Study of the effects of rabbit scleral fibroblasts on cellular biomechanical properties and MMP-2 expression using two modes of riboflavin/ultraviolet A wave collagen cross-linking.

OBJECTIVE: The aim of this study is to evaluate the cellular biomechanical properties and MMP-2 expression changes in rabbit scleral fibroblasts using two modes of riboflavin and ultraviolet A (UVA) collagen cross-linking (CXL). METHODS: Twenty-four New Zealand white rabbits were randomly divided into two groups, A and B. The left eye was chosen for the experimental group and the right eye for the control group. In group A, the eyes were irradiated for 30 min, with a power density of 3.0 mW/cm2. In group B, the eyes were irradiated for 9 min, with a power density of 10.0 mW/cm2. One week after CXL, full-field electroretinography was performed. Sixty days after CXL, the rabbits were sacrificed, and scleral fibroblasts were extracted from the CXL-treated sclera area and corresponding parts of control sclera and cultured. Cellular biomechanical properties were evaluated using the micropipette aspiration technique, and the MMP-2 protein expression was determined by Western blot analysis. RESULTS: There was no statistical difference in the amplitude and latency of the dark adaptation 3.0 and light adaptation 3.0 between the CXL and control eyes of groups A and B (P > 0.05). Compared with the control groups, the Young's modulus of the fibroblasts and apparent viscosity of the experimental eyes in groups A and B were increased after CXL (P < 0.05), but there was no significant difference between the two groups under different irradiation modes (P > 0.05). The MMP-2 expression in scleral fibroblasts from experimental eyes was significantly higher than that in scleral fibroblasts from control eyes in groups A and B. Under the two different irradiation modes, the MMP-2 expression in the scleral fibroblasts from experimental eyes in group A was significantly higher than that in the scleral fibroblasts from experimental eyes in group B. CONCLUSION: The riboflavin-UVA scleral CXL conducted in two different modes produced no significant side effects on the retina and could strengthen the cell biomechanical properties as well as increase the MMP-2 expression of scleral fibroblasts significantly.

[Rhizosphere microorganism evidence on geo-authentic features of traditional Chinese medicine from Moutan(Paeonia suffruticosa)].

Paeonia suffruticosa also named Moutan that cultivated in five geographic regions during different growth stages were chosen in this study. Biolog and 454 pyrosequencing technology were used to detect the whole microbial activity and fungal diversity for exploring the relationship between the geo-authentic features of the medicinal plant and the rhizosphere microorganism. The results suggest that the value of average well color development(AWCD) from the rhizosphere soil of P. suffruticosa in the five regions at the four growth stage have an increasing tendency. 9 703 operational taxonomic unit(OTU) were obtained from 272 463 high quality sequences according to the similarity of 97% by the pyrosequencing. Fungi in five phyla, twenty-two classes, seventy orders, one hundred and thirty-nine families and two hundred and sixty-six genera were detected in the five regions excluding twelve percent to fifty-eight percent unidentified fungi. They were divided into four branches, i.e. Blastocladiales, Chytridiomycota, Dikarya and Glomeromycetes. Twenty-four genera such as Leptosphaeria were found in the five regions while six genera such as Curvularia were only detected in the geo-authentic regions. The dominant genera were Guehomyces, Exophiala and Fusarium in geo-authentic regions, whereas genus Leptosphaeria, Cryptococcus, Exophiala, Fusarium and Ceratobasidium in non-authentic regions. The results from principal co-ordinates analysis (PCoA) showed that the fungi formations were similar in Tongling and Nanling region at four different growth stages, and the same in Heze at the leaf bud and fruiting stage. According to heatmap analysis, Tongling and Nanling region showed a close similarity in fungal community structures on phylogenetic analysis, while Bozhou, Heze and Luoyang showed the same. In brief, the whole microbial activity was higher in geo-authentic regions than the non-authentic. Fungi in rhizosphere soil of the medicinal peony presented diversity and region specificity. We found not only the abundant new species in the five regions, but also the phylogenetic similarity in the geo-authentic regions.

