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Lumbar spinal stenosis (LSS) is a common degenerative spinal condition in older individuals that causes impaired walking and other disabilities due to severe lower back and leg pain. Ligamentum flavum hypertrophy is a major LSS cause that may result from oxidative stress caused by degenerative cascades, including imbalanced iron homeostasis that leads to excessive reactive oxygen species production. We investigated the effects of Harpagophytum procumbens (HP) on iron-induced oxidative stress associated with LSS pathophysiology. Primary spinal cord neuron cultures were incubated in FeSO4-containing medium, followed by addition of 50, 100, or 200 μg/mL HP. Cell viability was assessed by CCK-8 and live/dead cell assays and by propidium iodide-live imaging. In an in vivo rat model of LSS, HP were administered at 100, 200, and 400 mg/kg, and disease progression was monitored for up to 3 weeks. We investigated the in vitro and in vivo effects of HP on iron-induced neurotoxicity by immunochemistry, real-time PCR, and flow cytometry. HP exerted neuroprotective effects and enhanced neurite outgrowths of iron-injured rat primary spinal cord neurons in vitro. HP treatment significantly reduced necrotic cell death and improved cells’ antioxidative capacity via the NRF2 signaling pathway in iron-treated neurons. At 1 week after HP administration in LSS rats, the inflammatory response and oxidative stress markers were substantially reduced through regulation of excess iron accumulation. Iron that accumulated in the spinal cord underneath the implanted silicone was also regulated by HP administration via NRF2 signaling pathway activation. HP-treated LSS rats showed gradually reduced mechanical allodynia and amelioration of impaired behavior for 3 weeks. We demonstrated that HP administration can maintain iron homeostasis within neurons via activation of NRF2 signaling and can consequently facilitate functional recovery by regulating iron-induced oxidative stress. This fundamentally new strategy holds promise for LSS treatment.

BACKGROUND CONTEXT Lumbar intervertebral disc herniation (LDH) is a common disease that causes low back pain, radiating leg pain, and sensory impairment. Preclinical studies rely heavily upon standardized animal models of human diseases to predict clinical treatment efficacy and to identify and investigate potential adverse events in human subjects. The current method for making the LDH model involves harvesting the nucleus pulposus (NP) from autologous coccygeal discs and applying to the lumbar nerve roots just proximal to the corresponding dorsal root ganglion. However, this surgical method generates a model that exhibits very different characteristics of disc herniation than that observed in human. PURPOSE To produce a rat LDH model that better resembles disc herniation in humans and a standardized and uniform LDH model using Interleukin-1 beta (IL-1β). STUDY DESIGN Experimental rat LDH model. METHODS We exposed the L5–6 disc dorsolaterally on the right side through hemi-laminectomy without nerve compression. Herniation was initiated by puncturing the exposed disc with a 30-gauge needle at a depth of 4 mm. Interleukin-1 beta (IL-1β) was injected simultaneously to heighten the pathological processes of disc degeneration, including inflammatory responses, matrix destruction, and herniation of the NP. We performed histological staining to assess morphological changes, immunohistochemistry to analyze inflammation- and pain-related expression within and around the puncture site of the L5–6 disc, and real-time polymerase chain reaction to examine expression of markers for degenerative processes. In addition, we performed locomotor tests on the rats. RESULTS We found that the IL-1β groups showed that the border between the annulus fibrosis and nucleus pulposus was severely interrupted compared to that of the control (puncture only) group. And, the injection of IL-1β leads to accelerated disc degeneration and inflammation in a more consistent manner in LDH model. Functional deficit was consistently induced by puncturing and injection of IL-1β in the exposed disc. CONCLUSIONS The method proposed here can be used as an index to control the severity of disc degeneration and inflammation through the injected IL-1β concentration concurrent with surgically induced herniation. CLINICAL SIGNIFICANCE Our proposed model may facilitate research in drug development to evaluate the efficacy of potential therapeutic agents for disc herniation and neuropathic pain and may also be used for nonclinical studies to more accurately assess the effectiveness of various treatment strategies according to the severity of disc degeneration.

