Experimental apical periodontitis alters salivary biochemical composition and induces local redox state disturbances in the salivary glands of male rats.

OBJECTIVES: The objective was to evaluate the effects of experimental apical periodontitis on the inflammatory, functional, biochemical, and redox parameters of the parotid and submandibular glands in rats. MATERIALS AND METHODS: Twenty 12-week-old male Wistar rats were randomly divided into two groups (n = 10): a control group and apical periodontitis group. After 28 days, the saliva was collected for salivary flow rate and biochemistry composition. Both glands were sampled for quantification of the tumor necrosis factor-alpha (TNF-α) and biochemical analyses of redox state. RESULTS: TNF-α concentrations were higher in both salivary glands adjacent to the periapical lesions in animals with apical periodontitis and also compared to the control group. The apical periodontitis group increased the salivary amylase, chloride, potassium, calcium, and phosphate. The total oxidant capacity increased in the parotid gland adjacent to the periapical lesions in the same rat and compared to the control group. Conversely, the total antioxidant capacity of the parotid glands on both sides in the apical periodontitis group was lower than that in the control group. Furthermore, glutathione peroxidase activity increased in the submandibular gland adjacent to the apical periodontitis group compared to the control group. CONCLUSIONS: Experimental apical periodontitis alters salivary biochemical composition, in addition to increasing inflammatory marker and inducing local disturbances in the redox state in the parotid and submandibular glands of male rats. CLINICAL RELEVANCE: Apical periodontitis could exacerbate the decline in oral health by triggering dysfunction in the salivary glands.

Analysis of the cognitive and functional behavior of female rats in the periestropause after hormone therapy with estrogen.

Perimenopause is a critical period, with severe cycle irregularity and lower estrogen secretion altering redox state biomarkers, leading to behavioral changes. The estrogen hormonal therapy (EHT) being commonly used to alleviate climacteric effects. Therefore, the aim of this study was to analyze anxiolytic profile, recognition memory (short and long term), ambulation, redox status, cell synaptic activity in locus coeruleus and hippocampus of Wistar rats in the periestropause after EHT. Forty rats participated in the study; 20 were treated with corn oil (group 21Mo/Veh; corn oil/0.2 mL/sc; 2x/week) and 20 were submitted to EHT (group 21Mo/E2; 17ß-estradiol/15 µg/Kg/sc; 2x/week) for 120 days. Open field, elevated plus maze, object recognition (RO), and footprint tests were performed immediately before and at the end of the treatment period. From the decapitated brains, isolated hippocampus were destined for biochemical analysis, in turn, perfused brains were destined for histological analysis. The 21Mo/E2 group had a significantly greater total time in the central region and a significantly greater number of entries into the open arms compared to the 21Mo/Veh group, as in crossing, rearing and grooming behaviors, evidencing an anxiolytic profile. In the RO test, the 21Mo/Veh group decreased long-term memory, and the 21Mo/E2 group maintained the same index as at 17 months of age, in addition to a better balance of the hippocampal redox state, prevention of neuronal cell loss and better gait. Based on the results, it appears that exogenous E2 supplementation during periestropause may help preserve neurological functions and potentially prevent neuropsychological and neurodegenerative disorders.

Aerobic physical exercise modifies the prostate tumoral environment.

Exercise is recognized for its potential role in reducing the risk of certain cancers. However, the molecular mechanisms behind this risk reduction are not fully understood. Here, we hypothesized that aerobic physical exercise induces cancer attenuating effects through the modulation of oxidative stress and inflammation. To test this hypothesis, twenty male Sprague Dawley rats with chemically induced prostate tumors were divided into two groups: Prostate cancer (PC) in the absence and presence of exercise (PC + Ex). Rats in the PC + Ex group performed exercises on a treadmill for 8 weeks, 5 sessions per week, at an intensity of 60 % of maximum capacity. Weight and feed efficiency, Ki-67, apoptosis, prostatic inflammation, and markers of oxidative stress were analyzed. We found that aerobic physical exercise significantly decreased prostate cell proliferation (p < 0.05) across modulation, tumor size, and prostate weight. The PC + Ex group also significantly reduced anti-apoptosis protein expression (p < 0.05) and increased pro-apoptotic protein expression. Furthermore, physical exercise increased enzymatic antioxidant defenses in the prostate, plasma, and whole blood. Moreover, PC + Ex reduced lipid peroxidation and protein carbonyl levels (p < 0.05). In the prostate, there was an increase in anti-inflammatory cytokines (IL-10), and a reduction in pro-inflammatory cytokines (IL-6, TNF-α, and NF-κB) after 8 weeks of physical exercise. In conclusion, we found that aerobic physical exercise is a functional, beneficial, and applicable approach to control PC progression, because it modifies the systemic environment, including the regulation of glucose and circulating lipids. This modification of the cancer cells environment has anti-inflammatory and antioxidant effects that attenuate tumor growth.

