Neuroprotection of Chrysanthemum indicum Linne against cerebral ischemia/reperfusion injury by anti-inflammatory effect in gerbils

In this study, we tried to verify the neuroprotective effect of Chrysanthemum indicum Linne（CIL） extract, which has been used as a botanical drug in East Asia, against ischemic damage and to explore the underlying mechanism involving the anti-inflammatory approach. A gerbil was given CIL extract for 7 consecutive days followed by bilateral carotid artery occlusion to make a cerebral ischemia/reperfusion model. Then, we found that CIL extracts protected pyramidal neurons in the hippocampal CA1 region（CA1） from ischemic damage using neuronal nucleus immunohistochemistry and Fluoro-Jade B histofluorescence. Accordingly, interleukin-13 immunoreactivities in the CA1 pyramidal neurons of CIL-pretreated animals were maintained or increased after cerebral ischemia/reperfusion. These findings indicate that the pre-treatment of CIL can attenuate neuronal damage/death in the brain after cerebral ischemia/reperfusion via an anti-inflammatory approach.

Proteomic analysis of the dorsal spinal cord in the mouse model of spared nerve injury-induced neuropathic pain

Peripheral nerve injury often causes neuropathic pain and is associated with changes in the expression of numerous proteins in the dorsal horn of the spinal cord. To date, proteomic analysis method has been used to simultaneously analyze hundreds or thousands of proteins differentially expressed in the dorsal horn of the spinal cord in rats or dorsal root ganglion of rats with certain type of peripheral nerve injury. However, a proteomic study using a mouse model of neuropathic pain could be attempted because of abundant protein database and the availability of transgenic mice. In this study, whole proteins were extracted from the ipsilateral dorsal half of the 4th-6th lumbar spinal cord in a mouse model of spared nerve injury（SNI）-induced neuropathic pain. In-gel digests of the proteins size-separated on a polyacrylamide gel were subjected to reverse-phase liquid-chromatography coupled with electrospray ionization ion trap tandem mass spectrometry（MS/MS）. After identifying proteins, the data were analyzed with subtractive proteomics using ProtAn, an in-house analytic program. Consequently, 15 downregulated and 35 upregulated proteins were identified in SNI mice. The identified proteins may contribute to the maintenance of neuropathic pain,and may provide new or valuable information in the discovery of new therapeutic targets for neuropathic pain.

Hyperglycemia Induced Apoptosis Changes in Salivary Gland Cells of Mice

If the function of salivary glands that secrete saliva is degraded, saliva secretion will decrease and symptoms of xerostomia will appear. Although symptoms of xerostomia are often felt by patients with diabetes, the relationship between diabetes and xerostomia is currently unclear. In relation to DM, there are studies on salivation flow and saliva composition, but there are not studies of apoptosis of salivary gland cells. The objective of this study was to investigate whether apoptosis in salivary glands of mice with hyperglycemic, a symptom of diabetes, might be altered based on immunohistochemical analysis. This study used mice with hyperglycemia. Immunohistochemistry and Western blot analyses were performed using Fas, Bax, and cleaved caspase-3 antibodies. These antibodies are used not only as death receptors, but also are antibodies that activate upstream and downstream signals of apoptosis. TUNEL assay was performed to detect apoptosis by immunofluorescence using TdT enzyme. It was observed that the expression level of apoptosis signaling molecules and TUNEL positive cells were increased in hyperglycemia group (HG). As a result, there are many apoptosis cells in the HG groups of the salivary gland. The results of this study, the function of salivary gland could occur deteriorated due to apoptosis on salivary gland cells by hyperglycemic, a characteristic of diabetes.

