A Brazilian regional basic diet-induced chronic malnutrition drives liver inflammation with higher ApoA-I activity in C57BL6J mice

Malnutrition is still considered endemic in many developing countries. Malnutrition-enteric infections may cause lasting deleterious effects on lipid metabolism, especially in children living in poor settings. The regional basic diet (RBD), produced to mimic the Brazilian northeastern dietary characteristics (rich in carbohydrate and low in protein) has been used in experimental malnutrition models, but few studies have explored the effect of chronic RBD on liver function, a central organ involved in cholesterol metabolism. This study aimed to investigate whether RBD leads to liver inflammatory changes and altered reverse cholesterol metabolism in C57BL6/J mice compared to the control group, receiving a standard chow diet. To evaluate liver inflammation, ionized calcium-binding adapter protein-1 (IBA-1) positive cell counting, interleukin (IL)-1β immunohistochemistry, and tumor necrosis factor (TNF)-α and IL-10 transcription levels were analyzed. In addition, we assessed reverse cholesterol transport by measuring liver apolipoprotein (Apo)E, ApoA-I, and lecithin-cholesterol acyltransferase (LCAT) by RT-PCR. Furthermore, serum alanine aminotransferase (ALT) was measured to assess liver function. RBD markedly impaired body weight gain compared with the control group (P<0.05). Higher hepatic TNF-α (P<0.0001) and IL-10 (P=0.001) mRNA levels were found in RBD-challenged mice, although without detectable non-alcoholic fatty liver disease. Marked IBA-1 immunolabeling and increased number of positive-IBA-1 cells were found in the undernourished group. No statistical difference in serum ALT was found. There was also a significant increase in ApoA mRNA expression in the undernourished group, but not ApoE and LCAT, compared with the control. Altogether our findings suggested that chronic RBD-induced malnutrition leads to liver inflammation with increased ApoA-I activity.

Amifostine reduces inflammation and protects against 5-fluorouracil-induced oral mucositis and hyposalivation

Oral mucositis (OM) is a common and dose-limiting side effect of cancer treatment, including 5-fluorouracil (5-FU) and radiotherapy. The efficacy of the therapeutic measures to prevent OM is limited and disease prevention is not fully observable. Amifostine is a cytoprotective agent with a described anti-inflammatory potential. It is clinically used to reduce radiotherapy and chemotherapy-associated xerostomia. This study investigated the protective effect of amifostine on an experimental model of OM. Hamsters were divided into six groups: saline control group (5 mL/kg), mechanical trauma (scratches) of the right cheek pouch; 5-FU (60 and 40 mg/kg, ip, respectively, administered on days 1 and 2); amifostine (12.5, 25, or 50 mg/kg) + 5-FU + scratches. Salivation rate was assessed and the animals were euthanized on day 10 for the analysis of macroscopic and microscopic injury by scores. Tissue samples were harvested for the measurement of neutrophil infiltration and detection of inflammatory markers by ELISA and immunohistochemistry. 5-FU induced pronounced hyposalivation, which was prevented by amifostine (P<0.05). In addition, 5-FU injection caused pronounced tissue injury accompanied by increased neutrophil accumulation, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) tissue levels, and positive immunostaining for TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS). Interestingly, amifostine prevented the inflammatory reaction and consequently improved macroscopic and microscopic damage (P<0.05 vs 5-FU group). Amifostine reduced inflammation and protected against 5-FU-associated oral mucositis and hyposalivation.

Impact of acute undernutrition on growth, ileal morphology and nutrient transport in a murine model

Undernutrition represents a major public health challenge for middle- and low-income countries. This study aimed to evaluate whether a multideficient Northeast Brazil regional basic diet (RBD) induces acute morphological and functional changes in the ileum of mice. Swiss mice (∼25 g) were allocated into two groups: i) control mice were fed a standard diet and II) undernourished mice were fed the RBD. After 7 days, mice were killed and the ileum collected for evaluation of electrophysiological parameters (Ussing chambers), transcription (RT-qPCR) and protein expression (western blotting) of intestinal transporters and tight junctions. Body weight gain was significantly decreased in the undernourished group, which also showed decreased crypt depth but no alterations in villus height. Electrophysiology measurements showed a reduced basal short circuit current (Isc) in the undernourished group, with no differences in transepithelial resistance. Specific substrate-evoked Isc related to affinity and efficacy (glutamine and alanyl-glutamine) were not different between groups, except for the maximum Isc (efficacy) induced by glucose. Transcription of Sglt1 and Pept1 was significantly higher in the undernourished group, while SN-2 transcription was decreased. No changes were found in transcription of CAT-1 and CFTR, while claudin-2 and occludin transcriptions were significantly increased in the undernourished group. Despite mRNA changes, SGLT-1, PEPT-1, claudin-2 and occludin protein expression showed no difference between groups. These results demonstrate early effects of the RBD on mice, which include reduced body weight and crypt depth in the absence of significant alterations to villus morphology, intestinal transporters and tight junction expression.

