Inhibition of phosphorylated calcium/calmodulin-dependent protein kinase IIα relieves streptozotocin-induced diabetic neuropathic pain through regulation of P2X3 receptor in dorsal root ganglia.

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7.

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate), a nonspecific P2X1-7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4-L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.

Electroacupuncture may alleviate diabetic neuropathic pain by inhibiting the microglia P2X4R and neuroinflammation.

Diabetic neuropathic pain (DNP) is a common and destructive complication of diabetes mellitus. The discovery of effective therapeutic methods for DNP is vitally imperative because of the lack of effective treatments. Although 2 Hz electroacupuncture (EA) was a successful approach for relieving DNP, the mechanism underlying the effect of EA on DNP is still poorly understood. Here, we established a rat model of DNP that was induced by streptozotocin (STZ) injection. P2X4R was upregulated in the spinal cord after STZ-injection. The upregulation of P2X4R was mainly expressed on activated microglia. Intrathecal injection of a P2X4R antagonist or microglia inhibitor attenuated STZ-induced nociceptive thermal hyperalgesia and reduced the overexpression of brain-derived neurotrophic factor (BDNF), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the spinal cord. We also assessed the effects of EA treatment on the pain hypersensitivities of DNP rats, and further investigated the possible mechanism underlying the analgesic effect of EA. EA relieved the hyperalgesia of DNP. In terms of mechanism, EA reduced the upregulation of P2X4R on activated microglia and decreased BDNF, IL-1ß and TNF-α in the spinal cord. Mechanistic research of EA's analgesic impact would be beneficial in ensuring its prospective therapeutic effect on DNP as well as in extending EA's applicability.

Electroacupuncture Alleviates Diabetic Neuropathic Pain and Downregulates p-PKC and TRPV1 in Dorsal Root Ganglions and Spinal Cord Dorsal Horn.

Diabetic neuropathic pain (DNP) is a common complication of diabetes. Streptozotocin (STZ)-induced changes of protein in dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) are critical for DNP genesis. However, which proteins change remains elusive. Here, the DNP model was established by a single intraperitoneal injection of STZ, accompanied by increased fasting blood glucose (FBG), decreased body weight (BW), and decreased paw withdrawal latency (PWL). Proteins change in L4-L6 DRGs and SCDH of rats were detected. Western blot and immunofluorescence results showed that expression levels of phosphorylated protein kinase C (p-PKC), transient receptor potential vanilloid-1 (TRPV1), Substance P (SP) and calcitonin gene-related peptide (CGRP) in the DRG and the SCDH of rats were increased after STZ injection. A preliminary study from our previous study showed that 2 Hz electroacupuncture (EA) effectively alleviates DNP. However, the analgesic mechanism of EA needs further elucidation. Here, EA at the bilateral Zusanli (ST36) and KunLun (BL60) acupoints was applied for one week, and to investigate the effect on DNP. EA reversed thermal hyperalgesia in DNP rats and downregulated the expression of p-PKC, TRPV1, SP, and CGRP in DRG and SCDH.

Unexpected Dynamic Binding May Rescue the Binding Affinity of Rivaroxaban in a Mutant of Coagulation Factor X.

A novel coagulation factor X (FX) Tyr319Cys mutation (Y99C as chymotrypsin numbering) was identified in a patient with severe bleeding. Unlike the earlier reported Y99A mutant, this mutant can bind and cleave its specific chromogenetic substrate at a normal level, suggesting an intact binding pocket. Here, using molecular dynamics simulations and MM-PBSA calculations on a FX-rivaroxaban (RIV) complex, we confirmed a much stronger binding of RIV in Y99C than in Y99A on a molecular level, which is actually the average result of multiple binding poses in dynamics. Detailed structural analyses also indicated the moderate flexibility of the 99-loop and the importance of the flexible side chain of Trp215 in the different binding poses. This case again emphasizes that binding of ligands may not only be a dynamic process but also a dynamic state, which is often neglected in drug design and screening based on static X-ray structures. In addition, the computational results somewhat confirmed our hypothesis on the activated Tyr319Cys FX (Y99C FXa) with an impaired procoagulant function to bind inhibitors of FXa and to be developed into a potential reversal agent for novel oral anticoagulants (NOAC).

E3 ubiquitin-protein ligase 2 inhibits cell proliferation, migration, and invasion of non-small cell lung cancer through ubiquitination of Notch1.

