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Mitochondrial oxidative phosphorylation (OXPHOS) system dysfunction in cancer cells has been exploited as a target for anti-cancer therapeutic intervention. The downregulation of CR6-interacting factor 1 (CRIF1), an essential mito-ribosomal factor, can impair mitochondrial function in various cell types. In this study, we investigated whether CRIF1 deficiency induced by siRNA and siRNA nanoparticles could suppress MCF-7 breast cancer growth and tumor development, respectively. Our results showed that CRIF1 silencing decreased the assembly of mitochondrial OXPHOS complexes I and II, which induced mitochondrial dysfunction, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential depolarization, and excessive mitochondrial fission. CRIF1 inhibition reduced p53-induced glycolysis and apoptosis regulator (TIGAR) expression, as well as NADPH synthesis, leading to additional increases in ROS production. The downregulation of CRIF1 suppressed cell proliferation and inhibited cell migration through the induction of G0/G1 phase cell cycle arrest in MCF-7 breast cancer cells. Similarly, the intratumoral injection of CRIF1 siRNA-encapsulated PLGA nanoparticles inhibited tumor growth, downregulated the assembly of mitochondrial OXPHOS complexes I and II, and induced the expression of cell cycle protein markers (p53, p21, and p16) in MCF-7 xenograft mice. Thus, the inhibition of mitochondrial OXPHOS protein synthesis through CRIF1 deletion destroyed mitochondrial function, leading to elevated ROS levels and inducing antitumor effects in MCF-7 cells. [ABSTRACT FROM AUTHOR]

Oxygenated water (OW) contains more oxygen than normal drinking water. It may induce oxygen enrichment in the blood and reduce oxidative stress. Hypoxia and oxidative stress could be involved in epilepsy. We aimed to examine the effects of OW-treated vs. control on four rodent models of epilepsy: (1) prenatal betamethasone priming with postnatal N-methyl-D-aspartate (NMDA)-triggered spasm, (2) no prenatal betamethasone, (3) repetitive kainate injection, and (4) intraperitoneal pilocarpine. We evaluated, in (1) and (2), the latency to onset and the total number of spasms; (3) the number of kainate injections required to induce epileptic seizures; (4) spontaneous recurrent seizures (SRS) (numbers and duration). In model (1), the OW-treated group showed significantly increased latency to onset and a decreased total number of spasms; in (2), OW completely inhibited spasms; in (3), the OW-treated group showed a significantly decreased number of injections required to induce epileptic seizures; and in (4), in the OW-treated group, the duration of a single SRS was significantly reduced. In summary, OW may increase the seizure threshold. Although the underlying mechanism remains unclear, OW may provide an adjunctive alternative for patients with refractory epilepsy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), affects 25% of the global population. Despite the prevalence of NAFLD worldwide, effective therapeutics are currently lacking. Amomum villosum var. xanthioides (Wall. ex Baker) T.L.Wu & S.J.Chen (AX) is a medicinal herb traditionally used for treating digestive tract disorders in countries across Asia. We aimed to examine the pharmacological effects of the ethyl acetate fraction of AX (AXEF) against tunicamycin (TM)-induced ER stress in a NASH mouse model using C57/BL6J male mice. Following TM injections (2 mg/kg), the mice were orally administrated AXEF (12.5, 25, or 50 mg/kg), silymarin (50 mg/kg), or distilled water daily for 5 days, and the outcomes for fatty liver, inflammation, and oxidative stress were measured in serum or liver tissue levels. AXEF drastically attenuated hepatic ER stress-induced NASH as indicated by decreases in lipid droplet accumulations, serum liver enzymes, hepatic inflammations, and cell death signals in the hepatic tissue and/or serum levels. Interestingly, AXEF showed potent antioxidant effects by quenching reactive oxidative stress and its final product lipid peroxide in the hepatic tissue, specifically an increase in metallothionein (MT). To confirm the underlying actions of AXEF, we observed that AXEF increases MT1 gene promoter activities in the physiological levels. Collectively, AXEF showed antioxidant properties on TM-induced ER stress in a NASH mice model through the improvement of MTs. [ABSTRACT FROM AUTHOR]

