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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2019, Vol. 13 Issue (4) : 451-460
Structural shifts in the intestinal microbiota of rats treated with cyclosporine A after orthotropic liver transplantation
Junjun Jia1,2,3, Xinyao Tian1,2,3, Jianwen Jiang1,2,3, Zhigang Ren1,2,3, Haifeng Lu2,4, Ning He1,2,3, Haiyang Xie1,2,3, Lin Zhou1,2,3, Shusen Zheng1,2,3()
1. Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
2. Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health
3. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
4. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
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Understanding the effect of immunosuppressive agents on intestinal microbiota is important to reduce the mortality and morbidity from orthotopic liver transplantation (OLT). We investigated the relationship between the commonly used immunosuppressive agent cyclosporine A (CSA) and the intestinal microbial variation in an OLT model. The rat samples were divided as follows: (1) N group (normal control); (2) I group (isograft LT, Brown Norway [BN] rat to BN); (3) R group (allograft LT, Lewis to BN rat); and (4) CSA group (R group treated with CSA). The intestinal microbiota was assayed by denaturing gradient gel electrophoresis profiles and by using real-time polymerase chain reaction. The liver histopathology and the alanine/aspartate aminotransferase ratio after LT were both ameliorated by CSA. In the CSA group, the numbers of rDNA gene copies of Clostridium cluster I, Clostridium cluster XIV, and Enterobacteriaceae decreased, whereas those of Faecalibacterium prausnitzii increased compared with the R group. Cluster analysis indicated that the samples from the N, I, and CSA groups were clustered, whereas the other clusters contained the samples from the R group. Hence, CSA ameliorates hepatic graft injury and partially restores gut microbiota following LT, and these may benefit hepatic graft rejection.

Keywords microbial community      liver transplantation      immunosuppressive agents      cyclosporine A     
Corresponding Authors: Shusen Zheng   
Just Accepted Date: 04 March 2019   Online First Date: 24 April 2019    Issue Date: 02 August 2019
 Cite this article:   
Junjun Jia,Xinyao Tian,Jianwen Jiang, et al. Structural shifts in the intestinal microbiota of rats treated with cyclosporine A after orthotropic liver transplantation[J]. Front. Med., 2019, 13(4): 451-460.
Target Sequence (5'–3') Sequence (3'–5') Annealing temperature (°C)
Tab.1  The primers of the dominating bacteria
Fig.1  Liver histopathology (hematoxylin and eosin stain, original magnification 200×).
Fig.2  ALT and AST levels in the N, I, R, and CSA groups (n = 6 per group). Values are expressed as mean±SEM. *P<0.05 versus N, ×P<0.05 versus I group, #P<0.05 versus R group.
Fig.3  Numbers of fecal-dominating bacteria in the N, I, R, and CSA groups (n = 8 per group). Values are expressed as mean±SEM. *P<0.05 versus N group, ×P<0.05 versus I group, #P<0.05 versus R group. FPRA, Faecalibacterium prausnitzii; ENCO, Enterococcus spp.; CG1, Clostridium cluster I; CG2, Clostridium cluster XI; CG3, Clostridium cluster XIV; ECO, Enterobacteriaceae; LAC, Lactobacillus spp.; BAC, BacteroidesPrevotella group; BIF, Bifidobacterium spp.
Fig.4  CSA improved the intestinal microbiota in rats after LT as shown in the DGGE profiles. (A) DGGE profiles of intestinal bacteria from rats. Numbers of sample above the lanes represent rats from various groups. 50, 45, 39, 26, 35, and 16 samples were from N group; 22, 21, 15, 19, 27, and 28 samples were from I group; 11, 23, 33, 24, 30, and 29 samples were from CSA group; and 46, 44, 48, 54, 13, and 9 samples were from the R group. Gel-to-gel comparison is marked by different marker lanes. Each bacterial clone indicates a band. Numbers of each band (corresponding to Fig. 6 band classes) expressed the position of bands abscised for analyses (e.g., 8 means band class 8). (B) Diversity of fecal microbial comparison (Shannon’s diversity index). Values are expressed as mean±SEM. ×P<0.05 versus I group.
Fig.5  DGGE profile cluster analysis assessed with universal primers V3, the utilizing Dice’s coefficient and UPGMA. (A) DGGE profiles cluster analysis from the different groups. Metric scale expresses the degree of similarity. (B) Cluster MDS analysis shown in (A). The plot shows an optimized three-dimensional expression of the similarity matrix obtained from the BioNumerics software; the x, y and z axes express three different dimensions separately: Dim 1, 2, and 3. Euclidean distance between 2 points reflects similarity. (C) Fecal microbial PCA of DGGE fingerprinting shown in (A). Reoriented plots maximize the variation among different lanes along the first 3 principal components with contributions of 34.7%, 16.1%, and 12.2% as obtained from the BioNumerics software.
Fig.6  Neighbor-joining method was used for phylogenetic tree sequencing. Black squares indicate band classes with increased intensity versus the N group; black triangles indicate band classes with decreased intensity versus the N group; black diamonds indicate band classes with increased intensity versus the R group; and black spots indicate band classes with decreased intensity versus the R group. The plot was generated from MEGA5 software.
Fig.7  The key band changed among the groups. (A) The R group increased the band intensity, but CSA restored it to the N group level. (B) The key bands changed in the CSA group compared with the N group, while the R group maintained a normal level (n = 6 in each group). Values are expressed as mean±SEM. *P<0.05 versus N group, #P<0.05 versus R group.
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