miR-26a-5p alleviates CFA-induced chronic inflammatory hyperalgesia through Wnt5a/CaMKII/NFAT signaling in mice

Background

Inflammation often leads to the occurrence of chronic pain, and many miRNAs have been shown to play a key role in the development of inflammatory pain. However, whether miR-26a-5p relieves pain induced by inflammation and its possible mechanism are still unclear.

Methods

The complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model was employed. Intrathecal or subcutaneous injection of miR-26a-5p agomir was performed after modeling to study its antinociceptive effect and the comparison of different administration methods. Bioinformatics analysis of miRNAs was performed to study the downstream mechanisms of miR-26a-5p. HE staining, RT-qPCR, Western blotting, and immunofluorescence were used for further validation.

Results

A single intrathecal and subcutaneous injection of miR-26a-5p both reversed mechanical hypersensitivity and thermal latency in the left hind paw of mice with CFA-induced inflammatory pain. HE staining and immunofluorescence studies found that both administrations of miR-26a-5p alleviated inflammation in the periphery and spinal cord. Bioinformatics analysis and dual-luciferase reporter gene analysis identified Wnt5a as a direct downstream target gene of miR-26a-5p. Wnt5a was mainly expressed in neurons and microglia in the spinal cord of mice with inflammatory pain. Intrathecal injection of miR-26a-5p could significantly reduce the expression level of Wnt5a and inhibit the downstream molecules of noncanonical Wnt signaling Camk2/NFAT, inhibiting the release of spinal cord inflammatory factors and alleviating the activation of microglia. In addition, miR-26a-5p could also inhibit lipopolysaccharide (LPS)-stimulated BV2 cell inflammation in vitro through a noncanonical Wnt signaling pathway.

Conclusions

miR-26a-5p is a promising therapy for CFA-induced inflammatory pain. Both intrathecal and subcutaneous injections provide relief for inflammatory pain. miR-26a-5p regulated noncanonical Wnt signaling to be involved in analgesia partly through antineuroinflammation, suggesting a pain-alleviating effect via noncanonical Wnt signaling pathway in the CFA-induced inflammatory pain model in vivo.     

MicroRNA-146a-5p attenuates neuropathic pain via suppressing TRAF6 signaling in the spinal cord

Glia-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. Our recent study demonstrated that TNF receptor associated factor-6 (TRAF6) is expressed in spinal astrocytes and contributes to the maintenance of spinal nerve ligation (SNL)-induced neuropathic pain. MicroRNA (miR)-146a is a key regulator of the innate immune response and was shown to target TRAF6 and reduce inflammation. In this study, we found that in cultured astrocytes, TNF-α, IL-1β, or lipopolysaccharide (LPS) induced rapid TRAF6 upregulation and delayed miR-146a-5p upregulation. In addition, miR-146a-5p mimic blocked LPS-induced TRAF6 upregulation, as well as LPS-induced c-Jun N-terminal kinase (JNK) activation and chemokine CCL2 expression in astrocytes. Notably, LPS incubation with astrocytes enhanced the DNA binding activity of AP-1 to the promoters of mir-146a and ccl2. TRAF6 siRNA or JNK inhibitor SP600125 significantly reduced LPS-induced miR-146a-5p increase in astrocytes. In vivo, intrathecal injection of TNF-α or LPS increased spinal TRAF6 expression. Pretreatment with miR-146a-5p mimic alleviated TNF-α- or LPS-induced mechanical allodynia and reduced TRAF6 expression. Finally, SNL induced miR-146a-5p upregulation in the spinal cord at 10 and 21 days. Intrathecal injection of miR-146a-5p mimic attenuated SNL-induced mechanical allodynia and decreased spinal TRAF6 expression. Taken together, the results suggest that (1) miR-146a-5p attenuates neuropathic pain partly through inhibition of TRAF6 and its downstream JNK/CCL2 signaling, (2) miR-146a-5p is increased by the activation of TRAF6/JNK pathway. Hence, miR-146a-5p may be a novel treatment for chronic neuropathic pain.     

