The effect of cacao bean extracts on the prevention of periodontal tissue breakdown in diabetic rats with orthodontic tooth movements.

Objective: Proper management of orthodontic treatment in diabetic patients is essential due to the heightened risk of periodontal tissue breakdown associated with hyperglycemia. Cacao bean extracts (CBE) are known to reduce the inflammatory response and increase synthesis and angiogenesis in periodontitis. Therefore, this study aims to examine the effect of CBE on preventing periodontal tissue breakdown in diabetes with orthodontic force. Methods: A total of 25 Wistar rats were divided randomly into 5 groups, including non-diabetes, diabetes, diabetes cacao 125, 250, and 500 mg/kg BW. Diabetic rats were induced with the stratified dose of Streptozotocin, and a 30-g-force from orthodontic device was applied in all groups. Diabetes cacao group was given CBE for 7 days using a gastric probe. GCF samples were used to analyze the eNOS level through the ELISA method. NFκB, Collagen-1, and FGF-2 expression were then assessed using the immunohistochemical method, while the number of fibroblasts and blood vessels was observed using hematoxylin-eosin stained tissue. The data obtained were analyzed with one-way ANOVA and post hoc tests, with p < 0.05. Results: CBE at a dose of 250 mg/kg BW significantly increased eNOS level, Collagen-1, and FGF-2 expression, and the number of fibroblasts and blood vessels in diabetes groups. Meanwhile, the treatment decreased NFκB expression in diabetes groups (p < 0.05). Conclusion: This study proved that CBE increased periodontal ligament synthesis and angiogenesis and decreased inflammatory response, thereby preventing periodontal tissue breakdown in diabetic rat models with tooth movement.

Box-Wilson Design for Optimization of in vitro Levan Production and Levan Application as Antioxidant and Antibacterial Agents.

Background: Levan or fructan, a polysaccharide of fructose, is widely used in various commercial industries. Levan could be produced by many organisms, including plants and bacteria. The cloning of the gene from Bacillus licheniformis, which expressed levansucrase in Escherichia coli host, was carried out successfully. In the present study, we performed the in vitro production of levan and analyzed its potential application as antibacterial and antioxidant agents. Methods: In vitro levan production catalyzed by heterologous-expressed levansucrase Lsbl-bk1 and Lsbl-bk2 was optimized with Box-Wilson design. The antibacterial activity of the produced levan was carried out using agar well diffusion method, while its antioxidant activity was tested by free radical scavenging assays. Results: The optimum conditions for levan production were observed at 36 °C and pH 7 in 12% (w/v) sucrose for levansucrase Lsbl-bk1, while the optimum catalysis of levansucrase Lsbl-bk2 was obtained at 32 oC and pH 8 in the same sucrose concentration. The in vitro synthesized levan showed an antibacterial activity within a concentration range of 10-20% (w/v) against Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa. The same levan was also able to inhibit the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with the antioxidant strength of 75% compared to ascorbic acid inhibition. Conclusion: Our study, therefore, shows that the optimized heterologous expression of levansucrases encoded by Lsbl-bk1 and Lsbl-bk2 could open the way for industrial levan production as an antibacterial and antioxidant agent.

Oxidants-Antioxidants Profile in the Breast Cancer Cell Line MCF-7

Reactive oxygen species (ROS) have various biological effects and they are non-linear in characteristic. In high oxidative stress, they may cause cytotoxicity, inhibit cell proliferation, and induce cell death in the form of apoptosis/necrosis; while in low or medium oxidative stress, ROS may cause DNA damage, cell mutation, inflammation, cell proliferation, and eventually they may induce carcinogenesis. Antioxidants are compounds with the ability to reduce ROS. Cell line MCF-7 is one of the breast cancer cell lines that is known to have small amount of antioxidant MnSOD compared to the other cell lines. Low antioxidant MnSOD level in breast cancer cell line MCF-7 leads to low concentration of hydrogen peroxide, because antioxidant MnSOD will convert radical superoxide to hydrogen peroxide. The aim of this research was to analyze oxidants and antioxidants profile in breast cancer cell line MCF-7 and their relationship with cell number. Observations were conducted for 5 days. The cell number was counted with tryphan blue method and haematometer. The concentration of radical superoxide was measured with DHE staining using LSCM tipe Olympus Fluoview FV 1000-Ver 1.7. MnSOD activity, hydrogen peroxide concentration, and catalase activity were measured with ELISA. The results showed that the longer of observation, the greater concentration of oxidants and MnSOD activity, but there was no change in catalase activity. Conclusion the increase in cancer cells number is influenced by radical superoxide.