Streptococcal SspB Peptide Analog Inhibits Saliva-Promoted Adhesion and Biofilm Formation of Streptococcus mutans

Background: Streptococcus gordonii, a pioneer colonizer of dental plaque biofilm, expresses surface protein adhesin SspB by which the bacteria bind to salivary agglutinin (gp340). SspB has extensive homology with PAc, a surface adhesin of Streptococcus mutans. Hence, SspB of S. gordonii competes with PAc of S. mutans for the same niche environment in the salivary pellicles. The aim of this study was to develop anti-adherence agents that enabled us to control cariogenic biofilms by using the streptococcal SspB peptide analog SspB (A4K-A11K). Methods: First, we performed ELISA to determine the S. mutans-saliva interaction and saliva-binding activities of SspB (A4K- A11K). The inhibitory effects of SspB (A4K-A11K) were then evaluated by examining S. mutans adhesion to saliva-coated hydroxyapatite disks (s-HA). To determine peptide interference with biofilm formation, S. mutans biofilms were quantified by counting CFUs on MS agar plates and by measuring the absorbance at 492 nm of safranin-stained biofilms on s-HA. Results: Saliva, particularly salivary gp340 peptide, promoted adherence of S. mutans to polystyrene surfaces. SspB (A4K-A11K) significantly bound to saliva and inhibited the adhesion of S. mutans to s-HA without bactericidal activity. Furthermore, biofilms of S. mutans on s-HA were successfully reduced by pretreatment with SspB (A4K-A11K). Conclusion: SspB (A4K-A11K) peptide competitively blocked S. mutans adhesion to experimental pellicles through SspB-gp340 interaction, thereby inhibiting biofilm formation. These findings will contribute to the control cariogenic biofilms.

Loss of Masticatory Function Affects Morphology of the Tooth Root in Rats

Masticatory hypofunction and soft food affect the tooth rows, occlusion, and jawbone. This study aimed to clarify the influence of tooth loss and a soft diet on morphology of the tooth root during the growth period. We divided 3-week-old Wistar rats into the following three groups: Hard diet group (rats raised on solid standard diet), Powder diet group (rats raised on powdered standard feed diet), and Extraction group (rats raised on powdered standard diet with maxillary molars extraction). Length, width, cross-sectional area, and volume of the root of the mandibular M1 and M2 were measured using micro-CT analysis. Non-decalcified thin-slice specimens of sagittal sections of the M1 were obtained at the age of 20 weeks, and the roots were observed. The root length of all roots in the Extraction group was significantly longer than that in the other groups. The root width and cross-sectional area at the apical side 1/4 of all roots in the Extraction group were significantly smaller than those in the other groups. The root volume of the M1 mesial root in the Extraction group was significantly smaller than that in the other groups. This study clarified that when masticatory stimulus in the immature teeth is reduced by the extraction of opposing teeth and a powder diet, the root length increases due to the promotion of cellular cementum addition at the apex, and the root width and cross-sectional area decrease due to the suppression of cellular cementum addition at the apical side 1/4 of the roots.

Influence of Masticatory Functional Loss on the Remodeling of Alveolar Bone in Rats

There is plenty of literature on masticatory function and its impact on maxillofacial development. However, the influence of masticatory hypofunction on bone turnover in the alveolar bone has hardly been studied. This study aimed to clarify the influence of tooth loss and soft diet on the alveolar bone turnover during the growth period. Three-week-old Wistar rats were randomly divided into the following three groups: Hard diet group (rats raised on solid standard diet), Powder diet group (rats raised on powdered standard feed diet), and Extraction group (rats raised on powdered standard diet with maxillary molars extraction). BV, BMC, and BMD in the cancellous bone of M1 were measured using micro-CT analysis. To analyze the histological bone turnover, we prepared non-decalcified thin sections of alveolar cancellous bone when rats were 20 weeks old. On three-dimensional constructed images, the experimental groups (the Powder diet and Extraction groups) showed expansion of the medullary cavity of the interradicular septum of the first molar compared to controls (the Hard diet group). BV, BMC, and BMD were significantly lower in the experimental groups, with the difference from controls being greater in the Extraction group. On histomorphometric analysis, the bone mass parameters, bone formation parameters, and bone mineralization parameters were significantly lower in the experimental groups compared to controls. The bone resorption parameters were significantly higher in the experimental groups. From this study, we found that soft diet and tooth loss might worsen the bone microstructure, reduce osteogenesis, and promote bone resorption in alveolar bone.