Metabonomics evaluation of urine from rats administered with phorate under long-term and low-level exposure by ultra-performance liquid chromatography-mass spectrometry.

The purpose of this study was to investigate the toxic effect of long-term and low-level exposure to phorate using a metabonomics approach based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Male Wistar rats were given phorate daily in drinking water at low doses of 0.05, 0.15 or 0.45 mg kg⁻¹ body weight (BW) for 24 weeks consecutively. Rats in the control group were given an equivalent volume of drinking water. Compared with the control group, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), urea nitrogen (BUN) and creatinine (CR) were increased in the middle- and high-dose groups whereas albumin (ALB) and cholinesterase (CHE) were decreased. Urine metabonomics profiles were analyzed by UPLC-MS. Compared with the control group, 12 metabolites were significantly changed in phorate-treated groups. In the negative mode, metabolite intensities of uric acid, suberic acid and citric acid were significantly decreased in the middle- and high-dose groups, whereas indoxyl sulfic acid (indican) and cholic acid were increased. In the positive mode, uric acid, creatinine, kynurenic acid and xanthurenic acid were significantly decreased in the middle- and high-dose groups, but 7-methylguanine (N7G) was increased. In both negative and positive modes, diethylthiophosphate (DETP) was significantly increased, which was considered as a biomarker of exposure to phorate. In conclusion, long-term and low-level exposure to phorate can cause disturbances in energy-related metabolism, liver and kidney function, the antioxidant system, and DNA damage. Moreover, more information can be provided on the evaluation of toxicity of phorate using metabonomics combined with clinical chemistry.

Complete Chloroplast Genome of Krascheninnikovia ewersmanniana: Comparative and Phylogenetic Analysis.

Krascheninnikovia ewersmanniana is a dominant desert shrub in Xinjiang, China, with high economic and ecological value. However, molecular systematics research on K. ewersmanniana is lacking. To resolve the genetic composition of K. ewersmanniana within Amaranthaceae and its systematic relationship with related genera, we used a second-generation Illumina sequencing system to detect the chloroplast genome of K. ewersmanniana and analyze its assembly, annotation, and phylogenetics. Total length of the chloroplast genome of K. ewersmanniana reached 152,287 bp, with 84 protein-coding genes, 36 tRNAs, and eight rRNAs. Codon usage analysis showed the majority of codons ending with base A/U. Mononucleotide repeats were the most common (85.42%) of the four identified simple sequence repeats. A comparison with chloroplast genomes of six other Amaranthaceae species indicated contraction and expansion of the inverted repeat boundary region in K. ewersmanniana, with some genes (rps19, ndhF, ycf1) differing in length and distribution. Among the seven species, the variation in non-coding regions was greater. Phylogenetic analysis revealed Krascheninnikovia ceratoides, Dysphania ambrosioides, Dysphania pumilio, and Dysphania botrys to have a close monophyletic relationship. By sequencing the K. ewersmanniana chloroplast genome, this research resolves the relatedness among 35 Amaranthaceae species, providing molecular insights for germplasm utilization, and theoretical support for studying evolutionary relationships.

Characterization of ribostamycin and its impurities using a nano-quantity analyte detector: Systematic comparison of performance among three different aerosol detectors.

