Downregulation of spinal angiotensin converting enzyme 2 is involved in neuropathic pain associated with type 2 diabetes mellitus in mice

Journal Pre-proofs Downregulation of spinal angiotensin converting enzyme 2 is involved in neuropathic pain associated with type 2 diabetes mellitus in mice Ryota Yamagata, Wataru Nemoto, Osamu Nakagawasai, Kohei Takahashi, Koichi Tan-No PII: DOI: Reference:

S0006-2952(20)30035-6 https://doi.org/10.1016/j.bcp.2020.113825 BCP 113825

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

31 October 2019 22 January 2020

Please cite this article as: R. Yamagata, W. Nemoto, O. Nakagawasai, K. Takahashi, K. Tan-No, Downregulation of spinal angiotensin converting enzyme 2 is involved in neuropathic pain associated with type 2 diabetes mellitus in mice, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp.2020.113825

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© 2020 Published by Elsevier Inc.

Downregulation of spinal angiotensin converting enzyme 2 is involved in neuropathic pain associated with type 2 diabetes mellitus in mice

Ryota Yamagata1, a, Wataru Nemoto1,

a,

*, Osamu Nakagawasai1, Kohei Takahashi1,

Koichi Tan-No1 1Department

of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and

Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan aThese

authors contributed equally to this study.

*Corresponding author: Wataru Nemoto, Ph.D. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan Phone: Tel.: +81 22 727 0123, Fax: +81 22 727 0123 E-mail: [email protected]

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Abstract We have previously reported that the spinal angiotensin (Ang) system is involved in the modulation of streptozotocin (STZ)-induced diabetic neuropathic pain in mice. An important drawback of this model however is the fact that the neuropathic pain is independent of hyperglycemia and produced by the direct stimulation of peripheral nerves. Here, using the leptin deficient ob/ob mouse as a type 2 diabetic model, we examined whether the spinal Ang system was involved in naturally occuring diabetic neuropathic pain. Blood glucose levels were increased in ob/ob mice at 5-15 weeks of age. Following the hyperglycemia, persistent tactile and thermal hyperalgesia were observed at 11-14 and 9-15 weeks of age, respectively, which was ameliorated by insulin treatment. At 12 weeks of age, the expression of Ang-converting enzyme (ACE) 2 in the spinal plasma membrane fraction was decreased in ob/ob mice. Spinal ACE2 was expressed in neurons and microglia but the number of NeuN-positive neurons was decreased in ob/ob mice. In addition, the intrathecal administration of Ang (1-7) and SB203580, a p38 MAPK inhibitor, attenuated hyperalgesia in ob/ob mice. The phosphorylation of spinal p38 MAPK was also attenuated by Ang (1-7) in ob/ob mice. These inhibitory effects of Ang (1-7) were prevented by A779, a Mas receptor antagonist. In conclusion, we revealed that the Ang (1-7)-generating system is downregulated in ob/ob mice and is accompanied by

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a loss of ACE2-positive neurons. Furthermore, Ang (1-7) decreased the diabetic neuropathic pain through inhibition of p38 MAPK phosphorylation via spinal Mas receptors.

Keywords: type 2 diabetes; neuropathic pain; angiotensin (1-7); angiotensin-converting enzyme 2; spinal cord

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1. Introduction Neuropathy is one of the major complications of diabetes and affects at least half of all diabetic patients [1, 2]. There are 425 million people with diabetes mellitus in the world. According to published articles, at least 20-34% of diabetic patients experience painful neuropathy [3, 4]. Several drugs such as amitriptyline, duloxetine, gabapentin and pregabalin are used to treat diabetic neuropathic pain but most of these are not optimal for pain management [5-7]. Hence, there is a need to develop novel therapeutic targets to improve the quality of life for patients who experience diabetic neuropathic pain. The renin-angiotensin (Ang) system (RAS) plays an important role in blood pressure regulation and hydro-electrolyte balance. Ang II, a main bioactive component in the RAS and a product of Ang-converting enzyme (ACE), activates both G protein-coupled Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors. The activation of the ACE/Ang II/AT1 receptor pathway is involved in inflammation [8], oxidative stress [9], fibrosis [10] and insulin resistance [11]. Ang (1-7), an N-terminal fragment produced by the direct action of ACE2 on Ang II, binds instead to the G protein-coupled Mas receptor [12]. The activation of the ACE2/Ang (1-7)/Mas receptor pathway leads to opposite physiological effects such as vasodilation [13, 14], reduced inflammation and fibrosis [15] as well as improved insulin resistance [16]. Indeed, it is well known that the ACE2/Ang (1-7)/Mas

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receptors pathway represents a counterregulatory mechanism to control the ACE/Ang II/AT1 receptors pathway [17]. In our previous study, we have shown that the i.t. administration of Ang (1-7) inhibits the AT1 receptor-mediated nociceptive behaviour via Mas receptors [18]. We previously demonstrated that the ACE/Ang II/AT1 receptor pathway facilitates spinal pain transmission, which led to diabetic neuropathic pain in the streptozotocin (STZ)-induced type 1 diabetic mouse model [19]. In addition, the neuropathic pain was attenuated by an intrathecal (i.t.) administration of Ang (1-7) via Mas receptors [20]. These results indicated that the spinal Ang system plays a pivotal role in the modulation of diabetic neuropathic pain. However, several studies suggest that the STZ mouse may not represent the most appropriate animal model for the study of diabetic neuropathic pain. Indeed, low doses of STZ that did not induce hyperglycemia, still activated the transient receptor potential (TRP) vanilloid 1 in dorsal root ganglia (DRG) neurons and caused hyperalgesia [21]. In addition, an intraplantar administration of STZ induced hyperalgesia by stimulating peripheral TRP ankyrin 1 [22]. These reports suggest that STZ can induce neuropathic pain by activating peripheral nerves directly and independently of hyperglycemia. Thus, the main purpose of this study was to determine whether an alteration in the

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spinal Ang system might also be involved in the appearance of diabetic neuropathic pain in a different diabetes model. For this purpose we used leptin-deficient ob/ob mice, a well-known model for type 2 diabetes. In addition, we determined the effect of Ang (17) on the neuropathic pain observed in type 2 diabetic mice.

