Diabetic Neuropathic Pain Group Research Articles

Glycogen synthesis kinase-3β involves in the analgesic effect of liraglutide on diabetic neuropathic pain

AimsExplore whether Glycogen synthesis kinase-3β (GSK3β) involved in the analgesic effect of liraglutide on diabetic neuropathic pain (DNP). MethodsDNP was induced by streptozocin (STZ) in WT and GSK3β(S9A) mice, which carried a constitutively active form of GSK3β. DNP mice were intracerebroventricularly injected with liraglutide 5 weeks after STZ injection. The behavior of neuropathic pain was evaluated 2 h after drugs administration. The microglial activation and the expression of NOD-like receptor protein 3 (NLRP3) in microglia in cortex were evaluated. The role of GSK3β in the inhibitory effect of liraglutide on the NLRP3 inflammasome was explored in BV2 microglia. ResultsIntracerebroventricular administration of liraglutide significantly relieved neuropathic pain and inhibited the activation of cortical microglia in WT mice with DNP. But the effect of liraglutide disappeared in GSK3β(S9A) mice. In BV2 microglia, GSK3β inhibitor significantly suppressed NLRP3 inflammasome activation. And activating GSK3β through GSK3β(S9A) lentivirus significantly blocked the inhibitory effect of liraglutide on NLRP3 inflammasome in BV2 microglia. Intracerebroventricular administration of liraglutide significantly inhibited the expression of NLRP3 in cortex microglia of DNP group in WT mice but failed in GSK3β(S9A) mice. ConclusionGSK3β involves in the analgesic effect of liraglutide on DNP through NLRP3 inflammasome in microglia.

Effects of Dietary Gingerol-Enriched Ginger Supplementation on Distribution of Colon Function Markers in Rats With Diabetic Neuropathic Pain

ObjectivesInflammation and leaky gut may contribute to the pathogenesis of diabetic neuropathic pain (DNP). Ginger has been used in the treatment of a variety of pain disorders for its anti-inflammatory, antioxidant, and analgesic effects. We previously reported that gingerol-enriched ginger (GEG) supplementation suppressed the DNP-induced mRNA expression levels of claudin-3 (tight junction protein), PINK1 (autophagy marker), DRP1 (mitochondrial fission protein), and GFAP (astrocyte marker) in colons. Thus, this study was to further investigate the effects of GEG supplementation on the distribution of function markers in the colon of DNP rats. MethodsMale Sprague-Dawley rats were assigned to three groups: low-fat diet (Control), high-fat diet (HFD)+ streptozotocin (DNP), and HFD + streptozotocin + 0.75% GEG w/w in diet (DNP + GEG). Colons were collected and preserved at -80 degrees. The localization and quantity of claudin-3, PINK1, DRP1, and GFAP in colons were determined by cryosectioning, immunohistochemistry and fluorescence confocal microscopy. The protein levels were quantified by the average fluorescence intensity in cross-sections of intestinal crypts within pre-determined regions of interest (ROIs), normalized by the background ROI. The comparisons between groups were made using an unpaired t-test. ResultsCompared to the Control group, the DNP group had higher expression levels of claudin-3 (Control, n = 6; DNP, n = 5; P < 0.005), PINK1 (Control, n = 7; DNP, n = 7; P < 0.05), and DRP1 (Control, n = 6; DNP, n = 7; P < 0.01) in colons of animals. GEG supplementation into diets significantly suppressed DNP-induced expression levels of claudin-3 (DNP, n = 7, DNP + GEG, N = 6; P < 0.05), PINK1 (DNP, n = 7, DNP + GEG, n = 5; P < 0.05), and DRP1 (DNP, n = 7, DNP + GEG, n = 6; P < 0.05) in colons of animals. There were no significant differences in PINK1 expression between the Control group and the DNP + GEG group (p > 0.05). Similar to PINK1 finding, we did not observe the difference in DRP1 expression between the Control and the DNP + GEG group. ConclusionsThese findings show that gingerol-enriched ginger reduces the physiological colonic disruption associated with diabetic neuropathy. Funding SourcesMedical Student Summer Research Program, Texas Tech University Health Sciences Center, Lubbock, TX.