Characterization of aminoglycoside antibiotics like ribostamycin is important due to the complex composition and common toxic impurities. Aerosol detectors are often employed for determination of these non-absorbent analytes. In this work, a robust and cost-effective method was developed for simultaneous detection of ribostamycin and its related substances using high-performance liquid chromatography (HPLC) with a relative new aerosol detector named nano-quantity analyte detector (NQAD). With the introduction of less toxic but more compatible ion-pairs pentafluoropropionic acid (PFPA) and trifluoroacetic acid (TFA) in the eluent, an optimized separation effect was achieved. Compared with the other two aerosol detectors namely ELSD (evaporative light scattering detector) and CAD (charged aerosol detector), method verification and quantitative detection results revealed that NQAD had higher sensitivity than ELSD with a 0.8 µg/mL limit of detection, as well as wider linear range (from 2 µg/mL to 1000 µg/mL) than both CAD (from 2 µg/mL to 200 µg/mL) and ELSD (from 8 µg/mL to 200 µg/mL) detector. The performance of NQAD helped to realize detection of ribostamycin and its impurities with significant concentration differences in a single run. With a cation suppressor to eliminate the ion-suppression caused by the ion-pairs in the eluent, the structure of nine impurities in ribostamycin sample was characterized by liquid chromatography-mass spectrum (LC-MS). Both external standard and area normalization calculation were investigated, and NQAD obtained more accurate results due to its full-range linear response-to-concentration relationship, providing an alternative for routine quality control of multi analyte systems.

The plasma metabolic profiling of chronic acephate exposure in rats via an ultra-performance liquid chromatography-mass spectrometry based metabonomic method.

This study aimed to investigate the toxic effects of long-term, low-dose acephate administration on rats using ultra-performance liquid chromatography-mass spectrometry. A total of 120 male Wistar rats were randomly assigned to different groups: control; low-dose acephate (0.5 mg kg(-1) bw(-1)); middle-dose acephate (1.5 mg kg(-1) bw(-1)); and high-dose acephate (4.5 mg kg(-1) bw(-1)). The rats continuously received acephate via drinking water for 24 weeks. Rat plasma samples were collected at different time points to measure metabonomic profiles. Liver tissues were subjected to histopathological examination. The results showed that 10 metabolites in the plasma were significantly changed in the treated groups compared with those in the control group (P < 0.05 or P < 0.01). Exposure to acephate resulted in increased lysoPC (15 : 0), lysoPC (16 : 0), lysoPC (O-18 : 0), lysoPC (18 : 1(9Z)), lysoPC (18 : 0), lysoPC (20 : 4(5Z, 8Z, 11Z, 14Z)), arachidonic acid, and 12-HETE as well as decreased tryptophan and indoleacrylic acid in rat plasma. Moreover, the contents of high-density lipoprotein, low-density lipoprotein, triglyceride, total cholesterol, free fatty acids, and malondialdehyde, as well as the activities of superoxide dismutase and phospholipaseA2 in the serum, were significantly changed in the middle- and high-dose groups compared with those in the control group (P < 0.05 or P < 0.01). Histopathological examination results revealed that exposure to acephate may induce vacuolar degeneration in the liver cell cytoplasm, fat degeneration, and liver cell necrosis. These results indicated that exposure to acephate disrupted metabolism of lipids and amino acids, induced oxidative stress, caused neurotoxicity, and resulted in liver dysfunction.

Effect of quercetin against dichlorvos induced nephrotoxicity in rats.

This study was carried out to determine the effect of quercetin against renal injury induced by dichlorvos (DDVP) in rats. Rats were randomly assigned to control, DDVP-treated (7.2mg/kg bw), three different doses of quercetin-treated (2mg/kg bw, 10mg/kg bw, 50mg/kg bw) and different doses of quercetin plus DDVP-treated groups. DDVP was administered daily to rats through their drinking water, and quercetin was administered by intragastric gavage for 90 days. By the end of the 90th day in the DDVP-treated group, the following indices significantly increased compared with the control (P<0.01): activities of catalase, glutathione peroxidase and superoxide dismutase; level of malondialdehyde in kidney tissues; serum levels of creatinine and urea nitrogen; and level of ß2-microglobulin, level of retinol-conjugated protein, and activity of N-acetyl-ß-d-glucosaminidase in urine; by contrast, urine uric acid levels significantly decreased. However, in the quercetin (50mg/kg bw) plus DDVP group, the aforementioned indices were significantly decreased compared with the DDVP-treated group (P<0.05), except the urine uric acid levels were significantly increased (P<0.05). Thus, rat exposure to DDVP caused renal injury, including renal tubular, glomerular filtration, and oxidative stress. These toxic effects were also regulated by high-dose quercetin. Histopathological examination revealed that exposure to DDVP induced extensive cell vacuolar denaturation, but milder histopathological alterations in the kidney tissues of rats co-treated with DDVP and quercetin (50mg/kg bw) were observed. These results indicated that quercetin at 50mg/kg bw can partly prevent the kidney injury induced by DDVP.