Analysis of salivary flow rate, biochemical composition, and redox status in orchiectomized spontaneously hypertensive rats.

OBJECTIVE: This study aimed to analyze the salivary flow rate, biochemical composition, and redox status in orchiectomized spontaneously hypertensive rats (SHR) compared to normotensive Wistar rats. DESIGN: Thirty-two young adult male SHR and Wistar (3-months-old) rats were randomly distributed into four groups; either castrated bilaterally (ORX) or underwent fictitious surgery (SHAM) as Wistar-SHAM, Wistar-ORX, SHR-SHAM, and SHR-ORX. Two months beyond castration, pilocarpine-induced salivary secretion was collected from 5-month-old rats to analyze salivary flow rate, pH, buffer capacity, total protein, amylase, calcium, phosphate, sodium, potassium, chloride, thiobarbituric acid reactive substances (TBARs), carbonyl protein, nitrite, and total antioxidant capacity. RESULTS: The salivary flow rate was higher in the Wistar-ORX compared to the Wistar-SHAM group, while remaining similar between the SHR-SHAM and SHR-ORX groups. ORX did not affect pH and salivary buffer capacity in both strains. However, salivary total protein and amylase were significantly reduced in the Wistar-ORX and SHR-ORX compared to the respective SHAM groups. In both ORX groups, salivary total antioxidant capacity and carbonylated protein were increased, while lipid oxidative damage (TBARs) and nitrite concentration were higher only in the Wistar-ORX than in the Wistar-SHAM group. In the Wistar-ORX and SHR-ORX, the salivary calcium, phosphate, and chloride were increased while no change was detected in the SHAM groups. Only salivary buffering capacity, calcium, and chloride in the SHR-ORX adjusted to values similar to Wistar-SHAM group. CONCLUSION: Hypertensive phenotype mitigated the orchiectomy-induced salivary dysfunction, since the disturbances were restricted to alterations in the salivary biochemical composition and redox state.

Dose-response effect of Montelukast on post-extraction dental socket repair and skeletal phenotype of mice.

Bone metabolism and repair are directly regulated by arachidonic acid metabolites. At present, we analyzed the dose-response effects of a selective cysteinyl leukotriene receptor type-1 antagonist during bone repair after tooth extraction and on non-injured skeleton. Sixty-three 129 Sv/Ev male mice composed the groups: C-Control (saline solution); MTK2-2 mg/Kg of Montelukast (MTK) and MTK4-4 mg/Kg of MTK, daily administered by mouth throughout all experimental periods set at 7, 14, and 21 days post-operative. Dental sockets were analyzed by computed microtomography (microCT), histopathology, and immunohistochemistry. Femurs, L5 vertebra and organs were also removed for observation. Blood was collected for plasma bone and liver markers. Histopathology and microCT analysis revealed early socket repair of MTK2 and MTK4 animals, with significant increased BV/TV at days 14 and 21 compared to C. Higher plasma calcium was detected at days 7 and 21 in MTK4 in comparison to C, while phosphate was significantly increased in MTK2 in the same periods in comparison to C and MTK4. No significant differences were found regarding plasma ALP and TRAP, neither for local TRAP and Runx2 immunolabeling at the healing sockets. Organs did not present histological abnormalities. Increased AST levels have been detected in distinct groups and periods. In general, femur phenotype was improved in MTK treated animals. Collectively, MTK promoted early bone formation after tooth extraction and increased bone quality of femurs and vertebra in a time-dose-dependent manner, and should be considered as an alternative therapy when improved post-extraction socket repair or skeleton preservation is required.

Melatonin decreases plasma TNF-α and improves nonenzymatic antioxidant defence and insulin sensitivity in rats with apical periodontitis fed a high-fat diet.

