In vitro-in vivo availability of metformin hydrochloride-PLGA nanoparticles in diabetic rats in a periodontal disease experimental model.

CONTEXT: Metformin is an important oral anti-hyperglycemic used in diabetes. Polylactic-co-glycolic acid (PLGA) has been widely used due to its reliability in controlling the release of drugs. OBJECTIVE: This study evaluates the in vitro-in vivo availability of metformin hydrochloride-loaded polylactic-co-glycolic acid. MATERIAL AND METHODS: In vitro metformin release (Met-free or PLGA + Met-12.5 mg/mL per 360 min) was evaluated using static Franz vertical diffusion cells. The in vivo study was performed with two control groups (validation bioanalytical method) and two experimental groups of diabetic male Wistar rats treated with PLGA + Met 10 mg/kg or Met 100 mg/kg by oral gavage. Diabetes was induced by streptozotocin (40 mg/kg) through the penile vein. Blood samples were collected 0.5, 1, 4, 7, 10, 12, 18, 24, 36, 48 and 72 h and analysed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: PLGA + Met 10 mg/kg was released in the in vitro assay suggesting a parabolic diffusion kinetic model (K -0.0619-0.5h) with a 100% release profile in 10 h by controlled diffusion. The in vivo assay showed the apparent volume of distribution Vz/F (PLGA + Met 10 mg/kg, 40971.8 mL/kg vs. Met 100 mg/kg, 2174.58 mL/kg) and mean residence time MRTinf (PLGA + Met 10 mg/kg, 37.66 h vs. Met 100 mg/kg, 3.34 h). DISCUSSION AND CONCLUSIONS: The formulation modifies pharmacokinetics parameters such as apparent distribution volume and mean residence time. The PLGA + Met 10 mg/kg had a slower elimination rate compared to Met 100 mg/kg in diabetic rats in a periodontal disease experimental model.

The involvement of mast cells in the irinotecan-induced enteric neurons loss and reactive gliosis.

BACKGROUND: The irinotecan (CPT-11) causes intestinal mucositis and diarrhea that may be related to changes in the enteric nervous system (ENS). In inflammatory condition, mast cells release a variety of pro-inflammatory mediators that can interact with the ENS cells. It has not been explored whether CPT-11 is able to alter the enteric glial and neuronal cell, and the role of mast cells in this effect. Therefore, this study was conducted to investigate the effect of CPT-11 on the enteric glial and neuronal cells, as well as to study the role of mast cells in the CPT-11-induced intestinal mucositis. METHODS: Intestinal mucositis was induced in Swiss mice by the injection of CPT-11 (60 mg/kg, i.p.) once a day for 4 days following by euthanasia on the fifth day. To investigate the role of mast cells, the mice were pretreated with compound 48/80 for 4 days (first day, 0.6 mg/kg; second day, 1.0 mg/kg; third day, 1.2 mg/kg; fourth day, 2.4 mg/kg) to induce mast cell degranulation before the CPT-11 treatment. RESULTS: Here, we show that CPT-11 increased glial fibrillary acidic protein (GFAP) and S100ß gene and S100ß protein expressions and decreased HuC/D protein expression in the small intestine segments. Concomitantly, CPT-11 enhanced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels and inducible nitric oxide synthase (iNOS) gene expression, associated with an increase in the total number macrophages (positive cells for ionized calcium-binding adapter molecule, Iba-1) and degranulated mast cells in the small intestine segments and caused significant weight loss. The pretreatment with compound 48/80, an inductor of mast cells degranulation, significantly prevented these CPT-11-induced effects. CONCLUSIONS: Our data suggests the participation of mast cells on the CPT-11-induced intestinal mucositis, macrophages activation, enteric reactive gliosis, and neuron loss.

Target Inhibition of IL-1 Receptor Prevents Ifosfamide Induced Hemorrhagic Cystitis in Mice.

