upregulated in the sciatic nerves of diabetic animals as compared with the control rats. LGK974 significantly downregulated the expression of b-catenin at 10 mmol/L (P < .05) and 30 mmol/L (P < .01), Dvl1 at 30 mmol/L (P < .01), and c-myc at 30 mmol/L (P < .01) in diabetic rats. However, decreases in

The Journal of Pain 7 Wnt3a expression were not significant with LGK974 treatment at either of these doses. LGK974 significantly downregulated the expression of GRP78 (P < .05) and MMP2 (P < .05) at 30 mmol/L in diabetic rats, whereas the changes at 10 mmol/L were not significant (Fig 4A). NSC668036 at the 30 mmol/L dose significantly downregulated the expression of b-catenin (P < .05), Dvl1 (P < .01), and c-myc (P < .05) in diabetic rats. NSC668036 treatment significantly downregulated the expression of MMP2 (P < .05) and GRP78 (P < .01) in diabetic rats only at the higher dose (30 mmol/L; Fig 5A). PNU74654 treatment significantly downregulated the expression of b-catenin at both 10 mmol/L (P < .05) and 30 mmol/L (P < .01) and also c-myc at both 10 mmol/L (P < .05) and 30 mmol/L (P < .01) in diabetic rats. PNU74654 treatment significantly downregulated the expression of MMP2 (P < .01) and GRP78 (P < .01) in diabetic rats at the higher dose 30 mmol/L (Fig 6A). Vehicle-treated diabetic

Figure 4. Effects of PORCN inhibitor (LGK974) on protein expressions. LGK974 treatment in diabetic rats reduced the expression of

Wnt3a, b-catenin, Dvl1, c-myc, GRP78, and MMP2 in the sciatic nerve (A). b-Catenin expression in DRG (B) and spinal cord (C). Bar graphs represent the densiometric analysis of the respective blots. Data are expressed as mean § standard error of the mean. **P < .01; *P < .05 versus control animals; ###P < .001; ##P < .01; # P < .05 versus diabetic group (n = 3 per group).

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Figure 5. Effects of Dvl inhibitor (NSC668036) on protein expressions. NSC668036 treatment in diabetic rats reduced the expression of b-catenin, Dvl1, c-myc, GRP78, and MMP2 significantly in the sciatic nerve (A), b-catenin in DRG (B), and spinal cord (C). Bar graphs represent the densiometric analysis of the respective blots. Data are expressed as mean § standard error of the mean. **P < .01; *P < .05 versus control animals; ##P < .01; #P < .05 versus the diabetic group (n = 3 per group).

rats and the control rats that received treatment of inhibitors did not show any significant effects on protein expression changes as compared with the diabetic and naive control animals. b-Catenin expression was also significantly upregulated in the spinal cord and DRGs of diabetic animals as compared with the control rats. LGK974 significantly downregulated the expression of b-catenin in the DRGs (Fig 4B; P < .05 at 10 mmol/L and P < .01 at 30 mmol/L) and at 30 mmol/L (P < .01) in the spinal cord (Fig 4C) of diabetic rats. NSC668036 treatment significantly downregulated the expression of b-catenin at 10 mmol/L (P < .05) and 30 mmol/L (P < .01) doses in both DRG (Fig 5B) and the spinal cord (Fig 5C) of diabetic rats. PNU74654 treatment significantly downregulated the expression of b-catenin at both 10 mmol/L (P < .05) and 30 mmol/L (P < .01) in both DRG (Fig 6B) and the spinal cord (Fig 6C) of diabetic rats.

Effects of PORCN Inhibitor (LGK974), Dvl Inhibitor (NSC668036), and b-Catenin Inhibitor (PNU74654) on Immunohistochemistry of b-Catenin Diabetic rats depicted significant increase in the b-catenin levels (P < .001) as compared with control group rats, which confirmed the results of Western blotting. This increase in the expression of b-catenin was found to be reduced significantly by intrathecal treatment of LGK974 at 30 mmol/L (P < .05), NSC668036 at 30 mmol/L (P > .01) and PNU74654 at both 10 (P < .01) and 30 mmol/L (P < .001) doses as compared with diabetic animals. Vehicle-treated diabetic rats and the control rats that received treatment of inhibitors did not show any significant effects on b-catenin expression as compared with the diabetic and control animals (Fig 7).

