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Lentiviral?mediated inducible silencing of TLR4 attenuates neuropathic pain in a rat model of chronic constriction injury.

ABSTRACT: An increasing body of evidence has indicated that spinal microglial Toll?like receptor 4 (TLR4) may serve a significant role in the development and maintenance of neuropathic pain (NP). In the present study, experiments were conducted to evaluate the contribution of a tetracycline inducible lentiviral?mediated delivery system for the expression of TLR4 small interfering (si)RNA to NP in rats with chronic constriction injury (CCI). Behavioral tests, including paw withdrawal latency and paw withdrawal threshold, and biochemical analysis of the spinal cord, including western blotting, reverse transcription?quantitative polymerase chain reaction and ELISA, were conducted following CCI to the sciatic nerve. Intrathecal administration of LvOn?si?TLR4 with doxycycline (Dox) attenuated allodynia and hyperalgesia. Biochemical analysis revealed that the mRNA and proteins levels of TLR4 were unregulated following CCI to the sciatic nerve, which was then blocked by intrathecal administration of LvOn?siTLR4 with Dox. The LvOn?siTLR4 was also demonstrated to have no effect on TLR4 or the pain response without Dox, which indicated that the expression of siRNA was Dox?inducible in the lentivirus delivery system. In conclusion, TLR4 may serve a significant role in neuropathy and the results of the present study provide an inducible lentivirus?mediated siRNA against TLR4 that may serve as a potential novel strategy to be applied in gene therapy for NP in the future.

SUBMITTER: Liu Y

PROVIDER: S-EPMC6236283 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Lentiviral‑mediated inducible silencing of TLR4 attenuates neuropathic pain in a rat model of chronic constriction injury.

Liu Yantao Y Zhang Yan Y Pan Ruirui R Chen Mo M Wang Xiaoqiang X Kong Erliang E Yu Weifeng W Sun Yuming Y Wu Feixiang F

Molecular medicine reports 20181016 6

An increasing body of evidence has indicated that spinal microglial Toll‑like receptor 4 (TLR4) may serve a significant role in the development and maintenance of neuropathic pain (NP). In the present study, experiments were conducted to evaluate the contribution of a tetracycline inducible lentiviral‑mediated delivery system for the expression of TLR4 small interfering (si)RNA to NP in rats with chronic constriction injury (CCI). Behavioral tests, including paw withdrawal latency and paw withdr ...[more]

PMID: 30365084

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Project description:Transmembrane member 16A (TMEM16A) is involved in many physiological functions, such as epithelial secretion, sensory conduction, nociception, control of neuronal excitability, and regulation of smooth muscle contraction, and may be important in peripheral pain transmission. To explore the role of TMEM16A in the persistent hyperalgesia that results from chronic constriction injury-induced neuropathic pain, a rat model of the condition was established by ligating the left sciatic nerve. A TMEM16A selective antagonist (10 ?g T16Ainh-A01) was intrathecally injected at L5-6. For measurement of thermal hyperalgesia, the drug was administered once at 14 days and thermal withdrawal latency was recorded with an analgesia meter. For measurement of other indexes, the drug was administered at 12 days, once every 6 hours, totally five times. The measurements were performed at 14 days. Western blot assay was conducted to analyze TMEM16A expression in the L4-6 dorsal root ganglion. Immunofluorescence staining was used to detect the immunoreactivity of TMEM16A in the L4-6 dorsal root ganglion on the injured side. Patch clamp was used to detect electrophysiological changes in the neurons in the L4-6 dorsal root ganglion. Our results demonstrated that thermal withdrawal latency was shortened in the model rats compared with control rats. Additionally, TMEM16A expression and the number of TMEM16A positive cells in the L4-6 dorsal root ganglion were higher in the model rats, which induced excitation of the neurons in the L4-6 dorsal root ganglion. These findings were inhibited by T16Ainh-A01 and confirm that TMEM16A plays a key role in persistent chronic constriction injury-induced hyperalgesia. Thus, inhibiting TMEM16A might be a novel pharmacological intervention for neuropathic pain. All experimental protocols were approved by the Animal Ethics Committee at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. A2017-170-01) on February 27, 2017.

Project description:BackgroundAnti-nerve growth factor (NGF) monoclonal antibodies (anti-NGF mAbs) have been reported to significantly attenuate pain, but the mechanism involved has not been fully elucidated, and the serious adverse events associated with mAbs seriously limit their clinical use. This study further investigated the mechanism by which peripheral NGF is involved in neuropathic pain and found safe, natural compounds that target NGF to attenuate neuropathic pain.MethodsNociception was assessed by the Von Frey hair and Hargreaves' methods. Western-blotting, qPCR and immunofluorescence were used to detect the cell signaling pathway. RAW264.7 macrophages and RSC96 Schwann cells were cultured for in vitro evaluation.ResultsIntraplantar administration of anti-NGF mAbs suppressed the expression of phosphorylated transforming growth factor-β-activated kinase 1 (TAK1) in the dorsal root ganglion (DRG) and sciatic nerve. Intraplantar administration of a TAK1 inhibitor attenuated CCI-induced neuropathic pain and suppressed the expression of phosphorylated mitogen-activated protein kinases (MAPKs) in the DRG and sciatic nerve. Perisciatic nerve administration of levo-corydalmine (l-CDL) on the operated side obviously attenuated CCI-induced neuropathic pain and suppressed the expression of mNGF and proNGF. In addition, l-CDL-induced antinociception was reversed by intraplantar administration of NGF. Further results indicated that l-CDL-induced suppression of phosphorylated TAK1, MAPKs, and p65 and expression of the proinflammatory cytokines TNF-α and IL-1β in the DRG and sciatic nerve were all abolished by NGF. In addition, in vitro experiments indicated that l-CDL suppressed the secretion of NGF and proNGF in RAW264.7 macrophages and RSC96 Schwann cells, which was abolished by AP-1 and CREB agonists, respectively.ConclusionsThis study showed NGF inhibition suppressed TAK1 in the periphery to attenuate CCI-induced neuropathic pain through inhibition of downstream MAPK and p65 signaling. The natural compound l-CDL inhibited NGF secretion by macrophages and Schwann cells and downstream TAK1-MAPK/NF-κB signaling in the periphery to attenuate CCI-induced neuropathic pain. Video abstract Proposed mechanisms underlying the effect of l-CDL in periphery of CCI rats. In CCI rats, macropahages and Schwann cells could secret NGF to act on the receptors in the periphery to activate TAK1-MAPK/NF-κB axis and promote the release of proinflammatory cytokines, including TNF-α and IL-1β to promote neuropathic pain. l-CDL decreased the secretion of NGF through inhibiting AP-1 and CREB respectively in RAW264.7 and RSC96 Schwann cells to attenuate CCI-induced neuropathic pain by inhibiting the TAK1-p38 MAPK/NF-κB signaling pathway.

Dataset Information

Lentiviral?mediated inducible silencing of TLR4 attenuates neuropathic pain in a rat model of chronic constriction injury.

Publications

Lentiviral‑mediated inducible silencing of TLR4 attenuates neuropathic pain in a rat model of chronic constriction injury.

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