LncRNA OIP5-AS1 knockdown is associated with attenuated nonylphenol-induced cardiac fibrosis

Figures

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  Fig. 1. Time stages and processes of rat treatment in each group.

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  Fig. 2. Effects of NP on H9C2 cell viability, and expression levels of fibrosis-related genes and LncRNA OIP5-AS1. A: Impact of NP at different concentration on the viability of H9C2 cells ( X¯±SD , n = 3, Kruskal–Wallis test). B: Relative expression of TGF-β1 mRNA ( X¯±SD , n = 3, t test) C: Relative expression of collagen I mRNA ( X¯±SD , n = 3, t test) D: Relative expression of collagen III mRNA ( X¯±SD , n = 3, t test) E: Relative expression of MMP-9 mRNA ( X¯±SD , n = 3, t test) F: Relative expression of LncRNA OIP5-AS1 ( X¯±SD , n = 3, t test) *P < 0.05; **P < 0.01; ***P < 0.001.

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  Fig. 3. Effects of NP exposure on the expression of fibrosis-related proteins in H9C2 cells. A–E: Fibrosis-related protein bands in Western blot. F: Relative expression of TGF-β1 protein ( X¯±SD , n = 4, t test). G: Relative expression of collagen I protein ( X¯±SD , n = 4, t test). H: Relative expression of collagen III protein ( X¯±SD , n = 4, t test). I: Relative expression of α-SMA protein ( X¯±SD , n = 4, t test). J: Relative expression of CTGF protein ( X¯±SD , n = 4, t test). *P < 0.05; ***P < 0.001.

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  Fig. 4. Effects of siRNA and NP on fibrosis in OIP5-AS1-knockdown H9C2 cells. A: Effects of siRNA and NP on cell membrane integrity ( X¯±SD , n = 3, ANOVA). B: siRNA interference effect on lncRNA OIP5-AS1 ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). C: Effect of NP on the proliferation of lncRNA OIP5-AS1 knockdown cells ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). D: Cell migration in each group. E: Effect of NP on the migration ability of lncRNA OIP5-AS1 knockdown cells ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). ns: P > 0.05, ***P < 0.001. avs control group, P < 0.05; bvs si-NC group, P < 0.05; cvs si-OIP5-AS1 group, P < 0.05; dvs NP, P < 0.05; evs si-NC+NP, P < 0.05.

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  Fig. 5. Effects of NP on the expression of fibrosis-related genes and proteins following knockdown of lncRNA OIP5-AS1. A: Relative expression of TGF-β1 mRNA ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). B: Relative expression of collagen I mRNA ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). C: Relative expression of collagen III mRNA ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). D: Relative expression of MMP-9 mRNA ( X¯±SD , n = 3, Kruskal–Wallis test followed by Bonferroni method was adopted for statistical analysis). E–I: Western blot protein band. J: Relative expression of TGF-β1 protein ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). K: Relative expression of collagen I protein ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). L: Relative expression of collagen III protein ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). M: Relative expression of α-SMA protein ( X¯±SD , n = 4,…

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  Fig. 6. Rat myocardial fibrosis modeling and lncRNA OIP5-AS1 interference detection. A: Naked eye view of rat heart tissue in each group. B: Interference effect of OIP5-AS1-RNAi on the expression of lncRNA OIP5-AS1 in rat heart ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). C: Masson and Sirius red staining (scale bar: 20 µm). D: Quantitative analysis of Masson and Sirius red staining ( X¯±SD , n = 3, t test). avs control group, P < 0.05; bvs NP group, P < 0.05; cvs AAV9-Vector+NP group, P < 0.05; dvs AAV9-sh-OIP5-AS1+NP group, P < 0.05.

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  Fig. 7. AAV9-mediated OIP5-AS1 knockdown and NP effects on cardiac fibrosis in rats. A: Masson staining of rat heart tissue (collagen fibers in rat heart tissue are blue; muscle fibers, cellulose, and red blood cells are red). B: Sirius red staining of rat heart tissue (collagen fibers in rat heart tissue are red with a yellow background under a light microscope). C: Quantitative analysis of Masson staining (CVF (%) = positive area/total area, X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). D: Quantitative analysis of Sirius red staining (CVF (%) = positive area/total area, X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). avs control group, P < 0.05; bvs NP group, P < 0.05; cvs AAV9-Vector+NP group, P < 0.05; dvs AAV9-sh-OIP5-AS1+NP group, P < 0.05.

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  Fig. 8. Effect of lncRNA OIP5-AS1 knockdown on NP-induced expression of fibrosis-related proteins in rat myocardium. A: Fluorescence expression of TGF-β1, collagen I, collagen III, α-SMA, and CTGF. B: Fluorescence expression of TGF-β1 ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). C: Fluorescence expression of collagen I ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). D: Fluorescence expression of collagen III ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). E: Fluorescence expression of α-SMA ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). F: Fluorescence expression of CTGF ( X¯±SD , n = 3, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). avs Control group, P < 0.05; bvs NP group, P < 0.05; cvs AAV9-Vector+NP group, P < 0.05; dvs AAV9-sh-OIP5-AS1+NP group, P < 0.05.

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  Fig. 9. Effect of lncRNA OIP5-AS1 knockdown on NP-induced expression of fibrosis-related proteins in rat myocardium. A–E: Western blot protein band. F: Relative expression of TGF-β1 protein ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). G: Relative expression of collagen I ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). H: Relative expression of collagen III ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). I: Relative expression of α-SMA ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). J: Relative expression of CTGF ( X¯±SD , n = 4, One-way ANOVA followed by Tukey’s HSD test was adopted for statistical analysis). avs Control group, P < 0.05; bvs NP group, P < 0.05; cvs AAV9-Vector+NP group, P < 0.05; dvs AAV9-sh-OIP5-AS1+NP group, P < 0.05.

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  Fig. 10. Scientific hypothesis mechanism diagram. The expression of LncRNA OIP5-AS1 was upregulated after NP exposure, which led to an increase in TGF-β1 expression. This further enhanced collagen and ECM secretion, ultimately resulting in the development of myocardial fibrosis. Inhibition of the high expression of LncRNA OIP5-AS1 reduced collagen and ECM secretion and attenuated NP-induced myocardial fibrosis.