Alanyl-glutamine attenuates 5-fluorouracil-induced intestinal mucositis in apolipoprotein E-deficient mice

Apolipoprotein E (APOE=gene, apoE=protein) is a known factor regulating the inflammatory response that may have regenerative effects during tissue recovery from injury. We investigated whether apoE deficiency reduces the healing effect of alanyl-glutamine (Ala-Gln) treatment, a recognized gut-trophic nutrient, during tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout (APOE-/-) and wild-type (APOE+/+) C57BL6J male and female mice (N=86) were given either Ala-Gln (100 mM) or phosphate buffered saline (PBS) by gavage 3 days before and 5 days after a 5-fluorouracil (5-FU) challenge (450 mg/kg, via intraperitoneal injection). Mouse body weight was monitored daily. The 5-FU cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt ratio, and villin expression were monitored to assess recovery of the intestinal absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1) and B-cell lymphoma 2 (Bcl-2) intestinal mRNA transcripts were used to evaluate intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss and leukopenia (P<0.001) in both mouse strains, which was not improved by Ala-Gln. Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05) were found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared with PBS controls. Ala-Gln improved villus height only in APOE-/- mice. Crypt cell apoptosis and necrotic scores were increased in all mice challenged by 5-FU, compared with untreated controls. Those scores were significantly lower in Ala-Gln-treated APOE+/+ mice than in controls. Bcl-2 and IGF-1 mRNA transcripts were reduced only in the APOE-/--challenged mice. Altogether our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU challenge.

In vivo and in vitro anti-inflammatory and anti-nociceptive activities of lovastatin in rodents

Statins are among the most prescribed drugs in recent clinical practice. They are also known for their pleiotropic actions, which are independent of their lipid-lowering properties. The effect of lovastatin was investigated against carrageenan-induced paw edema in male Wistar rats (200-250 g) and on leukocyte migration, as measured by carrageenan-induced peritonitis in male Swiss mice (20-25 g), which are models of acute inflammation. Lovastatin (administered 1 h prior to carrageenan), at oral doses of 2, 5, and 10 mg/kg, markedly attenuated paw edema formation in rats at the 4th hour after carrageenan injection (25, 43, and 37 percent inhibition, respectively). Inhibitions of 20, 45 and 80 percent were observed in the leukocyte migration, as evaluated by carrageenan-induced peritonitis in mice with lovastatin doses of 0.5, 1 and 5 mg/kg, as compared to controls. Furthermore, lovastatin (administered 1 h before initiation) reduced the nociceptive effect of the formalin test in mice, at both phases, at doses of 2, 5, and 10 mg/kg: first phase (51, 65, and 70 percent, respectively) and second phase (73, 57, and 66 percent inhibition of licking time, respectively). The anti-nociceptive activity of lovastatin was inhibited by naloxone (3 mg/kg, sc). Lovastatin (0.01, 0.1, and 1 µg/mL) inhibited by 23, 79, and 86 percent, respectively, the release of myeloperoxidase from human neutrophils. Leukocyte (predominantly neutrophils) infiltration was almost completely reduced by lovastatin treatment, as observed in the model of acute paw edema with hematoxylin and eosin staining. In addition, lovastatin decreased the number of cells expressing tumor necrosis factor-α (TNF-α) and the inducible form of nitric oxide synthase (iNOS) activity. Therefore, the alterations in leukocyte activity and cytokine release could contribute to the anti-inflammatory activity of lovastatin.

Antimicrobial activity of the essential oil from Lippia sidoides, carvacrol and thymol against oral pathogens

Dental caries and periodontal disease are associated with oral pathogens. Several plant derivatives have been evaluated with respect to their antimicrobial effects against such pathogenic microorganisms. Lippia sidoides Cham (Verbenaceae), popularly known as "Alecrim-pimenta" is a typical shrub commonly found in the Northeast of Brazil. Many plant species belonging to the genus Lippia yield very fragrant essential oils of potential economic value which are used by the industry for the commercial production of perfumes, creams, lotions, and deodorants. Since the leaves of L. sidoides are also extensively used in popular medicine for the treatment of skin wounds and cuts, the objective of the present study was to evaluate the composition and antimicrobial activity of L. sidoides essential oil. The essential oil was obtained by hydro-distillation and analyzed by GC-MS. Twelve compounds were characterized, having as major constituents thymol (56.7 percent) and carvacrol (16.7 percent). The antimicrobial activity of the oil and the major components was tested against cariogenic bacterial species of the genus Streptococcus as well as Candida albicans using the broth dilution and disk diffusion assays. The essential oil and its major components thymol and carvacrol exhibited potent antimicrobial activity against the organisms tested with minimum inhibitory concentrations ranging from 0.625 to 10.0 mg/mL. The most sensitive microorganisms were C. albicans and Streptococcus mutans. The essential oil of L. sidoides and its major components exert promising antimicrobial effects against oral pathogens and suggest its likely usefulness to combat oral microbial growth.