Mesenchymal stem cell-derived apoptotic bodies alleviate alveolar bone destruction by regulating osteoclast differentiation and function

Periodontitis is caused by overactive osteoclast activity that results in the loss of periodontal supporting tissue and mesenchymal stem cells(MSCs)are essential for periodontal regeneration.However,the hypoxic periodontal microenvironment during periodontitis induces the apoptosis of MSCs.Apoptotic bodies(ABs)are the major product of apoptotic cells and have been attracting increased attention as potential mediators for periodontitis treatment,thus we investigated the effects of ABs derived from MSCs on periodontitis.MSCs were derived from bone marrows of mice and were cultured under hypoxic conditions for 72 h,after which ABs were isolated from the culture supernatant using a multi-filtration system.The results demonstrate that ABs derived from MSCs inhibited osteoclast differentiation and alveolar bone resorption.miRNA array analysis showed that miR-223-3p is highly enriched in those ABs and is critical for their therapeutic effects.Targetscan and luciferase activity results confirmed that Itgb1 is targeted by miR-223-3p,which interferes with the function of osteoclasts.Additionally,DC-STAMP is a key regulator that mediates membrane infusion.ABs and pre-osteoclasts expressed high levels of DC-STAMP on their membranes,which mediates the engulfment of ABs by pre-osteoclasts.ABs with knock-down of DC-STAMP failed to be engulfed by pre-osteoclasts.Collectively,MSC-derived ABs are targeted to be engulfed by pre-osteoclasts via DC-STAMP,which rescued alveolar bone loss by transferring miR-223-3p to osteoclasts,which in turn led to the attenuation of their differentiation and bone resorption.These results suggest that MSC-derived ABs are promising therapeutic agents for the treatment of periodontitis.

Entacapone promotes hippocampal neurogenesis in mice

Entacapone,a catechol-O-methyltransferase inhibitor,can strengthen the therapeutic effects of levodopa on the treatment of Parkinson’s disease.However,few studies are reported on whether entacapone can affect hippocampal neurogenesis in mice.To investigate the effects of entacapone,a modulator of dopamine,on proliferating cells and immature neurons in the mouse hippocampal dentate gyrus,60 mice(7 weeks old)were randomly divided into a vehicle-treated group and the groups treated with 10,50,or 200 mg/kg entacapone.The results showed that 50 and 200 mg/kg entacapone increased the exploration time for novel object recognition.Immunohistochemical staining results revealed that after entacapone treatment,the numbers of Ki67-positive proliferating cells,doublecortin-positive immature neurons,and phosphorylated cAMP response element-binding protein(pCREB)-positive cells were significantly increased.Western blot analysis results revealed that treatment with tyrosine kinase receptor B(TrkB)receptor antagonist significantly decreased the exploration time for novel object recognition and inhibited the expression of phosphorylated TrkB and brain-derived neurotrophic factor(BDNF).Entacapone treatment antagonized the effects of TrkB receptor antagonist.These results suggest that entacapone treatment promoted hippocampal neurogenesis and improved memory function through activating the BDNF-TrkB-pCREB pathway.This study was approved by the Institutional Animal Care and Use Committee of Seoul National University(approval No.SNU-130730-1)on February 24,2014.

Time- and cell-type specific changes in iron, ferritin, and transferrin in the gerbil hippocampal CA1 region after transient forebrain ischemia

In the present study, we used immunohistochemistry and western blot analysis to examine changes in the levels and cellular localization of iron, heavy chain ferritin（ferritin-H）, and transferrin in the gerbil hippocampal CA1 region from 30 minutes to 7 days following transient forebrain ischemia. Relative to sham controls, iron reactivity increased significantly in the stratum pyramidale and stratum oriens at 12 hours following ischemic insult, transiently decreased at 1–2 days and then increased once again within the CA1 region at 4–7 days after ischemia. One day after ischemia, ferritin-H immunoreactivity increased significantly in the stratum pyramidale and decreased at 2 days. At 4–7 days after ischemia, ferritin-H immunoreactivity in the glial components in the CA1 region was significantly increased. Transferrin immunoreactivity was increased significantly in the stratum pyramidale at 12 hours, peaked at 1 day, and then decreased significantly at 2 days after ischemia. Seven days after ischemia, Transferrin immunoreactivity in the glial cells of the stratum oriens and radiatum was significantly increased. Western blot analyses supported these results, demonstrating that compared to sham controls, ferritin H and transferrin protein levels in hippocampal homogenates significantly increased at 1 day after ischemia, peaked at 4 days and then decreased. These results suggest that iron overload-induced oxidative stress is most prominent at 12 hours after ischemia in the stratum pyramidale, suggesting that this time window may be the optimal period for therapeutic intervention to protect neurons from ischemia-induced death.

