Nara Shin

Aim: Amitriptyline (AMI) has been used to treat neuropathic pain. However, the clinical outcomes remain unsatisfactory, presumably due to a limited understanding of the underlying molecular mechanisms. Here, we investigated a drug repositioning strategy using a low-dose of AMI encapsulated in poly (D, L lactic-co-glycolic acid) (PLGA) nanoparticles (AMI NPs) for neuropathic pain, since PLGA nanoparticles are known to enhance delivery to microglia. Methods: We evaluated the anti-allodynic effects of AMI and AMI NPs on neuropathic pain by assessing behaviors and inflammatory responses in a rat model of spinal nerve ligation (SNL). While the anti-allodynic effect of AMI (30 mu g) drug injection on SNL-induced neuropathic pain persisted for 12 h, AMI NPs significantly alleviated mechanical allodynia for 3 days. Results: Histological and cytokine analyses showed AMI NPs facilitated the reduction of microglial activation and pro-inflammatory mediators in the spinal dorsal horn. This study suggests that AMI NPs can provide a sustained anti-allodynic effect by enhancing the targeting of microglia and regulating the release of pro-inflammatory cytokines from activated microglia. Conclusion: Our findings suggest that the use of microglial-targeted NPs continuously releasing AMI (2 mu g) as a drug repositioning strategy offers long-term anti-allodynic effects.

Social isolation stress–derived depressive behavior and ethanol intake act as a vicious cycle.

Reactive oxygen species (ROS) exacerbate stroke-induced cell damage. We found that ShcA, a protein that regulates ROS, is highly expressed in a Rose Bengal photothrombosis model. We investigated whether ShcA is essential for mitophagy in ROS-induced cellular damage and determined whether ROS exacerbate mitochondrial dysfunction via ShcA protein expression. Ischemic stroke was generated by Rose Bengal photothrombosis in mice. To silence ShcA protein expression in the mouse brain, ShcA-targeting siRNA-encapsulated nanoparticles were intrathecally injected into the cisterna magna. Upon staining with antibodies against ShcA counterpart caspase-3 or NeuN, we found that the ShcA protein expression was increased in apoptotic neurons. In addition, mitochondrial dysfunction and excessive mitophagy were evident in photothrombotic stroke tissue. Infarct volumes were significantly reduced, and neurological deficits were diminished in the ShcA siRNA nanoparticle-treated group, compared with the negative control siRNA nanoparticle-treated group. We confirmed that the reduction of ShcA expression by nanoparticle treatment rescued the expression of genes, associated with mitochondrial dynamics and mitophagy mediation in a stroke model. This study suggests that the regulation of ShcA protein expression can be a therapeutic target for reducing brain damage with mitochondrial dysfunction caused by thrombotic infarction.

P66shc, a member of the shc adaptor protein family, has been shown to participate in regulation of mitochondrial homeostasis, apoptosis, and autophagosome formation. The present study was performed to investigate whether p66shc siRNA-encapsulated poly(d,l-lactic-co-glycolic acid) nanoparticles (p66shc siRNA-PLGA NPs) can attenuate spinal nerve ligation (SNL)-induced neuropathic pain in rats. The SNL-induced pain behavior was decreased in the p66shc siRNA-PLGA NP-treated group compared with the scrambled siRNA-PLGA NP-treated group. In the L5 spinal cord of the p66shc siRNA-PLGA NP-treated group, expression levels of phosphorylated p66shc, cleaved caspase-3, p62, and PINK1, as well as microglial activation, were also decreased. In addition, p66shc knockdown using p66shc siRNA reduced the expression levels of cleaved caspase-3, p62, and PINK1, as well as proinflammatory mediators in the H₂O₂-treated HT22 neuronal cells. These results suggest that downregulation of p66shc expression in the spinal cord using p66shc siRNA-PLGA NPs could reduce the SNL-induced neuropathic pain by attenuating the SNL-induced aberrant autophagic, mitophagic, and neuroinflammatory processes in rats.

Background: Osteoarthritis (OA) is the most common type of joint disease associated with cartilage breakdown. However, the role played by mitochondrial dysfunction in OA remains inadequately understood. Therefore, we investigated the role played by p66shc during oxidative damage and mitochondrial dysfunction in OA and the effects of p66shc downregulation on OA progression. Methods: Monosodium iodoacetate (MIA), which is commonly used to generate OA animal models, inhibits glycolysis and biosynthetic processes in chondrocytes, eventually causing cell death. To observe the effects of MIA and poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles, histological analysis, immunohistochemistry, micro-CT, mechanical paw withdrawal thresholds, quantitative PCR, and measurement of oxygen consumption rate and extracellular acidification rate were conducted. Results: p-p66shc was highly expressed in cartilage from OA patients and rats with MIA-induced OA. MIA caused mitochondrial dysfunction and reactive oxygen species (ROS) production, and the inhibition of p66shc phosphorylation attenuated MIA-induced ROS production in human chondrocytes. Inhibition of p66shc by PLGA-based nanoparticles-delivered siRNA ameliorated pain behavior, cartilage damage, and inflammatory cytokine production in the knee joints of MIA-induced OA rats. Conclusion: p66shc is involved in cartilage degeneration in OA. By delivering p66shc-siRNA-loaded nanoparticles into the knee joints with OA, mitochondrial dysfunction-induced cartilage damage can be significantly decreased. Thus, p66shc siRNA PLGA nanoparticles may be a promising option for the treatment of OA.