Comparison of etoricoxib and indomethacin for the treatment of experimental periodontitis in rats

We investigated the effect of etoricoxib, a selective cyclooxygenase-2 inhibitor, and indomethacin, a non-selective cyclooxygenase inhibitor, on experimental periodontitis, and compared their gastrointestinal side effects. A ligature was placed around the second upper left molars of female Wistar rats (160 to 200 g). Animals (6 per group) were treated daily with oral doses of 3 or 9 mg/kg etoricoxib, 5 mg/kg indomethacin, or 0.2 mL saline, starting 5 days after the induction of periodontitis, when bone resorption was detected, until the sacrifice on the 11th day. The weight and survival rate were monitored. Alveolar bone loss (ABL) was measured as the sum of distances between the cusp tips and the alveolar bone. The gastric mucosa was examined macroscopically and the periodontium and gastric and intestinal mucosa were examined by histopathology. The ongoing ABL was significantly inhibited (P < 0.05) by 3 and 9 mg/kg etoricoxib and by indomethacin: control = 4.08 ± 0.47 mm; etoricoxib (3 mg/kg) = 1.89 ± 0.26 mm; etoricoxib (9 mg/kg) = 1.02 ± 0.14 mm; indomethacin = 0.64 ± 0.15 mm. Histopathology of periodontium showed that etoricoxib and indomethacin reduced inflammatory cell infiltration, ABL, and cementum and collagen fiber destruction. Macroscopic and histopathological analysis of gastric and intestinal mucosa demonstrated that etoricoxib induces less damage than indomethacin. Animals that received indomethacin presented weight loss starting on the 7th day, and higher mortality rate (58.3 percent) compared to etoricoxib (0 percent). Treatment with etoricoxib, even starting when ABL is detected, reduces inflammation and cementum and bone resorption, with fewer gastrointestinal side effects.

A model of hemorrhagic cystitis induced with acrolein in mice

Acrolein is a urinary metabolite of cyclophosphamide and ifosfamide, which has been reported to be the causative agent of hemorrhagic cystitis induced by these compounds. A direct cytotoxic effect of acrolein, however, has not yet been demonstrated. In the present study, the effects of intravesical injection of acrolein and mesna, the classical acrolein chemical inhibitor, were evaluated. Male Swiss mice weighing 25 to 35 g (N = 6 per group) received saline or acrolein (25, 75, 225 æg) intravesically 3, 6, 12, and 24 h before sacrifice for evaluation of bladder wet weight, macroscopic and histopathological changes by Gray's criteria, and 3 and 24 h for assessment of increase in vascular permeability. In other animals, mesna was administered intravesically (2 mg) or systemically (80 mg/kg) 1 h before acrolein. Intravesical administration of acrolein induced a dose- and time-dependent increase in vascular permeability and bladder wet weight (within 3 h: 2.2- and 21-fold increases in bladder wet weight and Evans blue dye exuded, respectively, at doses of 75 æg/bladder), as confirmed by Gray's criteria. Pretreatment with mesna (2-mercaptoethanesulfonic acid), which interacts with acrolein resulting in an inactive compound, inhibited all changes induced by acrolein. Our results are the first demonstration that intravesical administration of acrolein induces hemorrhagic cystitis. This model of acrolein-induced hemorrhagic cystitis in mice may be an important tool for the evaluation of the mechanism by which acrolein induces bladder lesion, as well as for investigation of new uroprotective drugs.

