Fra-1 induces apoptosis and neuroinflammation by targeting S100A8 to modulate TLR4 pathways in spinal cord ischemia/reperfusion injury.

Spinal cord ischemia/reperfusion injury (SCII) is a severe complication driven by apoptosis and neuroinflammation. An increase in the expression of c-Fos, a member of the AP-1 family, is known as a neuronal activation marker in SCII. The AP-1 family is composed of Jun, Fos, and is associated with the regulation of cytokines expression and apoptosis. Fra-1 is a member of the Fos family, however, the contribution of Fra-1 to SCII is still unclear. In our study, Fra-1 was highly upregulated especially in neurons and microglia and promoted apoptosis by changing the expression of Bax/Bcl-2 after SCII. Furthermore, we found that Fra-1 directly regulated the transcription expression of S100A8. We demonstrated that knockdown of Fra-1 alleviated S100A8 mediated neuronal apoptosis and inflammatory factor release, thus improved motor function after SCII. Interestingly, we showed that administration of TAK-242, the TLR4 inhibitor, to the ischemia/reperfusion (I/R) injury induced rats suppressed the activation of the ERK and NF-κB pathways, and further reduced Fra-1 expression. In conclusion, we found that Fra-1-targeted S100A8 was expressed the upstream of Fra-1, and the Fra-1/S100A8 interaction formed a feedback loop in the signaling pathways activated by SCII.

The roles of microRNAs in spinal cord ischemia-reperfusion injury.

Spinal cord ischemia/reperfusion injury is a devastating medical disorder with poor prognosis that is associated with several pathophysiological conditions. However, multiple stimuli can trigger SCII, so the underlying mechanism of this pathology has not yet been fully established. MicroRNAs (miRNAs) are a class of non-coding RNAs that mediate a variety of nervous system diseases and regulate numerous physiological functions, including apoptosis, autophagy, inflammation, and blood-spinal cord barrier damage. miRNA expression profiles are known to be altered after spinal cord ischemia/reperfusion injury. Therefore, gaining a better understanding of the significant roles that miRNAs play in spinal cord ischemia/reperfusion injury could help develop potential preventive and therapeutic strategies for spinal cord ischemia/reperfusion injury. This review summarizes the current state of our knowledge about the relationship between miRNAs and spinal cord ischemia/reperfusion injury, as well as potential miRNAs that could be targeted to treat spinal cord ischemia/reperfusion injury.

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-1ß expression, 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.

Ketamine Induces Ferroptosis of Liver Cancer Cells by Targeting lncRNA PVT1/miR-214-3p/GPX4.

BACKGROUND: Liver cancer ranks the top four malignant cancer type worldwide, which needs effective and safe treatment. Ferroptosis is a novel form of regulated cell death driven by iron-dependent lipid peroxidation and has been regarded as a promising therapeutic target for cancers. In this work, we aimed to study the effects of anesthetic ketamine on proliferation and ferroptosis of liver cancer. METHODS: Cell viability and proliferation were detected by cell counting kit 8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assay. Ferroptosis was determined by levels of Fe2+, lipid reactive oxygen species (ROS), and malondialdehyde (MDA). RNA levels of lncPVT1, miR-214-3p, and glutathione peroxidase 4 (GPX4) were checked by real-time PCR assay. Clinical liver tumor samples were collected to detect the levels of long noncoding RNA lncPVT1, miR-214-3p, and GPX4, and their correlation was evaluated by Pearson comparison test. Luciferase reporter gene assay and RNA pulldown were conducted to determine the binding between lncPVT1, miR-214-3p, and GPX4 3'UTR. RESULTS: Ketamine significantly suppressed viability and proliferation of liver cancer cells both in vitro and in vivo, as well as stimulated ferroptosis, along with decreased expression of lncPVT1 and GPX4. LncPVT1 directly interacted with miR-214-3p to impede its role as a sponge of GPX4. Depletion of lncPVT1 accelerated the ferroptosis of live cancer cells, whereas miR-214-3p inhibition and GPX4 overexpression reversed this effect. Ketamine-induced cell growth suppression and ferroptosis were also suppressed by miR-214-3p inhibition and GPX4 overexpression. CONCLUSION: In this work, we determined that ketamine suppressed viability of liver cancer cells and induced ferroptosis and identified the possible regulatory mechanism of lncPVT1/miR-214-3p/GPX4 axis.