Combined effects of shear stress and glucose on the morphology, actin filaments, and VE-cadherin of endothelial cells in vitro.

OBJECTIVE: The purpose of the present study was to analyze the effects of glucose and shear stress on the morphology and density of endothelial cells, actin filamen, and the expression of VE-cadherin. METHODS: After confluency of endothelial cells (3-4 days), 22 mM of d-glucose was administered for 7 days. Endothelial cells were exposed to shear stress of 10 dyne/cm2 for varied durations of 5, 8, 12, and 15 min. Morphology of ECs was observed using an inverted microscope before and after shear stress exposure. VE-cadherin and actin filament were analyzed immunohistochemically. RESULTS: Exposure to high glucose induces more shrinkage and the cell density decreased at 15 min. High glucose reduced actin filaments and the more globular ones, especially around the nuclei. There was a decline in VE-cadherin scores with significant differences between treatments with 5 mM and 22 mM of glucose. CONCLUSION: Combination exposure of shear stress and high glucose changes morphology, reduces actin filament and VE-cadherin, of endothelial cells.

Neuropathy optic glaucomatosa induced by systemic hypertension through activation endothelin-1 signaling pathway in central retinal artery in rats.

AIM: To evaluate effect of hypertension on retinal ganglion cell (RGC) apoptosis, intraocular pressure (IOP), and the activation of endothelin-1 (ET-1) signaling pathway in central retinal artery (CRA) in rats. METHODS: The experimental study was performed on 20 male Sprague Dawley rats that were divided into control group, and hypertension groups. The hypertension was induced by subcutaneous deoxycorticoacetate (DOCA) 10 mg/kg twice a week and administered 0.9% NaCl solution daily for 2, 6, and 10wk. Blood pressure (BP) was measured using animal BP analyzer. IOP was measured by handheld tonometry. Retinal tissue preparations by paraffin blocks were made after enucleation. The expression of ET-1, eNOS, ET-1 receptor A (ETRA), ET-1 receptor B (ETRB), and phosphorylated myosin light chain kinase (MLCK), and caldesmon (CaD) in CRA and RGC apoptosis were evaluated through immunofluorescent staining method then observed using laser scanning confocal microscopy. RESULTS: BP significantly increased in all of the hypertension groups compared to control (P=0.001). Peak IOP elevation (7.78±4.14 mm Hg) and RGC apoptosis (576.15±33.28 Au) occurred on 2wk of hypertension. ET-1 expression (1238.6±55.1 Au) and eNOS expression (2814.2±70.7 Au) were found highest in 2wk of hypertension, although the ratio of ET-1/eNOS decreased since 2wk. ETRA reached peak expression in 10wk of hypertension (1219.4±6.3 Au), while ETRB significantly increased only in 2 weeks group (1069.2±9.6 Au). The highest MLCK expression (1190.09±58.32 Au), CaD (1670.28±18.36 Au) were also found in 2wk of hypertension. CONCLUSION: Hypertension effects to activation of ET-1 signaling pathway significantly in CRA, elevation of IOP, and RGC apoptosis. The highest value was achieved at 2wk, which is the development phase of hypertension.

Methanolic Extract of Ceplukan Leaf (Physalis minima L.) Attenuates Ventricular Fibrosis through Inhibition of TNF-α in Ovariectomized Rats.

The increase of heart failure prevalence on menopausal women was correlated with the decrease of estrogen level. The aim of this study is to investigate the effects of ceplukan leaf (Physalis minima L.), which contains phytoestrogen physalin and withanolides, on ventricular TNF-α level and fibrosis in ovariectomized rats. Wistar rats were divided into six groups (control (-); OVX 5: 5-week ovariectomy (OVX); OVX 9: 9-week ovariectomy; treatments I, II, and III: 9-weeks OVX + 4-week ceplukan leaf's methanolic extract doses 500, 1500, and 2500 mg/kgBW, resp.). TNF-α levels were measured with ELISA. Fibrosis was counted as blue colored tissues percentage using Masson's Trichrome staining. This study showed that prolonged hypoestrogen increases ventricular fibrosis (p < 0.05). Ceplukan leaf treatment also resulted in a decrease of ventricular fibrosis and TNF-α level in dose dependent manner compared to without treatment group (p < 0.05). Furthermore, the TNF-α level was normalized in 2500 mg/kgBW Physalis minima L. (p < 0.05) treatment. The reduction of fibrosis positively correlated with TNF-α level (p < 0.05, r = 0.873). Methanolic extract of ceplukan leaf decreases ventricular fibrosis through the inhibition of ventricular TNF-α level in ovariectomized rats.