This study aimed to explore the role of MIB2 in non-small cell lung cancer (NSCLC) and the underlying mechanism. Quantitative real-time PCR (QRT-PCR) and western blot were first performed to detect MIB2 expression in tumor tissues obtained from NSCLC patients (n = 30) and NSCLC cells, respectively. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and transwell assays were then used to examine the effect of MIB2 on the proliferation, migration and invasion of NSCLC cells. Western blot was further performed to examine the effect of Mind bomb 2 (MIB2), an E3 ligase on Notch1 protein and its ubiquitination. MIB2 was significantly down-regulated in NSCLC tissues and cells, both in mRNA and protein level. MIB2 also note worthily inhibited the proliferation, migration, and invasion of NSCLC cells. Furthermore, MIB2 only down-regulated Notch1 protein level, while facilitated the ubiquitination of Notch1. Additionally, Notch1 significantly relieved the repressed proliferation, migration and invasion of NSCLC cells induced by MIB2. Conclusively, MIB2 inhibited cell proliferation, migration and invasion via inducing Notch1 ubiquitination and degradation in NSCLC.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

An unexpected dynamic binding mode between coagulation factor X and Rivaroxaban reveals importance of flexibility in drug binding.

Rivaroxaban (RIV) is a direct oral anticoagulant (DOAC) targeting activated coagulation factor X (FXa). An earlier study reported the F174A mutant of FXa resistant to a RIV-like inhibitor, Apixaban. In current study, the detailed molecular mechanism of the resistance has been explored by molecular dynamics simulations on the impaired interactions between RIV and FXa in the damaged S4 pocket of F174A mutant. Besides, an unexpected relative stable binding mode of S1'S1 was revealed, which required dynamic motions of Gln192 and Gln61 to allow the morpholinone moiety of RIV to shift into the S1' pocket and form strong interactions. These dynamic motions of RIV and critical residues might be important in drug design for direct inhibitors of coagulation factors.

Enhancement of T Follicular Helper Cell-Mediated Humoral Immunity Reponses During Development of Experimental Autoimmune Myasthenia Gravis.

Myasthenia gravis (MG) is a prototypical antibody-mediated neurological autoimmune disease with the involvement of humoral immune responses in its pathogenesis. T follicular helper (Tfh) cells have been implicated in many autoimmune diseases. However, whether and how Tfh cells are involved in MG remain unclear. Here, we established and studied a widely-used and approved animal model of human MG, the rat model with acetylcholine receptor alpha (AChRα) subunit (R-AChR97-116)-induced experimental autoimmune myasthenia gravis (EAMG). This model presented mild body-weight loss 10 days after the first immunization (representing the early stage of disease) and more obvious clinical manifestations and body-weight loss 7 days after the second immunization (representing the late stage of disease). AChR-specific pre-Tfh cells and mature Tfh cells were detected in these two stages, respectively. In co-cultures of Tfh cells and B cells, the number of IgG2b-secreting B cells and the level of anti-AChR antibodies in the supernatant were higher in the cultures containing EAMG-derived Tfh cells. In immunohistochemistry and immunofluorescence assays, a substantial number of CD4+/Bcl-6+ T cells and a greater number of larger germinal centers were observed in lymph node tissues resected from EAMG rats. Based on these results, we hypothesize that an AChR-specific Tfh cell-mediated humoral immune response contributes to the development of EAMG.

The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X.

The Cys22-Cys27 disulfide bond of factor X (FX) protease domain is not conserved among coagulation factors and its contribution to the physiological haemostasis and implication in the pathogenesis of haemostatic and thrombotic disorders remain to be elucidated. Mutation p.Cys27Ser was identified in a pedigree of congenital FX deficiency and fluorescence labelling study of transiently transfected HEK293 cells showed accumulation of FX p.Cys27Ser within cell, indicating incompetent secretion partially responsible for the FX deficiency. The clotting activity of FX p.Cys27Ser was decreased to about 90% of wild-type, while amidolytic and pro-thrombinase activities (kcat/Km) determined with recombinant FXa mutant were 1.33- and 4.77-fold lower. Molecular dynamic simulations revealed no major change in global structure between FXa p.Cys27Ser and wild-type FXa; however, without the Cys22-Cys27 disulfide bond, the insertion of newly formed N terminal of catalytic domain after the activation cleavage is hindered, perturbing the conformation transition from zymogen to enzyme. The crystal structure of FXa shows that this disulfide bond is solvent accessible, indicating that its stability might be subject to the oxidation/reduction balance. As demonstrated with FX p.Cys27Ser here, Cys22-Cys27 disulfide bond may modulate FX clotting activity, with reduced FX pertaining less pro-coagulant activity.