Downregulation of miR-491-5p promotes neovascularization after traumatic brain injury

Micro RNA-491-5 p(miR-491-5 p) plays an important role in regulating cell proliferation and migration;however,the effect of miR-491-5 p on neovascularization after traumatic brain injury remains poorly understood.In this study,a controlled cortical injury model in C57 BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro,respectively.In the in vivo model,quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5 p increased or decreased following the intracerebroventricular injection of an miR-491-5 p agomir or antagomir,respectively,and the expression of miR-491-5 p decreased slightly after traumatic brain injury.To detect the neuroprotective effects of miR-491-p,neurological severity scores,Morris water maze test,laser speckle techniques,and immunofluorescence staining were assessed,and the results revealed that miR-491-5 p downregulation alleviated neurological dysfunction,promoted the recovery of regional cerebral blood flow,increased the number of lectin-stained microvessels,and increased the survival of neurons after traumatic brain injury.During the in vitro experiments,the potential mechanism of miR-491-5 p on neovascularization was explored through quantitative real-time-polymerase chain reaction,which showed that miR-491-5 p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5 p mimic or inhibitor,respectively.Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5 p.Cell counting kit 8(CCK-8) assay,flow cytometry,and 2′,7′-dichlorofluorescein diacetate(DCFH-DA) assay results confirmed that the downregulation of miR-491-5 p increased brain microvascular endothelial cell viability,reduced cell apoptosis,and alleviated oxidative stress under oxygen-glucose deprivation conditions.Cell scratch assay,Transwell assay,tube formation assay,and western blot assay results demonstrated that miR-491-5 p downregulation promoted the migration,proliferation,and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway.These findings confirmed that miR-491-5 p downregulation promotes neovascularization,restores cerebral blood flow,and improves the recovery of neurological function after traumatic brain injury.The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress.All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University,China(approval No.2020-304) on June 22,2020.     

Key genes expressed in different stages of spinal cord ischemia/reperfusion injury

The temporal expression of microRNA atfer spinal cord ischemia/reperfusion injury is not yet fully understood. In the present study, we established a model of spinal cord ischemia in Sprague-Dawley rats by clamping the abdominal aorta for 90 minutes, before allowing reperfusion for 24 or 48 hours. A sham-operated group underwent surgery but the aorta was not clamped. The damaged spinal cord was removed for hematoxylin-eosin staining and RNA extraction. Neuronal degeneration and tissue edema were the most severe in the 24-hour reperfusion group, and milder in the 48-hour reperfusion group. RNA ampliifcation, labeling, and hybridization were used to obtain the microRNA expression proifles of each group. Bioinformatics analysis conifrmed four differentially expressed microRNAs (miR-22-3p, miR-743b-3p, miR-201-5p and miR-144-5p) and their common target genes (Tmem69 and Cxcl10). Compared with the sham group, miR-22-3p was continuously upregulated in all three ischemia groups but was highest in the group with no reperfusion, whereas miR-743b-3p, miR-201-5p and miR-144-5p were downregulated in the three ischemia groups. We have successfully identiifed the key genes expressed at different stages of spinal cord ischemia/reperfusion injury, which provide a reference for future investigations into the mechanism of spinal cord injury.     

Key genes expressed in different stages of spinal cord ischemia/reperfusion injury ischemia/reperfusion injury

The temporal expression of microRNA after spinal cord ischemia/reperfusion injury is not yet fully understood.In the present study,we established a model of spinal cord ischemia in Sprague-Dawley rats by clamping the abdominal aorta for 90 minutes,before allowing reperfusion for 24 or 48 hours.A sham-operated group underwent surgery but the aorta was not clamped.The damaged spinal cord was removed for hematoxylin-eosin staining and RNA extraction.Neuronal degeneration and tissue edema were the most severe in the 24-hour reperfusion group,and milder in the 48-hour reperfusion group.RNA amplification,labeling,and hybridization were used to obtain the microRNA expression profiles of each group.Bioinformatics analysis confirmed four differentially expressed microRNAs(miR-22-3p,miR-743b-3p,miR-201-5p and miR-144-5p) and their common target genes(Tmem69 and CxcllO).Compared with the sham group,miR-22-3p was continuously upregulated in all three ischemia groups but was highest in the group with no reperfusion,whereas miR-743b-3p,miR-201-5p and miR-144-5p were downregulated in the three ischemia groups.We have successfully identified the key genes expressed at different stages of spinal cord ischemia/reperfusion injury,which provide a reference for future investigations into the mechanism of spinal cord injury.     