SspB Peptide Assay Reveals Saliva-Mediated <i>Porphyromonas gingivalis</i>Attachment

Background: Porphyromonas gingivalis is a major periodontal pathogen that binds efficiently to Streptococcus gordonii, which in turn binds to salivary agglutinin (gp340). The SspB of S. gordonii appears to mediate this association. We previously reported that the strepto-coccal SspB peptide analog, designated SspB (390-T400K-402), showed high binding activity with saliva. To understand the three-way interaction among S. gordonii, P. gingivalis and salivary gp340 as a unit, we established a peptide binding assay using SspB (390-T400K-402). Methods: The binding activity of the SspB (390-T400K-402) to P. gingivalis was detected by ELISA. Ninety-six well plates were coated with whole bacterial cell (P. gingivalis strains ATCC 33277, and W83;S. gordonii DL1) in Na2CO3 coating buffer. After blocking, bacterial cells were incubated with saliva or salivary agglutinin peptide (SRCRP2). Biotinylated SspB (390-T400K-402) was applied and incubated with 1:1000 streptoavidin-conjugated alkaline phosphatase. After development, A405 was recorded. Results: P. gingivalis 33277 showed the highest binding activity of the tested bacteria, whereas P. gingivalis W83, which was deficient in Mfa1 fimbriae, exhibited poor binding activity, as did S. gordonii. The binding of SspB (390-T400K-402) peptide in saliva- or SRCRP2-treated P. gingivalis was significantly higher than that in non-treated cells. Conclusion: The SspB (390-T400K-402) peptide binding assay revealed that initial attachment of P. gingivalis to the substrata of S. gordonii may require gp340-mediated SspB-Mfa1 interactions. The assay is available to assess the relationships among SspB, Mfa1 and salivary gp340 as a unit.

Oenanthe Javanica Extract Protects Against Experimentally Induced Ischemic Neuronal Damage via its Antioxidant Effects

Background： Water dropwort （Oenanthejavanica） as a popular traditional medicine in Asia shows various biological properties including antioxidant activity. In this study, we firstly examined the neuroprotective effect of Oenanthejavanica extract （OJE） in the hippocampal comus ammonis 1 region （CA 1 region） of the gerbil subjected to transient cerebral ischemia. Methods： Gerbils were established by the occlusion of common carotid arteries for 5 min. The neuroprotective effect of OJE was estimated by cresyl violet staining. In addition, 4 antioxidants （copper, zinc superoxide dismutase [SOD], manganese SOD, catalase, and glutathione peroxidase） immunoreactivities were investigated by immunohistochemistry. Results： Pyramidal neurons in the CA1 region showed neuronal death at 5 days postischemia; at this point in time, all antioxidants immunoreactivities disappeared in CA1 pyramidal neurons and showed 100 mg/kg, OJE protected CA 1 pyramidal neurons from ischemic damage in many nonpyramidal cells. Treatment with 200 mg/kg, not In addition, 200 mg/kg OJE treatment increased or maintained antioxidants immunoreactivities. Especially, among the antioxidants, glutathione peroxidase immunoreactivity was effectively increased in the CA 1 pyramidal neurons of the OJE-treated sham-operated and ischemia-operated groups. Conclusion： Our present results indicate that treatment with OJE can protect neurons from transient ischemic damage and that the neuroprotective effect may be closely associated with increased or maintained intracellular antioxidant enzymes by OJE.