L-arginine combination with 5-fluorouracil inhibit hepatocellular carcinoma cells through suppressing iNOS/NO/AKT-mediated glycolysis.

L-arginine can produce nitric oxide (NO) under the action of inducible nitric oxide synthase (iNOS), while 5-fluorouracil (5-FU) can induce the increase of iNOS expression. The present study was to investigate the mechanism of L-arginine combined with 5-FU regulating glucose metabolism of hepatocellular carcinoma (HCC) through iNOS/NO/AKT pathway. The combination of L-arginine and 5-FU resulted in decreased cell survival and exhibited synergistic cytotoxic effects in HepG2 and SMMC7721 cells. Meanwhile, L-arginine increased 5-FU inhibitory effect on HepG2 and SMMC7721 cells by increasing NO production. Co-treatment with L-arginine and 5-FU resulted in a significant decrease in both G6PDH and LDH enzymatic activities, as well as reduced levels of ATP and LD compared to treatment with L-arginine or 5-FU alone. Moreover, the combination of L-arginine and 5-FU resulted in a decrease in the expression of GLUT1, PKM2, LDHA, p-PI3K and p-AKT. Furthermore, the combination demonstrated a synergistic effect in downregulating the expression of HIF-1α and ß-catenin, which were further diminished upon the addition of shikonin, a specific inhibitor of PKM2. LY294002 treatment further reduced the expression of GLUT1, PKM2, and LDHA proteins induced by combined L-arginine and 5-FU treatment compared to the combined group. However, the reduction in p-PI3K, p-AKT, and GLUT1 expression caused by L-arginine and 5-FU combination was also reversed in HepG2 and SMMC7721 cells with iNOS knockdown, respectively. Additionally, the combination of L-arginine and 5-FU led to a greater reduction in the enzymatic activity of ALT, AST, G6PDH and LDH, as well as a significant reduction in hepatic index, AFP, AFP-L3, ATP and LD levels in a rat model of HCC. Moreover, the simultaneous administration of L-arginine and 5-FU significantly improved the gross morphology of the liver, reduced nuclear atypia, inhibited the proliferation of cancer cells, and decreased the expression levels of p-PI3K, p-AKT, GLUT1, PKM2, and LDHA, while iNOS expression was increased in the combination group. Taking together, L-arginine and 5-FU combination resulted in the inhibition of enzymes in aerobic glycolysis via the iNOS/NO/AKT pathway, which led to the suppression of glucose metabolism and downregulation of nuclear transcription factors, thereby impeding the proliferation of hepatocellular carcinoma cells.

Effects of NCSTN Mutation on Hair Follicle Components in Mice.

BACKGROUND AND OBJECTIVES: Hidradenitis suppurativa (HS)/acne inversa is an intractable skin disease that is characterized by destructive lesions - primarily on the flexural areas. Although its etiology is unknown, genetics is considered to be a factor of its pathology - mutations in γ-secretase genes have been identified in certain familial HS patients, and follicular occlusion is widely accepted as the primary cause of HS. But, no relationship between these mutations and the components of hair follicles has been reported. Thus, we examined changes in these components in mice with a mutation in NCSTN (a γ-secretase gene). METHODS: We generated C57BL/6 mice with an NCSTN mutation and examined their expression of hair cortex cytokeratin and trichohyalin by Western blot and immunohistochemistry, in addition to nicastrin, the product of NCSTN, and NICD compared with wild-type mice. The structure of hair follicles was analyzed by hematoxylin-eosin staining and transmission electron microscopy. RESULTS: In mice with an NCSTN mutation, HS-like skin lesions appeared after age 6 months, the pathological manifestations of which were consistent with the features of human HS. The structure of hair follicles was abnormal in mice with an NCSTN mutation versus wild-type mice, and hair cortex cytokeratin, trichohyalin, nicastrin, and NICD were downregulated in these mice. CONCLUSIONS: This NCSTN mutant mouse model could be an improved model to study early lesion development aspects of human HS pathogenesis and could perhaps be a better alternative for evaluating early-acting and preventive therapeutics for HS experimentally before clinical trials in HS patients. NCSTN mutations disrupt the development of hair follicles, leading to abnormal hair follicle structures, perhaps resulting in the onset of HS.

Epigenetic restoration of voltage-gated potassium channel Kv1.2 alleviates nerve injury-induced neuropathic pain.

