Biotechnic & Histochemistry最新文献

We investigated the potential neuroprotective effects of thymoquinone (TQ) on amikacin (AK) induced oxidative damage in rat brain. We used 21 male rats divided randomly into three equal groups. The control group was injected intraperitoneally (i.p.) with 0.5 ml 0.9% aqueous NaCl and given 1 ml 0.9% aqueous NaCl orally. The AK group was administered 1.2 g/kg aqueous AK i.p. as a single dose on the day 3 of the study. The AK + TQ group was given a single 1.2 g/kg dose of AK i.p. on the day 3 of the study plus 40 mg/kg/day TQ by oral gavage daily. Treatment with TQ increased serum ferritin and decreased serum calcium levels significantly. TQ also decreased NADPH oxidase-2, NADPH oxidase-4, and caspase-3 levels. Decreased malondialdehyde (MDA) levels and increased superoxide dismutase (SOD) and catalase (CAT) activities were detected in the AK + TQ group compared to the AK group. TQ administration inhibited lipid peroxide formation and blocked oxidative reactions, which reduced the MDA level and increased SOD and CAT activities induced by AK. Oxidative damage caused by AK was ameliorated by TQ treatment owing to its antioxidative and anti-apoptotic effects. TQ may be a potential therapeutic agent for reducing the severity of AK induced oxidative damage to the brain.

An aqueous 7-amino-4-methylcoumarin (AMC) solution exhibits strong fluorescence under ultraviolet (UV) light and can be used as a Schiff reagent to visualize aldehydes. We investigated hemalum and eosin (H & E) and AMC staining for histological and pathological analysis. Sections of normal and lesioned human tissues were stained with combined H & E/AMC staining. After H & E/AMC staining, the H & E morphology was preserved under bright field microscopy. The AMC fluorescent signals observed under UV light were intense and the staining pattern was identical to that obtained by periodic acid-Schiff (PAS) staining. AMC staining of archived H & E sections also was successful. Diastase digestion differentiated glycogen from other AMC positive elements. Using H & E/AMC staining, mucus-rich adenocarcinoma cells, amebic trophozoites and fungal hyphae were visualized clearly under UV excitation. Using H & E/AMC staining, H & E and PAS-like histological imaging can be obtained using a single tissue section. H & E/AMC is useful for pathologic diagnosis especially when information from PAS staining is critical, the number of tissue sections is limited and/or the lesion in question is small.

Doxorubicin (DOX) is used as an anticancer drug despite its many side effects. Thymoquinone (THQ) is a plant-derived substance that exhibits antioxidant and anti-inflammatory properties. We investigated the protective effects of THQ on DOX induced nephrotoxicity in rats. Rats were divided into five groups of eight: group 1, untreated control; group 2, olive oil group given olive oil intraperitoneally (i.p.) for 14 days; group 3, THQ group given 10 mg/kg THQ i.p. for 14 days; group 4, DOX group given a single dose of 15 mg/kg DOX i.p. on day 7 of experiment; group 5, DOX + THQ given 10 mg/kg THQ i.p. for 14 days and 15 mg/kg DOX i.p. on day 7. Kidney tissues were evaluated for histopathology. Caspase-3, IL-17, GRP78 and TNF-α immunostaining was used to determine the expression levels of these proteins among the groups. The TUNEL method was used to determine the apoptotic index. Total antioxidant status (TAS), total oxidant status (TOS), and TNF-α and TGF-β1 levels in kidney tissue were measured using ELISA assay. Histopathologic damage, caspase-3, IL-17, GRP78 and TNF-α immunoreactivity, TUNEL positive cells, TOS, TNF-α and TGF-β1 levels were increased in group 4 compared to group 1. The TAS of group 4 decreased compared to group 1. We found decreased caspase-3, IL-17, GRP78 and TNF-α expressions and TUNEL positive cells in group 5 compared to group 4. In rats given DOX, THQ reduced kidney damage by suppressing endoplasmic reticulum stress, inflammation and apoptosis pathways.