Effect of electroacupuncture and pretreatment of electroacupuncture on pain sensitization and expression of P2X7R in spinal dorsal horn in rats with diabetic neuropathic pain

To observe the occurrence time of neuralgia and the expression of purinergic ligand-gated ion channel 7 receptor (P2X7R) in the dorsal horn of the spinal cord after intraperitoneal injection of streptozotocin (STZ) in diabetic rats, and to explore the effect of electroacupuncture (EA) and pretreatment of EA on the heat pain threshold and expression of P2X7R in the spinal dorsal horn in rats with diabetic neuropathic pain (DNP), and to explore the possible mechanism of EA for DNP. PartⅠ: Thirty male SD rats were randomly selected from 64 male SD rats as the control group; the remaining rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model, and 30 rats were successfully modeled as the model group. The control group and the model group were divided into three subgroups respectively at 7, 14 and 21 days, with 10 rats in each subgroup. Body mass, fasting blood glucose (FBG) and thermal pain threshold were recorded at 7, 14 and 21 days after injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot. PartⅡ: Eight SD rats were randomly selected from 35 male SD rats as the blank group, and the remaining 27 rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model. The 24 rats with successful diabetes model were randomly divided into a DNP group, an EA group and a pre-EA group, 8 rats in each group. Fifteen to 21 days after STZ injection, the EA group received EA at "Zusanli" (ST 36) and "Kunlun" (BL 60), continuous wave, frequency of 2 Hz, 30 min each time, once a day; the intervention method in the pre-EA group was the same as that in the EA group. The intervention time was 8 to 14 days after STZ injection. The body mass, FBG and thermal pain threshold were recorded before STZ injection and 7, 14 and 21 days after STZ injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot 21 days after injection. PartⅠ: Compared with the control group, in the model group, the body mass was decreased and FBG was increased 7, 14 and 21 days after STZ injection (P<0.01), and the thermal pain threshold was decreased 14 and 21 days after STZ injection (P<0.05), and the expression of P2X7R in spinal dorsal horn was increased 7, 14 and 21 days after STZ injection (P<0.05, P<0.01). PartⅡ: Compared with the blank group, in the DNP group, the body mass was decreased and fasting blood glucose were increased 7, 14 and 21 days after STZ injection (P<0.01). Compared with the DNP group, in the pre-EA group, the heat pain threshold was increased 14 and 21 days after STZ injection (P<0.05), while in the EA group, the heat pain threshold was increased 21 days after STZ injection (P<0.01), and the expression of P2X7R in the dorsal horn in the EA group and the pre-EA group was decreased (P<0.01). The diabetic neuropathic pain is observed 14 days after STZ injection. EA could not only treat but also prevent the occurrence of DNP, and its mechanism may be related to down-regulation of P2X7R expression in the dorsal horn of the spinal cord.

Lower / upper extremity F-wave ratio for detecting early diabetic neuropathy.

Results of conventional nerve conduction studies may be within normal limits in early diabetic neuropathy. Previous studies demonstrated that F-wave latency should be used to detect this early neuropathic process. The aim of this study is to evaluate the sensitivity of lower/upper extremity F latency ratios in detecting the early neuropathy in patients with diabetic neuropathic pain. 44 patients with diabetic neuropathic pain (DNP) and 44 control subjects whose both conventional nerve conduction studies and F-wave latencies were within normal limits were included to the study. We compared the nerve conduction parameters and lower/upper extremity (tibial/ulnar) F latency ratios of the groups. Tibial F latency was significantly prolonged and tibial/ulnar F latency ratio was significantly higher in DNP group. Our results support that F-waves are useful for detecting early diabetic neuropathy and suggest that comparison with a control group will demonstrate a difference even when the individuals' F-wave latencies are within the normal limits. The difference was significant for tibial but not for ulnar F latency values supporting the length dependent involvement. The tibial/ ulnar F-wave latency ratio was significantly higher in the DNP group, suggesting that it might also be useful to detect early neuropathy and to demonstrate that the underlying process was predominant in lower extremity. Further studies may provide additional information about the utility of this ratio for detecting early neuropathy even when F-wave latencies are within normal limits.

The P2X7 Receptor Is Involved in Diabetic Neuropathic Pain Hypersensitivity Mediated by TRPV1 in the Rat Dorsal Root Ganglion.