PURPOSE: Hemorrhagic cystitis is an important dose limiting side effect of ifosfamide based cancer chemotherapy. Despite chemoprophylaxis inflammation can still be found in cystoscopy guided biopsies. Previous studies confirmed the role of TNF-α and IL-1ß. We evaluated the protective effect of the IL-1R antagonist anakinra and the anti-TNF-α antibody infliximab in experimental ifosfamide induced hemorrhagic cystitis. MATERIALS AND METHODS: Hemorrhagic cystitis was induced by an injection of ifosfamide (400 mg/kg intraperitoneally) in Swiss wild-type C57Bl/6, IL-1R-/-, TNFR1-/- or TNFR1/R2-/- mice. Mice were treated 30 minutes before ifosfamide with anakinra (100 mg/kg intraperitoneally), infliximab (5 mg/kg intraperitoneally) or vehicle. Visceral nociception was evaluated after hemorrhagic cystitis induction. At 12 hours the animals were sacrificed. Bladders were harvested to assess bladder wet weight, vascular permeability, macroscopic and microscopic findings, muscle contractility, and for cystometrography. Inflammatory cell infiltration was assessed by myeloperoxidase assay and flow cytometry. RESULTS: Anakinra attenuated hemorrhage, edema, neutrophil infiltration, visceral hyperalgesia and bladder dysfunction. IL-1R-/- mice also showed milder hemorrhagic cystitis. Infliximab inhibited bladder edema and visceral hyperalgesia without preventing hemorrhage, bladder dysfunction, neutrophils or accumulation. Additionally, the lack of TNFR1 decreased bladder edema but not cell infiltration whereas concomitant deficiency of TNFR1 and TNFR2 resulted in worse hemorrhagic cystitis. CONCLUSIONS: Anakinra is effective for preventing experimentally ifosfamide induced hemorrhagic cystitis. It seems that neutrophil and macrophage infiltration in this circumstance depends on IL-1 signaling through IL1R. Possibly TNFR2 has a protective role in hemorrhagic cystitis.

Polysacharide of Agaricus blazei gel mitigates bone necrosis in model of the jaws related to bisphosphonate via Wnt signaling.

To investigate de effect of PAb gel on the bone tissue of rats submitted to Bisphosphonate-related osteonecrosis of the jaws (BRONJ). Initially, 54 animals were submitted to BRONJ model by Zoledronic Acid (ZA) (0.1 mg/kg 3x/wk for 9 wk, ip), followed by the 1st upper left molar extraction at the 8th wk. After tooth removal, the animals were divided into 3 groups, ZA that received placebo gel or PAb gel that received 1% PAb gel, inside the dental alveolus. The control Group (CONTROL) received 0.1 mg/kg of 0.9% saline and then placebo gel. Three weeks after tooth extraction, the animals were euthanized, and maxillae were colleted for macroscopic, radiographic, histological and Raman spectomery assays. Additionally, GSK3b, beta-catenin, and Runx2 mRNA expressions were determined. Blood samples were collected for the analysis of Bone-specific alkaline phosphatase (BALP) levels. PAb gel improved mucosal healing, increased the number of viable osteocytes, while it reduced the number of empty lacunae, as well as the amount of bone sequestration. Furthermore, PAb gel positively influenced the number and functionality of osteoblasts by stimulating Wnt signaling, thereby inducing bone remodeling. Additionally, PAb gel contributed to improved bone quality, as evidenced by an increase in bone mineral content, a decrease in bone solubility, and an enhancement in the quality of collagen, particularly type I collagen. PAb gel mitigated bone necrosis by stimulating of bone remodeling through Wnt signaling and concurrently improved bone quality. PAb gel emerges as a promising pharmacological tool for aiding in BRONJ therapy or potentially preventing the development of BRONJ.

The antioxidant effects of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic painful peripheral neuropathy.

