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Rapamycin ameliorates CCl4-induced liver fibrosis in mice through reciprocal regulation of the Th17/Treg cell balance.

Gu L, Deng WS, Sun XF, Zhou H, Xu Q - Mol Med Rep (2016)

Bottom Line: It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues.Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances.These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, P.R. China.

ABSTRACT
Previous investigations have suggested that the activation of Th17 cells and/or deficiency of regulatory T cells (Tregs) are involved in the pathogenesis of liver fibrosis. The aim of the present study was to investigate the effect of rapamycin on immune responses in a carbon tetrachloride (CCl4)-induced murine liver fibrosis model. Liver fibrosis was induced by intraperitoneal administration with CCl4. Following injection of CCl4, the mice were treated intraperitoneally with rapamycin (1.25 mg/kg/day) for 8 weeks. Hematoxylin and eosin staining and Masson's trichrome staining were used for histological examination. The protein levels of forkhead/winged helix transcription factor P3, retinoic-acid-related orphan receptor (ROR)‑γt in liver tissue were determined by western blotting, the frequency of Th17 and Treg cells in the liver was evaluated by flow cytometry, and a suppression assay was measured by incorporating [3H]‑thymidine. In addition, to explore the effect of Tregs expanded with rapamycin on hepatic stellate cells (HSC), HSCs were co‑cultured with Tregs from rapamycin or phosphate‑buffered saline‑treated mice. It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues. Simultaneously, the results of the present study showed a significant increase in the frequency of Tregs and a marked enhancement in the expression of forkhead/winged helix transcription factor P3 in the rapamycin‑treated mice. Furthermore, the Tregs in rapamycin‑treated mice had significantly higher suppressive effects, compared with the cells from mice treated with phospphate‑buffered saline. Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances. These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

No MeSH data available.


Related in: MedlinePlus

Rapamycin suppresses the generation of Th17 cells in liver tissues. (A) Cells were isolated from spleen and liver tissues in each group of mice and subjected to intracellular IL-17A staining. The percentages of Th17 cells were determined using flow cytometry and were (B) quantified. Data represent one of three separate experiments with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of ROR-γt in the liver tissues were determined using western blot analysis and (D) expressed as the relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.05, vs. PBS-treated group. CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; IL-17A, interleukin-17A; ROR-retinoic-acid-related orphan receptor-γt.
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f2-mmr-14-02-1153: Rapamycin suppresses the generation of Th17 cells in liver tissues. (A) Cells were isolated from spleen and liver tissues in each group of mice and subjected to intracellular IL-17A staining. The percentages of Th17 cells were determined using flow cytometry and were (B) quantified. Data represent one of three separate experiments with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of ROR-γt in the liver tissues were determined using western blot analysis and (D) expressed as the relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.05, vs. PBS-treated group. CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; IL-17A, interleukin-17A; ROR-retinoic-acid-related orphan receptor-γt.

Mentions: To analyze the immunoregulatory effect of rapamycin on Th17 cells in CCl4-induced liver fibrosis, the percentages of Th17 cells in the mouse spleen and liver were measured using flow cytometry (Fig. 2A). As shown in Fig. 2B, the cells from the PBS-treated mice showed markedly higher percentages of Th17 cells, compared with the negative control group (oil without CCl4). Following rapamycin administration, the percentages of Th17 cells were significantly lower, compared with those in the PBS-treated mice, but were higher, compared with the negative control group (Fig. 2B). In addition, as a crucial transcription factor of Th17 cell differentiation, the expression of ROR-γt in the rapamycin-treated mice was decreased, compared with that in the PBS-treated mice (Fig. 2C). The relative protein levels are shown in Fig. 2D.


Rapamycin ameliorates CCl4-induced liver fibrosis in mice through reciprocal regulation of the Th17/Treg cell balance.

Gu L, Deng WS, Sun XF, Zhou H, Xu Q - Mol Med Rep (2016)

Rapamycin suppresses the generation of Th17 cells in liver tissues. (A) Cells were isolated from spleen and liver tissues in each group of mice and subjected to intracellular IL-17A staining. The percentages of Th17 cells were determined using flow cytometry and were (B) quantified. Data represent one of three separate experiments with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of ROR-γt in the liver tissues were determined using western blot analysis and (D) expressed as the relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.05, vs. PBS-treated group. CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; IL-17A, interleukin-17A; ROR-retinoic-acid-related orphan receptor-γt.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940054&req=5

f2-mmr-14-02-1153: Rapamycin suppresses the generation of Th17 cells in liver tissues. (A) Cells were isolated from spleen and liver tissues in each group of mice and subjected to intracellular IL-17A staining. The percentages of Th17 cells were determined using flow cytometry and were (B) quantified. Data represent one of three separate experiments with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of ROR-γt in the liver tissues were determined using western blot analysis and (D) expressed as the relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.05, vs. PBS-treated group. CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; IL-17A, interleukin-17A; ROR-retinoic-acid-related orphan receptor-γt.
Mentions: To analyze the immunoregulatory effect of rapamycin on Th17 cells in CCl4-induced liver fibrosis, the percentages of Th17 cells in the mouse spleen and liver were measured using flow cytometry (Fig. 2A). As shown in Fig. 2B, the cells from the PBS-treated mice showed markedly higher percentages of Th17 cells, compared with the negative control group (oil without CCl4). Following rapamycin administration, the percentages of Th17 cells were significantly lower, compared with those in the PBS-treated mice, but were higher, compared with the negative control group (Fig. 2B). In addition, as a crucial transcription factor of Th17 cell differentiation, the expression of ROR-γt in the rapamycin-treated mice was decreased, compared with that in the PBS-treated mice (Fig. 2C). The relative protein levels are shown in Fig. 2D.

Bottom Line: It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues.Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances.These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, P.R. China.

ABSTRACT
Previous investigations have suggested that the activation of Th17 cells and/or deficiency of regulatory T cells (Tregs) are involved in the pathogenesis of liver fibrosis. The aim of the present study was to investigate the effect of rapamycin on immune responses in a carbon tetrachloride (CCl4)-induced murine liver fibrosis model. Liver fibrosis was induced by intraperitoneal administration with CCl4. Following injection of CCl4, the mice were treated intraperitoneally with rapamycin (1.25 mg/kg/day) for 8 weeks. Hematoxylin and eosin staining and Masson's trichrome staining were used for histological examination. The protein levels of forkhead/winged helix transcription factor P3, retinoic-acid-related orphan receptor (ROR)‑γt in liver tissue were determined by western blotting, the frequency of Th17 and Treg cells in the liver was evaluated by flow cytometry, and a suppression assay was measured by incorporating [3H]‑thymidine. In addition, to explore the effect of Tregs expanded with rapamycin on hepatic stellate cells (HSC), HSCs were co‑cultured with Tregs from rapamycin or phosphate‑buffered saline‑treated mice. It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues. Simultaneously, the results of the present study showed a significant increase in the frequency of Tregs and a marked enhancement in the expression of forkhead/winged helix transcription factor P3 in the rapamycin‑treated mice. Furthermore, the Tregs in rapamycin‑treated mice had significantly higher suppressive effects, compared with the cells from mice treated with phospphate‑buffered saline. Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances. These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

No MeSH data available.


Related in: MedlinePlus