Protective Effect of Scopoletin Against Cerulein-Induced Acute Pancreatitis and Associated Lung Injury in Mice.

OBJECTIVE: The present study aimed to evaluate the protective effects of scopoletin (SC) on cerulein-induced acute pancreatitis (AP) and associated lung injury in mice. METHODS: Acute pancreatitis was induced in male Swiss mice by 6 consecutive hourly intraperitoneal injections of cerulein (50 µg/kg). Scopoletin was administered 1 hour (intraperitoneal, 10 mg/kg) after the first cerulein injection. RESULTS: Administration of SC attenuated the severity of AP and associated lung injury as shown by histology, reduced myeloperoxidase, and serum amylase activity. Further, the anti-inflammatory effect of SC was associated with a reduction of pancreatic and pulmonary proinflammatory cytokines (interleukin 1ß and tumor necrosis factor α) and hydrogen sulfide. Moreover, SC inhibited cerulein-induced nuclear factor κB activation in both pancreas and lung. Also, SC treatment further enhances the beneficial effect by reducing cerulein-induced mast cell activation as shown by reduced monocyte chemoattractant protein 1, interleukin 33, and preprotachykinin A expression (encodes neuropeptide substance P) in the pancreas and lungs. CONCLUSIONS: The present findings show for the first time that in AP SC may exhibit an anti-inflammatory effect by down-regulating substance P and hydrogen sulfide signaling via nuclear factor κB pathway.

Oxidative stress in experimental rodent corneas infected with aflatoxigenic and nonaflatoxigenic Aspergillus flavus.

PURPOSE: To seek a possible association between aflatoxigenicity and oxidative stress in keratitis caused by Aspergillus flavus in an experimental rodent model. METHODS: Wistar rats were divided into 3 groups of 6 each. Group 1 served as mock-inoculated controls. Experimental fungal keratitis was induced in group 2 and group 3 rats using aflatoxigenic and nonaflatoxigenic A. flavus conidial suspensions, respectively, and clinical features were scored for 5 days after inoculation. At this time, animals were killed, and test corneas were excised and examined histologically. Expression of IL-1ß and TNF-α genes was sought in excised corneas. Levels of malondialdehyde (MDA) and reduced glutathione (GSH) and activities of key antioxidant enzymes were measured in excised corneas and fungal mycelial homogenates. Antioxidant enzyme isoforms were sought in mycelial homogenates by native polyacrylamide gel electrophoresis. RESULTS: Mean levels of MDA and GSH and mean activities of catalase, superoxide dismutase, and glutathione peroxidase were significantly (P < 0.05) higher in a mycelial homogenate of aflatoxigenic A. flavus than in the nonaflatoxigenic mycelial homogenate. Increased numbers of well-stained isoforms were detected in aflatoxigenic mycelial homogenates. Significantly (P < 0.05) higher expression profiles of IL-1ß and TNF-α genes, MDA and GSH levels, and antioxidant enzyme activities were noted in group 2 rat corneas than in group 3 rat corneas. Clinical and histological scores suggested a more severe keratitis in group 2 rat corneas than in group 1 and group 3 rat corneas. CONCLUSIONS: Aflatoxigenicity is associated with more intense oxidative stress in experimental A. flavus keratitis.

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis.

PURPOSE: To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. METHODS: In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9-14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca(2+)-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. RESULTS: Significantly lower mean activities of Ca(2+)-ATPase and Na(+)/K(+) -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. CONCLUSIONS: The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.

Expression of genes of the aflatoxin biosynthetic pathway in Aspergillus flavus isolates from keratitis.

PURPOSE: To document transcriptional activation (expression) of key aflatoxin biosynthetic pathway genes in corneal isolates of Aspergillus flavus. METHODS: The expression of certain regulatory (aflatoxin regulatory [aflR] and aflatoxin J [aflJ]) and structural (polyketide synthase acetate [pksA] and norsolonic acid-1 [nor-1]) genes in four corneal A. flavus isolates was evaluated by reverse transcription PCR. The aflatoxin-producing potential of each strain was determined by thin-layer chromatography and quantified by spectrophotometry. Four environmental isolates were used for comparison. The mean expression levels of these genes were compared in the corneal and environmental A. flavus isolates. In addition, the mean expression levels were also correlated with the aflatoxin production levels. RESULTS: All isolates expressed aflJ, nor-1, and pksA, while all but one expressed aflR. Overall, significantly higher mean expression levels occurred in aflatoxigenic than in non-aflatoxigenic corneal isolates. A significant positive correlation was noted between the mean expression level of aflR and the quantum of aflatoxin production by the corneal isolates. Essentially similar patterns of expression of these genes were noted in four environmental A. flavus isolates used for comparison. CONCLUSIONS: For the first time, isolates of A. flavus from human keratitis patients have been shown to express regulatory and structural aflatoxin biosynthetic pathway genes. Further studies are needed to clarify the precise influence of the corneal microenvironment on expression of these genes and aflatoxin production by A. flavus infecting the cornea.

Keratitis due to Aspergillus flavus: clinical profile, molecular identification of fungal strains and detection of aflatoxin production.

PURPOSE: To document the clinical profile of patients with keratitis due to Aspergillus flavus and to elaborate on differences in the aflatoxin-producing potential of keratitis strains versus environmental strains of A. flavus. METHODS: Over a 6-month period, strains of Aspergillus flavus were isolated in culture from corneal scrape or biopsy material of patients who presented with suppurative keratitis (clinical isolates). The strains were confirmed to be A. flavus by molecular methods (amplification of the internal transcribed spacer 2 [ITS 2] region and direct sequencing followed by comparative GenBank analysis). The aflatoxin-producing potential of each strain was determined by thin-layer chromatography. The ability of each strain to form sclerotia in Czapek-Dox agar (CDA) after 7 days incubation at 30 degrees C in the dark and to produce a beige ring in yeast extract sucrose agar supplemented with methyl beta-cyclodextrin and sodium desoxycholate (YESD medium) after 3 days incubation at 30 degrees C was also assessed. For comparison, the tests were also run on 10 strains of A. flavus (identity confirmed by molecular methods) collected from local farming areas (environmental isolates). RESULTS: Aflatoxin B1 was detected in 16 (80%) of 20 culture filtrate or mycelial homogenate samples of the clinical isolates (mean concentration: 366.7+/-125.4 parts per billion [ppb]) but in only eight (40%) of 20 samples of environmental isolates (mean concentration: 306.6+/-125.4 ppb). Seven of the eight aflatoxin-producing clinical isolates and two of the four aflatoxin-producing environmental isolates formed sclerotia (>400 microm) and a beige ring in culture. CONCLUSIONS: Aflatoxin B1 was detected in a significantly higher percentage of growth samples of clinical isolates (80%) than growth samples of environmental isolates (40%) (chi(2)=6.667; p=0.0098); the therapeutic implications of this finding require further study. The production of sclerotia and a beige ring in culture appear to be useful markers of aflatoxin-producing potential in strains of A. flavus isolated from keratitis.