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2. Materials and methods 2.1. Animals Male leptin-deficient mice (B6.Cg-Lepob/J, ob/ob, JAXTM Mice Stock Number 000632), a model for type 2 diabetes, and age-matched lean (ob/+ or +/+) mice were purchased from Charles River Japan (Yokohama, Japan). Mice were housed in cages with free access to food and water under controlled conditions (temperature, 22 ± 2ºC; humidity, 55 ± 5%) and a 12/12 h light/dark cycle (lights on: 07:00 to 19:00). Groups of 6-13 mice for behavioral experiments, 4-12 mice for western blotting, and 7 mice for immunohistochemical experiments were used in single experiments. All experiments were conducted following the approval from the Ethics Committee of Animal Experiment at Tohoku Medical and Pharmaceutical University. Additionally, all procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to use the small possible number of animals and to minimize suffering. 2.2. Intrathecal injections The i.t. injections were performed in unanesthetized mice at the L5, L6 intervertebral space as described by Hylden and Wilcox [23]. Briefly, a volume of 5 µL was administered i.t. with a 28-gauge needle connected to a 50-µL Hamilton microsyringe, the animal being lightly restrained to ensure needle positioning. Puncture of the dura was 7

indicated behaviorally by a slight flick of the tail. The needles and microsyringe used for i.t. administration were siliconized with Sigmacote (Sigma-Aldrich, St. Louis, MO). 2.3. Drugs and antibodies The following drugs and reagents were used: Ang (1-7) (Peptide Institute, Osaka, Japan); [D-Ala7]-Ang (1-7) (A779) (Bachem, Bubendorf, Switzerland); SB203580 (Tocris Bioscience, Bristol, UK); rabbit anti-angiotensinogen (AGT) antibody (Proteintech, Rosemont, IL); goat anti-ACE antibody and rabbit anti-ACE2 antibody (Santa Cruz Biotechnology, Dallas, TX); rabbit anti-AT1 receptor antibody (Alpha Diagnostic International, San Antonio, TX); rabbit anti-Mas receptor antibody (Alomone Laboratories, Jerusalem, Israel); rabbit monoclonal antibodies against -tubulin, p38 MAPK, phospho-p38 MAPK, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody and HRP-conjugated horse anti-mouse IgG antibody (Cell Signaling Technology, Danvers, MA); rabbit anti-Na+/K+-ATPase monoclonal antibody and HRPconjugated rabbit anti-goat IgG antibody (Abcam, Cambridge, UK); mouse anti-neuronal nuclei (NeuN) antibody and mouse anti-glial fibrillary acidic protein (GFAP) antibody (Merck Millipore, Burlington, MA); rabbit anti-ionized calcium binding adaptor molecule 1 (Iba-1) antibody (Wako Chemicals, Osaka, Japan);

Alexa Fluor 488-

conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG

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(Molecular Probes, Eugene, OR); Alexa Fluor 488-conjugated AffiniPure Fab fragment goat anti-mouse IgG and Alexa 647-conjugated AffiniPure Fab fragment goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA); normal goat serum (NGS) (Invitrogen, Carlsbad, CA); 4 ′ ,6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan). For immunohistochemical staining, rabbit anti-ACE2 antibody and Alexa Fluor 488-conjugated AffiniPure Fab fragment goat anti-rabbit IgG were diluted 1:100 and 1:80, respectively, whereas all other antibodies were diluted 1:200 in phosphate-buffered saline (PBS) containing 1% NGS. For i.t. injections, Ang (1-7) and A779 were dissolved in Ringer’s solution. 2.4. Measure of blood glucose levels and insulin treatment Blood was taken from the tail vein of mice and glucose levels measured using the FreeStyle Precision Neo-Blood Glucose Monitoring Meter (Abbott Japan, Tokyo, Japan). For insulin treatment, neutral protamine Hagedorn (NPH) insulin (Humulin N; Eli Lilly, Indianapolis, IN) was administered subcutaneously (s.c.) twice daily into ob/ob mice at 5-16 weeks of age. The dose of insulin was adjusted (< 36 IU/day) to maintain individual blood glucose level between 140-170 mg/dl. Age-matched ob/ob mice injected with 0.2 mL saline (s.c.) were used as controls. 2.5. von Frey filament test Tactile hyperalgesia was evaluated by measuring paw withdrawal in the von Frey 9

filament test as previously described [19]. Briefly, von Frey filaments (pressure applied by the individual filament: 0.07, 0.16, 0.4, 0.6 and 1.0 g) were applied perpendicularly against the plantar surface of both hindpaws and held for 3 seconds with slight buckling of the filaments. A positive response was recorded if the paw was sharply withdrawn and was followed by the application of the next weakest von Frey hair. On the other hand, if a negative response occurred, the next strongest force was applied. The average of left and right paw responses was used for analysis. To assess the effect of A779 on Ang (1-7)-induced inhibition of tactile hyperalgesia, a pressure of equal intensity (0.4 g filament) was applied to the plantar surface of each hindpaw, and repeated 10 times at intervals of 5 seconds. The frequency of sharp withdrawal responses was measured. 2.6. Hargreaves test Thermal hyperalgesia was measured using plantar test system (Ugo basile, Gemonio, Italy) according to a previously described method [24]. Mice were placed in a transparent cage that was positioned on a glass plate and acclimatized to the measurement environment for at least 30 minutes before the test. Any fluid on the glass plate was immediately wiped away to keep the measurement environment clean. A mobile infrared (IR) heat source was placed under the glass floor and focused on the hindpaw. Paw