Long-term ethanol intoxication reduces inflammatory responses in rats

The anti-inflammatory effects of long-term ethanol intoxication were determined during ethanol treatment and withdrawal on the basis of neutrophil and eosinophil migration, hind paw edema and mast cell degranulation. Male Wistar rats (180-200 g, around 2 months of age) were exposed to increasing concentrations of ethanol vapor over a 10-day period. One group was evaluated immediately after exposure (treated group - intoxicated), and another was studied 7 h later (withdrawal group). Ethanol inhalation treatment significantly inhibited carrageenan- (62 percent for the intoxicated group, N = 5, and 35 percent for the withdrawal group, N = 6) and dextran-induced paw edema (32 percent for intoxicated rats and 26 percent for withdrawal rats, N = 5 per group). Ethanol inhalation significantly reduced carrageenan-induced neutrophil migration (95 percent for intoxicated rats and 41 percent for withdrawn rats, N = 6 per group) into a subcutaneous 6-day-old air pouch, and Sephadex-induced eosinophil migration to the rat peritoneal cavity (100 percent for intoxicated rats and 64 percent for withdrawn rats, N = 6 per group). A significant decrease of mast cell degranulation was also demonstrated (control, 82 percent; intoxicated, 49 percent; withdrawn, 51 percent, N = 6, 6 and 8, respectively). Total leukocyte and neutrophil counts in venous blood increased significantly during the 10 days of ethanol inhalation (leukocytes, 13, 27 and 40 percent; neutrophils, 42, 238 and 252 percent, respectively, on days 5, 9 and 10, N = 7, 6 and 6). The cell counts decreased during withdrawal, but were still significantly elevated (leukocytes, 10 percent; neutrophils, 246 percent, N = 6). These findings indicate that both the cellular and vascular components of the inflammatory response are compromised by long-term ethanol intoxication and remain reduced during the withdrawal period.

Low-dose doxycycline prevents inflammatory bone resorption in rats

Matrix metalloproteinases (MMP) are considered to be key initiators of collagen degradation, thus contributing to bone resorption in inflammatory diseases. We determined whether subantimicrobial doses of doxycycline (DX) (<=10 mg kg-1 day-1), a known MMP inhibitor, could inhibit bone resorption in an experimental periodontitis model. Thirty male Wistar rats (180-200 g) were subjected to placement of a nylon thread ligature around the maxillary molars and sacrificed after 7 days. Alveolar bone loss (ABL) was measured macroscopically in one hemiarcade and the contralateral hemiarcade was processed for histopathologic analysis. Groups of six animals each were treated with DX (2.5, 5 or 10 mg kg-1 day-1, sc, 7 days) and compared to nontreated (NT) rats. NT rats displayed significant ABL, severe mononuclear cell influx and increase in osteoclast numbers, which were significantly reduced by 5 or 10 mg kg-1 day-1 DX. These data show that DX inhibits inflammatory bone resorption in a manner that is independent of its antimicrobial properties

Pharmacological and histopathological study of cyclophosphamide-induced hemorrhagic cystitis - comparison of the effects of dexamethasone and Mesna

Chemotherapy with oxazaphosphorines, such as cyclophosphamide (CYP), is often limited by unacceptable urotoxicity. Without uroprotection, hemorrhagic cystitis (HC) becomes dose-limiting. To compare the uroprotective efficacy of classical 2-mercaptoethanesulfonic acid (Mesna) treatment with dexamethasone in CYP-induced HC, male Wistar rats (150-200 g; N = 6 in each group) were treated with saline or Mesna (40 mg/kg, ip) immediately and 4 and 8 h after ip administration of CYP (200 mg/kg). One, 2 or 3 doses of Mesna were replaced with dexamethasone (1 mg/kg, ip). The animals were sacrificed 24 h later. Cystitis was evaluated by determining the changes in bladder wet weight (BWW) and by macroscopic and microscopic analysis. CYP treatment induced a marked increased in BWW 162 percent 0.05, which was significantly inhibited by treatment with 3 doses of Mesna 0.05; 80 percent. The replacement of 1 or 2 doses of Mesna with dexamethasone reduced the increase in BWW by 83.3 and 95 percent, respectively. Macroscopic analysis of the bladder of rats with CYP-induced HC showed severe edema and hemorrhage, confirmed by microscopic analysis, that also showed mucosal erosion, inflammatory cell infiltration and ulcerations. The replacement of 1 or 2 doses of Mesna with dexamethasone inhibited the CYP-induced increase in BWW and almost abolished the macroscopic and microscopic alterations, with no significant difference between the effects of Mesna and dexamethasone, indicating that both drugs were efficient in blocking HC. However, although the replacement of all Mesna doses with dexamethasone reduced the edema, it did not prevent HC, suggesting that Mesna is necessary for the initial uroprotection

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ñ 0.19 and Dex = 0.35 ñ 0.13 vs saline (S) = 2.85 ñ 0.59; fMLP: Ptx = 0.43 ñ 0.09 and Dex 0.01 ñ 0.01 vs S = 1.08 ñ 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ñ 1.4 and Dex = 3.06 ñ 0.76 vs S = 15.94 ñ 3.97; fMLP: Ptx = 3.85 ñ 0.56 and Dex = 0.40 ñ 0.16 vs S = 7.15 ñ 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ñ 0.38 vs S = 4.20 ñ 1.01; fMLP: Dex = 0.25 ñ 0.11 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ñ 2.25 vs S = 4.20 ñ 1.01; fMLP: Ptx = 4.66 ñ 1.24 vs S = 2.20 ñ 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.