The purinergic 2X7 (P2X7) receptor expressed in satellite glial cells (SGCs) is involved in the inflammatory response, and transient receptor potential vanilloid 1 (TRPV1) participates in the process of neurogenic inflammation, such as that in diabetic neuropathic pain (DNP) and peripheral neuralgia. The main purpose of this study was to explore the role of the P2X7 receptor in DNP hypersensitivity mediated by TRPV1 in the rat and its possible mechanism. A rat model of type 2 diabetes mellitus-related neuropathic pain (NPP) named the DNP rat model was established in this study. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of DNP rats were increased after intrathecal injection of the P2X7 receptor antagonist A438079, and the mRNA and protein levels of TRPV1 in the dorsal root ganglion (DRG) were decreased in DNP rats treated with A438079 compared to untreated DNP rats; in addition, A438079 also decreased the phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) in the DNP group. Based on these results, the P2X7 receptor might be involved in DNP mediated by TRPV1.

Small Fiber Functionality in Patients with Diabetic Neuropathic Pain.

Diabetic neuropathic pain is associated with small fiber neuropathy. We aimed to assess the functionality of small fibers in patients with diabetes by using a practical method. Patients with impaired glucose tolerance (IGT), diabetic neuropathic pain (DNP), type II diabetes mellitus without neuropathic pain, and healthy control were included. Axon-reflex flare responses were induced by the intradermal application of capsaicin and histamine at the distal leg. The associated flare characteristics (flare areas and flare intensities) were recorded by using Laser Speckle Contrast Analysis (LASCA). The pain and itch responses were rated while performing LASCA. To verify the structural properties of the small fibers, proximal and distal skin biopsies were performed. DN4, MNSI, NRS, evoked-burning pain scores, and HbA1c levels were the highest in the DNP group. Compatible with length-dependent neuropathy, the distal skin PGP9.5-positive intraepidermal nerve fiber densities (IENFDs) were the lowest, whereas TRPV1-positive IENFDs were the highest in patients with DNP. The distal leg LASCA data showed hypo-functionality in both patients with IGT and DNP and association with disease severity. There is an unmet need to practically assess the functionality of small fibers in patients with pain. In this study, a practical and objective method that does not need special expertise for the measurement of the functional properties of small fibers by using axon-flare responses is presented. The LASCA method could potentially facilitate a practical, quick (within 5 minutes), and very early diagnosis of small fiber hypo-functionality in both patients with IGT and DNP.

Decreased amino acids in the brain might contribute to the progression of diabetic neuropathic pain

AimsThe pathophysiological alteration of diabetic neuropathic pain (DNP) in brain is unclear. Here we aimed to explore the metabolomic characteristics of brain in rats over the progression of DNP through metabolomic analysis. MethodsAdult rats were randomly divided into control group and DNP group. Body weight, blood glucose and behavioral assessment of neuropathic pain were measured every week after streptozotocin (STZ) injection. Finally, the brains of 2 rats from control group and 6 rats from DNP group were removed every 4 weeks after STZ injection for metabolomics analysis. ResultsAfter 4 weeks of STZ-injection, the rats with diabetes developed DNP, which was characterized as mechanical allodynia and thermal nociception. As for metabolomic analysis, differentially expressed metabolites (DE metabolites) showed a dynamic alteration over the development of DNP and affected several KEGG pathways associated with amino acid metabolism. Furthermore, the expression of l-Threonine, l-Methionine, d-Proline, l-Lysine and N-Acetyl-l-alanine were significantly decreased at all time points of DNP group. The amino acids which were precursor of analgesic neurotransmitters were downregulated over the progression of DNP, including l-tryptophan, l-histidine and l-tyrosine. ConclusionsThe impairment of amino acid metabolism in brain might contribute to the progression of DNP through decreasing analgesic neurotransmitters.

Kynurenine, Tetrahydrobiopterin, and Cytokine Inflammatory Biomarkers in Individuals Affected by Diabetic Neuropathic Pain.

