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Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice.

ABSTRACT: Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflammatory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroinflammation in a mouse model of transient middle cerebral artery occlusion (tMCAO). siRNA against TLR4 (siTLR4) complexed to the novel LNP (siTLR4/DoGo310), which was based on a dioleoyl-conjugated short peptidomimetic (denote DoGo310), was readily internalized by the oxygen-glucose-deprived (OGD) mouse primary microglia, knocked-down TLR4, and polarized the cell to the anti-inflammatory phenotype in vitro. Systemic administration of siTLR4/DoGo310 LNPs in the tMCAO mice model resulted in the accumulation of siRNA mainly in the Iba1 positive cells in the peri-infarct. Analysis of the peri-infarct brain tissue revealed that a single injection of siTLR4/DoGo310 LNPs led to significant knockdown of TLR4 gene expression, reversing the pattern of cytokines expression, and improving the neurological functions in tMCAO model mice. Our data demonstrate that DoGo310 LNPs could be a promising nanocarrier for CNS-targeted siRNA delivery for the treatment of CNS disorders.

SUBMITTER: Ganbold T

PROVIDER: S-EPMC9228890 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice.

Ganbold Tsogzolmaa T Bao Qingming Q Xiao Hai H Zurgaanjin Dolgorsuren D Liu Caifeng C Han Shuqin S Hasi Agula A Baigude Huricha H

Nanomaterials (Basel, Switzerland) 20220616 12

Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflammatory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroinflammation in a mouse model of transient middle cerebral artery occlusion (tMCAO). siRNA against TLR4 (siTLR4) complexed to the novel LNP (siTLR4/DoGo310), which was based on a dioleoyl-conjugated short peptidomimetic (denote DoGo3 ...[more]

PMID: 35745411

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Project description:BackgroundElectroacupuncture (EA) has been shown to promote functional recovery after cerebral ischemia-reperfusion (I/R) injury. However, the contribution of mitochondrial dynamics to recovery remains unclear. The aim of this study was to investigate whether mitochondrial dynamics are involved in the effects of EA on cerebral I/R injury.MethodsThe rats with cerebral I/R injury were established by the middle cerebral artery occlusion/reperfusion. Subsequently, EA was applied to Baihui (GV20) and Dazhui (GV14) acupoints, with 2 Hz/5 Hz in frequency, 1.0 mA in intensity, 20 min each time, once a day for seven consecutive days. The therapeutic outcomes were assessed by modified neurological severity score (mNSS), 2,3,5-Triphenyte-trazolium chloride (TTC) staining, and hematoxylin-eosin (HE) staining. Mitochondrial morphology was observed under transmission electron microscopy. Adenosine triphosphate (ATP) content and ATP synthases (ATPases) activity were evaluated to measure mitochondrial function using ELISA. Finally, mitochondrial dynamics-related molecules, including dynamin-related protein 1 (Drp1), fission 1 (Fis1), mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optic atrophy 1 (OPA1), were detected by Western blot and immunofluorescence staining.ResultsCerebral I/R injury induced neurological dysfunction, cerebral infarction and neuronal injury, all of which were ameliorated by EA. And EA improved mitochondrial morphology and function. Moreover, EA altered the balance of mitochondrial dynamics. Specifically, the data showed a significant decrease in the expression of Drp1 and Fis1, leading to the inhibition of mitochondrial fission. Additionally, Mfn1, Mfn2 and Opa1, which are related to mitochondrial fusion, were effectively promoted after EA treatment. However, sham EA did not show any neuroprotective effects in rats with cerebral I/R injury.ConclusionsIn summary, our study indicates that the balance of mitochondrial dynamics is crucial for EA therapy to treat cerebral I/R injury.

