Hope for vascular cognitive impairment: Ac-YVAD-cmk as a novel treatment against white matter rarefaction.

Vascular cognitive impairment (VCI) is the second leading cause of dementia with limited treatment options, characterised by cerebral hypoperfusion-induced white matter rarefaction (WMR). Subcortical VCI is the most common form of VCI, but the underlying reasons for region susceptibility remain elusive. Recent studies employing the bilateral cortical artery stenosis (BCAS) method demonstrate that various inflammasomes regulate white matter injury and blood-brain barrier dysfunction but whether caspase-1 inhibition will be beneficial remains unclear. To address this, we performed BCAS on C57/BL6 mice to study the effects of Ac-YVAD-cmk, a caspase-1 inhibitor, on the subcortical and cortical regions. Cerebral blood flow (CBF), WMR, neuroinflammation and the expression of tight junction-related proteins associated with blood-brain barrier integrity were assessed 15 days post BCAS. We observed that Ac-YVAD-cmk restored CBF, attenuated BCAS-induced WMR and restored subcortical myelin expression. Within the subcortical region, BCAS activated the NLRP3/caspase-1/interleukin-1beta axis only within the subcortical region, which was attenuated by Ac-YVAD-cmk. Although we observed that BCAS induced significant increases in VCAM-1 expression in both brain regions that were attenuated with Ac-YVAD-cmk, only ZO-1 and occludin were observed to be significantly altered in the subcortical region. Here we show that caspase-1 may contribute to subcortical regional susceptibility in a mouse model of VCI. In addition, our results support further investigations into the potential of Ac-YVAD-cmk as a novel treatment strategy against subcortical VCI and other conditions exhibiting cerebral hypoperfusion-induced WMR.

Hepatic Homeostasis of Metal Ions Following Acute Repeated Stress Exposure in Rats.

Essential metals such as copper, iron, and zinc are cofactors in various biological processes including oxygen utilisation, cell growth, and biomolecular synthesis. The homeostasis of these essential metals is carefully controlled through a system of protein transporters involved in the uptake, storage, and secretion. Some metal ions can be transformed by processes including reduction/oxidation (redox) reactions, and correspondingly, the breakdown of metal ion homeostasis can lead to formation of reactive oxygen and nitrogen species. We have previously demonstrated rapid biochemical responses to stress involving alterations in the redox state to generate free radicals and the resultant oxidative stress. However, the effects of stress on redox-active metals including iron and copper and redox-inert zinc have not been well characterised. Therefore, this study aims to examine the changes in these essential metals following exposure to short-term repeated stress, and to further elucidate the alterations in metal homeostasis through expression analysis of different metal transporters. Outbred male Wistar rats were exposed to unrestrained (control), 1 day, or 3 days of 6 h restraint stress (n = 8 per group). After the respective stress treatment, blood and liver samples were collected for the analysis of biometal concentrations and relative gene expression of metal transporter and binding proteins. Exposure to repeated restraint stress was highly effective in causing hepatic redox imbalance. Stress was also shown to induce hepatic metal redistribution, while modulating the mRNA levels of key metal transporters. Overall, this study is the first to characterise the gene expression profile of metal homeostasis following stress and provide insight into the changes occurring prior to the onset of chronic stress conditions.

Preventive effects of the novel antimicrobial peptide Nal-P-113 in a rat Periodontitis model by limiting the growth of Porphyromonas gingivalis and modulating IL-1ß and TNF-α production.

BACKGROUND: P-113 (AKRHHGYKRKFH-NH2) is a 12-amino-acid histidine-rich peptide derived from histatin 5 that is highly degradable in high salt concentrations and biological fluids such as serum, plasma and saliva. Nal-P-113, a novel antimicrobial peptide whose histidine residues are replaced by the bulky amino acids ß-naphthylalanine, causes the antimicrobial peptide to retain its bactericidal activity even in physiological environments. This study evaluated the effect of the novel antimicrobial peptide Nal-P-113 in a rat periodontitis model and the mechanisms of action of Nal-P-113 for suppressing periodontitis. METHODS: Periodontitis was induced in mandibular first molars in rats receiving a ligature and infected with Porphyromonas gingivalis. Animals were randomly divided into six groups: a, P. gingivalis W83 alone; b, P. gingivalis W83 with 6.25 µg/mL of Nal-P-113; c, P. gingivalis W83 with 25 µg/mL of Nal-P-113; d, P. gingivalis W83 with 100 µg/mL of Nal-P-113; e, P. gingivalis W83 with 400 µg/mL of Nal-P-113; and f, control without P. gingivalis W83 or Nal-P-113. Morphometric analysis was used to evaluate alveolar bone loss. Microbiological assessment of the presence of Porphyromonas gingivalis and total bacteria was performed using absolute quantitative real-time PCR and scanning electron microscopy. Gingival tissue was collected for western blot and immunohistochemical assays of IL-1ß and TNF-α levels. RESULTS: Alveolar bone loss was inhibited by 100 µg/mL or 400 µg/mL of Nal-P-113 compared to the control group (P < 0.05). Lower amounts of P. gingivalis and total bacteria were found in groups d and e compared with group a (P < 0.05). A decrease in the levels of IL-1ß and TNF-α was detected in group d and group e compared to the control group (P < 0.05). The amount of P. gingivalis was positively correlated with IL-1ß and TNF-α expression in periodontal tissue (P < 0.05). CONCLUSIONS: Nal-P-113 exhibited protective effects on Porphyromonas gingivalis-induced periodontitis in rats by limiting the amount of bacteria and modulating IL-1ß and TNF-α production. The use of Nal-P-113 in vivo might serve as a beneficial preventive or therapeutic approach for periodontitis.