Neuropathic pain is a common complication of diabetes with high morbidity and poor treatment outcomes. Accumulating evidence suggests the immune system is involved in the development of diabetic neuropathy, whilst neuro-immune interactions involving the kynurenine (KYN) and tetrahydrobiopterin (BH4) pathways have been linked to neuropathic pain pre-clinically and in several chronic pain conditions. Here, using a multiplex assay, we quantified serum levels of 14 cytokines in 21 participants with type 1 diabetes mellitus, 13 of which were classified as having neuropathic pain. In addition, using high performance liquid chromatography and gas chromatography-mass spectrometry, all major KYN and BH4 pathway metabolites were quantified in serum from the same cohort. Our results show increases in GM-CSF and IL-8, suggesting immune cell involvement. We demonstrated increases in two inflammatory biomarkers: neopterin and the KYN/TRP ratio, a marker of indoleamine 2,3-dioxygenase activity. Moreover, the KYN/TRP ratio positively correlated with pain intensity. Total kynurenine aminotransferase activity was also higher in the diabetic neuropathic pain group, indicating there may be increased production of the KYN metabolite, xanthurenic acid. Overall, this study supports the idea that inflammatory activation of the KYN and BH4 pathways occurs due to elevated inflammatory cytokines, which might be involved in the pathogenesis of neuropathic pain in type 1 diabetes mellitus. Further studies should be carried out to investigate the role of KYN and BH4 pathways, which could strengthen the case for therapeutically targeting them in neuropathic pain conditions.

Changes in endoplasmic reticulum stress in spinal cord of rats with diabetic neuropathic pain

Objective To evaluate the changes in the endoplasmic reticulum stress in the spinal cord of rats with diabetic neuropathic pain (DNP). Methods Clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 120-160 g, were fed a high-fat and high-glucose diet for 8 weeks, then diabetes mellitus was induced by intraperitoneal streptozotocin 35 mg/kg and confirmed by blood glucose level >16.7 mmol/L 3 days later.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at day 14 after injection.The establishment of DNP model was considered successful when MWT and TWL were lower than 85% of the baseline value.Fifteen rats in which the DNP model was successfully established served as DNP group, 15 rats in which the DNP model was not successfully established served as non-NDNP group (NDNP), and another 15 normal rats were selected and served as control group (group C). The MWT and TWL were measured at 3, 7 and 14 days after successful establishment of the model.The rats were then sacrificed, and the lumbar enlargement segments (L4-6) of the spinal cord were harvested to detect the expression of inositol-requiring enzyme-1α, phosphorylated JNK (p-JNK) and Beclin1 by Western blot. Results Compared with C and NDNP groups, the MWT was significantly decreased and the TWL was shortened at 3, 7 and 14 days after successful establishment of the model, and the expression of inositol-requiring enzyme-1α, p-JNK and Beclin1 was up-regulated in DNP group (P<0.05). Conclusion The mechanism of development and maintenance of DNP may be related to aggravated endoplasmic reticulum stress in the spinal cord and to autophagy induced by up-regulated expression of p-JNK in rats. Key words: Diabetic neuropathies; Endoplasmic reticulum stress; Spinal cord

Role of spinal cord TRAF6/NF-κB signaling pathway in development of diabetic neuropathic pain in rats

Objective To evaluate the role of spinal cord tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa B(NF-κB)signaling pathway in the development of diabetic neuropathic pain(DNP)in rats. Methods Clean-grade healthy adult male Sprague-Dawley rats, aged 2 months, weighing 180-230 g, in which IT catheters were implanted, were used in this study.Streptozotocin 60 mg/kg was intraperitoneally injected after IT catheterization to establish the model of DNP.Twelve DNP rats were divided into 2 groups (n=6 each) by a random number table method: DNP group and DNP plus TRAF6 inhibitor group (group DTR). Another 6 age-matched Sprague-Dawley rats were used as normal control group(group NC). The rats in group DC and group DTR received IT injection of dimethyl sulfoxide 10 μl and TRAF6 inhibition 10 μg, respectively, once a day for 7 consecutive days starting from day 21 after establishing the model.The mechanical paw withdrawal threshold (MWT) was determined before establishing the model(T1), on 7, 14 and 21 days after establishing the model(T2-4), and on 1, 4 and 7 days after IT injection (T5-7). The rats were sacrificed after the last MWT measurement, and the L3-5 segments of the spinal cord were removed for determination of the expression of TRAF6 and NF-κB p65 by Western blot. Results Compared with group NC, the MWT at T3-7 in group DC and at T3-6 in group DTR was significantly decreased, and the expression of spinal TRAF6 and NF-κB p65 was up-regulated in DC and DTR groups (P<0.05). Compared with group DC, the MWT was significantly increased at T6-7, and the expression of spinal TRAF6 and NF-κB p65 was down-regulated in group DTR (P<0.05). Conclusion Spinal cord TRAF6/NF-κB signaling pathway is involved in the development of DNP in rats. Key words: Spinal cord; Tumor necrosis factor receptor-associated peptides and proteins; NF-kappa B; Diabetes mellitus; Neuralgia

Caveolin-1 in Spinal Cord Modulates Type-2 Diabetic Neuropathic Pain Through the Rac1/NOX2/NR2B Signaling Pathway

The present study determines whether Cav-1 modulates the initiation, development and maintenance of type-2 DNP via the Rac1/NOX2-NR2B signaling pathway.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.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).The present data suggests that Cav-1 in the spinal cord modulates type-2 DNP via regulating the Rac1/NOX2-NR2B pathway.

