Crocin Inhibits Oxidative Stress and Pro-inflammatory Response of Microglial Cells Associated with Diabetic Retinopathy Through the Activation of PI3K/Akt Signaling Pathway.

Diabetic retinopathy (DR) is a serious microvascular complication of diabetes mellitus that is closely associated with the degeneration and loss of retinal ganglion cells (RGCs) caused by diabetic microangiopathy and subsequent oxidative stress and an inflammatory response. Microglial cells are classed as neurogliocytes and play a significant role in neurodegenerative diseases. Over-activated microglial cells may cause neurotoxicity and induce the death and apoptosis of RGCs. Crocin is one of the two most pharmacologically bioactive constituents in saffron. In the present study, we focused on the role of microglial cells in DR, suggesting that DR may cause the over-activation of microglial cells and induce oxidative stress and the release of pro-inflammatory factors. Microglial cells BV-2 and N9 were cultured, and high-glucose (HG) and free fatty acid (FFA) were used to simulate diabetes. The results showed that HG-FFA co-treatment caused the up-regulated expression of CD11b and Iba-1, indicating that BV-2 and N9 cells were over-activated. Moreover, oxidative stress markers and pro-inflammatory factors were significantly enhanced by HG-FFA treatment. We found that crocin prevented the oxidative stress and pro-inflammatory response induced by HG-FFA co-treatment. Moreover, using the PI3K/Akt inhibitor LY294002, we revealed that PI3K/Akt signaling plays a significant role in blocking oxidative stress, suppressing the pro-inflammatory response, and maintaining the neuroprotective effects of crocin. In total, these results provide a new insight into DR and DR-induced oxidative stress and the inflammatory response, which provide a potential therapeutic target for neuronal damage, vision loss, and other DR-induced complications.

Puerarin Attenuates N-Methyl-D-aspartic Acid-induced Apoptosis and Retinal Ganglion Cell Damage Through the JNK/p38 MAPK Pathway.

PURPOSE: To explore the protective effect of puerarin on N-methyl-D-aspartic acid (NMDA)-induced retinal ganglion cells (RGCs) injury and its underlying mechanism. MATERIALS AND METHODS: Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. NMDA was used to mimic the glutamate activation, cell apoptosis, reactive oxygen species (ROS), malondialdehyde levels, SOD and NO production, nNOS and iNOS expression, as well as caspase-3 activity, Bcl-2, and Bax expression in the RGCs were analyzed by ELISA, RT-PCR, and Western blotting. A rat model of retinal injury was used to detect the protective effect of puerarin. RESULTS: Puerarin protected against NMDA-induced RGCs injury in a dose-dependent manner. Compared with the NMDA-treated group, puerarin pretreatment significantly reduced ROS and malondialdehyde levels, promoted SOD and NO production, and downregulated nNOS and iNOS expression in the RGCs. Mechanism analysis showed that pretreatment with puerarin could effectively offset the increase of Bax expression and caspase-3 activity brought by NMDA, and promote Bcl-2 expression in the RGCs. Puerarin pretreatment also effectively inhibited NMDA-induced JNK and p38 phosphorylation in the RGCs, whereas pretreatment with either JNK agonist anisomycin or p38 agonist P79350 could significantly compensate the effects caused by puerarin. Furthermore, puerarin prevented RGCs loss in the retinal injury induced by intravitreal NMDA in a rat model. CONCLUSIONS: The present results of this study demonstrated that puerarin protected against NMDA-induced apoptosis and RGCs damage through the JNK/p38 MAPK pathway.

Crocin Upregulates CX3CR1 Expression by Suppressing NF-κB/YY1 Signaling and Inhibiting Lipopolysaccharide-Induced Microglial Activation.

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Microglial activation has been shown to be deleterious to RGCs and may participate in the progression of glaucoma. Crocin, one of the major active ingredients in saffron, has been found to inhibit microglial activation. However, the mechanism remains unclear. The aim of this study was to investigate whether crocin can inhibit lipopolysaccharide (LPS)-induced microglial activation and to clarify the mechanisms involved. The influence of crocin on primary RGCs and LPS-stimulated BV2 microglial cells survival was determined by the MTT and lactate dehydrogenase assays, or by flow cytometry. BV2 cells were pretreated with various concentrations of crocin for 2 h followed by 1 µg/mL LPS stimulation. Microglial markers and pro-inflammatory mediators were assessed by real-time PCR, western blot and ELISA. Furthermore, CX3CR1 expression was detected and the underlying mechanism was examined. The concentrations of crocin ranged from 0.1 to 1 µM, and did not show any cytotoxicity in RGC and BV2 cells. After crocin pretreatment, the expression of microglial markers (CD11b and Iba-1) and pro-inflammatory mediators (iNOS, COX-2, IL-1ß, and TNF-α) induced by LPS were significantly decreased in a dose-dependent manner. Additionally, CX3CR1 expression was remarkably increased by crocin via the suppression of NF-κB/Yin Yang 1 (YY1) signaling in BV2 cells. In conclusion, crocin effectively suppresses microglial activation and upregulates CX3CR1 expression by suppressing NF-κB/YY1 signaling.

