Objective Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in intervertebral disc homeostasis; however, its role in autophagy regulation remains unclear. This study aimed to investigate whether agonist-induced TRPV4 activation promotes autophagy and extracellular matrix (ECM) synthesis in rat intervertebral discs.
Methods In vitro, rat nucleus pulposus (NP) cells were treated with the TRPV4 agonist (GSK1016790) under normal, serum-deprived, or interleukin-1β-stimulated conditions. Cell viability, intracellular Ca2+ influx, adenosine monophosphate-activated protein kinase/mammalian target of rapamycin (mTOR) (AMPK/mTOR) pathway, autophagy, ECM metabolism, apoptosis, and senescence were evaluated. In vivo, TRPV4 agonist was injected into the caudal discs subjected to temporary static compression, and disc changes were assessed by radiography, histomorphology, and immunofluorescence.
Results In vitro, agonist-induced TRPV4 activation rapidly increased intracellular Ca2+ influx and enhanced AMPK phosphorylation. A noncytotoxic concentration of the TRPV4 agonist (10 nM) was selected after dose-response testing. Under the inflammatory stress, TRPV4 agonist enhanced autophagy, promoted ECM synthesis, and suppressed apoptosis and senescence, leading to improved NP cell viability. In vivo, TRPV4 agonist treatment preserved radiographic disc height (p<0.01), reduced histomorphological degeneration (p<0.01), and increased expression of COL2A1, Brachyury, p-AMPK (phosphorylated AMPK), and autophagy markers (p<0.01) compared with controls.
Conclusion These findings demonstrated that TRPV4 activation promotes autophagy and ECM synthesis via the AMPK/mTOR pathway in rat discs and attenuates stress-induced degeneration, suggesting TRPV4 as a potential therapeutic target for disc degeneration.
Objective Neuronal apoptosis is considered to be a critical process in spinal cord injury (SCI). Despite growing evidence of the antiapoptotic, anti-inflammatory, and modulation of ischemic injury tolerance effects of extracellular ubiquitin (eUb), existing studies have paid less attention to the impact of eUb in neurological injury disorders, particularly in SCI. This study aimed to investigate whether eUb can play a protective role in neurons, both in vitro and in vivo, and explores the underlying mechanisms.
Methods By utilizing an oxygen glucose deprivation cellular model and a SCI rat model, we firstly investigated the therapeutic effects of eUb on SCI and further explored its effects on neuronal autophagy and mitochondria-dependent apoptosis-related indicators, as well as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mechanical target of rapamycin (mTOR) signaling pathway.
Results In the SCI models both in vivo and in vitro, early intervention with eUb enhanced neuronal autophagy and inhibited mitochondrial apoptotic pathways, significantly mitigating SCI. Further studies had shown that this protective effect of eUb was mediated through its receptor, CXC chemokine receptor type 4 (CXCR4). Additionally, eUb-enhanced autophagy and antiapoptotic effects were possibly associated with inhibiting the PI3K/Akt/mTOR pathway.
Conclusion In summary, the study demonstrates that early eUb intervention can enhance autophagy and inhibit mitochondrial apoptotic pathways via CXCR4, protecting neurons and promoting SCI repair.
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Objective The study aimed to investigate the role of N6-methyladenosine (m6A) modification in spinal cord injury (SCI) and its underlying mechanism, focusing on the interplay between m6A methyltransferase-like 3 (METTL3), miR-30c, and autophagy-related proteins.
Methods An SCI model was established in rats, and changes in autophagy-related proteins, m6A methylation levels, and miR-30c levels were analyzed. Hydrogen peroxide (H2O2)-stimulated spinal cord neuron cells (SCNCs) were used to assess the impact of METTL3 overexpression. The effects of STM2457, an antagonist of METTL3, were evaluated on cell viability, apoptosis, and autophagy markers in H2O2-stimulated SCNCs.
Results In the SCI model, decreased levels of autophagy markers and increased m6A methylation, miR-30c levels, and METTL3 were observed. Overexpression of METTL3 in SCNCs led to reduced cell viability, increased apoptosis, and suppressed autophagy. Conversely, co-overexpression of autophagy-related protein 5 (ATG5) or miR-30c inhibition reversed these effects. Knocking out METTL3 yielded opposite results. STM2457 treatment improved cell viability, reduced apoptosis, and upregulated autophagy markers in SCNCs, which also enhanced functional recovery in rats as measured by the Basso-Beattie-Bresnahan score and inclined plate test.
Conclusion STM2457 alleviated SCI by suppressing METTL3-mediated m6A modification of miR-30c, which in turn induces ATG5-mediated autophagy. This study provides insights into the role of m6A modification in SCI and suggests a potential therapeutic approach through targeting METTL3.
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Objective Electroacupuncture (EA) has a definite effect on the treatment of spinal cord injuries (SCIs), but its underlying molecular mechanism remains unclear. Meanwhile, MiR106b-5p is an autophagy- and apoptosis-related microribonucleic acid, but whether it regulates the progression of autophagy and apoptosis in SCIs is yet undetermined. As such, this study aimed to elucidate the involvement of miR-106b-5p in the EA treatment of an SCI.
Methods The miR-106b-5p level was detected by quantitative real-time polymerase chain reaction. In vitro, SH-SY5Y cells were transfected with miR-106b-5p mimics or inhibitors to regulate the miR-106b-5p expression, while in vivo, SCI rats were treated with EA for 7 days at the bilateral Zusanli (ST36) and Jiaji (EX-B2) acupoints. The motor function was evaluated using the Basso-Beattie-Bresnahan (BBB) criteria. Further, autophagic vacuoles, pathological damage, and neuronal cell morphology were observed by transmission electron microscopy, as well as by hematoxylin and eosin and Nissl staining, respectively.
Results The miR-106b-5p level, which can interact directly with Beclin-1 by influencing its expression, as well as the expressions of P62, Caspase-3, and Bax, was upregulated after an SCI, but it decreased after EA. Moreover, the ratio of LC3-II to LC3-I was upregulated after EA. EA can enhance autophagy, reduce neuronal apoptosis, and minimize motor dysfunction and histopathological deficits after an SCI. More importantly, however, all the above effects induced by EA can be reversed after an injection of miR-106-5p agomir to produce an overexpression of miR-106b-5p.
Conclusion EA treatment could downregulate miR-106b-5p to alleviate SCI-mediated injuries by promoting autophagy and inhibiting apoptosis.
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