Effect of gbpapentin on the expression of Nrf2 in dorsal root ganglion neurons of diabetic neuropathic pain rats

Objective To investigate the effect of systemic administration of gabapentin on the expression of Nrf2 in dorsal root ganglion neurons in a rat model of diabetic neuropathic pain (DNP). Methods 60 male SD rats weighing 200-220 g were randomly divided into 4 groups (n=15 each): control group (group C), DNP group, saline group (SC+ DNP group) and gabapentin group (GBP+ DNP group). Diabetes was induced with streptozocin (70 mg/kg) subcutaneously. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at 1d before and 3, 7, 10, 14, 21, and 28 d after streptozocin injection, and 21 and 28 d after administration of gabapentin (50 mg·kg-1·d-1, once) intraperitoneally. Saline and gabapentin (50 mg·kg-1·d-1) were given intraperitoneally once a day for 7 d starting from 15 d after streptozocin injection in saline and gabapentin groups respectively. The expression of Nrf2 was determined at 21 d after streptozocin injection by immune-histochemistry and Western blot. Results Compared with group C, PWMT(3.95±0.57) was significantly decreased, PWTL(6.3±0.81) was significantly shorten, Nrf2 in expression was significantly decreased in DNP, SC+ DNP and group (P 0.05). Conclusion Systemic administration of gabapentin attenuates diabetic neuropathic pain by enhancing the expression of Nrf2 in DRG neurons. (Chin J Endocrinol Metab, 2018, 34: 951-954) Key words: Gabapentin; Diabetic neuropathic pain; Dorsal root ganglion; Nrf2

Neuroprotective effect of tanshinone IIA against neuropathic pain in diabetic rats through the Nrf2/ARE and NF-κB signaling pathways

The study aims to investigate whether tanshinone (TSN) IIA affects diabetic neuropathic pain (DNP) via the Nrf2/AR and NF-κB signaling pathways. Rats were randomly assigned into DNP group, TSN group (injected with TSN IIA), TSN + DRB group (injected with TSN IIA and 15 mg/kg Nrf2/ARE inhibitors), TSN + PDTC group (injected with TSN IIA and 60 mg/kg NF-κB inhibitors) or control group. The first four groups were successfully established as DNP models after injection of streptozotocin. The blood glucose level, mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), nerve conduction velocity (NCV) and antioxidase level were detected. Transmission electron microscopy and toluidine blue staining were utilized to observe the sciatic nerve. RT-qPCR and western blot were used to measure expression levels of Nrf2/ARE and NF-κB signaling pathway-related genes. Blood glucose, malondialdehyde (MDA) and erythrocyte glutathione peroxidase (GSH-Px) levels as well as expression of Keap1 and NF-κB were increased in the TSN, TSN + DRB and TSN + PDTC groups compared to control group. Furthermore, the MWT, TWL, NVC, and superoxide dismutase (SOD) levels and expression of Nrf2, heme oxygenase 1 (HO-1) and inhibitory kappa B (IκB) decreased in the treatment groups. The TSN + DRB and TSN + PDTC groups showed similar trend when compared with the TSN group, while the opposite trend was observed in the TSN group when compared with the DNP group. Our study demonstrates that TSN IIA alleviates neuropathic pain by activating the Nrf2/ARE signaling pathway and inhibiting the NF-κB signaling pathway in diabetic rats.