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Figure 6. Effects of b-catenin inhibitor (PNU74654) on protein expression in sciatic nerve. PNU74654 treatment in diabetic rats reduced the expression of, b-catenin, c-myc, GRP78, and MMP2 significantly in the sciatic nerve (A), and b-catenin in the DRG (B) and spinal cord (C). Bar graphs represent the densiometric analysis of the respective blots. Data are expressed as mean § standard error of the mean. ***P < .001; **P < .01; *P < .05 versus control animals; ##P < .01; #P < .05 versus the diabetic group (n = 3 per group).

Effects of PORCN Inhibitor (LGK974), Dvl Inhibitor (NSC668036), and b-Catenin Inhibitor (PNU74654) on IENFD Diabetic animals showed marked decreases in the number of sensory fibers immunostained with anti-Pgp 9.5 antibody in the epidermis of plantar surface of paw tissue as compared with the control rats (P < .001). LGK974 treatment showed a slight increase in the IENFD at both 10 and 30 mmol/L, but the changes were not significant. However, significant improvement in IENFD was observed in animals treated with a higher dose (30 mmol/L) of NSC6680356 (P < .01) and PNU74654 (P < .001) in comparison with the diabetic counterpart. Vehicle-treated diabetic rats and the control rats that received treatment with inhibitors did not show any significant effects on IENFD as compared with the diabetic and naive control animals (Fig 8).

Discussion This study demonstrates the neuroprotective potential of PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036),

and b-catenin inhibitor (PNU74654) in DPN. Streptozotocin-induced type 1 diabetic rats confirmed DPN as evident from reduced pain-associated parameters namely heat, cold, and mechanical hyperalgesia. Also, decreased MNCV, SNCV, and NBF further confirmed the development of peripheral neuropathy in these animals, which is consistent with the previous reports.12,31,37,38 Interestingly, we found a significant increase in the expression of Wnt pathway proteins in the sciatic nerves, spinal cord (L4−L6 segment), DRGs of diabetic animals as compared with the control animals. These observations clearly hinted at the activation of Wnt signaling in diabetic animals, which contributed to the pathology of DPN. Many reports have suggested that hyperglycemia in diabetes can trigger Wnt signaling, which contributes to tissue damage and disease progression.3,13,27,44,49,61,69 Recent studies have elucidated the involvement of Wnt signaling pathway in neuropathic pain in rodent models and that the inhibition of this pathway leads to amelioration of neuropathic pain in animals.14,28,51,67,68 Thus, we chose to investigate the involvement of Wnt signaling in DPN using pharmacologic approach.

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Figure 7. Effects of PORCN inhibitor, Dvl inhibitor, and b-catenin inhibitor on immunohistochemistry of b-catenin. The PORCN inhibitor, Dvl inhibitor, and b-catenin inhibitor significantly reduced the expression of b-catenin in sciatic nerve tissues as compared with the diabetic rats as evident from decreased immunostaining in the treatment group tissues. Arrows indicate the immunostaining of nerve fibers with b-catenin. Data are expressed as mean § standard error of the mean. ***P < .001 versus control animals; ###P < .001; ##P < .01; #P < .05 versus the diabetic group (n = 4−8 per group). Images were taken at 40£ magnification and the scale bars shown in the images represent 50 microns.

The synthesis of Wnt ligands occurs in the ER, which are palmitoylated at serine residue 209, a lipid modification required for the activity of almost all the Wnt ligands, by the membrane bound o-acyl transferase enzyme known

as PORCN, which further promotes the secretion and binding of Wnt ligands to the Frizzled receptors on the plasma membrane.33,64 LGK974 is a PORCN inhibitor that inhibits Wnt secretion without decreasing its synthesis, thereby