A metabonomic analysis of the effect of quercetin on toxicity induced by chronic exposure to low-level dichlorvos in rat plasma.

A previous study of ours has reported that chronic exposure to low-level dichlorvos (DDVP, 7.2 mg per kg bw) damages the liver, interferes with fatty acid metabolism, and disturbs the antioxidant defense system in rats. This study aims to investigate whether or not quercetin can protect against DDVP-induced toxicity through metabonomics and to elucidate the mechanism underlying this protective effect. Rats were randomly assigned into the control group, DDVP-treated group, quercetin-treated group, and quercetin plus DDVP-treated group. DDVP and quercetin were administered to the rats daily via drinking water and gavage, respectively, continuously for 90 d. The metabonomic profiles of rat plasma were analyzed using ultra-performance liquid chromatography-mass spectrometry. Finally, 11 metabolites were identified, including those of quercetin, isorhamnetin, and quercetin-3-glucuronide. The 11 metabolites showed significant changes in some treatment groups compared with the control group. Arachidonic acid, phytosphingosine, and C16 sphinganine significantly decreased while p-cresol, lysoPE (16:0/0:0), lysoPC (15:0/0:0), lysoPC (16:0/0:0), lysoPC (0:0/18:0), and tryptophan significantly increased in the DDVP-treated group compared with the control group. The tendency of the aforementioned metabolites to change was significantly ameliorated in the high-dose quercetin (50 mg per kg bw per day) plus DDVP-treated group compared with the DDVP-treated group. However, the levels of these metabolites in the high-dose quercetin plus DDVP-treated group were still significantly different from those in the control group. The results indicate that high-dose quercetin (50 mg per kg bw per day) elicits a partial protective effect on DDVP-induced toxicity. The histopathology of the liver tissues was consistent with the above results. Quercetin demonstrated regulatory effects on the metabolism of lipids and amino acids, the antioxidant defense system, etc. Therefore, increasing the daily intake of quercetin can ameliorate the toxicity induced by chronic exposure to low-level DDVP residue in food and/or water.

Metabonomic analysis of quercetin against the toxicity of chronic exposure to low-level dichlorvos in rats via ultra-performance liquid chromatography-mass spectrometry.

This study aims to determine whether quercetin elicits a protective effect against the toxicity of chronic exposure to low-level DDVP using metabonomic technology. Rats were randomly assigned into the control, DDVP-treated, quercetin-treated, and quercetin plus DDVP-treated groups. DDVP and quercetin were given to rats daily via drinking water and gavage respectively for 90 days. Eighteen metabolites, including the biomarkers of DDVP exposure (dimethyl phosphate, DMP) and quercetin exposure (quercetin and isorhamnetina), were identified from the metabonomic profiles of rat urine using ultra-performance liquid chromatography-mass spectrometry. Compared with the control group, the DDVP-treated group showed statistically significantly increased intensities of indoxyl sulfate, estrone sulfate, cholic acid, deoxycholic acid, p-cresol, p-cresol sulfate, and orotic acid but decreased intensities of suberic acid, citric acid, sebacic acid, hippuric acid, taurine, phosphocreatine, 3-hydroxyanthranilic acid, and kynurenic acid. The tendency of the aforesaid metabolites to change was significantly ameliorated in the quercetin (50mg/kg·bw) plus DDVP (7.2mg/kg·bw)-treated group compared with the DDVP-treated group. However, the levels of these metabolites in the quercetin plus DDVP-treated groups were still significantly different from those of the control group. These results indicate that quercetin has a partial protective effect on DDVP-induced toxicity.

