DEAD-box helicase 54 regulates microglial inflammatory response in rats with chronic constriction injuries through NF-κB/NLRP3 signaling axis.

Neuropathic pain (NP) is caused by damage to or disease of the somatosensory nervous system, but its mechanism is still not fully understood. In this study, DEAD-box helicase 54 (DDX54) was targeted, and its regulatory role was explored in a chronic constriction injury (CCI) rat model. Microglia and HMC3 cells were stimulated with LPS. The interaction between DDX54 and myeloid differentiation factor-88 adapter protein (MYD88) was verified. A CCI of sciatic nerve model in rats was established. Behavioral testing was performed before and after the CCI. The expressions of IL-1ß, TNF-α, and IL-6 were upregulated, and those of DDX54, MYD88, NF-κB, and NOD-like receptor 3 (NLRP3) were upregulated in microglia and HMC3 cells after LPS induction. DDX54 knockdown in microglia and HMC3 cells inhibited IL-1ß, TNF-α, and IL-6 expressions and downregulated the protein levels of MYD88, p-NF-κB p65 (p-p65), and NLRP3. DDX54 overexpression promoted the stability of MYD88 mRNA. DDX54 binds to the MYD88-3'-untranslated region (UTR). DDX54 interference in rats could alleviate the decrease of paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) induced by CCI, inhibit Iba1 expression, and reduce inflammatory factors as well as MYD88 and NF-κB expressions. DDX54 promotes the activation of NF-κB/NLRP3 signaling by regulating MYD88 mRNA stability, thereby affecting inflammatory response and NP progression in CCI rats.NEW & NOTEWORTHY The role of DDX54 protein in LPS-induced microglia and a chronic constriction injury (CCI) rat model was investigated for the first time. DDX54 interference can inhibit microglial activation and reduce the secretion of inflammatory factors. The interaction between DDX54 protein and MYD88 mRNA was explored for the first time. DDX54 promotes NF-κB/NLRP3 signaling activation by regulating MYD88 transcription in a CCI rat model.

Resolvin D1 attenuates mechanical allodynia after burn injury: Involvement of spinal glia, p38 mitogen-activated protein kinase, and brain-derived neurotrophic factor/tropomyosin-related kinase B signaling.

Resolvin D1 (RvD1) suppresses inflammatory, postoperative, and neuropathic pain. The present study assessed the roles and mechanisms of RvD1 in mechanical allodynia after burn injury. A rat model of burn injury was established for analyses, and RvD1 was injected intraperitoneally. Pain behavior and the expression levels of spinal dorsal horn Iba-1 (microglia marker), GFAP (astrocyte marker), p-p38 mitogen-activated protein kinase (MAPK), brain-derived neurotrophic factor (BDNF), and tropomyosin-related kinase B (TrkB) were detected by behavioral and immunocytochemical assays. The results showed that RvD1 attenuated mechanical allodynia after burn injury, prevented microglial and astroglial activation, and downregulated p-p38 MAPK in microglia and BDNF/TrkB following burn injury. Similarly, inhibition of p38 MAPK and BDNF/TrkB signaling attenuated mechanical allodynia after burn injury. In addition, inhibition of p38 MAPK prevented spinal microglial activation and downregulated BDNF/TrkB following burn injury. Furthermore, inhibition of BDNF/TrkB signaling prevented spinal microglial activation and downregulated p-p38 MAPK within spinal microglia. Taken together, this study demonstrated that RvD1 might attenuate mechanical allodynia after burn injury by inhibiting spinal cord glial activation, microglial p38 MAPK, and BDNF/TrkB signaling in the spinal dorsal horn.

Cav3.2 T-Type calcium channels downregulation attenuates bone cancer pain induced by inhibiting IGF-1/HIF-1α signaling pathway in the rat spinal cord.

Background: Bone cancer pain (BCP) is one of the most ubiquitous and refractory symptoms of cancer patients that needs to be urgently addressed. Substantial studies have revealed the pivotal role of Cav3.2 T-type calcium channels in chronic pain, however, its involvement in BCP and the specific molecular mechanism have not been fully elucidated. Methods: The expression levels of Cav3.2, insulin-like growth factor 1(IGF-1), IGF-1 receptor (IGF-1R) and hypoxia-inducible factor-1α (HIF-1α) were detected by Western blot in tissues and cells. X-ray and Micro CT used to detect bone destruction in rats. Immunofluorescence was used to detect protein expression and spatial location in the spinal dorsal horn. Electrophoretic mobility shift assay used to verify the interaction between HIF-1α and Cav3.2. Results: The results showed that the expression of Cav3.2 channel was upregulated and blockade of this channel alleviated mechanical allodynia and thermal hyperalgesia in BCP rats. Additionally, inhibition of IGF-1/IGF-1R signaling not only reversed the BCP-induced upregulation of Cav3.2 and HIF-1α, but also decreased nociceptive hypersensitivity in BCP rats. Inhibition of IGF-1 increased Cav3.2 expression levels, which were abolished by pretreatment with HIF-1α siRNA in PC12 cells. Furthermore, nuclear HIF-1α bound to the promoter of Cav3.2 to regulate the Cav3.2 transcription level, and knockdown of HIF-1α suppresses the IGF-1-induced upregulation of Cav3.2 and pain behaviors in rats with BCP. Conclusion: These findings suggest that spinal Cav3.2 T-type calcium channels play a central role during the development of bone cancer pain in rats via regulation of the IGF-1/IGF-1R/HIF-1α pathway.