BACKGROUND: Oxaliplatin, the third-generation platinum compound, has evolved as one of the most important therapeutic agents in colorectal cancer chemotherapy. The main limiting factor in oxaliplatin treatment is painful neuropathy that is difficult to treat. This side effect has been studied for several years, but its full mechanism is still inconclusive, and effective treatment does not exist. Data suggest that oxaliplatin's initial neurotoxic effect is peripheral and oxidative stress-dependent. A spinal target is also suggested in its mechanism of action. The flavonoids rutin and quercetin have been described as cell-protecting agents because of their antioxidant, antinociceptive, and anti-inflammatory actions. We proposed a preventive effect of these agents on oxaliplatin-induced painful peripheral neuropathy based on their antioxidant properties. METHODS: Oxaliplatin (1 mg/kg, i.v.) was injected in male Swiss mice, twice a week (total of nine injections). The development of sensory alterations, such as thermal and mechanical allodynia, was evaluated using the tail immersion test in cold water (10°C) and the von Frey test. Rutin and quercetin (25-100 mg/kg, i.p.) were injected 30 min before each oxaliplatin injection. The animals' spinal cords were removed for histopathological and immunohistochemical evaluation and malondialdehyde assay. RESULTS: Oxaliplatin significantly increased thermal and mechanical nociceptive response, effects prevented by quercetin and rutin at all doses. Fos immunostaining in the dorsal horn of the spinal cord confirmed these results. The oxidative stress assays mainly showed that oxaliplatin induced peroxidation in the spinal cord and that rutin and quercetin decreased this effect. The flavonoids also decreased inducible nitric oxide synthase and nitrotyrosine immunostaining in the dorsal horn of the spinal cord. These results suggest that nitric oxide and peroxynitrite are also involved in the neurotoxic effect of oxaliplatin and that rutin and quercetin can inhibit their effect in the spinal cord. We also observed the preservation of dorsal horn structure using histopathological analyses. CONCLUSIONS: Oxaliplatin induced painful peripheral neuropathy in mice, an effect that was prevented by rutin and quercetin. The mechanism of action of oxaliplatin appears to be, at least, partially oxidative stress-induced damage in dorsal horn neurons, with the involvement of lipid peroxidation and protein nitrosylation.

Effect of telmisartan on levels of IL-1, TNF-α, down-regulated COX-2, MMP-2, MMP-9 and RANKL/RANK in an experimental periodontitis model.

AIM: The aim of this study was to evaluate the effect of telmisartan (TELM) on inflammation, oxidation and the expression of matrix metalloproteinases (MMPs) and the expression RANKL/RANK/OPG in the periodontal tissue of a rat model for ligature-induced periodontitis. MATERIALS AND METHODS: Male Wistar albino rats were randomly divided into five groups of 10 rats each: (i) non-ligated, given water; (ii) ligated, given water; (iii) ligated, given 1 mg/kg TELM; (iv) ligated, given 5 mg/kg TELM; and (v) ligated, given 10 mg/kg TELM. All groups were treated with saline or TELM for 10 days. Periodontal tissue was analysed by histopathology; by the immunohistochemical examination of COX-2, MMP-2, MMP-9 and the RANKL/RANK/OPG pathway; and by ELISA analysis of the levels of IL-1ß, IL-10, TNF-α, myeloperoxidase (MPO), malonaldehyde (MDA) and glutathione (GSH). RESULTS: Treatment with 10 mg/kg TELM resulted in reduced concentrations of MPO, MDA (p < 0.05) and the pro-inflammatory cytokine IL-1ß (p < 0.05); reduced expression of MMP-2, MMP-9, RANK, RANKL and COX-2; and an increase in OPG. The levels of TNF-α were significantly reduced in all TELM-treated groups. CONCLUSIONS: These findings confirm the involvement of TELM in reducing the inflammatory response, oxidative stress and bone loss in ligature-induced periodontitis in rats.

Adenosine receptors differentially mediate enteric glial cell death induced by Clostridioides difficile Toxins A and B.

Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.

P2X7 receptor blockade decreases inflammation, apoptosis, and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice.