Effect of Scurrula atropurpurea on nitric oxide, endothelial damage, and endothelial progenitor cells of DOCA-salt hypertensive rats.

OBJECTIVES: To know whether Scurrula atropurpurea is able to modulate total plasma nitrate/nitrite levels, decrease endothelial damage, and increase endothelial progenitor cells (EPCs) in hypertensive rats. MATERIALS AND METHODS: The rats were divided in 5 groups: control (normotensive) group, Desoxy cortico sterone (DOCA)-salt hypertensive group, and three DOCA-salt hypertensive groups. All 5 groups received methanolic extract of S. atropurpurea (MESA) at a dosage of 50; 100; and 200 mg/KgBW. Serum nitric oxide (NO) was assayed by colorimetric. Circulating endothelial cells (CECs) and EPCs were assayed using flow cytometry. RESULTS: The administration of MESA100 and MESA200 elevated the total plasma nitrate/nitrite levels but cannot reach the level in control group. MESA100 and MESA200 also elevated the EPCs number compared with hypertensive group. The administration of MESA significantly (P< 0.05) decreased the CECs number compared to hypertensive groups. CONCLUSION: Methanolic extract of S. atropurpurea is able to modulate total plasma nitrate/nitrite levels and diminish endothelial damage via increasing EPCs.

Vasa vasorum anti-angiogenesis through H2O2, HIF-1α, NF-κB, and iNOS inhibition by mangosteen pericarp ethanolic extract (Garcinia mangostana Linn) in hypercholesterol-diet-given Rattus norvegicus Wistar strain.

BACKGROUND: Oxidative stress in atherosclerosis produces H2O2 and triggers the activation of nuclear factor kappa beta (NF-κB) and increase of inducible nitric oxide synthase (iNOS). The formation of vasa vasorum occurs in atherosclerosis. Vasa vasorum angiogenesis is mediated by VEGFR-1 and upregulated by hypoxia-inducible factor-1α (HIF-1α). The newly formed vasa vasorum are fragile and immature and thus increase plaque instability. It is necessary to control vasa vasorum angiogenesis by using mangosteen pericarp antioxidant. This study aims to demonstrate that mangosteen pericarp ethanolic extract can act as vasa vasorum anti-angiogenesis through H2O2, HIF-1α, NF-κB, and iNOS inhibition in rats given a hypercholesterol diet. METHODS: This was a true experimental laboratory, in vivo posttest with control group design, with 20 Rattus norvegicus Wistar strain rats divided into five groups (normal group, hypercholesterol group, and hypercholesterol groups with certain doses of mangosteen pericarp ethanolic extract: 200, 400, and 800 mg/kg body weight). The parameters of this study were H2O2 measured by using colorimetric analysis, as well as NF-κB, iNOS, and HIF-1α, which were measured by using immunofluorescence double staining and observed with a confocal laser scanning microscope in aortic smooth muscle cell. The angiogenesis of vasa vasorum was quantified from VEGFR-1 level in aortic tissue and confirmed with hematoxylin and eosin staining. RESULTS: Analysis of variance test and Pearson's correlation coefficient showed mangosteen pericarp ethanolic extract had a significant effect (P<0.05) in decreasing vasa vasorum angiogenesis through H2O2, HIF-1α, NF-κB, and iNOS inhibition in hypercholesterol-diet-given R. norvegicus Wistar strain. CONCLUSION: Mangosteen pericarp ethanolic extract 800 mg/kg body weight is proven to decrease vasa vasorum angiogenesis. Similar studies with other inflammatory parameters are encouraged to clarify the mechanism of vasa vasorum angiogenesis inhibition by mangosteen pericarp ethanolic extract.