AIM: To analyse the effects of melatonin (ME) treatment on oxidative stress and insulin resistance (IR) in rats with apical periodontitis (AP) fed a high-fat diet (HFD). METHODOLOGY: Eighty 60-day-old rats were divided into eight groups: control (CN), AP, HFD with AP (HFDAP), control with ME (CNME), AP with ME (APME), HFD with ME (HFDME) and HFD with AP+ME (HFDAPME). The animals from the HFD groups were fed a HFD throughout the experimental period. On day 7, the animals from the AP groups were subjected to experimental AP, and after 70 days, the ME groups were treated for 30 days. Glycaemia, insulinaemia, homeostatic model assessment for IR index, tumour necrosis factor-α (TNF-α), and interleukin-6 were analysed in plasma using biochemical tests and enzyme-linked immunosorbent assay. Thiobarbituric acid-reactive substances (TBARS), carbonyl protein (CP), superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione (GSH) and total antioxidant capacity (ferric reducing antioxidant power [FRAP]) were analysed in the gastrocnemius muscle. RESULTS: (1) Association of AP and HDF exacerbated IR, and ME treatment improved this alteration; (2) AP and HFD and their association showed increased TNF-α, and ME reversed it; (3) TBARS increased in the AP and HFDAP groups, and ME reversed only in the group with the association of disease and diet; (4) CP increased in all HFD groups and improved in the ME groups; (5) GSH activity decreased in all experimental groups, and ME increased this parameter only in the CN and AP groups; (6) FRAP did not change between the groups, but ME treatment increased its activity in the AP and HFD groups; (7) ME increased SOD in the CN and AP groups. CONCLUSION: Apical periodontitis and HFD promoted IR, and the association of AP with diet promoted IR exacerbation; this resistance might have been caused by an increase in TNF-α. AP promoted more intense changes in lipid oxidative damage than in protein oxidative damage. In non-enzymatic antioxidant defence, it was observed that both AP and HFD and their association promoted a decrease in GSH levels. Overall, ME treatment reversed changes such as oxidative stress and IR.

Assessment of redox state and biochemical parameters of salivary glands in streptozotocin-induced diabetic male rats treated with mate tea (Ilex paraguariensis).

OBJECTIVE: The study aimed to assess the effects of mate tea [Ilex paraguariensis] on the redox state and biochemical parameters of salivary glands in diabetic male rats. DESIGN: Twenty-four male Wistar rats (3 months old) were randomly divided into groups (n = 8 per group): control rats that received water (C); diabetic rats that received water (D); diabetic rats treated with mate tea (DMT). The treated streptozotocin-induced diabetic rats were given mate tea powder by intragastric gavage at a dose of 20 mg/kg daily for 28 days. Content of total protein, amylase, oxidative lipid damage, measured as thiobarbituric acid reactive substances (TBARs), oxidative protein damage, measured as protein carbonyl, total antioxidant capacity, uric acid, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were examined by the spectrophotometric method in the parotid and submandibular glands. RESULTS: The D group showed lower total protein, amylase, TBARs, protein carbonyl, total antioxidant capacity, GSH, uric acid, and GPx than the C group in both salivary glands, as well as higher SOD and CAT activities. The DMT group showed higher total protein, amylase, total antioxidant capacity, GSH, uric acid, and GPx than the D group in both salivary glands. Moreover, mate tea increased SOD in the parotid gland and CAT in the submandibular gland of diabetic rats but did not influence TBARs and protein carbonyl in either salivary gland compared to D group. CONCLUSION: Mate tea increased tissue protein synthesis and improved antioxidant defenses in the salivary glands of streptozotocin-induced diabetic male rats.

Analysis of the femoral neck from rats in the periestropause treated with oxytocin and submitted to strength training.