Voltage-gated potassium channels (Kv) are important regulators of neuronal excitability for its role of regulating resting membrane potential and repolarization. Recent studies show that Kv channels participate in neuropathic pain, but the detailed underlying mechanisms are far from being clear. In this study, we used siRNA, miR-137 agomir, and antagomir to regulate the expression of Kv1.2 in spinal cord and dorsal root ganglia (DRG) of naïve and chronic constriction injury (CCI) rats. Kv currents and neuron excitability in DRG neurons were examined by patch-clamp whole-cell recording to verify the change in Kv1.2 function. The results showed that Kv1.2 was down-regulated in DRG and spinal dorsal horn (SDH) by CCI. Knockdown of Kv1.2 by intrathecally injecting Kcna2 siRNA induced significant mechanical and thermal hypersensitivity in naïve rats. Concomitant with the down-regulation of Kv1.2 was an increase in the expression of the miR-137. The targeting and regulating of miR-137 on Kcna2 was verified by dual-luciferase reporter system and intrathecal injecting miR-137 agomir. Furthermore, rescuing the expression of Kv1.2 in CCI rats, achieved through inhibiting miR-137, restored the abnormal Kv currents and excitability in DRG neurons, and alleviated mechanical allodynia and thermal hyperalgesia. These results indicate that the miR-137-mediated Kv1.2 impairment is a crucial etiopathogenesis for the nerve injury-induced neuropathic pain and can be a novel potential therapeutic target for neuropathic pain management.

MicroRNA-182 Alleviates Neuropathic Pain by Regulating Nav1.7 Following Spared Nerve Injury in Rats.

The sodium channel 1.7 (Nav1.7), which is encoded by SCN9A gene, is involved in neuropathic pain. As crucial regulators of gene expression, many miRNAs have already gained importance in neuropathic pain, including miR-182, which is predicted to regulate the SCN9A gene. Nav1.7 expression in L4-L6 dorsal root ganglions (DRGs) can be up regulated by spared nerve injury (SNI), while miR-182 expression was down regulated following SNI model. Exploring the connection between Nav1.7 and miR-182 may facilitate the development of a better-targeted therapy. In the current study, direct pairing of miR-182 with the SCN9A gene was verified using a luciferase assay in vitro. Over-expression of miR-182 via microinjection of miR-182 agomir reversed the abnormal increase of Nav1.7 at both mRNA and protein level in L4-6 DRGs of SNI rats, and significantly attenuated the hypersensitivity to mechanical stimulus in the rats. In contrast, administration of miR-182 antagomir enhanced the Nav1.7 expression at both mRNA and protein level in L4-6 DRGs, companied with the generation of mechanical hypersensitivity in naïve rats. Collectively, we concluded that miR-182 can alleviate SNI- induced neuropathic pain through regulating Nav1.7 in rats.

Glucocorticoid receptor inhibit the activity of NF-κB through p38 signaling pathway in spinal cord in the spared nerve injury rats.

AIMS: The glucocorticoid receptors (GRs) are an active regulator in inflammatory responses. The inflammatory reaction plays an important role in neuropathic pain, but the underlying mechanisms that GR regulates the inflammatory responses in neuropathic pain are still unknown. The activation of GRs has been shown to participate in the p38MAPK-mediated suppression of transcription activation. An unanswered question is whether GRs take part in inflammatory responses in neuropathic pain through p38MAPK signaling pathway. MAIN METHODS: The spared nerve injury (SNI) in rats was used as a model of neuropathic pain. Pain sensitivity was tested by von Frey filaments. The expression of GR, p-p38 and NF-κB were detected by Western blot and immunofluorescence. Elisa was used to examine the expression of IL-6 and TNF-α. KEY FINDINGS: Nerve injury led to p38 activation and GR expression decline in spinal cord of SNI rats. Intrathecal injection of the p38MAPK antagonist SB203580 activated GR and decreased NF-κB, resulting in pain relief since 3 days post-operation in SNI rats. Moreover, Intrathecal injection of the GR antagonist RU38486 counteracted the effect of SB203580 on NF-κB expression along with the release of IL-6 and TNF-α. On the contrary, activation of the GR by intrathecal administration of dexamethasone, a GR agonist, inhibited the expression of NF-κB and the release of IL-6 and TNF-α, resulting in pain relief. SIGNIFICANCE: Activation of p38MAPK in spinal cord could downregulate the GR expression and thereby activate NF-κB, thus promoting the release of IL-6 and TNF-α and participating in the development of neuropathic pain.

MiR-30b Attenuates Neuropathic Pain by Regulating Voltage-Gated Sodium Channel Nav1.3 in Rats.