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withdrawal latencies were measured with a cutoff time of 20 sec to avoid tissue damage. The IR intensity was adjusted to 100 mW/cm2. Measurements were performed thrice for each hindpaw and the average latency was used for the statistical analysis. 2.7. Preparation of plasma membrane fractions Subcellular enrichment of the dorsal lumbar spinal cords was performed according to our recent report [20]. Briefly, tissues were homogenized at 4°C in buffer with the following composition: 0.32 M sucrose, 20 mM Tris, 0.1 mM CaCl2, 1 mM MgCl2, pH 6.0. Homogenates from three mice were pooled per tube to increase yield. Homogenized samples were centrifuged at 1,000 g for 10 min at 4°C. Supernatants were transferred to new tubes whereas pellets which contained nuclei and unbroken cells were discarded. This centrifugation step was repeated three times in order to clean the supernatants after which they were transferred to new tubes and centrifuged at 10,000 g for 15 min at 4°C to obtain membrane-enriched pellets. The membrane-enriched pellet was resuspended in homogenization buffer and solubilized by the addition of sodium dodecyl sulphate (SDS) to a final concentration of 1%. 2.8. Western blotting Following decapitation, whole spinal cords were released by applying pressure with a syringe filled with ice-cold physiological saline. The dorsal part of the spinal cord at the

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lumbar level was quickly dissected on an ice-cold glass dish. The western blotting procedure was conducted as previously described [25]. Polyacrylamide gel electrophoresis was performed on 6% or 10% gels. The separated proteins were transferred from the gel onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA) using a semi-dry blotting apparatus (Bio-Rad Laboratories). Blots were blocked with 5% skim-milk in Tris-buffered saline supplemented with 0.01% Tween-20 (TBST) for 30 min then incubated overnight at 4°C in buffer of the same composition but containing primary antibodies made in either rabbit against -tubulin, p38 MAPK, phospho-p38 MAPK, AGT, AT1 receptors, ACE2, Mas receptors, Iba-1 (all diluted 1:1,000) or Na+/K+-ATPase (diluted 1:5,000), or made in mouse against NeuN (diluted 1:1,000), or goat against ACE (diluted 1:1,000). Blots were then washed several times then incubated at room temperature for 2 h with HRP-conjugated anti-rabbit, antimouse or anti-goat IgG antibodies (diluted 1:5,000, 1:5,000 or 1:20,000, respectively, with TBST containing 5% skim-milk). An enhanced chemiluminescence assay kit (GE Healthcare, Chicago, IL) was used to develop the blots and immunoreactive proteins were visualized on Fuji Medical X-ray Film (Fujifilm, Tokyo, Japan). The densities of the corresponding bands were analyzed by densitometry (Image-J 1.43u, National Institute of Health).

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2.9. Immunohistochemical staining and confocal microscopy Mice were anesthetized with a mixture of medetomidine (50 mg/kg; Nihonzenyaku Industry, Fukushima, Japan), midazolam (4 mg/kg; Sando, Tokyo, Japan) and butorphanol (5 mg/kg; Meiji Seika Pharma, Tokyo, Japan) injected intraperitoneally, then perfused through the heart with ice-cold phosphate-buffered saline (PBS, pH 7.2) immediately followed by a fixative containing 4% paraformaldehyde (Sigma-Aldrich) in PBS. Tissues were postfixed with the same fixative solution at 4 ºC for 1 h then placed in a buffered 20% sucrose solution at 4 ºC for 12 h. Spinal cords (lumbar 5; L5) were frozen and cut into 40 m-thick coronal sections on a cryostat (Microm International GmbH, Walldorf, Germany). The immunohistochemical staining for ACE2 and cell specific markers (NeuN, GFAP or Iba-1) were performed as previously described [26], while the triple immunolabelling for ACE2, NeuN, and Iba-1 was performed as follows: (1) two washes of coronal sections in PBS for a total of 30 min; (2) blocking with PBS containing 10% NGS at room temperature for 30 min; (3) two washes in PBS for a total of 30 min; (4) overnight incubation at 4 °C with one of the primary antibodies (rabbit anti-Iba-1 antibody diluted in PBS containing 1% NGS) ; (5) four washes in PBS for a total of 1 h; (6) incubation with Alexa Fluor 488-conjugated AffiniPure Fab fragment goat anti-rabbit IgG (diluted in PBS containing 1% NGS) overnight at 4 °C ; (7) four washes in PBS for

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a total of 1 h; (8) incubation with the other two primary antibodies (mouse anti-NeuN antibody and rabbit anti-ACE2 antibody) (diluted in PBS containing 1% NGS) at 4 °C for 2 days; (9) four washes in PBS for a total of 1 h; (10) incubation with Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated AffiniPure Fab fragment goat anti-rabbit IgG and DAPI (diluted in PBS containing 1% NGS) overnight at 4 °C ; (11) four washes in PBS for a total of 1 h; and (12) embedding in ProLong diamond antifade mountant (Molecular Probes). The labelled sections were kept at 4 °C in the dark until measurements were carried out. The fluorescent signal was visualized using a Nikon C2 confocal microscope system (Nikon, Tokyo, Japan). For each mouse, the total number of labeled cells in laminae I-III on both sides of the superficial dorsal horn was counted then averaged from five sections. Background noise from secondary antibodies was determined by performing immunohistochemical stainings on the superficial dorsal horn without primary antibodies. A cell was considered positive if it’s immunoreactive fluorescence intensity was 10-fold or higher than the background signal. The number of immunoreactive-cells and the fluorescence intensities were measured using the Nikon C2 confocal microscope software.