Among the interventions used to prevent osteoporosis in female organisms, strength training (ST) and oxytocin (OT) stand out, as a promising hormone with anabolic action on bone. This study aimed to verify whether the combined action of OT and ST, compared to isolated interventions, potentiates the bone remodeling process of the femoral neck of Wistar rats during periestropause. Forty Wistar rats (18 months) with irregular estrous cycle were randomly distributed into groups: 1-Vehicle (Veh; NaCl 0.15 mol/L ip); 2-Oxytocin (Ot; 134 µg/kg/ip); 3-Strength training (St); 4-Ot + St. The animals of the 1, 2 and 4 groups received two intraperitoneal injections with an interval of 12 h every 30 days, totaling 8 injections at the end of the experimental period (18 to 21 months). The animals in the St and Ot + St groups performed ST on a ladder 3 times a week, maximal voluntary carrying capacity (MVCC) test monthly. After 120 days, the animals were euthanized; the femur was collected for analysis of biomechanical testing, densitometry, bone microtomography, Raman spectroscopy, tissue PCR, and blood for analysis of bone biomarkers, liver damage, and oxidative stress. The main effects in the Ot group were observed in the maximum load and energy in the compression testing (femoral head), and stiffness and energy in the three-points bending testing (femur diaphysis). In addition, the main effects occurred on the bone mineral density (BMD), cortical thickness (Ct.Th), number of pores (Po.N), polar moment of inertia (J), trabecular thickness (Tb.Th), and connectivity density (Conn.Dn), Bone alkaline phosphatase (Alp), Tumor necrosis factor receptor superfamily member 11b (Opg), Tumor necrosis factor ligand superfamily member 11 (Rankl) and Cathepsin K (Ctsk) expression. There was an effect in the tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP). In the St group, the main effect was observed on the energy (compression and the three-points bending), stiffness, aBMD, BMD, cortical bone area (Ct.Ar), Po.N, trabecular bone volume (BV/TV), Tb.Th and in the mineralization ratio (ѵ1PO4/proline), Runt-related transcription factor 2 (Runx2), Bone morphogenetic protein 2 (Bmp2), Alp, Osteopontin/secreted phosphoprotein 1 (Opn/Spp1), Opg, Tumor necrosis factor receptor superfamily member 11ª (Rank), Rankl, Ctsk expression. There was an effect in the TRAP and ALP. The interaction in the combination of therapies in the Ot + St group was verified in energy to maximum load (compression and three-points bending testing), stiffness, BMD, Ct.Th, J, Tb.Th and ѵ1PO4/proline. In the gene analysis there was interaction in the Runx2, Osterix/Sp7 transcription factor (Osx/Sp7), Bmp2, Alp, Osteocalcin/Bone gamma-carboxyglutamate protein (Ocn/Bglap), Opg, Rankl and Acid phosphatase 5, tartrate resistant (Trap/Acp5) expression. In addition, the combination of OT and ST resulted in a higher maximum load compared to the Veh group, with higher BV/TV than the Ot group, higher Rankl and Ctsk expression than Veh and Ot groups, and lower Po.N and lower activity of TRAP than the other groups. In oxidative stress, total antioxidant capacity (TAC) was lower. These results showed that the combination of interventions is a promising anabolic strategy for the prevention of osteoporosis in the period of periestropause, standing out from the effects of isolated interventions.

Assessment of redox state and biochemical parameters of salivary glands in rats treated with anti-obesity drug sibutramine hydrochloride.

OBJECTIVES: To investigate the effects of anti-obesity drug sibutramine hydrochloride (SB) on redox state and biochemical parameters in the salivary glands. MATERIALS AND METHODS: Adult male Wistar rats were randomly divided into the following groups (n = 8 per group): control rats treated with vehicle (C) and rats treated with SB (10 mg/kg/day) by intragastric gavage for 28 days. The parotid (PG) and submandibular (SMG) glands were processed using histomorphometric analysis, and total protein, amylase, mucin, and oxidative damage to lipids were determined by measuring the formation of thiobarbituric acid reactive substances (TBARS), total antioxidant capacity (TAC), uric acid (UA), total glutathione (tGSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and AKT phosphorylation. RESULTS: SB decreased the acinar area, and increased the stromal area in PG, while no effect on the morphometric parameters was observed in SMG. SB also increased oxidative damage to lipids (TBARs). The SB group showed lower total protein, amylase, TAC, UA, tGSH, SOD, CAT, and GPx than the C group in PG, while in SMG, SB decreased total protein, mucin, tGSH, SOD, CAT, and GPx. However, increased AKT phosphorylation observed in both salivary glands suggests that SB exerts low-intensity oxidative stress. CONCLUSIONS: SB impaired enzymatic and non-enzymatic antioxidant defenses in the salivary glands of rats. CLINICAL RELEVANCE: Chronic treatment with SB could mitigate salivary gland dysfunction due to disturbance of redox state.

Effects of orchiectomy and testosterone replacement therapy on redox balance and salivary gland function in Wistar rats.

The objective of this study was to investigate the effects of orchiectomy (ORX) and testosterone replacement therapy (TRT) on redox balance and function of salivary glands. Forty-five young adult male Wistar rats (3 months old) were either castrated bilaterally or underwent fictitious surgery (SHAM) and were subsequently distributed into 3 groups: SHAM, ORX, and TRT (castrated rats that received an intramuscular injection of testosterone cypionate 10 mg/kg/weekly). All treatments started 4 weeks after castration (4 months old) and lasted 4 weeks (5 months old). At the end of treatment, pilocarpine-induced salivary secretion was collected to analyze salivary flow rate and biochemistry composition, and the parotid (PG) and submandibular (SMG) glands were sampled for redox balance markers and histomorphometric analyses. ORX increased salivary flow rate, calcium, phosphate, and chloride, and decreased total protein and amylase, while not changing the salivary buffer capacity, pH, sodium, and potassium compared to SHAM. TRT restored all salivary parameters to SHAM values. ORX increased oxidative lipid and protein damage, total antioxidant capacity, and uric acid in both salivary glands compared to SHAM. Superoxide dismutase, catalase, and glutathione peroxidase activities were greater only in the SMG of the ORX group in relation to SHAM. ORX decreased duct and acini area, while increasing connective tissue in the PG. On the other hand, ORX reduced duct area and increased acini area in the SMG compared to SHAM. TRT restored the redox balance and histomorphometric parameters to close to SHAM values in both salivary glands. Orchiectomy-induced salivary gland dysfunction was characterized by an increase in the salivary flow rate and changes in the secretion of total protein, amylase, and electrolytes, which are key factors, considered important for maintaining oral health status. To sum up, orchiectomy impaired the redox balance of the salivary glands. Our results also showed that TRT reversed the oxidative damage, morphological alterations, and salivary gland dysfunction induced by orchiectomy. Therefore, these results suggest an important action of testosterone on the redox balance and secretory ability of salivary glands.