BACKGROUND: Clostridioides difficile (C. difficile) is the most common pathogen causing health care-associated infections. C. difficile TcdA and TcdB have been shown to activate enteric neurons; however, what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown. AIM: To characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation, cell death, and the changes in the enteric nervous system in mice. METHODS: Swiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA (50 µg/Loop) for 4 h. To investigate the role of the P2X7 receptor, Brilliant Blue G (50 mg/kg, i.p.), which is a nonspecific P2X7 receptor antagonist, or A438079 (0.7 µg/mouse, i.p.), which is a competitive P2X7 receptor antagonist, were injected one hour prior to TcdA challenge. Ileal samples were collected to analyze the expression of the P2X7 receptor (by quantitative real-time polymerase chain reaction and immunohistochemistry), the population of myenteric enteric neurons (immunofluorescence), histological damage, intestinal inflammation, cell death (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), neuronal loss, and S100B synthesis (immunohistochemistry). RESULTS: TcdA upregulated (P < 0.05) the expression of the P2X7 receptor gene in the ileal tissues, increasing the level of this receptor in myenteric neurons compared to that in control mice. Comparison with the control mice indicated that TcdA promoted (P < 0.05) the loss of myenteric calretinin+ (Calr) and choline acetyltransferase+ neurons and increased the number of nitrergic+ and Calr+ neurons expressing the P2X7 receptor. Blockade of the P2X7 receptor decreased TcdA-induced intestinal damage, cytokine release [interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α], cell death, enteric neuron loss, and S100B synthesis in the mouse ileum. CONCLUSION: Our findings demonstrated that TcdA induced the upregulation of the P2X7 receptor, which promoted enteric neuron loss, S100B synthesis, tissue damage, inflammation, and cell death in the mouse ileum. These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C. difficile infection.

Host and Clostridioides difficile-Response Modulated by Micronutrients and Glutamine: An Overview.

Changes in intestinal microbiota are integral to development of Clostridioides difficile (C. difficile)-associated nosocomial diarrhea. Certain diets, especially Western diets, increase susceptibility to C. difficile infection (CDI). Here, we discuss recent findings regarding how nutrients modulate response of the host and C. difficile during infection. Calcium has a role in the sporulation and germination process. Selenium is effective in reducing the total amount of C. difficile toxin A (TcdA) and toxin B (TcdB) and in decreasing its cytotoxicity. In addition, selenium phosphate synthetase deficiency reduces C. difficile growth and spore production. On the other hand, iron has a dual role in C. difficile growth. For instance, high intracellular levels can generate reactive hydroxyl radicals, whereas low levels can reduce its growth. In humans, zinc deficiency appears to be related to the recurrence of CDI, in contrast, in the CDI model in mice a diet rich in zinc increased the toxin's activity. Low vitamin D levels contribute to C. difficile colonization, toxin production, and inflammation. Furthermore, glutamine appears to protect intestinal epithelial cells from the deleterious effects of TcdA and TcdB. In conclusion, nutrients play an important role in modulating host and pathogen response. However, further studies are needed to better understand the mechanisms and address some controversies.

Role of Pannexin-1-P2X7R signaling on cell death and pro-inflammatory mediator expression induced by Clostridioides difficile toxins in enteric glia.

Clostridioides difficile (C. difficile) produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. Pannexin-1 (Panx1) channels allows the passage of messenger molecules, such as adenosine triphosphate (ATP), which in turn activate the P2X7 receptors (P2X7R) that regulate inflammation and cell death in inflammatory bowel diseases. The aim of this study was to verify the effect of C. difficile infection (CDI) in the expression of Panx1 and P2X7R in intestinal tissues of mice, as well as their role in cell death and IL-6 expression induced by TcdA and TcdB in enteric glial cells (EGCs). Male C57BL/6 mice (8 weeks of age) were infected with C. difficile VPI10463, and the control group received only vehicle per gavage. After three days post-infection (p.i.), cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry. In vitro, EGCs (PK060399egfr) were challenged with TcdA or TcdB, in the presence or absence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079), and Panx1 and P2X7R expression, caspase-3/7 activity and phosphatidylserine binding to annexin-V, as well as IL-6 expression were assessed. CDI increased the levels of Panx1 in cecum and colon of mice compared to the control group. Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not IL-6 gene expression in TcdA and TcdB-challenged EGCs. P2X7 receptor antagonist accentually reduced caspase-3/7 activity, phosphatidylserine-annexin-V binding, and IL-6 gene expression in TcdA and TcdB-challenged EGCs. In conclusion, Panx1 is increased during CDI and plays an important role in the effects of C. difficile toxins in EGCs, participating in cell death induced by both toxins by promoting caspase-3/7 activation via P2X7R, which is also involved in IL-6 expression induced by both toxins.

