Evidence has continually accumulated to illustrate that sirtuin 1 (SIRT1) is a major factor in multiple animal models of neuropathic pain, encompassing those due to drug-induced peripheral nerves, diabetes-induced neuropathy, and chronic constriction injury. This investigation sought to examine if upregulating SIRT1 expression through the CDK5-Kalirin-7 signaling pathway can reduce pain in type 2 diabetic rats. A rat model simulating peripheral nerve injury as an imitation of type 2 diabetes was integrated into the investigation. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were utilized to evaluate pain-related behavior. Our findings revealed spinal SIRT1 expression is diminished in DNP (diabetic neuropathic pain) rats, and SRT1720 (SIRT1 agonist) can alleviate pain behavior. The expression of Kalirin-7 and P-NR2B was markedly increased, while the expression of t-NR2B had no statistical difference in DNP rats. The acetylation level of CDK5 in the DNP cohort was notably elevated, and after intrathecal injection of SRT1720, Ac-CDK5 in the SRT cohort was markedly diminished in contrast to the DNP cohort, and pain behavior was improved. Roscovitine (CDK5 antagonist) was validated to be significantly decreased in CDK5, p35, Kalirin-7, and p-NR2B protein on STZ 17, 21, and 28 days, and there was no difference in t-NR2B among all cohorts. Meanwhile, the thermal hyperalgesia and mechanical allodynia were markedly diminished in cohort Ros (the cohorts were administered Roscovitine). In vitro, the protein levels of CDK5 and p35 were elevated in BV2 cells, and the expression of Kalirin-7, PSD95, and p-NR2B was increased in co-cultured PC12 cells under high-glucose conditions. All of these in vitro effects were significantly attenuated following treatment with Roscovitine. These findings indicate that SIRT1 serves a crucial function in DNP advancement via CDK5-Kalirin-7 signaling pathway.

BACKGROUND: The number of patients with diabetic neuropathic pain (DNP) continues to increase, but available treatments are limited. This study aimed to examine the influence of reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NOD–like receptor protein 3 (NLRP3)-N-methyl-D-aspartic acid receptor 2B (NR2B) pathway on type 2 DNP. METHODS: Male Sprague-Dawley rats were fed with a high-fat and high-sugar diet for 8 weeks. Then, rats were intraperitoneally injected with streptozotocin (STZ, 35 mg/kg) to induce type 2 diabetes mellitus in rats. Diabetic rats with <85% of their basic levels in mechanical withdrawal threshold and thermal withdrawal latency were classified as DNP rats on day 14 after STZ injection. DNP rats were treated with ROS scavenger N-tert-Butyl-α-phenylnitrone (PBN, 100 mg·kg–1·d–1) or TXNIP small interfering ribonucleic acid (10 μg/d) once daily for 14 days. The level of ROS, protein levels of NLRP3, TXNIP, cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1β (IL-1β), NR2B phosphorylation at Tyr1472 (p-NR2B), total NR2B (t-NR2B), and distribution of NLRP3 in the spinal cord were examined. In vitro experiments, BV2 cells and PC12 cells were individually cultured and cocultured in a high-glucose environment (35 mmol/L D-glucose). The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, and IL-1β in BV2 cells, and p-NR2B, t-NR2B in PC12 cells were detected. The level of ROS was detected by the flow cytometry approach. The protein levels were detected by the Western blot technique. The location of NLRP3 was observed by immunofluorescent staining. The interaction between TXNIP and NLRP3 was detected by coimmunoprecipitation assay. RESULTS: The level of spinal ROS increased in DNP rats. The mechanical allodynia and thermal hyperalgesia of DNP rats were alleviated after systemic administration of PBN. This administration decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1β, and p-NR2B and the coupling of TXNIP to NLRP3 in spinal cords of DNP rats. Furthermore, knockdown of spinal TXNIP alleviated nociceptive hypersensitivity and decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1β, and p-NR2B in DNP rats. The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, IL-1β, the coupling of TXNIP to NLRP3, and the IL-1β secretion increased in BV2 cells, and the protein expression of p-NR2B increased in cocultured PC12 cells in a high-glucose environment. All of these in vitro effects were significantly blocked after treatment of PBN. CONCLUSIONS: Our findings suggest that spinal ROS can contribute to type 2 DNP through TXNIP-NLRP3-NR2B pathway.

