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Hyunseong Kim,1 Jin Young Hong,1 Wan-Jin Jeon,1 Junseon Lee,1 Seung Ho Baek,2 In-Hyuk Ha1 1Jaseng Spine and Joint Research Institute, Jaseng Medical Foundation, Seoul, 135-896, Republic of Korea; 2College of Korean Medicine, Dongguk University, Goyang-si, 10326, Republic of KoreaCorrespondence: In-Hyuk HaJaseng Spine and Joint Research Institute, Jaseng Medical Foundation, Seoul, 135-896, Republic of KoreaTel +82-2-2222-2740Fax +82-2-527-1869Email hanihata@gmail.comPurpose: Lycopus lucidus Turcz (LLT) is a potent traditional medicinal herb that exerts therapeutic effects, regulating inflammatory disorders. However, the precise mechanisms by which LLT plays a potent role as an anti-inflammatory agent are still unknown, and in particular, the effects of LLT on cortical neurons and related mechanisms of neuroinflammation have not been studied. The NLRP3 inflammasome pathway is one of the most well known as an important driver of inflammation. We therefore hypothesized that LLT, as an effective anti-inflammatory agent, might have neurotherapeutic potential by inhibiting the NLRP3 inflammasome pathway in cortical neurons.Materials and Methods: Primary cortical neurons were isolated from the embryonic rat cerebral cortex, and H2O2 was used to stimulate neuron damage in vitro. After treatment with LLT at three concentrations (10, 25, and 50 μg/mL), the expression of iNOS, NLRP3, ASC, caspase-1, IL-1β, IL-18, IL-6, and IL-10 was determined by immunocytochemistry, qPCR, and ELISA. Neuron apoptosis was also evaluated using Annexin V-FITC/PI double staining FACS analysis. Neural regeneration-related factors (BDNF, NGF, synaptophysin, NT3, AKT, and mTOR) were analyzed by immunocytochemistry and qPCR.Results: LLT effectively protected cultured rat cortical neurons from H2O2-induced neuronal injury by significantly inhibiting NLRP3 inflammasome activation. In addition, it significantly reduced caspase-1 activation, which is induced by inflammasome formation and regulated the secretion of IL-1β/IL-18. We demonstrated that LLT enhances axonal elongation and synaptic connectivity upon H2O2-induced neuronal injury in rat primary cortical neurons.Conclusion: It was first demonstrated in vitro that LLT suppresses NLRP3 inflammasome activation, attenuates inflammation and apoptosis, and consequently promotes neuroprotection and the stimulation of neuron repair, suggesting that it is a promising therapeutic for neurological diseases.Keywords: Lycopus lucidus Turcz, cortical neuron, hydrogen peroxide, NLRP3 inflammasome, neuroinflammation

Gongjin-dan (GJD) is a multiherbal formula produced from 10 medicinal herbs and has been traditonally used as an oriental medicine to treat cardiovascular diseases, alcoholic hepatitis, mild dementia, and anemia. Additionally, increasing evidence suggests that GJD exerts neuroprotective effects by suppressing inflammation and oxidative stress-induced events to prevent neurological diseases. However, the mechanism by which GJD prevents oxidative stress-induced neuronal injury in a mature neuron remains unknown. Here, we examined the preventive effect and mechanism of GJD on primary cortical neurons exposed to hydrogen peroxide (H2O2). In the neuroprotection signaling pathway, Sirtuin1 is involved in neuroprotective action as a therapeutic target for neurological diseases. After pre-treatment with GJD at three concentrations (10, 25, and 50 µg/mL) and stimulation by H2O2 (30 µM) for 24 h, the influence of GJD on Sirtuin1 activation was assessed using immunocytochemistry, real-time PCR, western blotting, and flow cytometry. GJD effectively ameliorated H2O2-induced neuronal death against oxidative damage through Sirtuin1 activation. In addition, GJD-induced Sirtuin1 activation accelerated elongation of new axons and formation of synapses via increased expression of nerve growth factor and brain-derived neurotrophic factor, as well as regeneration-related genes. Thus, GJD shows potential for preventing neurological diseases via Sirtuin1 activation.

Apamin is a minor component of bee venom and is a polypeptide with 18 amino acid residues. Although apamin is considered a neurotoxic compound that blocks the potassium channel, its neuroprotective effects on neurons have been recently reported. However, there is little information about the underlying mechanism and very little is known regarding the toxicological characterization of other compounds in bee venom. Here, cultured mature cortical neurons were treated with bee venom components, including apamin, phospholipase A2, and the main component, melittin. Melittin and phospholipase A2 from bee venom caused a neurotoxic effect in dose-dependent manner, but apamin did not induce neurotoxicity in mature cortical neurons in doses of up to 10 µg/mL. Next, 1 and 10 µg/mL of apamin were applied to cultivate mature cortical neurons. Apamin accelerated neurite outgrowth and axon regeneration after laceration injury. Furthermore, apamin induced the upregulation of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated gene expression in mature cortical neurons. Due to its neurotherapeutic effects, apamin may be a promising candidate for the treatment of a wide range of neurological diseases.