S100B Inhibition Attenuates Intestinal Damage and Diarrhea Severity During Clostridioides difficile Infection by Modulating Inflammatory Response.

The involvement of the enteric nervous system, which is a source of S100B, in Clostridioides difficile (C. difficile) infection (CDI) is poorly understood although intestinal motility dysfunctions are known to occur following infection. Here, we investigated the role of S100B in CDI and examined the S100B signaling pathways activated in C. difficile toxin A (TcdA)- and B (TcdB)-induced enteric glial cell (EGC) inflammatory response. The expression of S100B was measured in colon tissues and fecal samples of patients with and without CDI, as well as in colon tissues from C. difficile-infected mice. To investigate the role of S100B signaling in IL-6 expression induced by TcdA and TcdB, rat EGCs were used. Increased S100B was found in colonic biopsies from patients with CDI and colon tissues from C. difficile-infected mice. Patients with CDI-promoted diarrhea exhibited higher levels of fecal S100B compared to non-CDI cases. Inhibition of S100B by pentamidine reduced the synthesis of IL-1ß, IL-18, IL-6, GMCSF, TNF-α, IL-17, IL-23, and IL-2 and downregulated a variety of NFκB-related genes, increased the transcription (SOCS2 and Bcl-2) of protective mediators, reduced neutrophil recruitment, and ameliorated intestinal damage and diarrhea severity in mice. In EGCs, TcdA and TcdB upregulated S100B-mediated IL-6 expression via activation of RAGE/PI3K/NFκB. Thus, CDI appears to upregulate colonic S100B signaling in EGCs, which in turn augment inflammatory response. Inhibition of S100B activity attenuates the intestinal injury and diarrhea caused by C. difficile toxins. Our findings provide new insight into the role of S100B in CDI pathogenesis and opens novel avenues for therapeutic interventions.

Increased Oxidative Stress in Gastric Cancer Patients and Their First-Degree Relatives: A Prospective Study from Northeastern Brazil.

BACKGROUND AND AIMS: First-degree relatives of gastric cancer patients are at increased risk of developing gastric cancer. Increased oxidative stress, including lipid peroxidation, has been associated with gastric carcinogenesis. Whether first-degree relatives of gastric cancer patients have increased oxidative stress remains unknown. We aimed to compare oxidative stress in patients with gastric cancer, their first-degree relatives, and dyspeptic controls. METHODS: A total of 155 patients undergoing upper endoscopy were prospectively enrolled, including 50 with gastric cancer, 49 first-degree relatives of gastric cancer patients, and 56 controls. Serum concentrations of malondialdehyde (MDA) and glutathione) and activities of superoxide dismutase (SOD) and catalase were measured. Multivariate analysis adjusting for sex, age, smoking status, and alcohol consumption was performed. RESULTS: Lipid peroxidation, as measured by concentration of MDA (nmol/mL), was higher (p = 0.04), and glutathione levels were lower (p < 0.001) in the gastric cancer group compared to controls. There was no difference in the catalase activity among the groups. There was no difference in glutathione and MDA concentration or catalase activity between the different stages of gastric cancer based on the TNM classification. Relatives of gastric cancer patients had higher glutathione concentration (µmol/mL) compared to gastric cancer patients (262.5 vs. 144.6; p = 0.018), while there was no difference in MDA concentration. Catalase and superoxide dismutase activity were lower in the gastric cancer group (3.82 vs. 0.91; p < 0.001 and 1.04 vs. 0.6; p < 0.001) compared to their first-degree relatives. Interestingly, MDA concentration in the first-degree relative group was higher than in the control group (7.9 vs. 5.1; p = 0.03). CONCLUSIONS: In this study, similarly to gastric cancer patients, their first-degree relatives were found to have increased oxidative stress compared to controls. Further studies are warranted to validate this observation and to better understand the role of oxidative stress as a possible biomarker in this population.