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2.10. Statistical methods Data were expressed as means ± SEM. Significant differences were analyzed by a one-way or two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Student’s t test was used for comparisons between two groups. All statistical analyses were performed using GraphPad Prism software, version 7.03 (GraphPad Software, USA). In all comparisons, P < 0.05 was considered statistically significant. 3. Results 3.1. Temporal changes in body weights, blood glucose levels, tactile and thermal hyperalgesia in ob/ob mice Body weights, blood glucose levels, tactile and thermal hyperalgesia were measured in ob/ob and lean mice between the ages of 5 and 20 weeks. As shown in Fig. 1A, the body weights were significantly increased in ob/ob mice compared with those of lean mice throughout the experimental period. Blood glucose levels were also significantly increased in ob/ob mice between 5-15 weeks of age but became similar afterwards (Fig. 1B). Persistent tactile hyperalgesia was observed in ob/ob mice at 11-14 weeks of age (Fig. 1C). Similarly, paw withdrawal latency to thermal stimuli was significantly decreased in ob/ob mice at 9-15 weeks of age (Fig. 1D). Our results show that tactile and thermal hyperalgesia appeared following continuous hyperglycemic state and lasted until 15

glucose levels returned to normoglycemic levels. 3.2. Effect of insulin on tactile and thermal hyperalgesia in ob/ob mice To determine whether the continuous hyperglycemic state was responsible for the onset of tactile and thermal hyperalgesia in ob/ob mice, we administered insulin (b.i.d., s.c.) between 5-16 weeks of age. Blood glucose levels were measured 1 day prior to the start of insulin treatment then every week afterwards. As expected, insulin decreased the glycemia observed in ob/ob mice which was maintained at normal levels during the whole treatment period i.e. between 6-16 weeks of age (Fig. 2A). In addition, this reversal of hyperglycemia was soon followed by the disappearance of tactile and thermal hyperalgesia in these mice (Fig. 2B and C). These results suggest that the hyperalgesia observed in ob/ob mice is related to diabetic neuropathic pain. 3.3. Expression levels of AGT, ACE, AT1 receptors, ACE2 and Mas receptors in the lumbar dorsal spinal cord of ob/ob mice. We performed western blotting to detect changes in RAS components in the lumbar dorsal spinal cord of ob/ob mice. As shown in Fig. 3A, there were no significant changes in spinal AGT levels in ob/ob mice compared with that of lean mice. Similarly, the expression of spinal ACE and AT1 receptors were also not changed (Fig. 3B and C). On the other hand, the level of ACE2 was significantly decreased in ob/ob mice (Fig. 3D), while Mas receptors showed a trend towards lower levels, although the difference was not significant (Fig. 3E). These results suggest that the ACE2/Ang (1-7)/Mas receptor 16

pathway may be down-regulated in the lumbar dorsal spinal cord of ob/ob mice. 3.4. Alteration in the number of ACE2 positive cells in the lumbar superficial dorsal horn of ob/ob mice To investigate the mechanism behind the decrease in ACE2 expression, we examined the superficial dorsal horn of ob/ob mice for signs of changes in the expression of ACE2 positive cells. As shown in Fig. 4, ACE2 was localized in NeuN-positive cells (neurons) and Iba-1-positive cells (microglias), but was absent from GFAP-positive cells (astrocytes) in the superficial dorsal horn of both lean and ob/ob mice. Next, we counted the number of NeuN- and Iba-1-positive cells on both sides of the laminae I-III region. Although the fluorescence intensities for NeuN (Fig. 5A, B and C), Iba-1 (Fig. 5A, B and D) and ACE2 (Fig. 5A, B and E) were not different between lean and ob/ob mice, the number of NeuN-positive cells was significantly decreased in the superficial dorsal horn of ob/ob mice compared with that of lean mice (Fig. 5A, B and F). In contrast, the number of Iba-1-positve cells was not changed (Fig. 5A, B and G). In addition, ACE2 and NeuN double-positive cells (ACE2 positive neurons) were also significantly decreased (Fig. 5H), while ACE2 and Iba-1 double-positive cells (ACE2 positive microglials) were not changed between ob/ob and lean mice (Fig. 5I). Similar to the results obtained from our immunohistochemical experiments, a decrease in NeuN in the lumbar dorsal spinal cord of ob/ob mice was also detected by western blotting (Fig. 5J and K). Moreover, the

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expression of Iba-1 was not different between ob/ob and lean mice (Fig. 5J and L). Together, these findings suggest that the decrease observed in the expression of ACE2 in the superficial dorsal horn of ob/ob mice follows a decrease in the number of neurons. 3.5. Effect of an i.t. administration of Ang (1-7) on tactile hyperalgesia in ob/ob mice To examine whether Ang (1-7) can inhibit the tactile hyperalgesia observed in ob/ob mice, we conducted a von Frey filament test after treatment at 12 weeks of age, which was the time point that exhibited the strongest hyperalgesia. Tactile hyperalgesia was assessed prior (pre) and 15, 30, 45, 60, 90, 120, 150 and 180 min after the i.t. administration of Ang (1-7) at the doses of 0.3, 3 or 30 pmol in both ob/ob and lean mice. In the latter, the i.t. administration of Ang (1-7) (0.3-30 pmol) did not affect the paw withdrawal threshold (Fig. 6A). In contrast, in ob/ob mice, Ang (1-7) significantly attenuated the tactile hyperalgesia with a maximum effect 45 min after administration at the doses of 3 or 30 pmol (Fig. 6B). To examine the effect of the Mas receptor antagonist A779 on Ang (1-7)-induced anti-tactile hyperalgesia, A779 was co-administered i.t. with Ang (1-7) 45 min before the measurement. As shown in Fig. 6C, although Ang (1-7) (0.330 pmol) dose-dependently attenuated the tactile hyperalgesia, this inhibition was significantly prevented by A779 (0.3 nmol). In contrast, these treatments did not affect the withdrawal responses in lean mice. Furthermore, neither the i.t. administration of Ang (1-7) (3 pmol) nor A779 (0.3 nmol) affected blood glucose levels in ob/ob mice 18