Aims CREB (cAMP response element-binding protein)-regulated transcription coactivator (CRTC2) has been reported to act as a coactivator of CREB during gluconeogenesis. The role of CRTC2 in osteoblastic differentiation has not yet been elucidated. The aim of this study is to identify the mechanism of CRTC2 in osteoblast differentiation. Main methods The mRNA expression was determined by RT-PCR and qPCR. Protein levels were measured using Western blot assay. Alkaline phosphatase (ALP) staining was performed to evaluate ALP activity. Alizarin red S (ARS) staining was performed to measure extracellular mineralization. Transcriptional activity was detected using a luciferase assay. Key findings In the present study, TNF-α was found to stimulate CRTC2 expression. However, TNF-α did not increase the gene expression of osteoblast differentiation markers and inhibited BMP2-induced osteoblastic differentiation. Overexpression of CRTC2 decreased the expression of osteogenic genes, ALP activity and extracellular matrix mineralization. Knockdown of CRTC2 restored BMP2-induced osteogenic gene expression and ALP activity. CRTC2 increased Smurf1 mRNA expression, Smurf 1 promoter activity, and protein level. Furthermore, Smurf 1 decreased Smad 1/5/9 protein levels. These results suggest that CRTC2 decreased BMP2-induced osteoblastic differentiation via Smurf 1 expression. Significance Our results indicate that CRTC2 regulates the expression of Smurf1 in osteoblast differentiation.

Lumbar spinal stenosis (LSS) is a common neurodegenerative condition. However, how neurogenic claudication develops with severe leg pain has not yet been clearly elucidated. Moreover, cerebrospinal fluid (CSF) physiology at the lumbosacral level is poorly understood because of the difficulties involved in quantification and visualization. Recent studies have suggested that assessment of mitochondrial function in CSF provides an indirect way to assess neurological disorders and an important feature of disease progression. In this study, we assessed the relevance of endogenous extracellular mitochondria in the CSF of rats after LSS. Mitochondrial changes within the CSF were analyzed following LSS at 1 week using flow cytometry. An increase in cell size and number was observed in CSF with LSS, and reactive oxygen species (ROS) levels were also increased within the CSF at 1 week in the LSS group. Elevated mitochondrial ROS and functional changes in the CSF are hallmarks of LSS. The present study is the first to demonstrate that elevated mitochondrial ROS within the CSF is a new index for the early detection of LSS. Moreover, it may represent a potential novel treatment target for LSS.

Inula britannica var. chinensis (IBC) has been used as a traditional medicinal herb to treat inflammatory diseases. Although its anti-inflammatory and anti-oxidative effects have been reported, whether IBC exerts neuroprotective effects and the related mechanisms in cortical neurons remain unknown. In this study, we investigated the effects of different concentrations of IBC extract (5, 10, and 20 µg/mL) on cortical neurons using a hydrogen peroxide (H2O2)-induced injury model. Our results demonstrate that IBC can effectively enhance neuronal viability under in vitro-modeled reaction oxygen species (ROS)-generating conditions by inhibiting mitochondrial ROS production and increasing adenosine triphosphate level in H2O2-treated neurons. Additionally, we confirmed that neuronal death was attenuated by improving the mitochondrial membrane potential status and regulating the expression of cytochrome c, a protein related to cell death. Furthermore, IBC increased the expression of brain-derived neurotrophic factor and nerve growth factor. Furthermore, IBC inhibited the loss and induced the production of synaptophysin, a major synaptic vesicle protein. This study is the first to demonstrate that IBC exerts its neuroprotective effect by reducing mitochondria-associated oxidative stress and improving mitochondrial dysfunction.

Inflammation is an essential biological response that eliminates pathogenic bacteria and repairs tissue after injury. Acute kidney injury (AKI) is associated with systemic and intrarenal inflammation as the inflammatory process decreases renal function and promotes progression to advanced chronic kidney disease. Macrophages are key mediators of the inflammatory response, their activation influences the immune system and may have various effects. Classically activated type I macrophages (M1) produce a variety of pro-inflammatory cytokines at the lesion site. However, anti-inflammatory type II macrophages (M2) are alternatively activated upon exposure to anti-inflammatory cytokines and are associated with wound healing and tissue repair following AKI. Here, we used melittin from bee venom to enhance the polarization of M2 macrophages and promote renal recovery after AKI. Melittin was administered to mice intraperitoneally for 5 days at various concentrations (10, 50, and 100 &micro, g/kg), serum creatinine and blood urea nitrogen (BUN) levels were analyzed 72 h after cisplatin administration to confirm renal dysfunction. Melittin inhibited the cisplatin-induced increase in creatinine and BUN, an indicator of renal dysfunction. The expression of M1 markers (CD16/32) decreased significantly, whereas that of M2 markers (CD206, Arg1nase I) increased after melittin administration. Consistently, tubular necrosis was substantially reduced in melittin-treated mice. Thus, melittin alleviates cisplatin-induced AKI by regulating M2 macrophage expression.