Evaluation of HIV protease and nucleoside reverse transcriptase inhibitors on proliferation, necrosis, apoptosis in intestinal epithelial cells and electrolyte and water transport and epithelial barrier function in mice.

BACKGROUND: Protease inhibitors (PI's) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable side-effects, such as diarrhea. This study aims to investigate the effects of selected antiretroviral agents on intestinal histopathology and function in vivo and on cell proliferation and death in vitro. METHODS: Selected antiretroviral drugs were given orally over 7 days, to Swiss mice, as follows: 100 mg/kg of nelfinavir (NFV), indinavir (IDV), didanosine (DDI) or 50 mg/kg of zidovudine (AZT). Intestinal permeability measured by lactulose and mannitol assays; net water and electrolyte transport, in perfused intestinal segments; and small intestinal morphology and cell apoptosis were assessed in treated and control mice. In vitro cell proliferation was evaluated using the WST-1 reagent and apoptosis and necrosis by flow cytometry analysis. RESULTS: NFV, IDV, AZT and DDI caused significant reductions in duodenal and in jejunal villus length (p < 0.05). IDV and AZT increased crypt depth in the duodenum and AZT increased crypt depth in the jejunum. NFV, AZT and DDI significantly decreased ileal crypt depth. All selected antiretroviral drugs significantly increased net water secretion and electrolyte secretion, except for DDI, which did not alter water or chloride secretion. Additionally, only NFV significantly increased mannitol and lactulose absorption. NFV and IDV caused a significant reduction in cell proliferation in vitro at both 24 h and 48 h. DDI and AZT did not alter cell proliferation. There was a significant increase in apoptosis rates in IEC-6 cells after 24 h with 70 ug/mL of NFV (control: 4.7% vs NFV: 22%) while IDV, AZT and DDI did not show any significant changes in apoptosis compared to the control group. In jejunal sections, IDV and NFV significantly increased the number of TUNEL positive cells. CONCLUSION: The PI's, NFV and IDV, increased cell apoptosis in vivo, water and electrolyte secretion and intestinal permeability and decreased villus length and cell proliferation. NFV was the only drug tested that increased cell apoptosis in vitro. The nucleoside reverse transcriptase inhibitors, AZT and DDI, did not affect cell apoptosis or proliferation. These findings may partly explain the intestinal side-effects associated with PI's.

Effect of venlafaxine on bone loss associated with ligature-induced periodontitis in Wistar rats.

BACKGROUND: The present study investigated the effects of venlafaxine, an antidepressant drug with immunoregulatory properties on the inflammatory response and bone loss associated with experimental periodontal disease (EPD). MATERIALS AND METHODS: Wistar rats were subjected to a ligature placement around the second upper left molar. The treated groups received orally venlafaxine (10 or 50 mg/kg) one hour before the experimental periodontal disease induction and daily for 10 days. Vehicle-treated experimental periodontal disease and a sham-operated (SO) controls were included. Bone loss was analyzed morphometrically and histopathological analysis was based on cell influx, alveolar bone, and cementum integrity. Lipid peroxidation quantification and immunohistochemistry to TNF-alpha and iNOS were performed. RESULTS: Experimental periodontal disease rats showed an intense bone loss compared to SO ones (SO = 1.61 +/- 1.36; EPD = 4.47 +/- 1.98 mm, p < 0.001) and evidenced increased cellular infiltration and immunoreactivity for TNF-alpha and iNOS. Venlafaxine treatment while at low dose (10 mg/kg) afforded no significant protection against bone loss (3.25 +/- 1.26 mm), a high dose (50 mg/kg) caused significantly enhanced bone loss (6.81 +/- 3.31 mm, p < 0.05). Venlafaxine effectively decreased the lipid peroxidation but showed no significant change in TNF-alpha or iNOS immunoreactivity. CONCLUSION: The increased bone loss associated with high dose venlafaxine may possibly be a result of synaptic inhibition of serotonin uptake.

Clostridioides difficile Toxin A-Induced Wnt/ß-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation.