Project description:Ischemic stroke is a devastating disease with long-term disability. However, the pathogenesis is unclear and treatments are limited. Meisoindigo, a second-generation derivative of indirubin, has general water solubility and is well-tolerated. Previous studies have shown that meisoindigo reduces inflammation by inhibiting leukocyte chemotaxis and migration. In the present study, we investigated the hypothesis that meisoindigo was also protective against ischemic stroke, then evaluated its underlying mechanisms. In vivo, adult male C57BL/6J wild-type mice were used to produce a middle cerebral artery occlusion (MCAO) stroke model. On day three after reperfusion, obvious improvement in neurological scores, infarct volume reduction and cerebral edema amelioration were observed in meisoindigo treatment. Moreover, immunofluorescence staining and western-blot showed that the expression of NLRP3 inflammasome and its associated proteins in neurons and microglia was inhibited by meisoindigo. The effects of Meisoindigo on NLRP3 inflammasome inactivation and increased the M2 phenotype of microglia/macrophage through shifting from a M1 phenotype, which was possibly mediated by inhibition of TLR4/NF-κB. Furthermore, we verified the inhibitory effect of meisoindigo on TLR4/NF-κB signaling pathway, and found that meisoindigo treatment could significantly suppressed the expression of TLR4/NF-κB pathway-associated proteins in a dose-dependent manner, meanwhile, which resulted in downregulation of HMGB1 and IL-1β. Next, we established an in vitro oxygen glucose deprivation/Reperfusion (OGD/R) model in HT-22 and BV2 cells to simulate ischemic conditions. Cytotoxicity assay showed that meisoindigo substantially improved relative cell vitality and in HT-22 and BV2 cells following OGD/R in vitro. After suffering OGD/R, the TLR4/NF-κB pathway was activated, the expression of NLRP3 inflammasome-associated proteins and M1 microglia/macrophage were increased, but meisoindigo could inhibit above changes in both HT-22 and BV2 cells. Additionally, though lipopolysaccharide stimulated the activation of TLR4 signaling in OGD/R models, meisoindigo co-treatment markedly reversed the upregulation of TLR4 and following activation of NLRP3 inflammasome and polarization of M1 microglia/macrophages mediated by TLR4. Overall, we demonstrate for the first time that meisoindigo post-treatment alleviates brain damage induced by ischemic stroke in vivo and in vitro experiments through blocking activation of the NLRP3 inflammasome and regulating the polarization of microglia/macrophages via inhibition of the TLR4/NF-κB signaling pathway.

Project description:Acacetin (5,7-dihydroxy-4'-methoxyflavone), a potential neuroprotective agent, has an inhibitory effect on lipopolysaccharide-induced neuroinflammatory reactions. However, whether acacetin has an effect on inflammatory corpuscle 3 (NLRP3) after cerebral ischemia-reperfusion injury has not been fully determined. This study used an improved suture method to establish a cerebral ischemia-reperfusion injury model in C57BL/6 mice. After ischemia with middle cerebral artery occlusion for 1 hour, reperfusion with intraperitoneal injection of 25 mg/kg of acacetin (acacetin group) or an equal volume of saline (0.1 mL/10 g, middle cerebral artery occlusion group) was used to investigate the effect of acacetin on cerebral ischemia-reperfusion injury. Infarct volume and neurological function scores were determined by 2,3,5-triphenyltetrazolium chloride staining and the Zea-Longa scoring method. Compared with the middle cerebral artery occlusion group, neurological function scores and cerebral infarction volumes were significantly reduced in the acacetin group. To understand the effect of acacetin on microglia-mediated inflammatory response after cerebral ischemia-reperfusion injury, immunohistochemistry for the microglia marker calcium adapter protein ionized calcium-binding adaptor molecule 1 (Iba1) was examined in the hippocampus of ischemic brain tissue. In addition, tumor necrosis factor-α, interleukin-1β, and interleukin-6 expression in ischemic brain tissue of mice was quantified by enzyme-linked immunosorbent assay. Expression of Iba1, tumor necrosis factor-α, interleukin-1β and interleukin-6 was significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Western blot assay results showed that expression of Toll-like receptor 4, nuclear factor kappa B, NLRP3, procaspase-1, caspase-1, pro-interleukin-1β, and interleukin-1β were significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Our findings indicate that acacetin has a protective effect on cerebral ischemia-reperfusion injury, and its mechanism of action is associated with inhibition of microglia-mediated inflammation and the NLRP3 signaling pathway.

Dataset Information

Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice.

Publications

Peptidomimetic Lipid-Nanoparticle-Mediated Knockdown of TLR4 in CNS Protects against Cerebral Ischemia/Reperfusion Injury in Mice.

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