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

Background: Lumbar spinal stenosis (LSS) is defined as the narrowing of the spinal canal, which compresses the nerves traveling through the lower back into the legs. Inflammation is the most common cause of LSS. Chronic pain induced by nerve damage results from chronic inflammation, and the inflammation response worsens with elevated iron stores. Furthermore, macrophage polarization to the M1 (inflammatory) or M2 (anti-inflammatory) type is essential for controlling host defense or repairing tissues. However, the precise function of macrophage polarization in iron release or retention in LSS pathophysiology is not well-understood. Here, we introduce melittin to modulate macrophage polarization related to iron metabolism for LSS treatment.Methods: Primary peritoneal macrophage were cultured in 200 or 500 ng/mL of melittin and FeSO4-containing medium for 24 h. Macrophage polarization was assessed by Immunofluorescence staining to CD86 or Arg1 antibodies. In an in vivo rat model of LSS, melittin were administered at 100 and 250 µg/kg, and in vivo effects of melittin on iron deposition-induced macrophage polarization was evaluated by immunochemistry, real time-PCR, and flow-cytometry. The locomotor functions were assessed by BBB, ladder scoring, and Von Frey test for up to 3 weeks. Results: In vitro experiments demonstrated that macrophages can be polarized toward an M2 phenotype after melittin treatment in iron-insulted primary macrophages. Treatment with 100 and 250 μg/kg melittin in a rat LSS model increased the proportion of M2 macrophages in the damaged spinal cord. Moreover, we found that melittin attenuated iron overload-induced M1 polarization via regulating iron metabolism-related genes in LSS rats. As a result, melittin improved locomotor recovery and stimulated axonal growth following LSS.Conclusion: Melittin can promote functional recovery in LSS models by activating M2 macrophages via controlling macrophage iron metabolism, suggesting the potential applications of melittin for treating LSS.

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.

Cervus elaphus sibericus (CES), commonly known as deer antler, has been used as a medicinal herb because of its various pharmacological activities, including its anti-infective, anti-arthritic, anti-allergic, and anti-oxidative properties. However, the precise mechanisms by which CES functions as a potent anti-oxidative agent remain unknown, particularly, the effects of CES on cortical neurons and its neurobiological mechanism have not been examined. We used primary cortical neurons from the embryonic rat cerebral cortex and hydrogen peroxide to induce oxidative stress and damage in neurons. After post-treatment of CES at three concentrations (10, 50, and 200 µg/mL), the influence of CES on the neurobiological mechanism was assessed by immunocytochemistry, flow cytometry, and real-time PCR. CES effectively prevented neuronal death caused by hydrogen peroxide-induced damage by regulating oxidative signaling. In addition, CES significantly induced the expression of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated genes. We also observed newly processing elongated axons after CES treatment under oxidative conditions. In addition, filopodia tips generally do not form a retraction bulb, called swollen endings. Thus, CES shows therapeutic potential for treating neurological diseases by stimulating neuron repair and regeneration.

Simple Summary Oxidative stress plays a crucial role in nerve injury-induced changes, including the cellular redox state, greater disease severity, and reduced function. Most studies have demonstrated that the modulation of oxidative damage to neurons leads to improved survival and functional recovery. Deer antlers are considered a potent natural source of antioxidants. Thus, we examined the neurotherapeutic effect of Cervus elaphus sibericus in the axon regeneration and growth cone reformation of cortical neurons. The effect of three doses of C. elaphus sibericus on cortical neurons was determined in a hydrogen peroxide-induced injury model using immunocytochemistry, flow-cytometry, and real-time PCR. We found that C. elaphus sibericus effectively inhibited neuronal death following hydrogen peroxide-induced injury by significantly inhibiting oxidative stress and consequently promoting neuron repair and axon regeneration in cortical neurons. Abstract Cervus elaphus sibericus (CES), commonly known as deer antler, has been used as a medicinal herb because of its various pharmacological activities, including its anti-infective, anti-arthritic, anti-allergic, and anti-oxidative properties. However, the precise mechanisms by which CES functions as a potent anti-oxidative agent remain unknown; particularly, the effects of CES on cortical neurons and its neurobiological mechanism have not been examined. We used primary cortical neurons from the embryonic rat cerebral cortex and hydrogen peroxide to induce oxidative stress and damage in neurons. After post-treatment of CES at three concentrations (10, 50, and 200 µg/mL), the influence of CES on the neurobiological mechanism was assessed by immunocytochemistry, flow cytometry, and real-time PCR. CES effectively prevented neuronal death caused by hydrogen peroxide-induced damage by regulating oxidative signaling. In addition, CES significantly induced the expression of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated genes. We also observed newly processing elongated axons after CES treatment under oxidative conditions. In addition, filopodia tips generally do not form a retraction bulb, called swollen endings. Thus, CES shows therapeutic potential for treating neurological diseases by stimulating neuron repair and regeneration.

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.

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.

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.

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.

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.

We explored prognostic roles of circulating microRNAs (miRs) in multiple myeloma (MM) treated with autologous stem cell transplantation (ASCT) following induction chemotherapy. In part I of the study (n = 40), we identified a decreasing dynamics of circulating miR-193a-5p expression from diagnosis to pre-ASCT. In patients who experienced early relapse within one year post ASCT (n = 9) these patterns were distinctive compared to those without early relapse in a 1:2 matched cohort (n = 18). In part II (n = 90), multivariate analyses showed that the International Staging System score and miR-193a-5p expression before ASCT were independent prognostic factors. Conclusively, expression of circulating miR-193a-5p before ASCT could be a prognostic biomarker for transplant-eligible MM.

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.

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.