Clostridioides difficile toxin A (TcdA) has been shown to inhibit cellular Wnt signaling, the major driving force behind the proliferation of epithelial cells in colonic crypts, likely through the inhibition of ß-catenin nuclear translocation. Herein, we aimed to advance the understanding of this mechanism by replicating the findings in vivo and by investigating the specific role of Rac1, a member of the Rho GTPase family, on the inhibition of the Wnt-induced ß-catenin nuclear translocation triggered by TcdA. To investigate the effects of TcdA on the Wnt/ß-catenin pathway in vivo, we injected the ileal loops of C57BL/6 mice with TcdA [phosphate-buffered saline (PBS) as the control] to induce C. difficile disease-like ileitis. After 4 h post-injection, we obtained ileum tissue samples to assess Wnt signaling activation and cell proliferation through Western blotting, immunohistochemistry, and qPCR. To assess the role of Rac1 on Wnt signaling inhibition by TcdA, we transfected rat intestinal epithelial cells (IEC-6) with either a constitutively active Rac1 plasmid (pcDNA3-EGFP-Rac1-Q61L) or an empty vector, which served as the control. We incubated these cells with Wnt3a-conditioned medium (Wnt3a-CM) to induce Wnt/ß-catenin pathway activation, and then challenged the cells with TcdA. We assessed Wnt signaling activation in vitro with TOP/FOPflash luciferase assays, determined nuclear ß-catenin translocation by immunofluorescence, measured cyclin D1 protein expression by Western blotting, and quantified cell proliferation by Ki67 immunostaining. In vivo, TcdA decreased ß-catenin, cyclin D1, and cMYC expression and inhibited the translocation of ß-catenin into the nucleus in the ileum epithelial cells. In addition, TcdA suppressed cell proliferation and increased Wnt3a expression, but did not alter Rac1 gene expression in the ileum tissue. In vitro, constitutively active Rac1 prevented Wnt signaling inhibition by enabling the ß-catenin nuclear translocation that had been blocked by TcdA. Our results show that TcdA inhibits Wnt/ß-catenin pathway in vivo and demonstrate that this inhibition is likely caused by a Rac1-mediated mechanism.

The Alpha-Lipoic Acid Improves Survival and Prevents Irinotecan-Induced Inflammation and Intestinal Dysmotility in Mice.

Irinotecan, an anticancer drug, induces diarrhea and intestinal inflammation, resulting in an increase in the cost of care and in treatment delays. In this study, we investigated whether alpha-lipoic acid (α-LA) could improve irinotecan-mediated intestinal inflammation, diarrhea and dysmotility. Intestinal mucositis was induced by irinotecan injection (75 mg/kg, i.p., for 4 days) in Swiss mice. α-LA (50, 100 or 200 mg/kg, gavage) was administered daily 1 h before the injection of irinotecan. Duodenum tissues were obtained for inflammation and proliferation analysis. The outcomes: diarrhea, intestinal dysmotility, weight body loss and survival were evaluated. Compared with the control condition, irinotecan diminished (p < 0.05) intestinal villus height, caused a loss of crypt integrity and intense inflammatory cell infiltration, increased myeloperoxidase (MPO), IL-6 and IL-1ß levels and decreased reduced glutathione (GSH) levels in duodenum segments and increased gastric retention and decreased liquid retention in the medial intestinal segment, resulting in increased intestinal transit, severe diarrhea and reduced survival (approximately 72%). Furthermore, α-LA (200 mg/kg) pretreatment ameliorated (p < 0.05) these irinotecan-induced effects. Our findings show that α-LA reduced irinotecan-induced inflammation, intestinal dysmotility and diarrhea, resulting in improved survival. α-LA may be a useful therapeutic agent for the treatment of gut dysmotility in patients with intestinal mucositis associated with irinotecan treatment.

5-Fluorouracil Induces Enteric Neuron Death and Glial Activation During Intestinal Mucositis via a S100B-RAGE-NFκB-Dependent Pathway.