Spinal Ninjurin2 contributes to the neuropathic pain via NF-κB-mediated neuroinflammation in the spared sciatic nerve injury rats.

OBJECT: Ninjurin2 (nerve injury induced protein 2, NINJ2) is a molecule which mediates cell-to-cell and cell-to-extracellular matrix interactions in the nervous system. Clinical study shows NINJ2 is associated with the development of postherpetic neuralgia. However, it is lack of direct evidence that NINJ2 participated in neuropathic pain. In this study, we aim to investigate the role of NINJ2 in the development of neuropathic pain in spared sciatic nerve injury rats and the underlying mechanism. METHOD: Spared sciatic nerve injury (SNI) models were established. The level of NINJ2 and p-p65 (a NF-κB family member) were measured in SNI rats by western blots and immunofluorescent staining. Lentivirus encoding small interfering RNA targeting NINJ2 (RNAi) was intrathecally injected into rats. Then the change of pain behavior of rats induced by NINJ2 RNAi was tested by Von-Frey hairs. The change of p-p65 in the spinal cord in rats after NINJ2 RNAi treatment was also measured by western blots. inhibitor of p-p65-induced change of TNF-α, IL-1ß, and IL-6 levels were measured by ELISA. RESULTS: NINJ2 and p-p65 were increased in the spinal cord of SNI rats on the 3, 7, 14th days after modeling. NINJ2 were mainly expressed in neurons, and co-located with p-p65 in the spinal dorsal horn. When down regulating the level of NINJ2 by RNAi, the development of pain in SNI rats was partially blocked. Phosphorylation of p65 was also inhibited by NINJ2 RNAi. Blocking the phosphorylation of NF-κB pathway could inhibit the increase of TNF-α, IL-1ß, and IL-6 in the spinal cord of SNI rats. CONCLUSION: NINJ2 protein was increased in the spinal cord of SNI rats. It participated in the development of nerve injury-induced neuropathic pain by activating neuroinflammation in the spinal cord via NF-κB pathway. This study provides a new target to investigate the mechanism of neuropathic pain.

The Roles of Chemokine CXCL13 in the Development of Bone Cancer Pain and the Regulation of Morphine Analgesia in Rats.

Chemokines are important regulators of immune, inflammatory, and neuronal responses in peripheral and central pain pathway. The aim of this study was to investigate whether chemokine (C-X-C motif) ligand 13 (CXCL13) and its receptor (C-X-C chemokine receptor type 5, CXCR5) involve in the development of bone cancer pain (BCP) and the regulation of morphine analgesia in rats. The change of pain behaviors in BCP rats were measured by testing paw withdrawal threshold (PWT). The levels of CXCL13, CXCR5 and signal pathway proteins (p-p38, p-ERK and p-AKT etc.) in the spinal cord were measured via western blots. The expression of CXCL13 and CXCR5 in spinal cord was increased in BCP rats. The BCP rats showed decrease of PWTs, which was relieved by CXCR5i. Intrathecally injection of murine recombinant CXCL13 (mrCXCL13) decreased the PWTs of BCP rats and opposed morphine-induced analgesia in BCP rats. In BCP rats, the signal pathway proteins (p38, ERK and AKT) in the spinal cord were activated. CXCL13 and morphine had contrary effect on the phosphorylation of these proteins. MrCXCL13 directly increased the levels of p-p38, p-ERK and p-AKT in BCP rats. However, morphine decreased the levels of these proteins in BCP rats. While blocking the activation of p-p38, p-ERK and p-AKT, morphine analgesia was enhanced. These results suggest CXCL13 participated in bone cancer pain and opposed morphine analgesia via p38, ERK and AKT pathways. It may be a target to enhance pain management in cancer pain patients.

Research on the bioactivity of isoquercetin extracted from marestail on bladder cancer EJ cell and the mechanism of its occurrence.