throughout the experimental period (Fig. 6D). These results suggest that Ang (1-7) attenuates the tactile hyperalgesia observed in ob/ob mice via spinal Mas receptors without affecting blood glucose levels. 3.6. Effect of an i.t. administration of Ang (1-7) on thermal hyperalgesia in ob/ob mice We next examined the effect of Ang (1-7) on the thermal hyperalgesia observed in ob/ob mice at 12 weeks of age using the Hargreaves test. Thermal hyperalgesia was assessed prior and 30, 60, 90, 120, 150 and 180 min after the i.t. administration of Ang (1-7) (3 pmol) in both ob/ob and lean mice. Ang (1-7) did not affect the withdrawal latency in lean mice during the observation period (Fig. 7A). In contrast, the thermal hyperalgesia observed in ob/ob mice was significantly inhibited by Ang (1-7) which showed a maximum effect after 60 min (Fig. 7B). As above, to examine the effect of A779 on Ang (1-7)-induced anti-thermal hyperalgesia, the antagonist was coadministered i.t. with Ang (1-7) 60 min before the measurement. As shown in Fig. 7C, the anti-hyperalgesic effect of Ang (1-7) (3 pmol) was completely antagonized by A779 (0.3 nmol). These results suggest that Ang (1-7) decreased the thermal hyperalgesia observed in ob/ob mice and that this effect was mediated by spinal Mas receptors. 3.7. Involvement of spinal p38 MAPK in the anti-hyperalgesic effect of Ang (1-7) in ob/ob mice To examine whether spinal p38 MAPK activation may be responsible for the

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hyperalgesia observed in ob/ob mice, the von Frey and Hargreaves tests were used following the i.t. administration of the p38 MAPK inhibitor SB203580 (1 nmol). SB203580 significantly attenuated both the tactile and thermal hyperalgesia at 15-150 min and 60-120 min after administration, respectively (Fig. 8A and B). These results suggest that the activation of intraspinal p38 MAPK contributes to the tactile and thermal hyperalgesia observed in ob/ob mice. Furthermore, the i.t. administration of SB203580 (1 nmol) did not affect blood glucose levels in ob/ob mice (Fig. 8C). Next, we used western blotting to examine the effect of Ang (1-7) on the phosphorylation of spinal p38 MAPK in ob/ob mice at 12 weeks of age. As shown in Fig. 9, the higher level of phosphorylated p38 MAPK in the lumbar dorsal spinal cord of ob/ob mice was significantly reduced by Ang (1-7) (3 pmol), which in turn was prevented by the coadministration of A779 (0.3 nmol). In contrast, Ang (1-7) or Ang (1-7) in combination with A779 did not affect the levels of phosphorylated p38 MAPK in lean mice. These results suggest that Ang (1-7) attenuates the phosphorylation of p38 MAPK in the lumbar dorsal spinal cord of ob/ob mice via Mas receptors.

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4. Discussion The major findings in our study are that tactile and thermal hyperalgesia in type 2 diabetic ob/ob mice results from diabetic neuropathy and is accompanied by p38 MAPK activation, an anticipated consequence of the decrease detected in spinal ACE2 expression. Moreover, this decrease is likely caused by the loss of ACE2 positive neurons we observed in the spinal cord dorsal horn. Leptin-deficient ob/ob mice are hyperglycemic and display morphological abnormalities of peripheral nerves leading to tactile and thermal hyperalgesia, some of the symptoms of diabetic neuropathy [27]. In this study, ob/ob mice showed transient hyperglycemia between the ages of 5- and 15 weeks. However, the blood glucose levels measured in the present study were slightly lower than those in a previous report [27]. The reason for this discrepancy is not clear but may be due to differences in housing conditions between the two studies. Clinical reports on diabetic neuropathy state that strict glycemic control is one of the most important means used in the management of neuropathy [28]. In our study, ob/ob mice displayed hyperglycemia at 5 weeks of age while tactile and thermal hyperalgesia were observed at 11-14 and 9-15 weeks of age, respectively. Moreover, these hyperalgesia were completely abolished when blood glucose levels were controlled with insulin treatment. Hence, it seems that the hyperalgesia observed in ob/ob mice is equivalent to diabetic neuropathic pain. 21

Although we have only examined the spinal cord for changes in RAS components, it has been reported that the hyperglycemic statestrongly influences the Ang system in other tissues also. Patients with proliferative diabetic retinopathy show increased levels of Ang II in the vitreous humor [29] while the inactivation of the retinal ACE/Ang II/AT1 receptor pathway is being considered as a therapeutic strategy for diabetic patients [30, 31]. Moreover, it was shown in diabetic models that there is an upregulation of the ACE/Ang II/AT1 receptor pathway and a downregulation of the ACE2/Ang (1-7)/Mas receptor pathway in retina [32, 33] and kidney [34, 35]. Similar observations have been reported for the kidneys of type 2 diabetic patients with diabetic nephropathy [36]. These data suggest an imbalance between the ACE/Ang II/AT1 receptor and ACE2/Ang (17)/Mas receptor pathways as a common basis for the onset and/or evolution of diabetic complications. Recently, we have revealed that the Ang II-generating system is upregulated [19], while the Ang (1-7)-generating system is down-regulated [20] in the spinal cord of STZ-mice. The fact that the neuropathic pain observed in STZ mice decreased following the i.t. administration of losartan or Ang (1-7) supports the hypothesis that the imbalance between the ACE/Ang II/AT1 receptor and ACE2/Ang (1-7)/Mas receptor pathways is critical for the induction of neuropathic pain [19, 20]. It is interesting to note that the present study did not find any significant change in the levels of ACE and AT1