Crocin protects retinal ganglion cells against H2O2-induced damage through the mitochondrial pathway and activation of NF-κB.

Glaucoma is a degenerative nerve disorder that results in irreversible blindness. It has been reported that the apoptosis of retinal ganglion cells (RGCs) is a hallmark of glaucoma. Oxidative stress is one of the major factors that cause apoptosis of RGCs. Crocin has many beneficial effects, including antioxidant and anti-apoptotic actions. However, the mechanism by which crocin protects against oxidative stress­induced damage to RGCs remains unclear. The present study aimed to investigate the mechanism by which crocin protects RGC-5 cells against H2O2-induced damage. H2O2 was used to establish a model of oxidative stress injury in RGC-5 cells to mimic the development of glaucoma in vitro. Different concentrations (0.1 and 1 µM) of crocin were added to test whether crocin was capable of protecting RGCs from H2O2-induced damage. WST-1, lactic dehydrogenase (LDH) release and Annexin V/FITC assays were then performed. Levels of reactive oxygen species (ROS) were detected using a ROS assay kit, mitochondrial membrane potential (ΔΨm) was analyzed by JC-1 staining, caspase-3 activity was examined using a Caspase-3 assay kit, and the protein levels of Bax, Bcl-1 and cytochrome c were measured using western blot analysis. In addition, the protein level of phosphorylated nuclear factor-κB (p-NF-κB) p65 was also evaluated using western blot analysis. The results showed that crocin protected RGC-5 cells from apoptosis, decreased LDH release and enhanced cell viability. Additional experiments demonstrated that crocin decreased ROS levels, increased ΔΨm, downregulated the protein expression of Bax and cytochrome c, promoted Bcl-2 protein expression and activated NF-κB. Taken together, the findings of this study indicate that crocin prevented H2O2­induced damage to RGCs through the mitochondrial pathway and activation of NF-κB.

Human Ermin (hErmin), a new oligodendrocyte-specific cytoskeletal protein related to epileptic seizure.

During the maturation of oligodendrocytes, cells are characterized by their morphological changes such as the number of process extensions and sheet-like membranes. This process relies critically on cytoskeleton rearrangement, but the molecular mechanisms underlying this are still unclear. Here, we identify human Ermin (hErmin), a novel cytoskeletal molecule that is expressed exclusively in oligodendrocytes in human brain, as a regulator of cytoskeleton rearrangement. In vitro, full-length hErmin expression, but not its truncated mutants lacking the actin-binding domain, promote arborization of cultured COS-7 cells and induce marked changes in cell morphology. The most important is that expression of hErmin in specimens of epileptic patients is much lower than that of control, implying that hErmin may be involved in epileptogenic process. These findings suggest a role for hErmin as a novel cytoskeleton-related oligodendroglial protein in human brain myelination and human epileptogenesis, and provide new evidence for the relationship between oligodendrocytes and epilepsy.

Activation of serotonin 1A receptors in ventrolateral orbital cortex depresses persistent nociception: a presynaptic inhibition mechanism.

The present study examined the effect of serotonin 1A (5-HT(1A)) receptor activation in the ventrolateral orbital cortex (VLO) upon formalin-evoked flinching behavior and spinal Fos expression, and further determined whether activation of 5-HT(1A) receptors affected the spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in rat VLO slice by pharmacologically separated neurons to understand the possible mechanism underlying this effect. Microinjection of the 5-HT(1A) receptors agonist 8-OH-DPAT (8-hydro-2-(di-n-propylamino) tetralin) into the VLO depressed the formalin-evoked nociceptive behavior flinching response and the Fos expression in the lumbar spinal cord dorsal, which was antagonized by pre-treatment with 5-HT(1A) receptors antagonist NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide). Furthermore, application of 8-OH-DPAT into VLO slice inhibited GABAergic mIPSC frequency in a dose-dependent manner without effects on amplitude of the GABAergic mIPSCs, this effect was blocked by NAN-190. These results provide evidence for the involvement of 5-HT(1A) receptors in VLO in the modulation of persistent inflammatory nociception, and suggest that a presynaptic inhibition of the GABA release may contribute to the 5-HT(1A) receptor-mediated descending antinociception.