Effect of testosterone replacement therapy and mate tea (Ilex paraguariensis) on biochemical, functional and redox parameters of saliva in orchiectomized rats.

OBJECTIVE: Evaluate the effects of testosterone replacement therapy (TRT) and mate tea (MT) [Ilex paraguariensis] on biochemical, functional, and redox parameters of saliva in orchiectomized rats (ORX) DESIGN: Sixty young adult male Wistar rats (3 months old) were either castrated bilaterally or underwent fictitious surgery (SHAM) and were distributed into 5 groups: SHAM, ORX, TU (castrated rats that received a single intramuscular injection of testosterone undecanoate 100 mg/kg), MT (castrated rats that received MT 20 mg/kg, via intragastric gavage, daily), and TU + MT. All treatments started 4 weeks after castration (4 months old) and lasted 4 weeks (5 months old). At the end of treatment, pilocarpine-induced salivary secretion was collected to analyze salivary flow rate (SFR) and biochemistry composition through determination of total protein (TP), amylase (AMY), electrolyte, and biomarkers of oxidative stress. RESULTS: ORX increased SFR, salivary buffering capacity, calcium, phosphate, chloride, total antioxidant capacity, thiobarbituric acid reactive substances (TBARs), and carbonyl protein, reduced TP and AMY activity, and did not change pH, sodium, and potassium compared to SHAM. TU and TU+MT restored all salivary parameters to values of SHAM, while only TBARs and AMY returned to SHAM levels in the MT group. CONCLUSIONS: TRT with long-acting TU restored the biochemical, functional, and redox parameters of saliva in orchiectomized rats. Although MT did not have a TRT-like effect on salivary gland function, the more effective reduction in lipid oxidative damage in the MT and TU + MT groups could be considered as adjuvant to alleviate the salivary oxidative stress induced by orchiectomy.

Excessive caffeine intake increases bone resorption associated with periapical periodontitis in rats.

AIM: To evaluate the effect of excessive caffeine intake on the inflammation/resorption processes associated with periapical periodontitis (PP) in rats. METHODOLOGY: Sixteen Wistar rats were used. Periapical periodontitis was induced in the four first molars in each animal. The animals were arranged into two groups: control (C)-rats with periapical periodontitis; and caffeine (CAF)-rats with periapical periodontitis under caffeine administration protocol. The CAF animals received 10 mg/100 g of body weight/day of caffeine via gavage starting fifteen days before PP induction and continuing for thirty more days until euthanasia. On the 30th day, the animals were euthanized and the jaws removed for microcomputed tomography, histological and immunohistochemical analysis for RANKL, OPG, TRAP, IL-10, TNF-⍺ and IL-1ß. The Mann-Whitney test was performed for nonparametric data, and Student's t test was performed for parametric data, using p < .05. RESULTS: There was no significant difference in the weight change between the groups. The median score of the inflammatory process was significantly greater in the CAF group (3) compared with the C group (2), p = .0256. Bone resorption was greater in the group consuming caffeine (1.08 ± 0.15 mm3 ) compared with the C group (0.88 ± 0.10 mm3 ), p = .0346. The immunolabelling for RANKL, TRAP and IL-1ß was significantly higher in the CAF group when compared to the control, p < .05. No differences were found for the OPG, IL-10 and TNF-⍺ immunolabelling. CONCLUSION: Excessive caffeine exposure via gavage in rats was able to exacerbate the volume of periapical bone destruction, and the inflammatory pattern deriving from periapical periodontitis altering the expression of RANKL, IL-1ß and TRAP.