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receptors in the spinal cord of ob/ob mice, which is in contrast with the results obtained with STZ-induced type 1 diabetic mice. However, similar to STZ mice, ob/ob mice showed a significant reduction in ACE2 expression in the plasma membrane fraction from the lumbar dorsal spinal cord when compared with lean mice. Furthermore, the expression of spinal Mas receptors in ob/ob mice showed a tendency towards lower levels. Together, our results suggest that the ACE2/Ang (1-7)/Mas receptor pathway is downregulated, whereas the ACE/Ang II/AT1 receptor pathway is not changed, in the lumbar dorsal spinal cord of ob/ob mice. In this report, we demonstrated the expression of ACE2 in neurons and microglia of the superficial dorsal horn in both ob/ob and lean mice. While the number of microglial cells or ACE2-positive microglia in lamina I-III was not different between lean and ob/ob mice, the number of both ACE2-positive neurons and NeuN-positive neurons, as well as total NeuN levels, were all significantly decreased in ob/ob mice. Since almost all NeuNpositive cells in the superficial dorsal horn were also positive for ACE2, the decrease in NeuN-positive cells was accompanied by the loss of ACE2-positive spinal neuron in ob/ob mice. Peripheral nerve injury is known to induce apoptosis in the dorsal horn of the spinal cord, whereas the loss of spinal inhibitory mechanisms lead to neuropathic pain [37-39]. Scholz et al. have found that spinal dorsal horn neurons including GABAergic

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inhibitory interneurons are lost following peripheral nerve injury in rats　[40]. Together, these findings suggest that neuronal loss in the spinal dorsal horn can contribute to neuropathic pain. Recently, using a mouse model of peripheral nerve injury, it has been shown that the activation of N-methyl-D-aspartate (NMDA) receptors is necessary for the neuronal loss in the dorsal horn and the transition to persistent pain [41]. It has also been reported that reactive oxygen species (ROS) contribute to pain through the activation of NMDA receptors [42] while hyperglycemia-induced increase in ROS production leads to neuronal cell death [43, 44]. It is thus tempting to speculate that an activation of spinal NMDA receptors in diabetes may be involved in the neuronal loss observed in the spinal cord of ob/ob mice. Recently, we have shown that Ang (1-7) decreases the STZ-induced diabetic neuropathic pain via spinal Mas receptors [20]. Cancer-induced bone pain is also alleviated by Ang (1-7) via Mas receptors in DRG and femoral neoplasm [45]. Furthermore, the intraplantar injection of Ang (1-7) produces an antinociceptive effect via peripheral Mas receptors in the prostaglandin E2-induced inflammatory pain model [46]. In our present study, the tactile and thermal hyperalgesia observed in ob/ob mice were completely attenuated by an i.t. administration of Ang (1-7), and this effect was prevented by A779. Neurons and microglia in the superficial layer most probably

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contribute to the anti-hyperalgesic effect of Ang (1-7) as these are the cells that were shown to express Mas receptors in the dorsal horn [26]. Since the analgesic effect of Ang (1-7) was brief, we believe that a sustained activation of Mas receptors may represent an efficient and novel approach in the treatment of neuropathic pain associated with type 2 diabetes mellitus. The activation of spinal p38 MAPK results in neuropathic pain [47-50] as well as inflammatory pain [51-53]. In STZ-induced type 1 diabetic mice, spinal p38 MAPK was phosphorylated through AT1 receptor activation [19] and this phosphorylation could be attenuated by Ang (1-7) acting via Mas receptors [20]. Our study is the first to demonstrate the involvement of spinal p38 MAPK activation in the hyperalgesia in ob/ob mice as well as the attenuation of tactile and thermal hyperalgesia by the i.t. administration of SB203580.

Ang (1-7) attenuated the phosphorylation of p38 MAPK

in the lumbar dorsal spinal cord of ob/ob mice by acting on Mas receptors. Thus, we suggest that the anti-hyperalgesic effect produced by Ang (1-7) is accompanied by an inhibition of p38 MAPK phosphorylation and is mediated through spinal Mas receptors. In conclusion, we reveal that the neuropathic pain observed in type 2 diabetic mice involves the downregulation of ACE2/Ang (1-7)/Mas receptor pathway. This downregulation is believed to be caused by a loss of neurons in the superficial dorsal horn

25

that express ACE2. Since the activation of the ACE2/Ang (1-7)/Mas receptor pathway could alleviate hyperalgesia, an exogenous supplementation with Ang (1-7) may be an effective therapeutic strategy for the relief of neuropathic pain associated with type 2 diabetes mellitus. Author contributions: R.Y., W.N. and K.T-N. designed the experiments. R.Y., W.N. and O.N. performed the experiments. R.Y., W.N., O.N. and K.T. analyzed the data. R.Y., W.N. and K.T-N. wrote the manuscript. Conflict of interest: The authors report no conflicts of interests. Acknowledgements This study was supported in part by JSPS KAKENHI (grant Number 17K08313 to K.T-N., W.N., and O.N.; grant number 19K16376 to W.N.), Matching Fund Subsidy for Private University from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant number S1511001L) and Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan. We thank Mr. Ryota Yamagishi, Ms. Yuria Wada and Ms. Mizuki Watanabe at Tohoku Medical and Pharmaceutical University for their technical assistance.

26

Funding sources This work was funded by grants 17K08313 and 19K16376 from the JSPS KAKENHI, S1511001L from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan.