Activation of nuclear factor-kappa B (NF-κB) in microglia plays a decisive role in the progress of neuropathic pain, and the inhibitor of kappa B (IκB) is a protein that blocks the activation of NF-κB and is degraded by the inhibitor of NF-κB kinase subunit beta (IKBKB). The role of IKBKB is to break down IκB, which blocks the activity of NF-kB. Therefore, it prevents the activity of NK-kB. This study investigated whether neuropathic pain can be reduced in spinal nerve ligation (SNL) rats by reducing the activity of microglia by delivering IKBKB small interfering RNA (siRNA)-encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles. PLGA nanoparticles, as a carrier for the delivery of IKBKB genes silencer, were used because they have shown potential to enhance microglial targeting. SNL rats were injected with IKBKB siRNA-encapsulated PLGA nanoparticles intrathecally for behavioral tests on pain response. IKBKB siRNA was delivered for suppressing the expression of IKBKB. In rats injected with IKBKB siRNA-encapsulated PLGA nanoparticles, allodynia caused by mechanical stimulation was reduced, and the secretion of pro-inflammatory mediators due to NF-κB was reduced. Delivering IKBKB siRNA through PLGA nanoparticles can effectively control the inflammatory response and is worth studying as a treatment for neuropathic pain. [ABSTRACT FROM AUTHOR]

Nuclear factor of activated T cells (NFAT5) is a well-known transcription factor that regulates the expression of genes involved in osmotic stress. However, the role of NFAT5 in inflammatory pain remains unknown. Here, we studied the function of NFAT5 in inflammatory pain using NFAT5-heterozygous (Het) mice. To study inflammatory pain, we injected 10 µL of 2% formalin into the right hind paws of mice and monitored pain behaviors, such as licking, lifting, and flinching, for 60 min. After the first 15 min (phase I), there were no significant differences in pain behaviors between wild-type (WT) and NFAT5-Het mice. However, from 15–60 min (phase II), NFAT5-Het mice displayed significantly fewer pain behaviors compared to WT mice. Further, the expression levels of inflammatory-pain-related factors, including c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated n-methyl-D-aspartate receptor subunit 2B (p-NR2B), were significantly elevated in the spinal dorsal neurons of formalin-treated WT mice but was not elevated in NFAT5-Het mice. Similarly, c-Fos, p-ERK, and p-NR2B levels were significantly higher in glutamate-treated PC12 neuronal cells but were not affected by Nfat5 silencing in glutamate-treated PC12 cells. Altogether, our findings suggest that NFAT5 deficiency may mitigate formalin-induced inflammatory pain by upregulating mammalian target of rapamycin (mTOR) expression and downregulating its downstream factors in spinal dorsal neurons. Therefore, NFAT5 is a potential therapeutic target for the treatment of inflammatory pain. [ABSTRACT FROM AUTHOR]

Visceral pain frequently produces referred pain at somatic sites due to the convergence of somatic and visceral afferents. In skin overlying the referred pain, neurogenic spots characterized by hyperalgesia, tenderness and neurogenic inflammation are found. We investigated whether neurogenic inflammatory spots function as acupoints in the rat model of bile duct ligation-induced liver injury. The majority of neurogenic spots were found in the dorsal trunk overlying the referred pain and matched with locations of acupoints. The spots, as well as acupoints, showed high electrical conductance and enhanced expression of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP). Electroacupuncture at neurogenic spots reduced serum hepatocellular enzyme activities and histological patterns of acute liver injury in bile duct ligation (BDL) rats. The results suggest that the neurogenic spots have therapeutic effects as acupoints on hepatic injury in bile-duct ligated rats. [ABSTRACT FROM AUTHOR]