The relationship between autophagy activation in spinal cord and type 2 diabetic neuropathic pain in rats

To investigate the relationship between autophagy function in spinal cord and type 2 diabetic neuropathic pain in rats. Forty-two male Sprague-Dawley rats were fed with a high-sugar, high-fat diet for 8 weeks to induce the insulin resistance, and then received a single intraperitoneal streptozocin (STZ) injection to establish type 2 diabetes rat model. Two weeks after STZ injection, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats were detected, the rats with MWT and TWL decreasing to below 80% compared to baseline were chosen as type 2 diabetic neuropathic pain rats (group DNP, n=24), the rest of the rats were chosen as type 2 diabetic non-neuropathic pain rats (group DA, n=18). And another 18 normal rats randomly selected from the total were classified as control group (group C) and fed with common forage for 8 weeks. The MWT and TWL were measured again on the 3rd, 7th and 14th day after determining the grouping of DA and DNP, and then, the lumbar segments 4~6 of the spinal cord were removed from the executed rats for determination of the expressions of microtubule-associated protein light chain 3 (LC3)、Beclin-1and P62 by Western blot. The co-expressions of P62 with GFAP or OX-42 or NeuN in spinal dorsal horn were detected in another 6 lumbar segments of diabetic neuropathic pain (DNP) rats on the 7th day by immunofluorescence double dye method. Compared with group C, the insulin level was increased and ISI decreased in SD rats fed with high-sugar, high-fat diet, that meant the rats in insulin-resistance. After STZ injection, blood glucose rose to the standard of type 2 diabetes mellitus, i.e. ≥ 16.7 mmol/L. Compared with group C and group DA, MWT was significantly decreased, TWL shortened and the expression of LC3-Ⅱ and Beclin-1 in the spinal dorsal horn up-regulated, P62 expression down-regulated on the 3rd, 7th and 14th day in group DNP (P<0.05). P62 was mainly localized in spinal dorsal horn and coexisted with neurons, and spots of P62 immunoreactivity could be detected in a few microglia but not observed in astrocyte. The changes in expression of LC3-Ⅱ、Beclin-1 and P62 in spinal cord of type 2 diabetes neuropathic pain rats means autophagy activation of spinal, up-regulated autophagy of neurons in spinal dorsal horn mainly involves in the formation and development of type 2 diabetic neuropathic pain in rats.

Changes in expression of H3K9Ac and MeCP2 in spinal cord and dorsal root ganglion of rats with diabetic neuropathic pain

Objective To evaluate the changes in the expression of acetylated histone 3 lysine 9(H3K9Ac)and methyl-CpG-binding protein 2(MeCP2)in the spinal cord and dorsal root ganglion(DRG)of rats with diabetic neuropathic pain(DNP). Methods Pathogen-free male Sprague-Dawley rats, weighing 120-160 g, were fed a high-fat and high-sucrose diet for 8 weeks, then streptozocin 35 mg/kg was intraperitoneally injected, and type 2 diabetes mellitus was confirmed by blood glucose level≥16.7 mmol/L 3 days later.DNP model was considered successful when the decrease in pain threshold <85% of the baseline value on 14 days after injection, otherwise it was considered as non-DNP(NDNP). Eighteen rats with DNP and NDNP served as DNP and NDNP groups, respectively, and another 18 normal rats served as control group(group C). At 3, 7 and 14 days after successful establishment of the model, the mechanical paw withdrawal threshold and thermal paw withdrawal latency were measured in group DNP and at the corresponding time points in C and NDNP groups, and then rats were sacrificed, and the lumbar segment of the spinal cord and DRGs were removed for determination of the expression of H3K9Ac and MeCP2 by Western blot. Results Compared with C and NDNP groups, mechanical paw withdrawal threshold was significantly decreased, thermal paw withdrawal latency was shortened, the expression of H3K9Ac in the spinal cord and DRGs was up-regulated, and the expression of MeCP2 in the spinal cord and DRGs was down-regulated at 3, 7 and 14 days after successful establishment of the model in group DNP(P<0.05). Conclusion The maintenance mechanism of DNP may be related to up-regulated expression of H3K9Ac and down-regulated expression of MeCP2 in rats with DNP. Key words: Diabetes Mellitus; Neuralgia; Histones; Methyl-CpG-binding protein 2; Spinal cord; Ganglia, spinal

Role of microRNA9 in spinal dorsal horn neurons in over-expression of calcium homeostasis modulator 1 in rats with diabetic neuropathic pain

