Caveolin-1 Scaffolding Domain Peptides Alleviate Liver Fibrosis by Inhibiting TGF-β1/Smad Signaling in Mice
<p>Reduced expression of Cav1 in livers of WT mice after CCl<sub>4</sub> injection. (<b>A</b>) Cav1 mRNA was significantly lower in WT mice than control mice 3, 7, 14, and 28 days after CCl<sub>4</sub> injection. Gene expression was assessed by qRT-PCR and normalized to β-actin. WT control group value has been used for normalization among study groups. (<b>B</b>) Measurement of Cav-1 protein expression in livers of control and 3, 17, 14, and 28 days post CCl<sub>4</sub> injection. In insert, a representative Western blot from which these data were obtained. (<b>C</b>) Representative immunohistochemistry staining for Cav-1. (<b>D</b>) Area density of Cav1 staining in representative images for each group. This protein levels dramatically decreased at 7 (middle) and 14 days (right) after CCl<sub>4</sub> injection. <span class="html-italic">n</span> = 10, bar represents mean ± SD, ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05, compared with control animals, bar = 100 μm.</p> "> Figure 1 Cont.
<p>Reduced expression of Cav1 in livers of WT mice after CCl<sub>4</sub> injection. (<b>A</b>) Cav1 mRNA was significantly lower in WT mice than control mice 3, 7, 14, and 28 days after CCl<sub>4</sub> injection. Gene expression was assessed by qRT-PCR and normalized to β-actin. WT control group value has been used for normalization among study groups. (<b>B</b>) Measurement of Cav-1 protein expression in livers of control and 3, 17, 14, and 28 days post CCl<sub>4</sub> injection. In insert, a representative Western blot from which these data were obtained. (<b>C</b>) Representative immunohistochemistry staining for Cav-1. (<b>D</b>) Area density of Cav1 staining in representative images for each group. This protein levels dramatically decreased at 7 (middle) and 14 days (right) after CCl<sub>4</sub> injection. <span class="html-italic">n</span> = 10, bar represents mean ± SD, ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05, compared with control animals, bar = 100 μm.</p> "> Figure 2
<p>Increased inflammatory injury in Cav-1<sup>−/−</sup> livers. (<b>A</b>) Representative photomicrographs of HE-stained liver sections from CCl<sub>4</sub> treated WT and Cav1<sup>−/−</sup> mice. Degenerated (middle) and necrotic (right) hepatocytes were observed at three and seven days after CCl<sub>4</sub> injection, respectively. (<b>B</b>) The quantitative analysis of liver inflammation. Significantly increased inflammatory areas were present in Cav1<sup>−/−</sup> livers compared to WT. ALT (<b>C</b>); and AST (<b>D</b>) levels were detected by BS-200 Chemistry Analyzer (MINDRAY, Shenzhen, China). TGF-α (<b>E</b>); and IL-1β (<b>F</b>) levels were measured by ELISA kits (R&D Systems, Shanghai, China). <span class="html-italic">n</span> = 10, bar represents mean ± SD, ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05, compared with WT at the same time point, bar = 200 μm.</p> "> Figure 2 Cont.
<p>Increased inflammatory injury in Cav-1<sup>−/−</sup> livers. (<b>A</b>) Representative photomicrographs of HE-stained liver sections from CCl<sub>4</sub> treated WT and Cav1<sup>−/−</sup> mice. Degenerated (middle) and necrotic (right) hepatocytes were observed at three and seven days after CCl<sub>4</sub> injection, respectively. (<b>B</b>) The quantitative analysis of liver inflammation. Significantly increased inflammatory areas were present in Cav1<sup>−/−</sup> livers compared to WT. ALT (<b>C</b>); and AST (<b>D</b>) levels were detected by BS-200 Chemistry Analyzer (MINDRAY, Shenzhen, China). TGF-α (<b>E</b>); and IL-1β (<b>F</b>) levels were measured by ELISA kits (R&D Systems, Shanghai, China). <span class="html-italic">n</span> = 10, bar represents mean ± SD, ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05, compared with WT at the same time point, bar = 200 μm.</p> "> Figure 3
<p>Increased collagen deposition in CCl<sub>4</sub> treated Cav1<sup>−/−</sup> mice. (<b>A</b>) Representative photomicrographs of Sirius red stained liver sections from CCl<sub>4</sub> treated WT and Cav-1<sup>−/−</sup> mice. The collagen network is stained in red. The extent of collagen deposition was increased from day 7 to day 28. (<b>B</b>) Quantitative analysis demonstrated that Cav1<sup>−/−</sup> mice had significantly more collagen deposition than WT. (<b>C</b>) The mRNA expression of fibrotic markers were quantified using real-time RT-PCR. 18s rRNA was used for normalization of PCR data for the genes of α-SMA, Col α1(I) and Col α1(III). (<b>D</b>) Immunoblotting for α-SMA, Col α1(I), Col α1(III) in liver tissues of Cav1<sup>−/−</sup> and WT animals at 14 days post CCl<sub>4</sub> treatment. (<b>E</b>) Histogram showing densitometry analysis and quantification of α-SMA, Col α1(I), Col α1(III). The signals for α-SMA, Col α1(I), Col α1(III) were normalized to respective bands for β-actin. <span class="html-italic">n</span> = 10, bar represents mean ± SD, ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05, compared with WT at the same time point, bar = 200 μm.</p> "> Figure 4