Several studies have shown that brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1), an important molecule for maintaining circadian rhythms, inhibits the growth and metastasis of tumor cells in several types of cancer, including lung, colon, and breast cancer. However, its role in glioblastoma has not yet been established. Here, we addressed the function of BMAL1 in U87MG glioblastoma cells with two approaches—loss and gain of function. In the loss of function experiments, cell proliferation in U87MG cells transfected with small interfering RNA (siRNA) targeting BMAL1 was increased by approximately 24% (small interfering (si)-NC 0.91 ± 0.00 vs. si-BMAL1 1.129 ± 0.08) via upregulation of cyclin B1. In addition, cell migration and invasion of BMAL1 siRNA-treated glioblastoma cells were elevated by approximately 20% (si-NC 51.00 ± 1.53 vs. si-BMAL161.33 ± 0.88) and 209% (si-NC 21.28 ± 1.37 vs. si-BMAL1 44.47 ± 3.48), respectively, through the accumulation of phosphorylated-AKT (p-AKT) and matrix metalloproteinase (MMP)-9. Gain of function experiments revealed that adenovirus-mediated ectopic expression of BMAL1 in U87MG cells resulted in a 19% (Adenovirus (Ad)-vector 0.94± 0.03 vs. Ad-BMAL1 0.76 ± 0.03) decrease in cell proliferation compared with the control via downregulation of cyclin B1 and increased early and late apoptosis due to changes in the levels of BCL2-associated X protein (BAX), B-cell lymphoma 2 (BCL-2), and cleaved caspase-3. Likewise, cell migration and invasion were attenuated by approximately 24% (Ad-vector 55.00 ± 0.00 vs. Ad-BMAL1 41.83 ± 2.90) and 49% (Ad-vector 70.01 ± 1.24 vs. Ad-BMAL1 35.55 ± 1.78), respectively, in BMAL1-overexpressing U87MG cells following downregulation of p-AKT and MMP-9. Taken together, our results suggest that BMAL1 acts as an anti-cancer gene by altering the proliferation, migration, and invasion of glioblastoma cells. Therefore, the BMAL1 gene could be a potential therapeutic target in the treatment of glioblastoma.

Osteoarthritis (OA) is the most common joint disorder that has had an increasing prevalence due to the aging of the population. Recent studies have concluded that OA progression is related to oxidative stress and reactive oxygen species (ROS). ROS are produced at low levels in articular chondrocytes, mainly by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and ROS production and oxidative stress have been found to be elevated in patients with OA. The cartilage of OA-affected rat exhibits a significant induction of p47phox, a cytosolic subunit of the NADPH oxidase, similarly to human osteoarthritis cartilage. Therefore, this study tested whether siRNA p47phox that is introduced with poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (p47phox si_NPs) can alleviate chondrocyte cell death by reducing ROS production. Here, we confirm that p47phox si_NPs significantly attenuated oxidative stress and decreased cartilage damage in mono-iodoacetate (MIA)-induced OA. In conclusion, these data suggest that p47phox si_NPs may be of therapeutic value in the treatment of osteoarthritis.

Microglia, the resident macrophages, act as the first and main form of active immune defense in the central nervous system. Arginase 2 (Arg2) is an enzyme involved in L-arginine metabolism and is expressed in macrophages and nervous tissue. In this study, we determined whether the absence of Arg2 plays a beneficial or detrimental role in the neuroinflammatory process. We then investigated whether the loss of Arg2 potentiated microglia activation and pain behaviors following nerve injury-induced neuropathic pain. A spinal nerve transection (SNT) experimental model was used to induce neuropathic pain in mice. As a result of the peripheral nerve injury, SNT induced microgliosis and astrogliosis in the spinal cord, and upregulated inflammatory signals in both wild-type (WT) and Arg2 knockout (KO) mice. Notably, inflammation increased significantly in the Arg2 KO group compared to the WT group. We also observed a more robust microgliosis and a lower mechanical threshold in the Arg2 KO group than those in the WT group. Furthermore, our data revealed a stronger upregulation of M1 pro-inflammatory cytokines, such as interleukin (IL)-1β, and a stronger downregulation of M2 anti-inflammatory cytokines, including IL4 and IL-10, in Arg2 KO mice. Additionally, stronger formation of enzyme-inducible nitric oxide synthase, oxidative stress, and decreased expression of CD206 were detected in the Arg2 KO group compared to the WT group. These results suggest that Arg2 deficiency contributes to inflammatory response. The reduction or the loss of Arg2 results in the stronger neuroinflammation in the spinal dorsal horn, followed by more severe pain behaviors arising from nerve injury-induced neuropathic pain.

Cartilage loss is a central event in the pathogenesis of osteoarthritis (OA), though other than mechanical loading, the biochemical mechanisms underlying OA pathology remain poorly elucidated. We investigated the role of Pink1-mediated mitophagy in mitochondrial fission, a crucial process in OA pathogenesis. We used a monosodium iodoacetate (MIA)-induced rodent model of OA, which inhibits the activity of articular chondrocytes, leading to disruption of glycolytic energy metabolism and eventual cell death. The OA rat cartilage exhibits significant induction of autophagy-related proteins LC3B and p62, similar to human osteoarthritic cartilage. Moreover, expression of Pink1 and Parkin proteins were also increased in OA. Here, we confirm that Pink1-mediated mitophagy leads to cell death in chondrocytes following MIA treatment, while deficiency in Pink1 expression was associated with decreased cartilage damage and pain behaviors in MIA-induced OA. Finally, we found that autophagy and mitophagy-related genes are highly expressed in human osteoarthritic cartilage. These results indicate that OA is a degenerative condition associated with mitophagy, and suggest that targeting the Pink1 pathway may provide a therapeutic avenue for OA treatment.