MiR-126-3p-Enriched Extracellular Vesicles from Hypoxia-Preconditioned VSC 4.1 Neurons Attenuate Ischaemia-Reperfusion-Induced Pain Hypersensitivity by Regulating the PIK3R2-Mediated Pathway.

Recent evidence suggests that hypoxia preconditioning can alter the microRNA (miRNA) profile of extracellular vesicles (EVs) and has better neuroprotective effects when enriched miRs are delivered to recipients. However, the roles of exosomal miRNAs in regulating ischaemia-reperfusion (IR)-induced pain hypersensitivity are largely unknown. Thus, we isolated EVs from normoxia-conditioned neurons (Nor-VSC EVs) and Hypo-VSC EVs by ultracentrifugation. After the initial screening by a microarray analysis and quantitative RT-PCR (qRT-PCR), miR-126-3p, which was detected as the most altered miR in the Hypo-VSC EVs, was further confirmed by applying GW4869 to inhibit exosomal secretion. Moreover, transfection with a miR-126 mimic obviously increased miR-126-3p expression in Nor-VSC EVs, whereas a miR-126 inhibitor prevented the increase in miR-126-3p in Hypo-VSC EVs. A rat model of pain was established by performing 8-min occlusion of the aorta. Following IR, compared with the Nor-VSC EVs- or antagomir-126-injected rats, the Hypo-VSC EVs-injected rats displayed improved pain hypersensitivity demonstrated as higher PWT and PWL values. Mechanistically, PIK3R2 is a target of miR-126-3p and might be a modulator of the phosphoinositide 3-kinase (PI3K)/Akt pathway as the PIK3R2 and PI3K immunoreactivities in each group were changed in opposite directions. Compared with the controls, higher protein levels of PI3K and phosphorylated Akt but lower levels of phosphorylated nuclear factor-κ B (NF-κB), tumour necrosis factor (TNF)-α and interleukin (IL)-1ß were detected in the spinal cords of the Hypo-VSC EVs-injected rats, and these effects were impaired by an injection of Hypo-VSC EVs combined with antagomir-126. Collectively, the miR-126-3p-enriched Hypo-VSC EVs attenuated IR-induced pain hypersensitivity by restoring miR-126-3p expression in the injured spinal cord and subsequently modulating PIK3R2-mediated PI3K/Akt and NF-κB signalling pathways.

Inhibiting aberrant p53-PUMA feedback loop activation attenuates ischaemia reperfusion-induced neuroapoptosis and neuroinflammation in rats by downregulating caspase 3 and the NF-κB cytokine pathway.

BACKGROUND: Ischaemia reperfusion (IR) induces multiple pathophysiological changes. In addition to its classical role in regulating tumourigenesis, the feedback loop formed by p53 and its driven target p53-upregulated modulator of apoptosis (PUMA) was recently demonstrated to be the common node tightly controlling various cellular responses during myocardial IR. However, the roles of the p53-PUMA feedback loop in the spinal cord remain unclear. This study aimed to elucidate the roles of p53-PUMA feedback interactions in the spinal cord after IR, specifically investigating their regulation of caspase 3-mediated apoptosis and nuclear factor (NF)-κB-mediated cytokine release. METHODS: SD rats subjected to 12 min of aortic arch occlusion served as IR models. Neurological assessment as well as p53 and PUMA mRNA and protein expression analyses were performed at 12-h intervals during a 48-h reperfusion period. The cellular distributions of p53 and PUMA were determined via double immunofluorescence staining. The effects of the p53-PUMA feedback loop on modulating hind-limb function; the number of TUNEL-positive cells; and protein levels of caspase 3, NF-κB and cytokines interleukin (IL)-1ß and tumour necrosis factor (TNF)-α, were evaluated by intrathecal treatment with PUMA-specific or scramble siRNA and pifithrin (PFT)-α. Blood-spinal cord barrier (BSCB) breakdown was examined by Evans blue (EB) extravasation and water content analyses. RESULTS: IR induced significant behavioural deficits as demonstrated by deceased Tarlov scores, which displayed trends opposite those of PUMA and p53 protein and mRNA expression. Upregulated PUMA and p53 fluorescent labels were widely distributed in neurons, astrocytes and microglia. Injecting si-PUMA and PFT-α exerted significant anti-apoptosis effects as shown by the reduced number of TUNEL-positive cells, nuclear abnormalities and cleaved caspase 3 levels at 48 h post-IR. Additionally, p53 colocalized with NF-κB within the cell. Similarly, injecting si-PUMA and PFT-α exerted anti-inflammatory effects as shown by the decreased NF-κB translocation and release of IL-1ß and TNF-α. Additionally, injecting si-PUMA and PFT-α preserved the BSCB integrity as determined by decreased EB extravasation and spinal water content. However, injecting si-Con did not induce any of the abovementioned effects. CONCLUSIONS: Inhibition of aberrant p53-PUMA feedback loop activation by intrathecal treatment with si-PUMA and PFT-α prevented IR-induced neuroapoptosis, inflammatory responses and BSCB breakdown by inactivating caspase 3-mediated apoptosis and NF-κB-mediated cytokine release.