Experimental gel containing bioactive glass-ceramic to minimize the pulp damage caused by dental bleaching in rats

Abstract Objectives This study evaluated if the use of a bioactive glass-ceramic-based gel, named Biosilicate (BS), before, after or mixed with bleaching gel, could influence the inflammation of the dental pulp tissue of rats' molars undergoing dental bleaching with hydrogen peroxide (H2O2). Methodology The upper molars of Wistar rats (Rattus norvegicus, albinus) were divided into Ble: bleached (35% H2O2, 30-min); Ble-BS: bleached and followed by BS-based gel application (20 min); BS-Ble: BS-based gel application and then bleaching; BS/7d-Ble: BS-based gel applications for 7 days and then bleaching; Ble+BS: blend of H2O2 with BS-based gel (1:1, 30-min); and control: placebo gel. After 2 and 30 days (n=10), the rats were euthanized for histological evaluation. The Kruskal-Wallis and Dunn statistical tests were performed (P<0.05). Results At 2 days, the Ble and Ble-BS groups had significant alterations in the pulp tissue, with an area of necrosis. The groups with the application of BS-based gel before H2O2 had moderate inflammation and partial disorganization in the occlusal third of the coronary pulp and were significantly different from the Ble in the middle and cervical thirds (P<0.05). The most favorable results were observed in the Ble+BS, which was similar to the control in all thirds of the coronary pulp (P>0.05). At 30 days, the pulp tissue was organized and the bleached groups presented tertiary dentin deposition. The Ble group had the highest deposition of tertiary dentin, followed by the Ble-BS, and both were different from control (P<0.05). Conclusion A single BS-based gel application beforehand or BS-based gel blended with a bleaching gel minimize the pulp damage induced by dental bleaching.

Soluble yerba mate (Ilex Paraguariensis) extract enhances in vitro osteoblastic differentiation of bone marrow-derived mesenchymal stromal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Yerba mate (Ilex paraguariensis) consumption has been associated with beneficial effects on bone health. AIM OF THE STUDY: The purpose of this study was to evaluate the mechanism by which soluble yerba mate (SYM) stimulates osteoblast differentiation of bone marrow-derived mesenchymal stromal cells (BM-MSCs). MATERIALS AND METHODS: BM-MSCs from male Wistar rats were induced towards osteoblastic differentiation with different concentrations of SYM (10, 20, and 50 µg/mL). Osteoblastic differentiation was evaluated by measuring proliferation rates, alkaline phosphatase activity, MMP-2 activity, mineralization, and gene expression of Runx2, Osterix, ß-catenin (Catnb), collagen type I (Col1a1), osteopontin (Opn), osteocalcin (Ocn), bone sialoprotein (Bsp), bone morphogenetic protein-2 (Bmp2), osteoprotegerin (Opg), and Rankl. We also analyzed cytokine production and MAP kinase pathways. RESULTS: SYM (10 µg/mL) did not show a cytotoxic effect and induced a slight increase in ALP activity; however, a great increase in mineralization was observed. SYM was also able to reduce TNF-α and IL-10 production; increase the expression of transcription factors Runx2, Osterix, and Catnb; and increase matrix proteins Opn, Bsp, Ocn, and Bmp2. We also observed a decrease in intracellular signaling of ERK, JNK, and p38 MAPK, which seemed to be related to the SYM response. CONCLUSIONS: Together, these results help to explain the promoting effect on osteoblast differentiation produced by a low SYM concentration. However, a higher SYM concentration presented deleterious effects, including cytotoxicity, decreased ALP activity, increased cytokine production, decreased bone marker gene expression, increased MAPK signaling, and significant mineralization reduction. In conclusion, our results suggest a concentration-specific direct stimulatory effect of SYM on osteoblastic differentiation in vitro.

Oxytocin effects on osteoblastic differentiation of Bone Marrow Mesenchymal Stem Cells from adult and aging female Wistar rats.

Recently, it has been suggested that oxytocin (OT) might play a role in the control of bone remodeling and in bone health of young and adult females. The purpose of this study was to evaluate the effect of osteogenic medium (OM) plus OT (OM + OT; 100 nmol/L) on osteoblastic differentiation of bone marrow mesenchymal stem cells (BMMSCs) from cyclic adult (12 months old) and acyclic aging (24 months old) female Wistar rats. After 14 days, OM + OT increased the oxytocin and oxytocin receptor in the BMMSCs from animals of both age groups relative to OM controls. Alkaline phosphatase activity was higher in the OM + OT than OM group in BMMSCs from 24-month-old female rats. OM + OT improved osteogenic differentiation, observed by anticipated mineralization and increased gene expression of bone morphogenetic protein 2, bone sialoprotein, osteopontin and osteocalcin in both aged relative to OM controls. These findings suggest a role for OT as an adjuvant to induce osteoblastic differentiation of BMMSCs from aged female rat.