Abstract Purpose Liraglutide (LIRA), a Glucagon-like peptide-1 receptor agonist (GLP-1RA), showed potent cardioprotective effects of diabetic cardiomyopathy (DCM) with the mechanism remained incompletely understood. Methods T2DM rats were used as study subjects and randomly divided into four groups: 1) CON group, 2) CON + L group, 3) DM group and 4) DM + L group. All rats received either saline or LIRA 0.2 mg/kg (by i.p injection) per day for 4 weeks. After the model was successfully established, cardiac function was determined by invasive hemodynamic evaluation methods. Immunohistochemistry and western blot were performed to understand the molecular mechanism between cardiac function and LIRA. Cultured H9C2 cells with small interfering RNA (siRNA) of Cav3 under high glucose (HG), western blot was performed to understand the molecular mechanism between Cav3 and RyR2 with LIRA. Results Based on our results, LIRA treatment showed a trend to enhance LVSP (110.76 ± 5.61 mmHg) and ± dp/dtmax (5860.41 ± 200.32 mmHg and 3996.8 ± 179.3 mmHg), decreased LVEDP (7.23 ± 0.58 mmHg). The expression of Cav3, eNOS and RyR2 was significantly decreased in the myocardium in DM group, which increased in DM + L group after LIRA administrated. LIRA improved cardiac systolic and diastolic function, attenuate diabetic cardiomyopathy injury by improving Cav3/eNOS/NO signaling and increasing interaction of Cav3 and ryanodine receptor 2 (RyR2) in diabetic cardiac tissues. Conclusion In summary, we found that Liraglutide ameliorates cardiac dysfunction in rats with type 2 diabetes mellitus via improving Cav3/eNOS/NO signaling and increasing interaction of Cav3 and RyR2.

Abstract Background Liraglutide (LIRA), a Glucagon-like peptide-1 receptor agonist (GLP-1RA), showed potent cardioprotective effects with the mechanism remained incompletely understood. Caveolin-3 (Cav3) is the cardiomyocytes specific caveolae structural protein, decreased in the diabetic heart. Therefore, this study aimed to investigate whether LIRA exerts its effect on cardiac function in rats with type 2 diabetes mellitus (T2DM) via enhance Cav3 expression. Methods T2DM rats were used as study subjects and randomly divided into four groups: 1) CON group, 2) CON+L group, 3) DM group and 4) DM+L group. All rats received either saline or LIRA 0.2 mg/kg (by i.p injection) per day for 4 weeks. After the model was successfully established, cardiac function was determined by invasive hemodynamic evaluation methods. Immunohistochemistry and western blot were performed to understand the molecular mechanism between cardiac function and LIRA. Results Based on our results, DM group displayed higher blood glucose than Con group (20.57±2.75 mol/L vs. 4.34±0.21 mol/L), while blood glucose level in DM+L group was lower than DM group after received LIRA (10.36±1.84 mol/L). LVSP (91.39±4.98 mmHg), LV +dp/dtmax (4040.74±197.72 mmHg/s) were significantly reduced in DM group, and diabetic rats also exhibited reduced -dp/dtmax (2926.5±142.3 mmHg/s) and elevated LVEDP (10.87±0.83 mmHg). LIRA treatment showed a trend to enhance LVSP (110.76±5.61 mmHg) and ± dp/dtmax (5860.41±200.32 mmHg and 3996.8±179.3 mmHg), decreased LVEDP (7.23±0.58 mmHg). The expression of Cav3, eNOS and RyR2 was significantly decreased in the myocardium in DM group, which increased in DM+L group after LIRA administrated. Hemodynamic data showed DM rats exhibited impairment of myocardial function, while LIRA improved cardiac systolic and diastolic function, attenuate diabetic cardiomyopathy injury by improving Cav3/eNOS/NO signaling, reducing ROS level in cardiac tissues, and increasing interaction of Cav3 and ryanodine receptor 2 (RyR2). Conclusions Liraglutide ameliorates cardiac dysfunction in rats with type 2 diabetes mellitus via reducing ROS level in cardiac tissues, improving Cav3/eNOS/NO signaling and increasing interaction of Cav3 and RyR2. Keywords Type-2 diabetes Mellitus, liraglutide, caveolin-3, ryanodine receptor2, myocardial dysfunction