Male reproductive dysfunction is a common complication of diabetes mellitus. Trifolium pratense exhibits antioxidant and antidiabetic effects. We investigated the effects of an extract of T. pratense on serum antioxidant status, sperm characteristics, testicular tissue changes and testosterone level in diabetic rats. Male Wistar rats were divided into six groups: 1, untreated control; 2, diabetic; 3 and 4, 100 or 200 mg/kg T. pratense extract treated, respectively; 5 and 6, diabetic 100 or 200 mg/kg T. pratense extract treated, respectively. Diabetes was induced by intraperitoneal injection of streptozotocin. After 3 weeks, serum glucose, testosterone and nitric oxide (NO); sperm parameters; testicular histology and total antioxidant capacity (TAC) were evaluated. In diabetic rats treated with T. pratense extract, sperm motility, count and viability, as well as TAC and testosterone were increased significantly compared to untreated diabetic rats, while serum NO and bcl-2 and p53 expression was decreased significantly compared to untreated diabetic rats. T. pratense extract reduced testicular tissue destruction caused by diabetes.

We investigated the effects of caffeic acid phenethyl ester (CAPE) and low-dose doxycycline (LDD) on sclerostin and bone morphogenic protein (BMP)-2 expression in experimental periodontitis. We used male rats in groups as follows: control group (C), periodontitis + CAPE group (PC), periodontitis + LDD group (PD), periodontitis + LDD + CAPE group (PCD) and periodontitis group (P). We administered 10 µmol/kg/day CAPE by an intraperitoneal (i.p.) injection and 10 mg/kg/day LDD by oral gavage. Histopathological changes among groups were evaluated and compared. Sclerostin and BMP-2 expression was analyzed using immunohistochemistry. LDD and/or CAPE treatment ameliorated pathology. The highest sclerostin and lowest BMP-2 expressions were found in P group. Group PC exhibited the highest BMP-2 expression scores and the most significant improvement among the treatment groups. The lowest sclerostin expression was observed in the PD group. We found that preventing sclerostin activity may be a useful treatment alternative for bone resorption, especially in cases of periodontitis and peri-implantitis. We found that CAPE and/or LDD may act as anti-sclerostin agents.

Hepatotoxicity is a common side effect of doxorubicin (Dox) treatment of cancer. Liv-52 is an ayurvedic medicine that is reported to ameliorate liver injury due to oxidative stress. We investigated the effects of Liv-52 on Dox induced oxidative damage to liver tissues of rats using biochemical and histopathological techniques. Thirty male rats were assigned randomly into three equal groups: control (CG), Dox group (DG) Liv-52 + Dox group (LD). Rats in the LD group received 50 mg/kg Liv-52 in distilled water via gastric gavage. Distilled water was given via the same route to the rats in the DG and CG groups. Rats in the LD and DG groups were injected intraperitoneally with 5 mg/kg Dox 1 h after administration of Liv-52 or distilled water. The procedure was repeated daily for 7 days. On day 8, the animals were sacrificed, and serum and tissue biochemical and histopathological assays were performed. The malondialdehyde level was increased significantly in the DG group, while glutathione and superoxide dismutase levels were significantly lower in the DG group compared to the LD and CG groups. The highest levels of alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were found in the DG group, while the lowest levels were found in the CG group, which exhibited levels similar to those of the LD group. Treatment with Liv-52 prior to Dox treatment reduced the histopathologic changes in the Dox group. Therefore, pre-treatment with Liv-52 protected against Dox induced oxidative stress and hepatotoxicity.