Research studies in recent years have found that isoquercetin has an inhibiting effect on multiple carcinogens, but research studies filed on isoquercetin in bladder cancer are quite few. This paper observed the influence of isoquercetin on biological activity of the EJ cell of bladder cancer through HC dyeing and trypan blue counting, studied the EJ cell cycle by flow cytometry (FCM), and then analyzed the influence of isoquercetin and its effect on the protein expression of STAT3 and STAT3-inhibiting factors (PIAS3) in EJ cells. Research has shown that isoquercetin has an inhibitory effect on the EJ cells of bladder cancer, but it is not obvious.

[Resolvin D1 inhibits the injury of PC12 cells induced by activated microglia].

Objective To investigate the effect of resolvin D1 (RvD1) on the injury of PC12 cells induced by activated BV-2 microglia and the related mechanisms. Methods BV-2 cells were divided into control group, lipopolysaccharide (LPS)-treated group, RvD1-treated group and RvD1 combined with LPS (RvD1-LPS)-treated group. After BV-2 cells were incubated with the corresponding substances for 12 and 24 hours, the levels of interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α) in the supernatants were determined by ELISA. The culture supernatants of BV-2 cells were collected at 24 hours and added into PC12 cells for another 24-hour culture. Thereafter, the survival rate of PC12 cells was tested by MTT assay. The expression of NF-κB p65 protein in BV-2 cells was deteced by Western blotting. Results Compared with the control group, the survival rate of PC12 cells in the LPS group significantly decreased; the levels of IL-1ß, IL-6 and TNF-α in the supernatant of BV-2 cells and the nuclear translocation of NF-κB p65 significantly increased in the LPS group. Compared with the LPS group, the survival rate of PC12 cells in RvD1-LPS group was significantly elevated; the levels of IL-1, IL-6, TNF-α and the nuclear translocation of NF-κB p65 were significantly reduced in RvD1-LPS group. Conclusion RvD1 can inhibit the injury of PC12 cells induced by activated BV-2 microglia through inhibiting the nuclear translocation of NF-κB p65 and inflammatory factor levels in BV-2 cells.

Pharmacodynamics and Pharmacokinetics of Levobupivacaine Used for Epidural Anesthesia in Patients with Liver Dysfunction.

The objective of this study was to study the pharmacodynamics and pharmacokinetics of levobupivacaine used for epidural anesthesia in patients with liver dysfunction. Twenty patients aged 20-60, American Society of Anesthesiologists (ASA) graded I-III according to the ASA guidelines, scheduled for elective upper abdominal surgery, were included in the study. They were divided into two groups of ten each. In group I, the patients with liver dysfunction were included, whereas group II was composed of those with normal liver function. In both groups, anesthesia was induced by general anesthesia combined with epidural block, given by T 8-9 interspace injection of 1.8 mg kg(-1) levobupivacaine (0.75 %) with 5 µg mL(-1) of adrenaline in 1.5 min. The sensory and motor blockade indices were recorded for 30 min after the injection. The plasma concentration of levobupivacaine was determined by high performance liquid chromatography from 0 to 1440 min after the injection and pharmacokinetics of the drug were calculated. The onset and recovery time from the sensory block in the two groups were similar with no significant difference (P > 0.05). The maximum spread of anesthetic effect, the number of spinal segments regressed, onset time, and degree of motor block after the injection were also insignificantly different in the two groups. The plasma levobupivacaine concentration/time curve of the liver dysfunction (group I) was significantly higher than that of the controls (group II). In the liver dysfunction patients, the volume of distribution (V/F) was significantly increased, the elimination rate, i.e., half-life (t 1/2ß ), was prolonged, and the elimination rate constants (K 12 and K 10) were significantly decreased (P < 0.05 or P < 0.01). The patients with liver dysfunction injected with 0.75 % levobupivacaine exhibited normal onset and recovery time of the sensory and motor blocks within 30 min. However, in these patients, the metabolism of levobupivacaine was significantly slower as evidenced by the higher blood concentration of the drug than in cases with normal functioning liver.

The relationship between the target effective site concentration of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients.

OBJECTIVE: To study the relationship between the target effective site concentration (Ce) of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients. METHODS: 50 elderly patients (ASA grade II) scheduled for selective surgical procedure under general anaesthesia were randomly divided into two groups, A and B, with 25 cases in each group. The Ce of rocuronium for intubation was 3 µg·ml(-1) in both groups, and the Ce during operation were 0.8 and 1.0 µg·ml(-1) in group A and B, respectively. When target controlled infusion of rocuronium was stopped, without the administration of reversal agents for neuromuscular blockade, the relationship between Ce and the first twitch height (T1) was studied by regression analysis. RESULTS: There was a significant linear relationship between Ce and T1, and there was no statistical difference in regression coefficient and interception between group A and B (P>0.05). CONCLUSION: The degree of recovery from neuromuscular blockade could be judged by the target effective site concentration of rocuronium at the time of reversal from neuromuscular blockade in the elderly patients.