<p>Enhanced activation of TGF-β signaling in Cav1<sup>−/−</sup> miceActivation of TGF-β signaling was assessed by measuring the protein expression levels of p-Smad2 and total Smad2 in mouse livers following CCl<sub>4</sub> treatment. The increased ratio of p-Smad2 to Smad2 indicates the activation of TGF-β1/Smad signaling. Enhanced activation of TGF-β signaling was present in Cav1<sup>−/−</sup> mice at 3 and 14 days post CCl4 treatment. <span class="html-italic">n</span> = 10, bar represents mean ± SD, ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05, compared with control for the same strain; <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with WT for the same time point.</p> "> Figure 5
<p>CSD inhibits activation of HSCs. (<b>A</b>) Immunoblotting for p-Smad2, α-SMA, Col α1(I) and Col α1(III) following stimulation with TGF-β1 (5 ng/mL) for 24 h in the presence or absence of Cav1 scaffolding domain peptide (CSD, 10 mM). (<b>B</b>) Histogram showing densitometry analysis and quantification of p-Smad2, α-SMA, Col α1(I), and Col α1(III). The signals for α-SMA, Col α1(I), Col α1(III) and Smad2 were normalized to respective bands for β-actin and/or Smad2. Bar represents mean ± SD. ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05, compared to control for the same strain; <sup>##</sup> <span class="html-italic">p</span> < 0.01, <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with TGF-β1 group for the same strain, <span class="html-italic">n</span> = 3.</p> "> Figure 6
<p>Enhanced activity of TGF-β1 signaling in Cav1<sup>−/−</sup> HSCs Primary mouse HSCs extracted from WT and Cav1<sup>−/−</sup> mice were cotransfected with SMAD luciferase reporter plasmid and pRL-TK vector. Twenty-four hours after transfection, cells were treated with TGF-β1 (5 ng/mL) for another 24 h. Luciferase activity was determined with the commercial luciferase reporter assay system. Each value represents the mean ± SD of at least three independent transfection experiments, each performed in triplicate. * <span class="html-italic">p</span> < 0.05 compared with no TGF-β1 group for the same strain; <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with TGF-β1-treated HSCs in WT mice.</p> "> Figure 7
<p>CSD alleviates liver fibrosis by inhibition of Smad2 phosphorylation. (<b>A</b>) Representative photomicrographs of Sirius red stained livers from CCl<sub>4</sub>-treated WT and Cav1<sup>−/−</sup> mice. The collagen network is stained in red. The scar contained a large amount of collagen at 14 days post injury when the mice were treated with the scrambled peptide (SCR). There is less collagen deposition in the livers of mice treated with the CSD peptide for both strains. (<b>B</b>) Qualitative analysis of Sirius red stained livers demonstrated that Cav1<sup>−/−</sup> mice had significantly more collagen deposition than WT animals at 14 days when treated with SCR. Administration of the CSD peptide prevented the accumulation of collagen. (<b>C</b>) The p-Smad2 expression following CCl<sub>4</sub> injection treated with CSD was reduced in both Cav1<sup>−/−</sup> and WT mice. <span class="html-italic">n</span> = 10, bar represents mean ± SD. ** <span class="html-italic">p</span> < 0.01 compared with 14-day SCR group in WT mice; <sup>##</sup> <span class="html-italic">p</span> < 0.01, <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with 14-day SCR group for the same strain, bar = 200 μm.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Downregulation of Cav1 after CCl4 Injection
2.2. Enhanced Inflammation Response in Cav1−/− Mice
2.3. Increased Collagen Production in Cav1−/− Mice
2.4. Enhanced Activation of TGF-β1/Smad Signaling in Cav1−/− Mice
2.5. Enhanced Activation of HSCs in Cav1−/− Mice
2.6. CSD Alleviates CCl4-Induced Liver Fibrosis
3. Discussion
4. Materials and Methods
4.1. Mouse Strain
4.2. Mouse Models and CSD Treatment
4.3. Isolation and Culture of Primary Mouse HSCs
4.4. Biochemical Measurements
4.5. Assessment of Liver Fibrosis
4.6. qRT-PCR
4.7. Immunohistochemical Staining
4.8. Western Blot
4.9. Reporter Gene Assays
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Lu, J.; Zhang, J.; Wang, Y.; Sun, Q. Caveolin-1 Scaffolding Domain Peptides Alleviate Liver Fibrosis by Inhibiting TGF-β1/Smad Signaling in Mice. Int. J. Mol. Sci. 2018, 19, 1729. https://doi.org/10.3390/ijms19061729
Lu J, Zhang J, Wang Y, Sun Q. Caveolin-1 Scaffolding Domain Peptides Alleviate Liver Fibrosis by Inhibiting TGF-β1/Smad Signaling in Mice. International Journal of Molecular Sciences. 2018; 19(6):1729. https://doi.org/10.3390/ijms19061729
Chicago/Turabian StyleLu, Jing, Jie Zhang, Yan Wang, and Quan Sun. 2018. "Caveolin-1 Scaffolding Domain Peptides Alleviate Liver Fibrosis by Inhibiting TGF-β1/Smad Signaling in Mice" International Journal of Molecular Sciences 19, no. 6: 1729. https://doi.org/10.3390/ijms19061729