27

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Figure legends Figure 1. Variations in body weights, blood glucose levels, tactile and thermal hyperalgesia in ob/ob compared to lean mice. (A) Body weights, (B) blood glucose levels, (C) tactile hyperalgesia and (D) thermal hyperalgesia were measured between 5 and 20 weeks of age. (A) Two-way ANOVA: group (F1,19 = 87.07, P < 0.01); time (F15,285 = 69.23, P < 0.01); group × time (F15,285 = 15.78, P < 0.01). (B) Two-way ANOVA: group (F1,19 = 8.47, P < 0.01); time (F15,285 = 3.96, P < 0.01); group × time (F15,285 = 3.38, P < 0.01). (C) Two-way ANOVA: group (F1,19 = 17.95, P < 0.01); time (F15,285 = 1.30, P > 0.05); group × time (F15,285 = 1.37, P > 0.05). (D) Two-way ANOVA: group (F1,19 = 69.34, P < 0.01); time (F15,285 = 1.52, P > 0.05); group × time (F15,285 =3.37, P < 0.01). Values represent the means ± SEM for 8 (lean) or 13 (ob/ob) mice per group. *P < 0.05, **P < 0.01 compared with lean mice. Figure 2. Effect of insulin on blood glucose levels, tactile and thermal hyperalgesia observed in ob/ob mice. (A) Blood glucose levels, (B) tactile hyperalgesia and (C) thermal hyperalgesia were measured in ob/ob mice between 5-16 weeks of age following the administration of insulin or vehicle (s.c., b.i.d.) at age 5 weeks. (A) Two-way ANOVA: treatment (F1,8 = 5.39, P < 0.05); time (F11,88 = 6.89, P < 0.01); treatment × time (F11,88 = 4.65, P < 0.01). (B) Two-way ANOVA: treatment (F1,8 = 39.59, P < 0.01); 33

time (F11,88 = 1.99, P < 0.05); treatment × time (F11,88 = 3.48, P < 0.01). (C) Two-way ANOVA: treatment (F1,8 = 30.71, P < 0.01); time (F11,88 = 1.84, P > 0.05); treatment × time (F11,88 = 2.55, P < 0.01). Values represent the means ± SEM for 5 mice per group. *P < 0.05, **P < 0.01

compared with vehicle-treated ob/ob mice.

Figure 3. Changes in AGT, ACE, AT1 receptors, ACE2 and Mas receptors in the lumbar dorsal spinal cord of ob/ob mice. Spinal cord samples from the lumbar dorsal area were taken from ob/ob mice and age-matched lean mice at 12 weeks of age. AGT levels from whole tissue and other proteins from the plasma membrane (PM) fraction were compared using western blotting. (A-E) Representative blots and quantifications relative to -tubulin or Na+/K+-ATPase for AGT, ACE, AT1 receptor, ACE2, and Mas receptor, with ratios set as 1.0 for the lean mice. Values represent the means ± SEM of 4-6 mice per group. *P < 0.05 compared with lean mice. Figure 4. Double immunohistochemical staining for ACE2 and cell specific markers for neuron (NeuN), microglia (Iba-1) or astrocyte (GFAP) in the lumbar superficial dorsal horn of ob/ob mice. Photomicrographs showing fluorescent labeling for ACE2 (red), NeuN (green), Iba-1 (green), GFAP (green) or nuclei with DAPI (blue), as well as merged images (right panels), in the superficial dorsal horn of (A) lean and (B) ob/ob

34

mice. Scale bar = 50 μm. Figure 5. Decrease in ACE2 positive cells in the lumbar superficial dorsal horn of ob/ob mice. (A, B) Triple immunofluorescence staining for NeuN, Iba-1 and ACE2 in the lumbar superficial dorsal horn (laminae I-III) of (A) lean and (B) ob/ob mice at 12 weeks of age. Photomicrographs showing DAPI staining (blue), NeuN-positive cells (red), Iba1-positive cells (green), ACE2-positive cells (magenta) and merged images. Scale bar = 100 m. (C-E) Fluorescence intensity for (C) NeuN, (D) Iba-1 and (E) ACE2 in the lumbar superficial dorsal horn (laminae I-III) of lean and ob/ob mice. Values represent the means ± SEM of 7 mice per group. (F-I) Comparison between lean and ob/ob mice in total counts from both sides of the superficial dorsal horn (laminae I-III) for (F) NeuNpositive cells, (G) Iba-1-positive cells, (H) NeuN/ACE2 double positive cells and (I) Iba1/ACE2 double positive cells. Values represent the means ± SEM of 7 mice per group. **P

< 0.01 compared with lean mice. (J) Representative western blots showing NeuN,

Iba-1 and -tubulin in the lumbar dorsal spinal cord of lean and ob/ob mice at 12 weeks of age, with relative quantifications of (K) NeuN and (L) Iba-1 to -tubulin, set as 1.0 in lean mice. Values represent the means ± SEM of 6 mice per group. *P < 0.05 compared with lean mice. Figure 6. Effect of Ang (1-7) on tactile hyperalgesia in ob/ob mice. Tactile hyperalgesia

35

and blood glucose levels were measured in ob/ob and lean mice at 12 weeks of age. Time courses of the effect of the Ang (1-7) (0.3-30 pmol) administration on the paw withdrawal responses to von Frey filaments in (A) lean and (B) ob/ob mice. (A) Two-way ANOVA: treatment (F3,20 = 0.12, P > 0.05); time (F8,160 = 0.28, P > 0.05); treatment × time (F24,160 = 0.43, P > 0.05). Values represent the means ± SEM for 6 mice per group. (B) Two-way ANOVA: treatment (F3,22 = 3.99, P < 0.05); time (F8,176 = 16.31, P < 0.01); treatment × time (F24,176 = 3.79, P < 0.01). Values represent the means ± SEM for 6-8 mice per group. *P < 0.05, **P < 0.01

compared with vehicle-injected ob/ob mice. (C) Effect of A779 on

the Ang (1-7)-induced inhibition of tactile hyperalgesia was measured by assessing the frequency of the paw withdrawal response to a 0.4 g tactile stimuli in lean and ob/ob mice. Vehicle, Ang (1-7) (3 pmol), or Ang (1-7) (3 pmol) in combination with A779 (0.3 nmol) were administered 45 min prior to measurements. Two-way ANOVA: group (F1,52 = 63.42, P < 0.01), treatment (F4,52 = 8.22, P < 0.01), group × treatment (F4,52 = 11.78, P < 0.01). One-way ANOVA: F9,52 = 10.38, P < 0.01. Values represent the means ± SEM for 6–8 mice per group. mice,

