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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

Treg expansion by rapamycin suppresses the activation of HSCs. According to previous methods, Tregs were isolated from rapamycin- or PBS-treated mice, and co-cultured with HSCs. (A-C) Expression levels of α-SMA were detected using immunofluorescence staining (magnification, ×200) and western blot analysis. (D) Fluorescence intensity reflecting the relative levels of intracellular α-SMA. Values are expressed as the mean ± standard error of the mean of triplicate experiments. *P<0.01, vs. control; **P<0.01, vs. HSC+Treg (PBS). (E) Western blot analysis and (F) quantification following treatment of HSCs with Tregs from different groups. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P>0.05, vs. control; **P<0.01, vs. HSC+Treg (PBS). HSCs, hepatocellular stellate cells; Treg, regulatory T cell; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; α-SMA, α-smooth muscle actin.
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f4-mmr-14-02-1153: Treg expansion by rapamycin suppresses the activation of HSCs. According to previous methods, Tregs were isolated from rapamycin- or PBS-treated mice, and co-cultured with HSCs. (A-C) Expression levels of α-SMA were detected using immunofluorescence staining (magnification, ×200) and western blot analysis. (D) Fluorescence intensity reflecting the relative levels of intracellular α-SMA. Values are expressed as the mean ± standard error of the mean of triplicate experiments. *P<0.01, vs. control; **P<0.01, vs. HSC+Treg (PBS). (E) Western blot analysis and (F) quantification following treatment of HSCs with Tregs from different groups. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P>0.05, vs. control; **P<0.01, vs. HSC+Treg (PBS). HSCs, hepatocellular stellate cells; Treg, regulatory T cell; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; α-SMA, α-smooth muscle actin.

Mentions: It is known that regulatory immune cells, including CD4+CD25+ T cells, are important in the pathogenesis of liver fibrosis and the activation of HSCs (9,11). To examine the effect of the Treg cell expansion by rapamycin on HSCs, HSCs were isolated from the mouse liver, and then cultured with splenic Tregs from the rapamycin- or PBS-treated mice. Immunofluorescence staining and western blot analysis were performed to evaluate changes in the expression of α-SMA. As shown in Fig. 4A–C, immunofluorescence staining showed that the expression of α-SMA was significantly reduced following exposure to the Tregs from the rapamycin-treated mice, compared with those from the PBS-treated mice. Fig. 4D shows the fluorescence intensity of each group, which shows the trends described above. The results of the western blot analysis showed that, compared with the control group, the expression of α-SMA was reduced following exposure to the Tregs, however, the difference was not significant. The protein expression of α-SMA was also markedly decreased following exposure to Tregs from the rapamycin-treated mice, compared with the PBS-treated mice (Fig. 4E). The expression levels of α-SMA are shown in Fig. 4F. These data showed that the expansion of the Treg cell population by rapamycin increased the capacity to inhibit the activation of HSCs.


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)

Treg expansion by rapamycin suppresses the activation of HSCs. According to previous methods, Tregs were isolated from rapamycin- or PBS-treated mice, and co-cultured with HSCs. (A-C) Expression levels of α-SMA were detected using immunofluorescence staining (magnification, ×200) and western blot analysis. (D) Fluorescence intensity reflecting the relative levels of intracellular α-SMA. Values are expressed as the mean ± standard error of the mean of triplicate experiments. *P<0.01, vs. control; **P<0.01, vs. HSC+Treg (PBS). (E) Western blot analysis and (F) quantification following treatment of HSCs with Tregs from different groups. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P>0.05, vs. control; **P<0.01, vs. HSC+Treg (PBS). HSCs, hepatocellular stellate cells; Treg, regulatory T cell; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; α-SMA, α-smooth muscle actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-mmr-14-02-1153: Treg expansion by rapamycin suppresses the activation of HSCs. According to previous methods, Tregs were isolated from rapamycin- or PBS-treated mice, and co-cultured with HSCs. (A-C) Expression levels of α-SMA were detected using immunofluorescence staining (magnification, ×200) and western blot analysis. (D) Fluorescence intensity reflecting the relative levels of intracellular α-SMA. Values are expressed as the mean ± standard error of the mean of triplicate experiments. *P<0.01, vs. control; **P<0.01, vs. HSC+Treg (PBS). (E) Western blot analysis and (F) quantification following treatment of HSCs with Tregs from different groups. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P>0.05, vs. control; **P<0.01, vs. HSC+Treg (PBS). HSCs, hepatocellular stellate cells; Treg, regulatory T cell; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; α-SMA, α-smooth muscle actin.
Mentions: It is known that regulatory immune cells, including CD4+CD25+ T cells, are important in the pathogenesis of liver fibrosis and the activation of HSCs (9,11). To examine the effect of the Treg cell expansion by rapamycin on HSCs, HSCs were isolated from the mouse liver, and then cultured with splenic Tregs from the rapamycin- or PBS-treated mice. Immunofluorescence staining and western blot analysis were performed to evaluate changes in the expression of α-SMA. As shown in Fig. 4A–C, immunofluorescence staining showed that the expression of α-SMA was significantly reduced following exposure to the Tregs from the rapamycin-treated mice, compared with those from the PBS-treated mice. Fig. 4D shows the fluorescence intensity of each group, which shows the trends described above. The results of the western blot analysis showed that, compared with the control group, the expression of α-SMA was reduced following exposure to the Tregs, however, the difference was not significant. The protein expression of α-SMA was also markedly decreased following exposure to Tregs from the rapamycin-treated mice, compared with the PBS-treated mice (Fig. 4E). The expression levels of α-SMA are shown in Fig. 4F. These data showed that the expansion of the Treg cell population by rapamycin increased the capacity to inhibit the activation of HSCs.

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