Molecular pathways underlying inhibitory effect of antimicrobial peptide Nal-P-113 on bacteria biofilms formation of Porphyromonas gingivalis W83 by DNA microarray.

BACKGROUND: Wound-related infection remains a major challenge for health professionals. One disadvantage in conventional antibiotics is their inability to penetrate biofilms, the main protective strategy for bacteria to evade irradiation. Previously, we have shown that synthetic antimicrobial peptides could inhibit bacterial biofilms formation. RESULTS: In this study, we first delineated how Nal-P-113, a novel antimicrobial peptide, exerted its inhibitory effects on Porphyromonas gingivalis W83 biofilms formation at a low concentration. Secondly, we performed gene expression profiling and validated that Nal-P-113 at a low dose significantly down-regulated genes related to mobile and extrachromosomal element functions, transport and binding proteins in Porphyromonas gingivalis W83. CONCLUSIONS: These findings suggest that Nal-P-113 at low dose is sufficient to inhibit the formation of biofilms although Porphyromonas gingivalis W83 may maintain its survival in the oral cavity. The newly discovered molecular pathways may add the knowledge of developing a new strategy to target bacterial infections in combination with current first-line treatment in periodontitis.

Gene expression changes in Porphyromonas gingivalis W83 after inoculation in rat oral cavity.

BACKGROUND: The development of chronic periodontitis was due to not only periodontal pathogens, but also the interaction between periodontal pathogens and host. The aim of this study is to investigate the alterations in gene expression in Porphyromonas gingivalis (P.gingivalis) W83 after inoculation in rat oral cavity. RESULTS: P.gingivalis W83 inoculation in rat oral cavity caused inflammatory responses in gingival tissues and destroyed host alveolar bone. Microarray analysis revealed that 42 genes were upregulated, and 22 genes were downregulated in the detected 1786 genes in the inoculated P.gingivalis W83. Real-time quantitative PCR detection confirmed the expression alterations in some selected genes. Products of these upregulated and downregulated genes are mainly related to transposon functions, cell transmembrane transportation, protein and nucleic acid metabolism, energy metabolism, cell division and bacterial pathogenicity. CONCLUSIONS: P.gingivalis W83 has a pathogenic effect on host oral cavity. Meanwhile, inflammatory oral environment alters P.gingivalis W83 gene expression profile. These changes in gene expression may limit the proliferation and weaken the pathogenicity of P.gingivalis W83, and favor themselves to adapt local environment for survival.

[A study of frequency of TNF alpha gene with type 2 diabetes mellitus with chronic periodontitis].

PURPOSE: To detect the frequency of TNF alpha gene in patients of type 2 diabetes mellitus with chronic periodontitis, periodontitis without any systemic diseases and healthy controls. METHODS: The case series were consisted of 112 patients with moderate, severe type 2 diabetes mellitus with chronic periodontitis, 99 patients with moderate, severe periodontitis without any systemic disease, 50 age- and gender-matched subjects with healthy periodontal conditions were enrolled. Clinical parameters were measured and recorded including probing depth(PD), clinical attachment loss(CAL), bleeding index(BI), and tooth movement(TM). The polymorphism of TNF-α-308 genotype (TNF1/2) was examined after electrophoresis on agarose gel and ethidium bromide staining. The difference between the case and healthy groups was analysed by Chi-square test, the difference in clinical index among groups which had different allele was analyzed for ANOVA with SPSS13.0 software package. RESULTS: We divided DM and CP groups into moderate and severe groups. There were significant difference between severe DM group and severe, moderate CP group, moderate DM group and chronic periodontitis of severe,moderate group. The probing depth and clinical attachment loss of the patients who took TNF-α-308 allele II were significantly higher than the patients who took TNF-α-308 allele I in DM and CP group. CONCLUSIONS: TNF-α-308 allele II might increase the susceptivity of periodontitis in population. TNF-α-308 allele II may play an important role in synergistic reaction of periodontitis and type 2 diabetes.