miR-30a-5p inhibition promotes interaction of Fas+ endothelial cells and FasL+ microglia to decrease pathological neovascularization and promote physiological angiogenesis

Ischemia-induced angiogenesis contributes to various neuronal and retinal diseases, and often results in neurodegeneration and visual impairment. Current treatments involve the use of anti-VEGF agents but are not successful in all cases. In this study we determined that miR-30a-5p is another important mediator of retinal angiogenesis. Using a rodent model of ischemic retinopathy, we show that inhibiting miR-30a-5p reduces neovascularization and promotes tissue repair, through modulation of microglial and endothelial cell cross-talk. miR-30a-5p inhibition results in increased expression of the death receptor Fas and CCL2, to decrease endothelial cell survival and promote microglial migration and phagocytic function in focal regions of ischemic injury. Our data suggest that miR-30a-5p inhibition accelerates tissue repair by enhancing FasL–Fas crosstalk between microglia and endothelial cells, to promote endothelial cell apoptosis and removal of dead endothelial cells. Finally, we found that miR-30a levels were increased in the vitreous of patients with proliferative diabetic retinopathy. Our study identifies a role for miR-30a in the pathogenesis of neovascular retinal disease by modulating microglial and endothelial cell function, and suggests it may be a therapeutic target to treat ischemia-mediated conditions.     

MicroRNA-31 regulating apoptosis by mediating the phosphatidylinositol-3 kinase/protein kinase B signaling pathway in treatment of spinal cord injury

Apoptosis is a highly conservative energy demand program for non-inflammatory cell death, which is extremely significant in normal physiology and disease. There are many techniques used for studying apoptosis. MicroRNA (miRNA) is closely related to cell apoptosis, and especially microRNA-31 (miR-31) is involved in apoptosis by regulating a large number of target genes and signaling pathways. In many neurological diseases, cell apoptosis or programmed cell death plays an important role in the reduction of cell number, including the reduction of neurons in spinal cord injuries. In recent years, the phosphoinositol 3-kinase/AKT (PI3K/AKT) signal pathway, as a signal pathway involved in a variety of cell functions, has been studied in spinal cord injury diseases. The PI3K/AKT pathway directly or indirectly affects whether apoptosis occurs in a cell, thereby affecting a significant intracellular event sequence. This paper reviewed the interactions of miR-31 target sites in the PI3K/AKT signaling pathway, and explored new ways to prevent and treat spinal cord injury by regulating the effect of miR-31 on apoptosis.     

MicroRNA-101a-3p mimic ameliorates spinal cord ischemia/reperfusion injury

miR-101a-3p is expressed in a variety of organs and tissues and plays a regulatory role in many diseases,but its role in spinal cord ischemia/reperfusion injury remains unclear.In this study,we established a rat model of spinal cord ischemia/reperfusion injury by clamping the aortic arch for 14 minutes followed by reperfusion for 24 hours.Results showed that miR-101a-3p expression in L4-L6 spinal cord was greatly decreased,whereas MYCN expression was greatly increased.Dual-luciferase reporter assay results showed that miR-101a-3p targeted MYCN.MYCN immunoreactivity,which was primarily colocalized with neurons in L4-L6 spinal tissue,greatly increased after spinal cord ischemia/reperfusion injury.However,intrathecal injection of an miR-101a-3p mimic within 24 hours before injury decreased MYCN,p53,caspase-9 and interleukin-1βexpression,reduced p53 immunoreactivity,reduced the number of MYCN/NeuN-positive cells and the number of necrotic cells in L4-L6 spinal tissue,and increased Tarlov scores.These findings suggest that the miR-101a-3p mimic improved spinal ischemia/reperfusion injury-induced nerve cell apoptosis and inflammation by inhibiting MYCN and the p53 signaling pathway.Therefore,miR-101a-3p mimic therapy may be a potential treatment option for spinal ischemia/reperfusion injury.     