##P < 0.01

**P < 0.01

compared with vehicle-injected lean

compared with vehicle-injected ob/ob mice and

$$P

< 0.01 compared

with Ang (1-7) (3 pmol)-injected ob/ob mice. (D) Time courses of the effect of i.t. administration of Ang (1-7) (3 pmol) or A779 (0.3 nmol) on blood glucose levels in ob/ob

36

mice. Two-way ANOVA: treatment (F2,10 = 0.10, P > 0.05); time (F2,25 = 2.87, P < 0.05); treatment × time (F10,650 = 0.39, P > 0.05). Values represent the means ± SEM for 6 mice per group. Figure 7. Effect of Ang (1-7) on thermal hyperalgesia in ob/ob mice. Thermal hyperalgesia was measured in ob/ob mice at 12 weeks of age. Time courses of the effect of a 3 pmol administration of Ang (1-7) on the paw withdrawal latency to radiant heat stimuli in (A) lean and (B) ob/ob mice. (A) Two-way ANOVA: treatment (F1,10 = 1.44, P > 0.05); time (F6,60 = 0.93, P > 0.05); treatment × time (F6,60 = 0.69, P > 0.05). (B) Two-way ANOVA: treatment (F1,14 = 6.51, P < 0.05); time (F6,84 = 5.47, P < 0.01); treatment × time (F6,84 = 5.30, P < 0.01). Values represent the means ± SEM for 6-10 mice per group. *P < 0.05,

**P < 0.01

compared with vehicle-injected ob/ob mice. (C)

Effect of A779 on the Ang (1-7)-induced inhibition of thermal hyperalgesia in lean and ob/ob mice. Vehicle, Ang (1-7) (3 pmol) or Ang (1-7) (3 pmol) in combination with A779　 (0.3 nmol) were administered 60 min prior to measurements. Two-way ANOVA: group (F1,34 = 48.21, P < 0.01), treatment (F2,34 = 18.66, P < 0.01), group × treatment (F2,34 = 28.00, P < 0.01). One-way ANOVA: F5,34 = 31.42, P < 0.01. Values represent the means ± SEM for 6–10 mice per group. **P < 0.01 compared with vehicle-injected lean mice,

##P < 0.01

compared with vehicle-injected ob/ob mice and

37

$$P

< 0.01 compared

with Ang (1-7) (3 pmol)-injected ob/ob mice. Figure 8. Effect of p38 MAPK inhibition on tactile and thermal hyperalgesia in ob/ob mice. Tactile and thermal hyperalgesia as well as blood glucose levels were measured in ob/ob mice at 12 weeks of age. Time courses of the effect of the i.t. administration of SB203580 (1 nmol) on the paw withdrawal responses to (A) von Frey filaments or (B) radiant heat stimuli in ob/ob mice. (A) Two-way ANOVA: treatment (F1,10 = 22.30, P < 0.01); time (F8,80 = 5.78, P < 0.01); treatment × time (F8,80 = 4.66, P < 0.01). (B) Twoway ANOVA: treatment (F1,10 = 20.57, P < 0.01); time (F6,60 = 3.55, P < 0.01); treatment × time (F6,60 = 2.70, P < 0.05). Values represent the means ± SEM for 6 mice per group. *P < 0.05, **P < 0.01

compared with vehicle-injected ob/ob mice. (C) Time courses of the

effect of i.t. administration of SB203580 (1 nmol) on blood glucose levels in ob/ob mice. Two-way ANOVA: treatment (F1,5 = 0.25, P > 0.05); time (F5,25 = 0.86, P > 0.05); treatment × time (F5,25 = 0.33, P > 0.05). Values represent the means ± SEM for 6 mice per group. Figure 9. Phosphorylation of p38 MAPK in the lumbar dorsal spinal cord of ob/ob mice. Lumbar dorsal spinal cord of lean and ob/ob mice at 12 weeks of age were collected 45 min after the i.t. administration of either vehicle, Ang (1-7) (3 pmol) alone or in combination with A779 (1 nmol). Top: representative western blots of total- and phospho-

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p38 MAPK. Bottom: quantitative analysis of the western blots showing the fold changes in phospho-p38 MAPK relative to total-p38 MAPK set as 1.0 in the vehicle-injected lean mice. Two-way ANOVA: group (F2,30 = 5.26, P < 0.05), treatment (F1,30 = 14.40, P < 0.01), group ×treatment (F2,30 = 3.57, P < 0.05). One-way ANOVA: F5,30 = 6.40, P < 0.01. Values represent the means ± SEM for groups of 6 mice. **P < 0.01 compared with vehicle-injected lean mice, ##P < 0.01 compared with vehicle-injected ob/ob mice, $P < 0.05 compared with Ang (1-7)-injected ob/ob mice.

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CRediT author statement Ryota Yamagata: Validation, Formal analysis, Writing - Original Draft, Writing Review & Editing. 　 Wataru Nemoto: Conceptualization, Writing - Original Draft, Writing - Review & Editing, Project administration, Funding acquisition. Osamu Nakagawasai: Methodology, Formal analysis, Writing - Review & Editing. Kohei Takahashi: Investigation. Koichi Tan-No: Conceptualization, Writing - Review & Editing, Supervision, Project administration, Funding acquisition.

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