Nav1.3 is a tetrodotoxin-sensitive isoform among voltage-gated sodium channels that are closely associated with neuropathic pain. It can be up-regulated following nerve injury, but its biological function remains uncertain. MicroRNAs (miRNAs) are endogenous non-coding RNAs that can regulate post-transcriptional gene expression by binding with their target mRNAs. Using Target Scan software, we discovered that SCN3A is the major target of miR-30b, and we then determined whether miR-30b regulated the expression of Nav1.3 by transfecting miR-30b agomir through the stimulation of TNF-α or by transfecting miR-30b antagomir in primary dorsal root ganglion (DRG) neurons. The spinal nerve ligation (SNL) model was used to determine the contribution of miR-30b to neuropathic pain, to evaluate changes in Nav1.3 mRNA and protein expression, and to understand the sensitivity of rats to mechanical and thermal stimuli. Our results showed that miR-30b agomir transfection down-regulated Nav1.3 mRNA stimulated with TNF-α in primary DRG neurons. Moreover, miR-30b overexpression significantly attenuated neuropathic pain induced by SNL, with decreases in the expression of Nav1.3 mRNA and protein both in DRG neurons and spinal cord. Activation of Nav1.3 caused by miR-30b antagomir was identified. These data suggest that miR-30b is involved in the development of neuropathic pain, probably by regulating the expression of Nav1.3, and might be a novel therapeutic target for neuropathic pain. Perspective: This study is the first to explore the important role of miR-30b and Nav1.3 in spinal nerve ligation-induced neuropathic pain, and our evidence may provide new insight for improving therapeutic approaches to pain.

MicroRNA-30b regulates expression of the sodium channel Nav1.7 in nerve injury-induced neuropathic pain in the rat.

Voltage-gated sodium channels, which are involved in pain pathways, have emerged as major targets for therapeutic intervention in pain disorders. Nav1.7, the tetrodotoxin-sensitive voltage-gated sodium channel isoform encoded by SCN9A and predominantly expressed in pain-sensing neurons in the dorsal root ganglion, plays a crucial role in nociception. MicroRNAs are highly conserved, small non-coding RNAs. Through binding to the 3' untranslated region of their target mRNAs, microRNAs induce the cleavage and/or inhibition of protein translation. Based on bioinformatics analysis using TargetScan software, we determined that miR-30b directly targets SCN9A To investigate the roles of Nav1.7 and miR-30b in neuropathic pain, we examined changes in the expression of Nav1.7 in the dorsal root ganglion by miR-30b over-expression or knockdown in rats with spared nerve injury. Our results demonstrated that the expression of miR-30b and Nav1.7 was down-regulated and up-regulated, respectively, in the dorsal root ganglion of spared nerve injury rats. MiR-30b over-expression in spared nerve injury rats inhibited SCN9A transcription, resulting in pain relief. In addition, miR-30b knockdown significantly increased hypersensitivity to pain in naive rats. We also observed that miR-30b decreased Nav1.7 expression in PC12 cells. Taken together, our results suggest that miR-30b plays an important role in neuropathic pain by regulating Nav1.7 expression. Therefore, miR-30b may be a promising target for the treatment of chronic neuropathic pain.

Intrathecal injection of fluorocitric acid inhibits the activation of glial cells causing reduced mirror pain in rats.

BACKGROUND: Growing evidence has shown that unilateral nerve injury results in pain hypersensitivity in the ipsilateral and contralateral sides respective to the injury site. This phenomenon is known as mirror image pain (MIP). Glial cells have been indicated in the mechanism of MIP; however, it is not clear how glial cells are involved in MIP. METHODS: To observe phenomenon MIP and the following mechanism, 20 adult male Sprague-Dawley rats (weighing 180-220 g) were separated into two groups: Sham Group (n = 10) and left L5 spinal nerve ligated and sectioned (SNL) group (n = 10). Thermal hyperalgesia and mechanical hypersensitivity were measured for both groups to determine if the SNL model had Mirror image of Pain (MIP). Nav1.7 protein expression in DRG was analyzed using immunohistochemistry and western-blotting. And then to observe the effect of fluorocitrate on MIP, 15 rats were separated into three Groups: Sham Group (n = 5); SNL + FC group: intrathecal injection of Fluorocitric acid(FC) 1 nmol/10 µL (n = 5); SNL + NS group: intrathecal injection of 0.9% Normal Saline (n = 5). Behavior testing, immunocytochemistry, and western-blotting using dorsal root ganglion (DRG) from both sides were then conducted. RESULTS: The results showed pain hypersensitivity in both hind-paws of the SNL animals, Mechanical tests showed the paw withdrawal threshold dropped from 13.30 ± 1.204 g to 2.57 ± 1.963 g at 14 d as will as the ipsilateral paw thermal withdrawal threshold dropped from 16.5 ± 2.236 s to 4.38 ± 2.544 s at 14 d. Mechanical tests showed the contralateral paw withdrawal threshold dropped from 14.01 ± 1.412 to 4.2 ± 1.789 g at 7d will the thermal withdrawal threshold dropped from 16.8 ± 2.176 s to 7.6 ± 1.517 s at 7d. Nav1.7 expression increased and glial cells actived in bilateral side DRG after SNL compared with sham group. After intrathecal injection of fluorocitrate, the glial cell in bilateral DRG were inhibited and the pain behavior were reversed in both hindpaws too. CONCLUSIONS: Fluorocitrate can inhibit the activation of glial cells in spinal cord and DRG, and reduce MIP.