MicroRNA expression in animal models of amyotrophic lateral sclerosis and potential therapeutic approaches

A review of recent animal models of amyotrophic lateral sclerosis showed a large number of mi RNAs had altered levels of expression in the brain and spinal cord,motor neurons of spinal cord and brainstem,and hypoglossal,facial,and red motor nuclei and were mostly upregulated.Among the mi RNAs found to be upregulated in two of the studies were mi R-21,mi R-155,mi R-125 b,mi R-146 a,mi R-124,mi R-9,and mi R-19 b,while those downregulated in two of the studies included mi R-146 a,mi R-29,mi R-9,and mi R-125 b.A change of direction in mi RNA expression occurred in some tissues when compared(e.g.,mi R-29 b-3 p in cerebellum and spinal cord of wobbler mice at 40 days),or at different disease stages(e.g.,mi R-200 a in spinal cord of SOD1(G93 A)mice at 95 days vs.108 and 112 days).In the animal models,suppression of mi R-129-5 p resulted in increased lifespan,improved muscle strength,reduced neuromuscular junction degeneration,and tended to improve motor neuron survival in the SOD1(G93 A)mouse model.Suppression of mi R-155 was also associated with increased lifespan,while lowering of mi R-29 a tended to improve lifespan in males and increase muscle strength in SOD1(G93 A)mice.Overexpression of members of mi R-17~92 cluster improved motor neuron survival in SOD1(G93 A)mice.Treatment with an artificial mi RNA designed to target h SOD1 increased lifespan and improved muscle strength in SOD1(G93 A)animals.Further studies with animal models of amyotrophic lateral sclerosis are warranted to validate these findings and identify specific mi RNAs whose suppression or directed against h SOD1 results in increased lifespan,improved muscle strength,reduced neuromuscular junction degeneration,and improved motor neuron survival in SOD1(G93 A)animals.     

Identification of potential oxidative stress biomarkers for spinal cord injury in erythrocytes using mass spectrometry

Oxidative stress is a hallmark of secondary injury associated with spinal cord injury.Identifying stable and specific oxidative biomarkers is of important significance for studying spinal cord injury-associated secondary injury.Mature erythrocytes do not contain nuclei and mitochondria and cannot be transcribed and translated.Therefore, mature erythrocytes are highly sensitive to oxidative stress and may become a valuable biomarker.In the present study, we revealed the proteome dynamics of protein expression in erythrocytes of beagle dogs in the acute and subacute phases of spinal cord injury using mass spectrometry-based approaches.We found 26 proteins that were differentially expressed in the acute(0–3 days) and subacute(7–21 days) phases of spinal cord injury.Bioinformatics analysis revealed that these differentially expressed proteins were involved in glutathione metabolism, lipid metabolism, and pentose phosphate and other oxidative stress pathways.Western blot assays validated the differential expression of glutathione synthetase, transaldolase, and myeloperoxidase.This result was consistent with mass spectrometry results, suggesting that erythrocytes can be used as a novel sample source of biological markers of oxidative stress in spinal cord injury.Glutathione synthetase, transaldolase, and myeloperoxidase sourced from erythrocytes are potential biomarkers of oxidative stress after spinal cord injury.This study was approved by the Experimental Animal Centre of Ningxia Medical University, China(approval No.2017-073) on February 13, 2017.     