Topographical distributions of endomorphinergic pathways from nucleus tractus solitarii to periaqueductal gray in the rat.

In the central nervous system (CNS), endomorphin 1 (EM1)- and endomorphin 2 (EM2)-containing neuronal cell bodies have been found in the nucleus tractus sollitarii (NTS) and the hypothalamus, and EMergic fibers and terminals are distributed widely in many regions of the CNS, including the periaqueductal gray (PAG). The aim of the present study was to examine whether EM-expressing neurons in the NTS of the rat send their axons to the PAG, and determine whether the EMergic pathway from the NTS to the PAG is topographic by using. Immunofluorescent staining for EM1 or EM2 combined with retrograde and anterograde tract-tracing methods. The results showed that after injecting tetramethyl rhodamine dextran-amine (TMR) into the ventrolateral or lateral column of the PAG, some EM1- or EM2-immunoreactive (IR) neurons in the NTS were retrogradely labeled with TMR, and the majority of the EM-IR/TMR double-labeled neurons were mainly distributed in the medial and commissural subnuclei of the NTS. Following injection of biotinylated dextran amine (BDA) into the medial or commissural subnucleus of the NTS, EM1-IR/BDA and EM2-IR/BDA double-labeled fibers and terminals were mainly distributed in the ventrolateral or lateral column of the PAG, respectively. The results indicate that EMergic pathway from the NTS to PAG is topographically organized, and suggest that EMs released from NTS to PAG projecting terminals may bind to mu-opioid receptor on the PAG neurons, and thereby contribute to various functions.

A novel scaffold with longitudinally oriented microchannels promotes peripheral nerve regeneration.

Longitudinally oriented microstructures are essential for a nerve scaffold to promote significant regeneration of injured peripheral axons across nerve gaps. Extensive attention has been devoted to develop scaffolds with inner structures mimicking the nerve-guiding basal lamina microchannels in autografts. However, to date, little information has been obtained about scaffolds with similar inner microstructures, and the efficacy of such scaffolds in bridging peripheral nerve gaps in vivo has never been examined. In the present study, we describe a novel nerve-guiding collagen-chitosan (CCH) scaffold with inner dimensions resembling the basal lamina microchannels of normal nerves. The scaffold has a number of structural advantages, including longitudinally orientated microchannels and extensive interconnected pores between the parallel microchannels. We evaluated the efficacy of the CCH scaffold to bridge a 15-mm-long sciatic nerve defect in rats using a combination of morphological and functional techniques. The in vivo animal study showed that the CCH scaffold achieved nerve regeneration and functional recovery equivalent to that of an autograft, without the exogenous delivery of regenerative agents or cell transplantation. These findings demonstrate that CCH scaffolds may be used as alternatives to nerve autografts for peripheral nerve regeneration.

Synaptic connections between GABAergic elements and serotonergic terminals or projecting neurons in the ventrolateral orbital cortex.

The ventrolateral orbital cortex (VLO) is part of an endogenous analgesic system, consisting of the spinal cord-thalamic nucleus submedius-VLO periaqueductal gray (PAG)-spinal cord loop. The present study examined morphological connections of GABAergic (gamma-aminobutyric acidergic) neurons and serotonergic projection terminals from the dorsal raphe nucleus (DR), as well as the relationship between GABAergic terminals and VLO neurons projecting to the PAG, by using anterograde and retrograde tracing combined with immunofluorescence, immunohistochemistry, and electron microscopy methods. Results indicate that the majority (93%) of GABAergic neurons in the VLO also express the 5-HT(1A) (5-hydroxytryptamine 1A) receptor, and serotonergic terminals originating from the DR nucleus made symmetrical synapses with GABAergic neuronal cell bodies and dendrites within the VLO. GABAergic terminals also made symmetrical synapses with neurons expressing GABA(A) receptors and projecting to the PAG. These results suggest that a local neuronal circuit, consisting of 5-HTergic terminals, GABAergic interneurons, and projection neurons, exists in the VLO, and provides morphological evidence for the hypothesis that GABAergic modulation is involved in 5-HT(1A) receptor activation-evoked antinociception.