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Figure 8. Effects of PORCN inhibitor, Dvl inhibitor, and b-catenin inhibitor on IENFD. PORCN inhibitor, Dvl inhibitor, and b-catenin inhibitor significantly increased the Pgp9.5 immunofluorescence in the intraepidermal layer of paw tissue of diabetic rats. Arrows indicate intraepidermal nerve fibers. The white line drawn demarcates the dermal−epidermal border. Data are expressed as mean § standard error of the mean. ***P < .001 versus control animals; ###P < .001; #P < .05 versus the diabetic group (n = 4−8 per group). Images were taken at 10£ magnification and the scale bars shown in the images represent 200 microns. inhibiting Wnt signaling activation. LGK974 is in phase I clinical trials conducted by Novartis Pharmaceuticals for multiple malignancies associated with activation of Wnt signaling pathway.18

In our study, LGK974 treatment at intrathecal doses of 10 and 30 mmol/L in diabetic animals ameliorated the pain-associated behavioral parameters as well as the nerve functional parameters where the 30 mmol/L dose

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was found to effectively ameliorate both the pain-associated behavioral and functional parameters as compared with the 10 mmol/L dose. LGK974 significantly downregulated the expression of Wnt pathway proteins, namely, b-catenin, Dvl, and c-myc in the sciatic nerves of diabetic rats, thereby attenuating neuropathy in diabetic animals. However, the change in the expression of Wnt3a protein by LGK974 treatment was not significant, which was otherwise upregulated in diabetic animals. The probable explanation for this could be that LGK974 basically blocks the secretion of Wnt ligands, so it is possible that the overall synthesis and expression of Wnt3a in the cell might not get altered by LGK974 treatment. LGK974 also inhibited Wnt signaling in the spinal cord and DRGs by reducing the expression of b-catenin, the central effector protein in Wnt signaling. Inhibition of the Wnt signaling pathway by another PORCN inhibitor, IWP-2, was shown to decrease neuropathic pain by improving both the mechanical and thermal hyperalgesia thresholds in a chronic constriction injury model, which further supports our findings.67,68 The next crucial step after the binding of Wnt to frizzled receptor is the recruitment of a cytoplasmic scaffolding protein, Dvl, which inhibits the b-catenin destruction complex (composed of casein kinase 1, adenomatous polyposis coli, and glycogen synthase kinase3b), thereby increasing the accumulation and subsequent nuclear translocation of b-catenin.17,9 Dvl is composed of 3 highly conserved domains: an aminoterminal DIX domain, a central PDZ domain, and a carboxy-terminal DEP domain. The PDZ domain has been postulated to be crucial for interaction between frizzled receptor and Dvl.59 NSC668036 has been shown to bind to the Dvl-PDZ domain and blocks the PDZ-mediated interactions of Dvl, which further decreases the activation of Wnt signaling pathway.4,11,46,57 Interestingly, the results of our study showed that NSC668036 treatment significantly decreased the painassociated behavioral parameters. Also, NSC668036treated animals showed improvement in both nerve conduction velocities and NBF at the higher dose as compared with their diabetic counterparts. Moreover, NSC668036 decreased the expression of Wnt pathway proteins in the sciatic nerves of treatment group animals significantly as compared with diabetic group animals. Also, it decreased the expression of b-catenin in the spinal cord and DRGs in diabetic rats. Taken together, the data suggest that the protective effects of NSC668036 are mediated through the antagonizing Dvl-mediated downstream effects of the Wnt signaling in diabetic animals. Our results support an earlier study that showed that intraplantar pretreatment with NSC668036 completely blocked Wnt-mediated neuropathic pain in na€ıve mice by inhibiting Wnt3a-mediated thermal as well as mechanical hypersensitivity in mice.48 The accumulation and nuclear translocation of b-catenin is the next major step of the Wnt signaling, which in turn leads to the synthesis of Wnt target genes upon interaction with the T-cell/lymphoid enhancer factor transcription factors in the nucleus. b-Catenin has been found to be located at the synapse of neurons in the

spinal cord dorsal horn and modulate synaptic plasticity as well as neuronal remodeling.1,32 However, aberrant upregulation of b-catenin has been shown to contribute to the development of neuropathic pain.14 PNU74654 is a b-catenin inhibitor that destabilizes b-catenin and prevents its interaction with the T-cell/lymphoid enhancer factor transcription factors as reported in several cancer cell studies.25,50,56 Our results suggest that PNU74654 treatment at 10 and 30 mmol/L significantly reversed the pain-associated behaviors and nerve functional parameters as compared with the diabetic group rats. Also, PNU74654 treatment at both the doses downregulated the expression of b-catenin and c-myc significantly in the sciatic nerves of treatment group animals as compared with the diabetic animals. Moreover, it also decreased the expression of b-catenin in the spinal cord and DRGs of diabetic rats. Consistent with the blotting data, b-catenin expression was found to increase in the sciatic nerves of diabetic animals when compared with that of control rats as evident from increased b-catenin immunostaining. This increased b-catenin expression was reduced in the LGK974, NSC668036, and PNU74654 treatment groups as compared with diabetic animals.