Objective To evaluate the role of microRNA9 (miR-9) in spinal dorsal horn neurons in over-expression of calcium homeostasis modulator 1 (CALHM1) in rats with diabetic neuropathic pain (DNP). Methods Ninety-three healthy male Sprague-Dawley rats, aged 2 months, weighing 180-200 g, were used in the study.Diabetes mellitus was induced by intraperitoneal 1% streptozocin (STZ) 60 mg/kg.The experiment was performed in two parts.Experiment Ⅰ The rats were divided into control group (group C, n=10) and DNP group (n=83) using a random number table.The mechanical paw withdrawal threshold (MWT) was measured before STZ injection and at 1, 2, 3, 4, 5 and 6 weeks after STZ injection.The expression of miR-9 in the spinal dorsal horn was determined using the in situ hybridization at 6 weeks after STZ injection.Experiment Ⅱ The rats with DNP were selected at 6 weeks after STZ injection, and the spinal dorsal horn neurons were isolated and cultured.The neurons were seeded in culture plates at the density of 5×106 cells/ml (2 ml/well) and divided into 2 groups (n=18 each) using a random number table: control group (group C) and miR-9 antisense oligonucleotide group (group ASO). The neurons were cultured in normal culture atmosphere in group C. In group ASO, the single nucleotide sequence of miR-9 antisense oligonucleotide sequence 5′-UUCUCCGAACGUGUCACGUTT-3′ was added with the final concentration of 100 pmol/L.The expression of miR-9 and CALHM1 mRNA was detected using quantitative real-time polymerase chain reaction at 48 h of incubation.The expression of CALHM1 was detected by Western blot at 72 h of incubation. Results Experiment Ⅰ Compared with group C, the MWT was significantly decreased at 2-6 weeks after STZ injection, and the expression of miR-9 in the spinal dorsal horn was up-regulated in group DNP (P<0.05). Experiment Ⅱ Compared with group C, the expression of miR-9 and CALHM1 protein and mRNA in spinal dorsal horn neurons was significantly down-regulated in group ASO (P<0.05). Conclusion miR-9 in spinal dorsal horn neurons probably mediates the over-expression of CALHM1 in rats with DNP. Key words: microRNAs; Calcium channels; Spinal cord; Neurons; Diabetes mellitus; Neuralgia

Influences of intraperitoneal injection of dexmedetomidine on activation of spinal glial cells in diabetic neuropathic pain rats

Objective Exploring the influences of intraperitoneal injection of dexmedetomidine(Dex) on activation of spinal glial cells and expression of inflammatory factors in rats with diabetic neuropathic pain(DNP). Methods Thirty two health male SD rats, weight 180-200 g, were divided into four groups (n=8) according the random number table method. The four groups included the control group(group C), diabetic neuropathic pain group(group DNP), group Dex and α2 receptor antagonist group(group YOH). Streptozotocin(STZ) was used to establish the DNP model in rats. Starting from 28 d after STZ injection, Dex group was given intraperitoneal injection of Dex(50 μg/kg) for consecutive 7 d, while group YOH was given intraperitoneal injection of Yohimbine(0.1 mg/kg) 30 min before intraperitoneal injection of Dex(50 μg/kg) for consecutive 7 d. Mechanical withdrawal threshold (MWT) was determined in each group of rats 29-35 d after STZ injection. Rats were sacrificed after MWT detection on 35 d. Immunofluorescence double-staining was used to detect activation of microglia and astrocytes in rat spinal cord of each group. ELISA was used to measure TNF-α and IL-1 expression in rat spinal cord of each group. Results Compared with group C, rat body weights were decreased while blood glucose values were increased in group DNP, group Dex and group YOH(P 0.05). Compared with group C, MWT at all time points decreased in group DNP and group YOH(P 0.05). Conclusions Dex can relieve hyperalgesia in rats with diabetic neuropathy pain. The underlying mechanism may be related to inhibition of microglia activation and reduction of the inflammatory reaction in spinal cord. Key words: Dexmedetomidine; Diabetic neuropathic pain; Microglia; Astrocyte; Inflammatory reaction

Effects of Duloxetine on the Toll-Like Receptor 4 Signaling Pathway in Spinal Dorsal Horn in a Rat Model of Diabetic Neuropathic Pain.