Mate tea (Ilex paraguariensis) improves bone formation in the alveolar socket healing after tooth extraction in rats.

OBJECTIVES: The objective of this study was to investigate the effects of mate tea (MT) [Ilex paraguariensis] on alveolar socket healing after tooth extraction. MATERIALS AND METHODS: Sixteen male rats were divided into MT and control groups. MT was administered by intragastric gavage at a dose of 20 mg/kg/day for 28 days before and 28 days after right maxillary incisor extraction. The control group received an equal volume of water. Histopathological and histometric analysis of the neoformed bone area and osteocyte density were performed, as well as immunohistochemical analysis of osteocalcin (OCN), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), tartrate-resistant acid phosphatase (TRAP), and manganese superoxide dismutase (MnSOD) in the alveolar socket. Calcium, phosphorus, alkaline phosphatase (ALP) activity, total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured in plasma, whereas TRAP activity was determined in serum. RESULTS: Histometry evidenced an increase in bone area (P < 0.0001) and osteocyte density (P < 0.0001). MT increased immunolabeling of MnSOD (P < 0.001), OCN (P < 0.0001), RANKL (P < 0.001), OPG (P < 0.0001), and TRAP (P < 0.001). Calcium and phosphorus concentrations did not differ between the groups. In addition, MT enhanced ALP (P < 0.05) and TRAP (P < 0.0001) activities. MT increased the TAC (P < 0.001), whereas it reduced MDA concentrations (P < 0.0001). CONCLUSIONS: MT increases bone area and osteocyte density in the alveolar socket healing on day 28 after tooth extraction. CLINICAL RELEVANCE: Regular MT ingestion improves the antioxidant defenses and bone formation, which is beneficial for alveolar socket bone healing after tooth extraction.

Effects of strength training on osteogenic differentiation and bone strength in aging female Wistar rats.

The effects of strength training (ST) on the mechanical bone strength and osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) from adult, aged and exercised aged rats were determined. The exercised aged animals displayed higher values of areal bone mineral density, compression test, alkaline phosphatase activity (ALP) and biological mineralization, while oil red O staining for adipocytes was lower. ST increased gene expression of runt-related transcription factor 2 (Runx2), osterix (Osx) as well as bone matrix protein expression, and reduced expression of peroxisome proliferator-activated receptor gamma (Pparγ). The production of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) was lower in BMSCs of the aged exercised group. The ST practice was able to improve the bone mechanical properties in aged female rats, increasing the potential for osteogenic differentiation of BMSCs, reducing the adipogenic differentiation and pro-inflammatory cytokine level. In summary, the data achieved in this study showed that strength training triggers physiological responses that result in changes in the bone microenvironment and bring benefits to biomechanical parameters of bone tissue, which could reduce the risk of fractures during senescent.

Tracheal Smooth Muscle Cells Stimulated by Stem Cell Factor-c-Kit Coordinate the Production of Transforming Growth Factor-ß1 and Fibroblast Growth Factor-2 Mediated by Chemokine (C-C Motif) Ligand 3.

The aim of this study was to evaluate the mechanism involved in the stem cell factor (SCF)-induced production of fibroblast growth factor-2 (FGF-2), transforming growth factor-ß1 (TGF-ß1), and chemokine (C-C motif) ligand 3 (CCL3) in tracheal smooth muscle cells (tSMCs) and the signaling pathway involved in the process. tSMC primary cultures were stimulated with SCF and evaluated at 24 h. Cells treated with specific antibodies did not show any immunolabeling for cytokeratin or fibroblast activation protein, but were positive for α-smooth muscle actin, indicating the purity of the primary cell line. Western blot analysis showed constitutive phosphorylation of c-Kit, as well as increased total protein and phosphorylated c-Kit levels in tSMCs after SCF stimulation. Flow cytometry analysis also showed an increase in cell-surface c-Kit expression in the presence of SCF. SCF induced TGF-ß mRNA expression in tSMCs, as well as the production of TGF-ß1, CCL3, and FGF-2. Pretreatment with anti-CCL3 antibody blocked TGF-ß1 expression and partially inhibited FGF-2 production. On the other hand, anti-c-Kit antibody blocked TGF-ß1 expression and FGF-2 production. Thus, TGF-ß1 and FGF-2 production were mediated by CCL3 production through c-Kit. Pretreatment with mitogen-activated protein kinase kinase 1, p38, and Jun N-terminal kinase inhibitors showed that the effects mediated by SCF were involved with the modulation of mitogen-activated protein kinase (MAPK) pathways. Development of inhibitors targeting CCL3 through MAPK activation could thus be an attractive strategy to inhibit tSMC activation during asthma.