[A survey on the periodontal status in type 2 diabetic patients].

OBJECTIVE: To investigate the type 2 diabetic patient's periodontal condition and to analyze the influencing factors of periodontitis. METHODS: A total of 182 type 2 diabetic patients were included in the survey and requested to fill out a questionnaire, and their periodontal status was evaluated by measuring probing depth (PD), attachment level (AL), sulcus bleeding index (SBI), simplified oral hygiene index (OHI-S). RESULTS: The prevalence of periodontitis in this group of patients was 96.7% (176/182), including 20 patients with mild periodontitis, 156 with moderate to advanced periodontitis. The mean PD and AL of the 182 patients were (2.92 +/- 0.67) mm and (2.87 +/- 1.31) mm. At least one tooth was lost in 57.1% (104/182) of the patients. The factors related to periodontitis were age, gender, smoking, living in town or country, and 2 h plasma glucose of oral glucose tolerance test (OGTT). There was no relationship between the severity of periodontitis and education level. The majority of patients did not receive any periodontal therapy. CONCLUSIONS: Periodontal status was bad in patients with type 2 diabetes. It is important to develop an education program on oral health for type 2 diabetic patients.

[Combination use of rh-bFGF and Bio-Oss collagen to enhance periodontal regeneration: an experimental study in dogs].

PURPOSE: To evaluate the effectiveness of combined use of rh-bFGF and Bio-Oss collagen compounds on regeneration of the canine periodontal tissues. METHODS: Four adult hybrid canine were selected and bone defect of 5mmx5mmx5mm in size was created in the mesio and distal alveolar crest where the premolars were lost in both sides of the maxilla and mandible. Three methods were used in 3 groups,in the experimental group 1, rh-bFGF and Bio-Oss collagen compounds were implanted,Bio-Gide were overlaid; in the experimental group 2,Bio-Oss collagen compounds were implanted, Bio-Gide were overlaid;in the control group only Bio-Gide were overlaid. The therapeutic results were evaluated by clinical and imaging examination as well as histological index. SAS 6.2 software package was used for paired t test. RESULTS: 8 weeks post operation, there were various amounts of newly formed alveolar bone,cement and connective tissues in each 3-wall intrabony defect. There was significant difference in alveolar osteogenesis between the control group(1.2mm+/-0.1mm) and the 2 experimental groups (3.7mm+/-0.3mm, 2.3mm+/-0.2mm).The difference was also significant between the two experimental groups (P<0.01). There was no significant difference in newly formed cement and connective tissues among the 3 groups (P>0.05). CONCLUSIONS: Application of rh-bFGF/ Bio-Oss collagen compounds benefits to the regeneration of the alveolar bone and avoid the side effects of root resorption ,bone malunion which often occur after conventional periodontal regeneration therapy.Addition of rh-bFGF/ Bio-Oss collagen compounds was useless to cement genesis and periodontal ligament formation.

[Construction of tissue engineered bone by osteoblasts from canine bone marrow mesenchymal stem cells and Bio-Oss: an in vitro study].

PURPOSE: Using the MSCs-Bio-Oss tissue engineered bone which was constructed by MSCs as seed cells and the Bio-Oss calf inorganic bone grains as scaffold materials to determine the canine bone formation activity and the feasibility of Bio-Oss combined MSCs to construct tissue engineered bone. METHODS: Gybrid canine MSCs were dissociated, cultivated, bone formation induced and differentiated into osteoblast in vitro; The bone formation induced MSCs were allowed to grow onto Bio-Oss calf inorganic bone grains at 10(6) cell/ml, and then incubated and cultivated, under light pressure without other treatments. Inverted phase contrast microscope and scanning electron microscope were used to observe their morphological changes, immunofluorescent labeling of cell surface factor CD44,calcium nodus staining,qualitative and quantitative detection of ALP were carried out, and SPSS 12.0 software package was used for statistical analysis. RESULTS: The MSCs were uniform with compact alignment and shape of prosenchymatous cells, CD44's surface antigen was positive; Osteoblast activity was present after induction and differentiation, alizarin Bordeaux S stain of calcium nodus was positive, ALP's Gomori staining was also positive. ALP content in the experimental group were (3.307 +/- 0.217) U/g, (5.929 +/- 0.781) U/g and (9.739 +/- 0.547)U/g respectively at 3rd day, 7th day, 14th day after induction and differentiation, which were significantly different (P < 0.01) from the control group: (0.442 +/- 0.087) U/g, (0.581 +/- 0.027)U/g and (0.768 +/- 0.126) U/g; the MSCs stuck compactly on the surface of Bio-Oss, grew well, and formed tissue engineered bone. CONCLUSION: Using canine MSCs which were induced by bone formation as seed cells combined with Bio-Oss as scaffold materials to construct tissue engineered bone is feasible. Supported by Liaoning Provincial Natural Science