Interest is increasing in certain parts of the world in replacing synthetic dyes with dyes from natural sources, particularly from plants. Although textile dyers have used various groups of natural dyes, microscopists generally have restricted their use to anthocyanins. Recently, however, another class of plant-based dyes has found some favor, the betacyanins. Betacyanins are a group of red and violet betalain dyes found only in certain plants of the order Caryophyalles and in Basidiomycetes mushrooms. Although the chemical structures of betacyanins are known, little use has been made of that information to understand or predict their behavior with biomedical specimens. We investigated two common, widely distributed betacyanin-containing plants, edible beets (Beta vulgaris) and wild pokeweed (Phytolacca americana). Aqueous alcoholic extracts were made from beet root and pokeweed berries, adjusted to pH 4.1 or 5.3 and used together with Harris' hematoxylin to stain histological sections. We used a methanolic extract of pokeweed berries, pH 3.0, to stain cultured mycological specimens. Both extracts produced satisfactory staining that was equivalent to that of eosin Y, although the colors were more muted with the beet root extract. Epithelial cytoplasm, muscle, collagen and erythrocytes were well demonstrated. Betanin is the predominant component of beet root extract; it possesses one delocalized positive charge and three carboxylic acid substituents. The dyes are weak acids and the carboxylate anions are more diffuse than for eosin Y; this produces weaker bonding to tissue cations. The principal colored component of pokeweed berries, prebetanin, possesses a sulfonic acid group as well as carboxylic acids, which favors acid dyeing and more intense coloration. Both dyes show potential for hydrogen bonding and to a much lesser extent for some types of van der Waals forces. Complex formation with metals such as aluminum to create a nuclear stain is not likely with beet root dyes nor is it possible with pokeweed dyes. Betacyanins are suitable for staining microscopy preparations in place of other red acid dyes such as eosin. Of the two dyes tested here, prebetanin from pokeweed berries was superior to betanin from red beet roots. These berries are widely distributed and readily collected; the extraction procedure is simple and does not require expensive solvents.

The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.

We investigated the function of cytochrome P450 (CYP450) genes in degradation of the diazo dye, Congo red, by white-rot fungus Lenzites gibbosa. Hyphae treated with Congo red at different times were sequenced to obtain transcription data. CYP450 genes in transcriptomes were identified using a gene-encoding protein functional search and analyzed in Cluster of Orthologous Genes, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes Databases. Differentially expressed genes (DEGs) in different groups were analyzed using EdgeR. We present the relation between transcription factors (TFs) and CYP450 regulation by analysis of the co-expression network. One hundred sixty CYP450 genes obtained by a functional annotation search were related to the oxido-reduction reaction of secondary metabolism, defense mechanism and aromatic compound degradation. The fastest decolorization and the greatest expression of CYP450 genes, which were related to the decolorization effect, occurred at 0-3 h. Seven CYP450 genes (7522, 6568, 4482, 9118, 10935, 7521 and 10926) were identified. The key TFs that regulate these genes belong to the zinc finger family. CYP450 genes and their products in L. gibbosa participated in degradation of Congo red and stress resistance. We provide a reference value for degradation and decolorization of Congo red.

We investigated the potential gastroprotective effects of chrysin on indomethacin induced gastric ulcers in rats. We used six groups of animals: control; indomethacin (Indo); reference (Ulcuran®); indomethacin + 25 mg/kg chrysin (Indo + CHR25); indomethacin + 50 mg/kg chrysin (Indo + CHR50); indomethacin + 100 mg/kg chrysin (Indo + CHR100). All doses of chrysin were given orally to rats before indomethacin. Gastric lesions were examined macroscopically and microscopically. The effects of treatment with chrysin were assessed versus a single dose of 30 mg/kg Ulcuran® (generic ranitidine) as reference standard. We also investigated gastric mucosal superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT), malonaldehyde (MDA) and arginase activities, and COX-2, PGE₂, iNOS, TNF-α, IL-1β, NFκB, MPO, Bax, caspase-3 and 8-OHdG levels. We assessed caspase-3 and Bax levels using immunohistochemistry. Compared to the control and reference groups, SOD, CAT, GPx and arginase activities and GSH levels decreased, and MDA levels increased in the indomethacin induced gastric ulcer group. iNOS, TNF-α, IL-1β, NFκB, MAPK-14, MPO, Bax and 8-OHdG levels were increased in the indomethacin treated gastric group, while COX-2 activity and PGE₂ levels were decreased. The three doses of chrysin co-administered with indomethacin increased COX-2 activity and PGE₂ levels in rats with ulcers. Chrysin exhibited gastroprotective effects on indomethacin induced gastric ulcer due to its antioxidant, anti-inflammatory and anti-apoptotic activities.