Circ_0101874 overexpression strengthens PDE4D expression by targeting miR-335-5p to promote neuronal injury in ischemic stroke

BackgroundIschemic stroke has been a public concern, while its pathogenesis is not fully understood. Increasing evidence suggests that circular RNAs (circRNAs) are involved in this disorder. The purpose of this study was to explore the role of circ_0101874 in ischemic stroke.MethodsThe in vivo model of ischemic stroke was established in mice with middle cerebral artery occlusion (MCAO) treatment. The in vitro model of ischemic stroke was established in SK-N-SH cells with oxygen-glucose deprivation (OGD) treatment. The expression of circ_0101874, miR-335-5p and phosphodiesterase 4D (PDE4D) mRNA was measured by quantitative real-time PCR (qPCR). The release of inflammatory factors was checked by ELISA. Cell viability, cell proliferation and cell apoptosis were detected using CCK-8 assay, EdU assay and flow cytometry assay, respectively. The protein levels of cyclinD1, cleaved-caspase-3 and PDE4D were detected by western blot. The interaction between miR-335-5p and circ_0101874 or PDE4D was validated by dual-luciferase reporter assay and RIP assay.ResultsCirc_0101874 was highly expressed in MCAO animal models and OGD-induced SK-N-SH cells. Circ_0101874 knockdown suppressed OGD-enhanced inflammation, cell apoptosis and oxidative stress and promoted OGD-inhibited cell viability and cell proliferation in SK-N-SH cells. Circ_0101874 directly bound to miR-335-5p, and miR-335-5p inhibition reversed the effects of circ_0101874 knockdown. PDE4D was a target gene of miR-335-5p, and PDE4D overexpression recovered OGD-promoted SK-N-SH cell injuries that were blocked by miR-335-5p enrichment. Circ_0101874 bound to miR-335-5p to enhance the expression of PDE4D.ConclusionCirc_0101874 knockdown alleviated OGD-induced neuronal cell injury by suppressing PDE4D via regulating miR-335-5p.     

长链非编码RNA MALAT1对神经母细胞瘤系细胞生物学特性的影响

目的 探讨长链非编码RNA MALAT1对神经母细胞瘤系细胞生物学特性的影响及作用机制。方法 体外培养神经母细胞瘤系SHEP2细胞，细胞分为Control、sh-MALAT1、miR-181a-5p inhibitor和sh-MALAT1+ miR-181a-5p inhibitor组，其中sh-MALAT1组转染sh-MALAT1，miR-181a-5p inhibitor组转染miR-181a-5p inhibitor，sh-MALAT1+inhibitor组共同转染sh-MALAT1与miR-181a-5p inhibitor，Control组加入等量空载体。PCR检测mRNA水平；生物信息预测MALAT1与miR-181a-5p的靶向关系，荧光素酶实验鉴定；CCK8法检测细胞增殖能力；Hoechst法检测细胞凋亡；划痕实验测试细胞迁移；Transwell实验检测细胞侵袭；免疫印迹法检测蛋白表达。结果 sh-MALAT1明显降低MALAT1并提高miR-181a-5p在神经母细胞瘤细胞系SHEP2细胞mRNA水平。miR-181a-5p mimic明显降低MALAT1 wt荧光素酶活性。sh-MALAT1抑制SHEP2细胞增殖、侵袭及迁移，促进细胞凋亡；miR-181a-5p inhibitor促进细胞增殖、侵袭及迁移，抑制细胞凋亡，并减弱sh-MALAT1产生的影响。同时，sh-MALAT1抑制PI3K/Akt信号通路，而miR-181a-5p inhibitor可激活此信号通路并减弱sh-MALAT1的抑制作用。结论 MALAT1靶向下调miR-181a-5p表达促进神经母细胞瘤细胞系SHEP2细胞增殖、迁移和侵袭。     

miR-138对脊髓全横断大鼠行为学的影响

目的探讨miR-138在脊髓全横断大鼠运动功能恢复过程中是否发挥调控作用。方法打开SD大鼠T9-T11椎板,脊髓内分别注射miR-138、DEPC处理水后制备脊髓全横断模型实验组与对照组。术后3d、65d取材,观察脊髓红肿范围、瘢痕长度;期间进行BBB运动功能评分;术后30d应用体感诱发电位(SEP)检测神经功能恢复情况。结果第5～9周miR-138组大鼠BBB评分明显低于对照组(P<0.05)。对照组后肢有运动的大鼠所占比例逐渐增多,而miR-138组该比例未见增加(27.03±16.2,6.5±11.72)(P<0.05)。SEP显示miR-138组大鼠左后肢均未测出潜伏期和波幅,右后肢潜伏期和波幅检出率均为16.7%,明显低于对照组(P<0.05)。术后3dmiR-138组脊髓横断处头端红肿长度较对照组增加(P<0.05)。术后65d脊髓横断处瘢痕明显,与周围软组织及椎骨粘连。miR-138组与对照组比较,瘢痕长度(1.07±0.21,1.03±0.12)cm及周长(1.00±0.17,1.05±0.09)cm均无显著性差异(P>0.05)。结论脊髓全横断大鼠局部应用miR-138后运动功能恢复较对照组...     