5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis associated with intestinal motility alterations maybe due to an effect on the enteric nervous system (ENS), but the underlying mechanism remains unclear. In this report, we used an animal model to investigate the participation of the S100B/RAGE/NFκB pathway in intestinal mucositis and enteric neurotoxicity caused by 5-FU (450 mg/kg, IP, single dose). 5-FU induced intestinal damage observed by shortened villi, loss of crypt architecture and intense inflammatory cell infiltrate as well as increased GFAP and S100B co-expression and decreased HuC/D protein expression in the small intestine. Furthermore, 5-FU increased RAGE and NFκB NLS immunostaining in enteric neurons, associated with a significant increase in the nitrite/nitrate, IL-6 and TNF-α levels, iNOS expression and MDA accumulation in the small intestine. We provide evidence that 5-FU induces reactive gliosis and reduction of enteric neurons in a S100B/RAGE/NFκB-dependent manner, since pentamidine, a S100B inhibitor, prevented 5-FU-induced neuronal loss, enteric glia activation, intestinal inflammation, oxidative stress and histological injury.

Role of cytokines (TNF-alpha, IL-1beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide.

INTRODUCTION: Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. PURPOSE: Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. MATERIALS AND METHODS: Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and KC ELISA. RESULTS: CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. CONCLUSION: These results suggest an important role of TNF-alpha, IL-1beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.

Role of retinol in protecting epithelial cell damage induced by Clostridium difficile toxin A.

Vitamin A (retinol), a fat-soluble vitamin, is an essential nutrient for the normal functioning of the visual system, epithelial cell integrity and growth, immunity, and reproduction. Our group has investigated the effect of high doses of oral vitamin A on early childhood diarrhea in our prospective community-based studies from Northeast Brazil and found a beneficial role in reducing the mean duration but not incidence of diarrheal episodes. In this study, we explored the role of retinol supplementation in intestinal cell lines following Clostridium difficile toxin A (TxA) challenge. C. difficile is the most common anaerobic pathogen borne with antibiotic-borne diarrhea and pseudomembranous colitis. Since retinol is critical for the integrity of tight junctions and to modulate the cell cycle, we have focused on changes in transepithelial electrical resistance (TEER) in Caco-2, a more differentiated intestinal cell line, and on models of cell proliferation, migration and viability in IEC-6 cells, an undifferentiated crypt cell line, following TxA injury. In this model, retinol therapy reduced apoptosis, improved cell migration and proliferation, and prevented the reduction in TEER, following C. difficile TxA challenge in a glutamine-free medium. These results suggest the role of retinol in protecting intestinal epithelial barrier function from C. difficile TxA enterotoxic damage.

The Effect of Calendula officinalis on Oxidative Stress and Bone Loss in Experimental Periodontitis.

Periodontitis is associated with reduced antioxidant capacity and increased oxidative damage. Oxidative stress induces inflammation and bone loss contributing to the pathological progression of periodontal disease. Calendula officinalis (CLO) has demonstrated anti-inflammatory and anti-oxidant activities. Therefore, the aim of this study was to evaluate the effect of CLO on oxidative stress and bone loss in rats subjected to experimental periodontitis (EP). For this, 72 male Wistar rats were divided into groups: Naïve, Saline (SAL) and CLO. Rats received SAL or CLO (90 mg/kg) 30 min before ligature and daily until the 11th day. Naïve group experienced no manipulation. After 11 days, the animals were euthanized and left maxillae collected for macroscopic analysis of alveolar bone loss (ABL). Periodontium was analyzed by macroscopy, scanning electron microscopy; confocal and light polarized microscopy. Immunohistochemical examination of DKK1, WNT 10b and ß-catenin was performed. The gingival tissue was collected to reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) analyses. The 11 days of ligature induced bone loss, breakdown of collagen fibers, increased the immunostaining DKK-1 while reduced WNT 10b and ß-catenin expressions. Periodontitis reduced GSH, SOD, CAT and increase MDA. All findings were reversed by 90 mg/kg of CLO. In summary our findings demonstrated that CLO reduced oxidative stress and bone loss and preserved collagen fibers in rats with EP, with participation of WNT signaling pathway.