OBJECTIVE:Diabetic neuropathic pain (DNP) is one of the common complications in type 2 Diabetes Mellitus (DM) patients. However, molecular mechanisms in underlying diabetic neuropathic pain are still poorly understood. Kalirin-7, a multifunctional Rho GDP/GTP exchange factor, located at the excitatory synapses, was reported to modulate the neuronal cytoskeleton. Therefore, in this study, we explored the effects of Kalirin-7 on type 2 diabetic neuropathic pain and the mechanisms in spinal cord in rats.METHODS:The type 2 diabetic neuropathic pain model was established in rats by feeding them with a high-sugar and high-fat diet for 8 weeks, and then fasting them for 12 hours, followed by a single intraperitoneal injection of STZ. Kalirin-7 was knocked down in the spinal cord by an intrathecal administration of Kalirin-7 siRNA.RESULTS:The levels of Kalirin-7, p-NR2B and PSD-95 as well as the PSD-95-NR2B coupling were significantly increased in the spinal cord of type 2 DM rats. The knockdown of Kalirin-7 expression in the spinal cord by the intrathecal administration of Kalirin-7 siRNA not only reduced the levels of p-NR2B and the PSD-95-NR2B coupling in the spinal cord, but also relieved mechanical allodynia and thermal hyperalgesia in type 2 DM rats.CONCLUSIONS:Our findings suggest that spinally expressed Kalirin-7 likely contributes to type 2 diabetic neuropathic pain through regulating the PSD-95/NR2B interaction-dependent NR2B phosphorylation in the spinal cord.

The mechanisms underlying type-2 diabetic neuropathic pain (DNP) are unclear. This study investigates the coupling of postsynaptic density-95 (PSD-95) to N-methyl-D-aspartate receptor subunit 2B (GluN2B), and the subsequent phosphorylation of GluN2B (Tyr1472-GluN2B) in the spinal cord in a rat model of type-2 DNP. Expression levels of PSD-95, Tyr1472-GluN2B, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its phosphorylated counterpart (Thr286-CaMKII), and α-amino-3-hydroxy-5-methyl-4-soxazole propionic acid receptor subtype 1 (GluR1) and its phosphorylated counterpart (Ser831-GluR1) were significantly increased versus controls in the spinal cord of type-2 DNP rats whereas the expression of total spinal GluN2B did not change. The intrathecal injection of Ro25-6981 (a specific antagonist of GluN2B) or Tat-NR2B9c (a mimetic peptide disrupting the interaction between PSD-95 and GluN2B) induced an antihyperalgesic effect and blocked the increased expression of Tyr1472-GluN2B, CaMKII, GluR1, Thr286-CaMKII, and Ser831-GluR1 in the spinal cords; the increase in spinal cord PSD-95 was not affected. These findings indicate that the PSD-95-GluN2B interaction may increase phosphorylation of GluN2B, and subsequently induce the expression of phosphorylation of CaMKII and GluR1 in the spinal cord of type-2 DNP rats. Targeting the interaction of PSD-95 with GluN2B may provide a new therapeutic strategy for type-2 DNP.

OBJECTIVE The present study determines whether Cav-1 modulates the initiation, development and maintenance of type-2 DNP via the Rac1/NOX2-NR2B signaling pathway. METHODS After regular feeding for three days, these rats were randomly divided into two groups: control group with normal-diet (maintenance feed) (n=8); type-2 DM group (n=8). In the type-2 DM group, the rats were fed with a high-fat and high-sugar diet, and received a single intraperitoneal streptozotocin (STZ) injection (35 mg/kg). At two weeks after STZ injection, these diabetic neuropathic pain (DNP) rats were treated with daidzein (0.4 mg/kg/day) and N-tert-Butyl-α-phenylnitrone (PBN, 100 mg/kg/day) for 14 days. After the type-2 DNP model was successfully established, the rats were assigned into four groups: DNP group, DNP+Da group (DNP rats with Cav-1 specific inhibitor daidzein), DNP+PBN group (DNP rats treated with ROS scavenger PBN), and SC group (solvent control group). Then, the mechanical and thermal hyperalgesia were assayed to evaluate the function of the caveolin 1-Recombinant Human Ras-Related C1/nicotinamide adenosine diphosphate oxidase 2-NR2B gene (Cav-1-Rac1/NOX2-NR2B) signaling pathway. In the mechanism study, the protein expression levels of p-Caveolin-1, Rac1, NOX2, p-NR2B and t-NR2B, the production of ROS, and the distribution of Cav-1 and NOX2 in the spinal cord were observed. RESULTS The present study revealed that p-Cav-1 was persistently upregulated and activated in the spinal cord microglia in type-2 DNP rats. The use of the pharmacological inhibitor of Cav-1 and a ROS scavenger resulted to a significantly relieved mechanical allodynia and thermal hyperalgesia. In addition, it was demonstrated that Cav-1 promoted ROS generation via the activation of Rac1-dependent NADPH oxidase (NOX). CONCLUSION The present data suggests that Cav-1 in the spinal cord modulates type-2 DNP via regulating the Rac1/NOX2-NR2B pathway.