Although duloxetine has been approved for clinical therapy for diabetic peripheral neuropathic pain, the exact mechanism underlying the anti-allodynic effects in rat models of diabetes mellitus remains obscure. We attempted to identify whether duloxetine exerts anti-allodynic effects via inhibition of the TLR4-Myd88-dependent pathway in diabetic neuropathic pain (DNP) rats. An animal model of type 1 diabetic neuropathic pain was induced by intraperitoneal streptozotocin in 108 rats randomized into four groups: control, DNP, solvent control + DNP, and DNP + duloxetine. The DNP model establishment was validated, providing the MWT and TWL measurements were less than 80% of the baseline value on d14 after streptozotocin administration. The expressions of TLR4, Myd88, and NF-κB in the spinal dorsal horn were determined 21 days after streptozotocin injection by immunohistochemical assay and Western blot. The results revealed that MWT and TWL in DNP, SC + DNP, and DLX + DNP groups were significantly decreased 14 days after STZ administration vs control (P < 0.05), while the pain thresholds in the DLX + DNP group were partially reversed. The expressions of TLR4, Myd88, and NF-κB in groups C, DNP, and SC + DNP were significantly increased, whereas duloxetine administration significantly downregulated the expressions of TLR4, Myd88, and NF-κB (P < 0.05). Our findings indicated that duloxetine mitigated mechanical and thermal withdrawal thresholds in STZ-injected rats and rescued the overexpression of the TLR4-Myd88-dependent pathway in the spinal dorsal horn in these rats. Whether these changes directly contributed to the reduction of thermal and mechanical withdrawal behavior needs to be further explored.

The mechanism of RvD1 alleviates type 2 diabetic neuropathic pain by influencing microglia polarization in rats

To study the relationship between microglia polarization in the spinal dorsal horn and type 2 diabetic neuropathic pain (DNP). And explore the mechanism of RvD1 alleviating type 2 diabetic neuropathic pain. Type 2 diabetes mellitus (T2DM) rats came from the male SD rats which were fed by high-fat and high-sucrose diet and given intraperitoneal streptozotocin(STZ), then detected fa sting blood glucose level, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), which was to prepare the type 2 DNP model rats. And they were randomly divided into 3 groups:type 2 DNP group (group D), type 2 DNP and RvD1 group (group R), type 2 DNP and solvent control group (group S), 36 rats in each group. After being given STZ 14 days, the rats of group D, R, S were placed a catheter in subarachnoid cavity. Three days later, the RvD1 10μl (10 ng/μl) and 100% ethanol 10μl were injected into subarach-noid cavity through the catheter once a day for 14 consecutive days. Another 36 normal rats were served as normal control group (group N) and were fed with common forage. MWT and TWL were measured at 1, 3, 7, 14 days after Subarachnoid injection, then the nine rats'spinal cord of the lumbar segment 4~6 were removed to detect the expression of inducible nitric oxide synthase(iNOS) and arginase 1(Arg1) by Western blot, the marker of microglia M1 and M2 polarization. Compared with group N, MWT was decreased significantly and TWL was shortened, the expression of Arg1 was down-regulated and the expression of iNOS was up-regulated in spinal dorsal horn at the 1, 3, 7, 14 days in groups D and S (P < 0.05). Compared with group D, MWT was significantly increased and TWL was prolonged, the expression of Arg1 was up-regulated and the expression of iNOS was down-regulated in spinal dorsal horn at the 7, 14 days in group R (P < 0.05). There was no significant difference in the MWT, TWL and expression of Arg1 and iNOS between D and S groups. RvD1 promotes mi-croglia toward M2 polarization and alleviates type 2 diabetic neuropathic pain in rats.

Effects of LncRNA BC168687 siRNA on Diabetic Neuropathic Pain Mediated by P2X7 Receptor on SGCs in DRG of Rats.

Diabetic neuropathic pain (DNP), one of the early symptoms of diabetic neuropathy, relates to metabolic disorders induced by high blood glucose, neurotrophic vascular ischemia and hypoxia, and autoimmune factors. This study was aimed at exploring the effects of long noncoding RNA (lncRNA) BC168687 siRNA on DNP mediated by P2X7 receptor on SGCs in DRG of rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats, the expression levels of P2X7 mRNA and protein in the DRG, and nitric oxide (NO) in the serum were, respectively, detected in our study. Our experimental results showed that the level of BC168687 mRNA in DNP group was markedly higher than that of control group; the MWT and TWL of DNP + BC168687 si group were significantly increased, and the expression levels of P2X7 in DRG and the concentrations of NO in serum of DNP + BC168687 si group were decreased compared to those of the DNP group. In conclusion, lncRNA BC168687 may participate in the pathogenesis of DNP mediated by P2X7 receptor, which will provide a novel way for the study of the pathogenesis of diabetes mellitus complicated with neuropathic pain and its prevention and treatment.