Inhibitory Potential of Shen-Shuai-Ling Formulation on Renal Interstitial Fibrosis via Upregulation of PLZF

Figure 1. Analysis of key components and core targets of SSLF therapy for RIF based on network pharmacology. (a) Venn diagram of the intersection gene between SSLF and RIF target. (b) Venn diagram of the target intersection gene. (c) Protein-protein interaction network. Edges represent protein-protein interactions. (d) SSLF-compound-target-RIF network. In this network, the blue diamonds represent candidate targets of SSLF against RIF, and the yellow hexagon represents active compounds in SSLF.

Figure 2. GO and KEGG pathway enrichment analyses of SSLF for RIF. (a–c) The bubble graph shows biological process, cellular component, molecular function, and the intersection gene GO analysis. The left Y-axis is the name of the GO path, and the abscissa is p value. The larger the circle is, the greater the number of genes compared to this path. The darker the color is, the higher the proportion of genes compared to this path. (d) KEGG pathway enrichment analysis circle diagram. In the outermost layer of the circle, signaling pathway names are displayed in the right side and gene names are displayed in the left side. The inner ring on the left indicates the significant p value of gene corresponding pathway.

Figure 3. Luteolin and core targets for molecular docking. (a–d) The molecular docking of AKT1, PLZF, STAT3, VEGFA, and luteolin. The yellow line represents the hydrogen bonds; the blue sticks represent amino acid residues. (e) Coimmunoprecipitation assay shows PLZF-luteolin interactions in HK-2 cells.

Figure 4. SSLF and luteolin upregulation of PLZF levels. (a, b) Protein and gene expression of PLZF, PRR, Ang-1, Ang-2, and TGF-β. Western blot and qPCR analyses were performed after treatment with different concentrations of medicated rat serum containing SSLF. (c, d) Protein and gene expression of PLZF, PRR, Ang-1, Ang-2, and TGF-β. Western blot and qPCR analyses were performed after treatment with different concentrations of luteolin (5 μM, 10 μM, and 20 μM). Data are represented as mean ± SD (n ≥ 3 experiments). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 as determined using Student's t-test (two groups) or one-way ANOVA, followed by Tukey's test (more than two groups).

Figure 5. PLZF inhibits PRR expression in HK-2 cells. (a, b) Protein and gene expression of PLZF, PRR, Ang-1, Ang-2, and TGF-β. Western blot and qPCR results of overexpression of PLZF and PLZF upon knockdown. (c, d) Protein and gene expression of PLZF, PRR, Ang-1, Ang-2, and TGF-β. Western blot and qPCR results of overexpression of PRR. Data are represented as mean ± SD (n ≥ 3 experiments). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 as determined using Student's t-test (two groups) or one-way ANOVA, followed by Tukey's test (more than two groups).