Metabonomic analysis of the joint toxic action of long-term low-level exposure to a mixture of four organophosphate pesticides in rat plasma.

In previously published articles, we evaluated the toxicity of four organophosphate (OP) pesticides (dichlorvos, dimethoate, acephate, and phorate) in rats using metabonomic technology at their corresponding no observed adverse effect levels (NOAELs). The results show that a single pesticide did not elicit a toxic response. The joint toxic action of four pesticides (at their corresponding NOAELs) was evaluated by metabolomic analysis of rat plasma under experimental conditions similar to those of the four single OP pesticides. The pesticides were administered daily to rats through drinking water for 24 weeks. The mixture of four pesticides showed a joint toxic action at the NOAELs of each pesticide. The 19 metabolites were statistically significantly changed in all the treated groups compared with those in the control group (p < 0.05 or p < 0.01). Exposure to OP pesticides resulted in increased lysoPC (15 : 0/0 : 0), lysoPC (16 : 0/0 : 0), lysoPC (O-18 : 0/0 : 0), lysoPC (P-19 : 1(12Z)/0 : 0), lysoPC (18 : 1(9Z)/0 : 0), lysoPC (18 : 0/0 : 0), lysoPC (20 : 4(5Z, 8Z, 11Z, 14Z)/0 : 0), lysoPE (16 : 0/0 : 0), lysoPC (17 : 0/0 : 0), 4-pyridoxic acid, glutamic acid, glycocholic acid, and arachidonic acid, as well as decreased C16 sphinganine, C17 sphinganine, phytosphingosine, indoleacrylic acid, tryptophan, and iodotyrosine in rat plasma. The results indicate that the mixture of OP pesticides induced oxidative stress, liver and renal dysfunction, disturbed the metabolism of lipids and amino acids, and interfered with the function of the thyroid gland. The present plasma results provided complementarities with our previous metabolomic analysis of the rat urine profile exposed to a mixture of four OP pesticides, and also contributed to the understanding of the mechanism of joint toxic action.

Application of ultraperformance liquid chromatography/mass spectrometry-based metabonomic techniques to analyze the joint toxic action of long-term low-level exposure to a mixture of organophosphate pesticides on rat urine profile.

In previously published articles, we evaluated the toxicity of four organophosphate (OP) pesticides (dichlorvos, dimethoate, acephate, and phorate) to rats using metabonomic technology at their corresponding no observed adverse effect level (NOAEL). Results show that a single pesticide elicits no toxic response. This study aimed to determine whether chronic exposure to a mixture of the above four pesticides (at their corresponding NOAEL) can lead to joint toxic action in rats using the same technology. Pesticides were administered daily to rats through drinking water for 24 weeks. The above mixture of the four pesticides showed joint toxic action at the NOAEL of each pesticide. The metabonomic profiles of rats urine were analyzed by ultraperformance liquid chromatography/mass spectrometry. The 16 metabolites statistically significantly changed in all treated groups compared with the control group. Dimethylphosphate and dimethyldithiophosphate exclusively detected in all treated groups can be used as early, sensitive biomarkers for exposure to a mixture of the OP pesticides. Moreover, exposure to the OP pesticides resulted in increased 7-methylguanine, ribothymidine, cholic acid, 4-pyridoxic acid, kynurenine, and indoxyl sulfate levels, as well as decreased hippuric acid, creatinine, uric acid, gentisic acid, C18-dihydrosphingosine, phytosphingosine, suberic acid, and citric acid. The results indicated that a mixture of OP pesticides induced DNA damage and oxidative stress, disturbed the metabolism of lipids, and interfered with the tricarboxylic acid cycle. Ensuring food safety requires not only the toxicology test data of each pesticide for the calculation of the acceptable daily intake but also the joint toxic action.