Maternal periodontitis decreases plasma membrane GLUT4 content in skeletal muscle of adult offspring.

AIMS: The fetal programming hypothesis suggests that intrauterine stimuli can induce metabolic changes in offspring, increasing the disease risk in adulthood. Periodontal disease may enhance serum cytokine levels. Cytokines such as tumor necrosis factor-alpha (TNF-α) have been associated with reduced glucose transporter type 4 (GLUT4) expression, decreased protein kinase B (Akt) phosphorylation, and insulin resistance. This study aimed to evaluate GLUT4 content, and Akt serine phosphorylation status in the gastrocnemius skeletal muscle (GSM), glycemia, insulinemia and change in body weight in offspring of rats with periodontal disease. MAIN METHODS: Female Wistar rats were distributed into a control group (CN) and an experimental periodontal disease group (PD), in which a ligature was placed around the mandibular first molars. Seven days after ligature placement, both groups were mated with normal male rats. The ligatures remained throughout pregnancy until weaning, after which the male offspring were distributed into groups: CN-o, control rat offspring; and PD-o, periodontal disease rat offspring. The body weight from 0 to 75days of age was measured. At 75days, the glycemia, insulinemia, TNF-α levels, Akt serine phosphorylation, and GLUT4 content in the GSM were measured in the offspring. KEY FINDINGS: The PD-o group showed a low birth weight (LBW), unchanged glycemia, increased insulinemia, insulin resistance, increased TNF-α levels, decreased Akt serine phosphorylation status, and reduced GLUT4 content in the plasma membrane and translocation index after insulin stimulation. SIGNIFICANCE: Maternal periodontal disease causes LBW, insulin resistance, and alterations in the final stage of insulin signaling in the GSM of adult offspring.

The effects of strength training and raloxifene on bone health in aging ovariectomized rats.

The aim of this study was to investigate the effects of strength training (ST) and raloxifene (Ral), alone or in combination, on the prevention of bone loss in an aging estrogen-deficient rat model. Aging Wistar female rats were ovariectomized at 14months and allocated to four groups: (1) non-trained and treated with vehicle, NT-Veh; (2) strength training and treated with vehicle, ST-Veh; (3) non-trained and treated with raloxifene, NT-Ral; and (4) strength training and treated with raloxifene, ST-Ral. ST was performed on a ladder three times per week and Ral was administered daily by gavage (1mg/kg/day), both for 120days. Areal bone mineral density (aBMD), strength, microarchitecture, and biomarkers (osteocalcin, OCN; osteoprotegerin, OPG; and tartrate-resistant acid phosphatase, TRAP) were assessed. Immunohistochemistry was performed for runt-related transcription factor 2 (RUNX2), osterix (OSX), OCN, OPG, TRAP, and receptor activator of nuclear factor kappa-B ligand (RANKL). The rats that performed ST (ST-Veh) or were treated with Ral (NT-Ral) showed significant improvements in aBMD (p=0.001 and 0.004), bone strength (p=0.001), and bone microarchitecture, such as BV/TV (%) (p=0.001), BS/TV (mm(2)/mm(3)) (p=0.023 and 0.002), Conn.Dn (1/mm(3)) (p=0.001), Tb.N (1/mm) (p=0.012 and 0.011), Tb.Th (1/mm) (p=0.001), SMI (p=0.001 and 0.002), Tb.Sp (p=0.001), and DA (p=0.002 and 0.007); there was also a significant decrease in plasma levels of OCN (p=0.001 and 0.002) and OPG (p=0.003 and 0.014), compared with animals in the NT-Veh group. Ral, with or without ST, promoted an increased immunolabeling pattern for RUNX2 (p=0.0105 and p=0.0006) and OSX (p=0.0105), but a reduced immunolabeling pattern for TRAP (p=0.0056) and RANKL (p=0.033 and 0.004). ST increased the immunolabeling pattern for RUNX2 (p=0.0105), and association with Ral resulted in an increased immunolabeling pattern for OPG (p=0.0034) and OCN (p=0.0024). In summary, ST and Ral administration in aged, estrogen-deficient Wistar female rats is associated with a decrease in bone turnover marker plasma levels, increased activity of cells that promote osteoblastogenesis, and decreased activity of cells that promote osteoclastogenesis; these are correlated with higher aBMD, bone strength, and bone microarchitecture at the femoral neck. The results indicate that strength training and Ral are potential tools to reduce the risk of fractures at clinically relevant sites.