A Study of Roundup-Induced Gut Dysbiosis, Irrelevant to Aromatic Amino Acid Deficiency, Impairs the Gut Function in Rats, Tianchi Duan et.al.

Description

We evaluated the impacts of chronic exposure to Roundup on gut health with the addition of AAA and explored the mechanism behind Roundup-induced gut dysfunction by performing fecal microbiota transplantation. 500 mg/kg·bw of Roundup, irrelevant to AAA deficiency, caused severe damage to gut function, as characterized by gut microbial dysbiosis, oxidative stress damage, intestinal inflammation and histopathological injury, particularly in female rats. Notably, similar to Roundup, Roundup-shaped gut microbiome evidently damaged serum, cecum and colon profiling of oxidative stress biomarkers (MDA, GSH-Px, SOD, GSSG and H2O2). Moreover, it induced 0.65-fold, 3.29-fold and 2.36-fold of increases in colonic IL-1β, IL-6 and TNF-α levels, and 0.34-fold of decrease in IL-10 level. Upon transplanting healthy fecal microbiota to Roundup-treated rats, they exhibited a healthier gut microenvironment with mitigated inflammation, oxidative damage and intestinal injury.

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Detection of oxidative markers Colon and cecum samples were prepared by adding 9 mL of cold saline in 1 g of tissue samples. Specimens were homogenized and centrifuged for 15 minutes at 1200 g. The obtained supernatants along with serum were used to carry out the SOD, H2O2, GSH-Px, GSH and MDA analyses. All the procedures were performed following the instructions. The protein contents of the colon and cecum samples were detected with the Coomassie Brilliant Blue method Histopathological observations The histological disparities on the colon were identified by H&E staining. First, the samples were conserved in 10% of formalin solution. These preserved specimens were further fixed with paraffin, sectioned (5-7 μm), and stained with H&E dye. Slides of dyed colon specimens were inspected and photographed using an Olympus light optical microscope . Finally, the images were examined for chronic changes (aberrant crypt, dysplasia, injury to the luinal surface and infiltration of the inflammatory cells) . 16S rRNA gene sequencing The gut microbial composition was examined by 16S rRNA gene sequencing. To remove the microbial DNA from fecal samples, E.Z.N.ATM Mag-Bind Soil DNA Kits (Omega, USA) were utilized. Moreover, agarose gel electrophoresis was performed to check the reliability, and the fluorometer was used to examine the DNA concentration. The V3-V4 universal primers of 341F and 805R were employed to run the PCR reaction and then to build the PCR-based library for 16S rRNA amplicon sequencing. To remove the indefinite products, the PCR products were washed with Agencourt AMPure XP (Beckman, USA). The qualified libraries were pair-end sequenced on the MiSeq System with the sequencing strategy PE300. Raw sequences were cleaned by in-house practice, and the combined end reads were fused to tags by fast length adjustment of short reads (version 1.2.3). The tags were then examined with USEARCH (version 5.2.236). Precisely, the tags were grouped into an operational taxonomic unit (OTU) with a 97% threshold by using UPARSE. The unique and emblematic sequences for each OTU were attained. Chimeras were removed by using UCHIME (version 4.2.40). The descriptive sequences for each OTU were taxonomically classified using Ribosomal Database Project (RDP) Classifier (version 2.12) trained on the Green-genes database (version 201305) by QIIME (version 1.8.0)15 . The OTU table was used to estimate the beta diversities and deliver taxonomic outlines. Resulting statistical analysis was performed on R software (version 3.0.1). Statistical Analysis Results were presented as mean ± SEM for three or more biological replicates. The levels of oxidative indicators and inflammatory factors, and the abundance of gut bacteria were statistically analyzed by one-way analysis of variance (ANOVA) using the GraphPad Prime 8 (GraphPad Software, San Diego, CA).

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