Figure 9. Schematic representation of effects of PORCN inhib-

itor, Dvl inhibitor, and b-catenin inhibitor on DPN. PORCN inhibitor (LGK974), Dvl inhibitor (NSC668036), and b-catenin inhibitor (PNU74654) decrease the aberrant expression of Wnt pathway proteins namely Wnt3a, Dvl1, b-catenin, and c-myc induced by hyperglycemia in diabetes. This further leads to a decrease in the expression of the proinflammatory mediator, MMP2, the ER stress marker, GRP78, and an improvement in the IENFD in diabetic rats resulting in the attenuation of behavioral pain and nerve functional parameters in DPN.

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Wnt signaling pathway activation and the accumulation of b-catenin has been shown by various reports to induce and intersect with pathogenic pathways like ER stress and the activation of proinflammatory cytokine mediators like MMP2 and MMP9 in different in vitro and in vivo cancer studies.5,41,52,21,34 ER stress is a pathogenic response of cell against misfolded protein synthesis, which ultimately results in the activation of caspases and apoptosis. GRP78 is a heat shock protein located inside the ER lumen that is responsible for binding to newly synthesized proteins in the ER. Under ER stress, its synthesis increases to meet the need of increased misfolded proteins in the ER.26 Several reports have suggested the implications of an upregulation of ER stressrelated proteins in DPN. Moreover, ER stress inhibitors have found to be effective in ameliorating DPN both in vitro and in vivo.30,47,60,65 Upregulated b-catenin has been reported to augment ER stress response in cells and conversely the silencing of b-catenin decreases the latter.41,21 Also, MMPs, specifically MMP2, have been suggested to lead to a persistence of neuropathic pain and treatment with MMP2 inhibitors is associated with an attenuation of DPN in experimental models.10,16,19 Another study has suggested that the activation and upregulation of MMP2 is associated with spinal upregulation of b-catenin in a spinal contusion injury model of neuropathic pain.34 Hence, these findings provide us with a connecting link between the regulation of MMP2 and ER stress by Wnt signaling pathway. Thus, to delineate the mechanism of the neuroprotective effects exhibited by Wnt inhibitors

used in our study, we looked into the effect of Wnt inhibitors on alterations in the expression of GRP78 and MMP2 in sciatic nerves of diabetic animals. Both GRP78 and MMP2 were found to be upregulated in the sciatic nerves of diabetic animals as compared with control animals. Treatment with the Wnt inhibitors, LGK974, NSC668036, and PNU74654 at both the doses decreased the expression of both GRP78 and MMP2 significantly in the sciatic nerves of diabetic animals. After looking into the effect of Wnt inhibitors on the expression of Wnt pathway proteins, ER stress and MMP2, we then determined the effect of these inhibitors on the specific marker of DPN, that is, IENFD. Multiple reports have suggested that DPN leads to reduction in IENFD and a loss of peripheral sensory fibers in the skin of animals as well as humans suffering from DPN.24,40,55 In this study, diabetic animals showed a marked decrease in the IENFD as evident from Pgp9.5 immunofluorescence staining. Treatment with NSC668036 and PNU74654 significantly reversed the decreased IENFD in diabetic animals at the higher dose. However, the increase in the LGK974-treated groups was not significant, which could be due to the presence of some Wnt ligand subtypes not requiring PORCN for their secretion.43 Taken together, the results of this study suggest novel insights into the pathogenic involvement of Wnt/b-catenin signaling in DPN in rats. Also, it highlights the neuroprotective potential of the Wnt signaling inhibitors in DPN (Fig 9), which provide a platform to further explore their clinical efficacy in DPN.

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