Exogenous platelet-derived growth factor improves neurovascular unit recovery after spinal cord injury

The blood-spinal cord barrier plays a vital role in recovery after spinal cord injury. The neurovascular unit concept emphasizes the relationship between nerves and vessels in the brain, while the effect of the blood-spinal cord barrier on the neurovascular unit is rarely reported in spinal cord injury studies. Mouse models of spinal cord injury were established by heavy object impact and then immediately injected with plateletderived growth factor(80 μg/kg) at the injury site. Our results showed that after platelet-derived growth factor administration, spinal cord injury, neuronal apoptosis, and blood-spinal cord barrier permeability were reduced, excessive astrocyte proliferation and the autophagyrelated apoptosis signaling pathway were inhibited, collagen synthesis was increased, and mouse locomotor function was improved. In vitro, human umbilical vein endothelial cells were established by exposure to 200 μM H_2O_2. At 2 hours prior to injury, in vitro cell models were treated with 5 ng/mL platelet-derived growth factor. Our results showed that expression of blood-spinal cord barrier-related proteins, including Occludin, Claudin 5, and β-catenin, was significantly decreased and autophagy was significantly reduced. Additionally, the protective effects of platelet-derived growth factor could be reversed by intraperitoneal injection of 80 mg/kg chloroquine, an autophagy inhibitor, for 3 successive days prior to spinal cord injury. Our findings suggest that platelet-derived growth factor can promote endothelial cell repair by regulating autophagy, improve the function of the blood-spinal cord barrier, and promote the recovery of locomotor function post-spinal cord injury. Approval for animal experiments was obtained from the Animal Ethics Committee, Wenzhou Medical University, China(approval No. wydw2018-0043) in July 2018.     

Bone morphogenetic proteins mediate cellular response and, together with Noggin, regulate astrocyte differentiation after spinal cord injury

Bone morphogenetic proteins (BMPs) play a critical role in regulating cell fate determination during central nervous system (CNS) development. In light of recent findings that BMP-2/4/7 expressions are upregulated after spinal cord injury, we hypothesized that the BMP signaling pathway is important in regulating cellular composition in the injured spinal cord. We found that BMP expressions were upregulated in neural stem cells (NSCs), neurons, oligodendrocytes and microglia/macrophages. Increased expression levels of pSmad1/5/8 (downstream molecules of BMP) were detected in neurons, NSCs, astrocytes, oligodendrocytes and oligodendroglial progenitor cells (OPCs). Active astrocytes which form the astroglial scar were probably derived from NSCs, OPCs and resident astrocytes. Since quiescent NSCs in the normal adult spinal cord will proliferate and differentiate actively into neural cells after traumatic injury, we proposed that BMPs can regulate cellular components by controlling NSC differentiation. Neurosphere culture from adult mouse spinal cord showed that BMP-4 promoted astrocyte differentiation from NSCs while suppressing production of neurons and oligodendrocytes. Conversely, inhibition of BMP-4 by Noggin notably decreased the ratio of astrocyte to neuron numbers. However, intrathecal administration of Noggin in the injured spinal cord failed to attenuate glial fibrillar acidic protein (GFAP) expression even though it effectively reduced pSmad expression. Noggin treatment did not block phosphorylation of Stat3 and the induction of GFAP in the injured spinal cord, suggesting that in addition to the BMP/Smad pathway, the JAK/STAT pathway may also be involved in the regulation of GFAP expression after spinal cord injury.