Pain can be induced by tissue injuries, diseases and infections. The interactions between the peripheral nervous system (PNS) and immune system are primary actions in pain sensitizations. In response to stimuli, nociceptors release various mediators from their terminals that potently activate and recruit immune cells, whereas infiltrated immune cells further promote sensitization of nociceptors and the transition from acute to chronic pain by producing cytokines, chemokines, lipid mediators and growth factors. Immune cells not only play roles in pain production but also contribute to PNS repair and pain resolution by secreting anti-inflammatory or analgesic effectors. Here, we discuss the distinct roles of four major types of immune cells (monocyte/macrophage, neutrophil, mast cell, and T cell) acting on the PNS during pain process. Integration of this current knowledge will enhance our understanding of cellular changes and molecular mechanisms underlying pain pathogenies, providing insights for developing new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Neonatal ischemic stroke has a higher incidence than childhood stroke. Seizures are the first sign for the need for clinical assessment in neonates, but many questions remain regarding treatments and follow-up modalities. In the absence of a known pathophysiological mechanism, only supportive care is currently provided. Stroke-induced microglia activation and neuroinflammation are believed to play a central role in the pathological progression of neonatal ischemic stroke. We induced a photothrombotic infarction with Rose Bengal in neonatal rats to investigate the effects of pre- and post-treatment with Aspirin (ASA), Clopidogrel (Clop), and Coenzyme Q10 (CoQ10), which are known for their neuroprotective effects in adult stroke. Pre-stroke medication ameliorates cerebral ischemic injury and reduces infarct volume by reducing microglia activation, cellular reactive oxygen species (ROS) production, and cytokine release. Post-stroke administration of ASA, Clop, and CoQ10 increased motor function and reduced the volume of infarction, and the statistical evidence was stronger than that seen in the pre-stroke treatment. In this study, we demonstrated that ASA, Clop, and CoQ10 treatment before and after the stroke reduced the scope of stroke lesions and increased behavioral activity. It suggests that ASA, Clop, and CoQ10 medication could significantly have neuroprotective effects in the neonates who have suffered strokes. [ABSTRACT FROM AUTHOR]

Activation of CX3CR1 in microglia plays an important role in the development of neuropathic pain. Here, we investigated whether neuropathic pain could be attenuated in spinal nerve ligation (SNL)-induced rats by reducing microglial activation through the use of poly(D,L-lactic-co-glycolic acid) (PLGA)-encapsulated CX3CR1 small-interfering RNA (siRNA) nanoparticles. After confirming the efficacy and specificity of CX3CR1 siRNA, as evidenced by its anti-inflammatory effects in lipopolysaccharide-stimulated BV2 cells in vitro, PLGA-encapsulated CX3CR1 siRNA nanoparticles were synthesized by sonication using the conventional double emulsion (W/O/W) method and administered intrathecally into SNL rats. CX3CR1 siRNA-treated rats exhibited significant reductions in the activation of microglia in the spinal dorsal horn and a downregulation of proinflammatory mediators, as well as a significant attenuation of mechanical allodynia. These data indicate that the PLGA-encapsulated CX3CR1 siRNA nanoparticles effectively reduce neuropathic pain in SNL-induced rats by reducing microglial activity and the expression of proinflammatory mediators. Therefore, we believe that PLGA-encapsulated CX3CR1 siRNA nanoparticles represent a valuable new treatment option for neuropathic pain. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Upon peripheral nerve injury, vesicular ATP is released from damaged primary afferent neurons. This extracellular ATP subsequently activates purinergic receptors of the spinal cord, which play a critical role in neuropathic pain. As an inhibitor of the vesicular nucleotide transporter (VNUT), Evans blue (EB) inhibits the vesicular storage and release of ATP in neurons. Thus, we tested whether EB could attenuate neuropathic pain behavior induced by spinal nerve ligation (SNL) in rats by targeting VNUT. An intrathecal injection of EB efficiently attenuated mechanical allodynia for five days in a dose-dependent manner and enhanced locomotive activity in an SNL rat model. Immunohistochemical analysis showed that EB was found in VNUT immunoreactivity on neurons in the dorsal root ganglion and the spinal dorsal horn. The level of ATP in cerebrospinal fluid in rats with SNL-induced neuropathic pain decreased upon administration of EB. Interestingly, EB blocked ATP release from neurons, but not glial cells in vitro. Eventually, the loss of ATP decreased microglial activity in the ipsilateral dorsal horn of the spinal cord, followed by a reduction in reactive oxygen species and proinflammatory mediators, such as interleukin (IL)-1β and IL-6. Finally, a similar analgesic effect of EB was demonstrated in rats with monoiodoacetate-induced osteoarthritis (OA) pain. Taken together, these data demonstrate that EB prevents ATP release in the spinal dorsal horn and reduces the ATP/purinergic receptor-induced activation of spinal microglia followed by a decline in algogenic substances, thereby relieving neuropathic pain in rats with SNL. [ABSTRACT FROM AUTHOR]