Oxidative stress plays a key role in the pathogenesis of several diseases, including neurodegenerative diseases. Recent studies have reported that mitochondrial dysfunction is a leading cause of the overproduction of reactive oxygen species and oxidative stress. Mitochondrial changes play an important role in preventing oxidative stress. However, there is a lack of experimental evidence supporting this hypothesis. Thymus quinquecostatus CELAK (TQC) extract is a plant from China belonging to the thymus species, which can mediate the inflammatory response and prevent cell damage through its antioxidant activities. This study examines whether TQC can scavenge excess ROS originating from the mitochondria in RAW 264.7 macrophages. We used lipopolysaccharide (LPS) to induce inflammation and oxidative stress in RAW 264.7 macrophages and performed an immunocytochemistry dot blot of 8-hydroxy-2&prime, deoxyguanosine (8-OHdG) and real-time PCR to analyze the expression levels of genes involved in mitochondrial biogenesis and oxidative metabolism. TQC was found to significantly reduce the intensity of immunostained MitoSOX and 8-OHdG levels in the total genomic DNA within the mitochondria in RAW 264.7 macrophages. The HO-1 and Nrf2 mRNA levels were also significantly increased in the TQC groups. Therefore, we verified that TQC improves mitochondrial function and attenuates oxidative stress induced by LPS. Our results can provide reference for the effect of TQC to develop new therapeutic strategies for various diseases.

A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 108/cm2. The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.

Lumbar spinal stenosis (LSS), one of the most commonly reported spinal disorders, can cause loss of sensation and dyskinesia. In currently used animal models of LSS, the spinal cord is covered entirely with a silicone sheet, or block-shaped silicone is inserted directly into the spinal canal after laminectomy. However, the effects of differences between these implant materials have not been studied. We assessed the degree of damage and locomotor function of an LSS model in Sprague-Dawley rats using silicone blocks of varying hardness (70, 80, and 90 kPa) implanted at the L4 level. In sham rats, the spinal cord remained intact; in LSS rats, the spinal cord was increasingly compressed by the mechanical pressure of the silicone blocks as hardness increased. Inflammatory cells were not evident in sham rats, but numerous inflammatory cells were observed around the implanted silicone block in LSS rats. CD68+ cell quantification revealed increases in the inflammatory response in a hardness-dependent manner in LSS rats. Compared with those in sham rats, proinflammatory cytokine levels were significantly elevated in a hardness-dependent manner, and locomotor function was significantly decreased, in LSS rats. Overall, this study showed that hardness could be used as an index to control the severity of nerve injury induced by silicone implants.

Aims Costunolide is a sesquiterpene lactones used in many herbal medicines, with well-established anti-inflammatory and anti-oxidant functions modulating endoplasmic reticulum (ER) stress pathways, and which promotes the expression of anti-oxidant genes. The aim of this study is to investigate whether costunolide is involved in osteoblast differentiation and, determine the mechanisms of differentiation in mesenchymal stem cells. Main methods The cytotoxicity of costunolide was identified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression levels of osteogenic genes were determined by RT-PCR and Western blot analysis. Alkaline phosphate (ALP) staining and Alizarin red S (ARS) staining were performed to evaluate ALP activity and matrix mineralization. Transcriptional activity was detected using a luciferase reporter assay. Key findings In this study, we determined that costunolide increased the expression of distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC) in C3H10T 1/2 cells. Furthermore, costunolide increased ALP activity and matrix mineralization. Interestingly, costunolide increased ER stress by Bip, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, it did not exert effects on expression of activating transcription factor 6 (ATF6). ATF4 activation has a protective role in oxidative stress, and its transcription induces anti-oxidant genes in cells. Heme oxygenase-1 (HO-1) is a major anti-oxidant enzyme, and is regulated by ATF4. We showed that costunolide treatment increased HO-1 expression. Furthermore, the HO-1 inhibitor, Sn(IV) Protoporphyrin IX dichloride (SnPP) was blocked costunolide-induced Runx2 expression. Significance Our results revealed that costunolide-induced osteoblast differentiation is regulated by ATF4-dependent HO-1 expression.