Elevated microRNA-129-5p level ameliorates neuroinflammation and blood-spinal cord barrier damage after ischemia-reperfusion by inhibiting HMGB1 and the TLR3-cytokine pathway.

BACKGROUND: Ischemia-reperfusion (IR) affects microRNA (miR) expression and causes substantial inflammation. Multiple roles of the tumor suppressor miR-129-5p in cerebral IR have recently been reported, but its functions in the spinal cord are unclear. Here, we investigated the role of miR-129-5p after spinal cord IR, particularly in regulating high-mobility group box-1 (HMGB1) and the Toll-like receptor (TLR)-3 pathway. METHODS: Ischemia was induced via 5-min occlusion of the aortic arch. The relationship between miR-129-5p and HMGB1 was elucidated via RT-PCR, western blotting, and luciferase assays. The cellular distribution of HMGB1 was determined via double immunofluorescence. The effect of miR-129-5p on the expression of HMGB1, TLR3, and downstream cytokines was evaluated using synthetic miRs, rHMGB1, and the TLR3 agonist Poly(I:C). Blood-spinal cord barrier (BSCB) permeability was examined by measuring Evans blue (EB) dye extravasation and the water content. RESULTS: The temporal miR-129-5p and HMGB1 expression profiles and luciferase assay results indicated that miR-129-5p targeted HMGB1. Compared with the Sham group, the IR group had higher HMGB1 immunoreactivity, which was primarily distributed in neurons and microglia. Intrathecal injection of the miR-129-5p mimic significantly decreased the HMGB1, TLR3, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α levels and the double-labeled cell count 48 h post-surgery, whereas rHMGB1 and Poly(I:C) reversed these effects. Injection of miR-129-5p mimic preserved motor function and prevented BSCB leakage based on increased Basso Mouse Scale scores and decreased EB extravasation and water content, whereas injection rHMGB1 and Poly(I:C) aggravated these injuries. CONCLUSIONS: Increasing miR-129-5p levels protect against IR by ameliorating inflammation-induced neuronal and BCSB damage by inhibiting HMGB1 and TLR3-associated cytokines.

Role of autophagy in the bimodal stage after spinal cord ischemia reperfusion injury in rats.

Autophagy plays an important role in spinal cord ischemia reperfusion (I/R) injury, but its neuroprotective or neurodegenerative role remains controversial. The extent and persistence of autophagy activation may be the critical factor to explain the opposing effects. In this study, the different roles and action mechanisms of autophagy in the early and later stages after I/R injury were investigated in rats. Thespinal cord I/R injury was induced by 14-min occlusion of the aortic arch, after which rats were treated with autophagic inhibitor (3-methyladenine, 3-MA) or agonist (rapamycin) immediately or 48h following the injury. Autophagy markers, microtubule-associated protein light chain 3-II (LC3-II) and Beclin 1 increased and peaked at the early stage (8h) and the later stage (72h) after spinal cord I/R injury. Beclin 1 was mostly expressed in neurons, but was also expressed to an extent in astrocytes, microglia and vascular endothelial cells. 8h after injury, rats treated with 3-MA showed a decrease in the hind-limb Basso-Beattie-Bresnahan (BBB) motor function scores, surviving motor neurons, and B-cell lymphoma-2 (Bcl-2) expression, and increase in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, Bcl-2-associated X protein (Bax), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) expression, and activation of microglia, while those treated with rapamycin showed opposing effects. However, 72h after injury, rats treated with 3-MA improved the BBB scores, and the surviving motor neurons, and reduced the autophagic cell death, while those treated with rapamycin had adverse effects. These findings provide the first evidence that early activated autophagy alleviates spinal cord I/R injury via inhibiting apoptosis and inflammation; however later excessively elevated autophagy aggravates I/R injury through inducing autophagic cell death.