Isoliensinine Attenuates Renal Fibrosis and Inhibits TGF-β1/Smad2/3 Signaling Pathway in Spontaneously Hypertensive Rats

Figures

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  Figure 1. Isoliensinine effectively improved hypertensive renal injury in SHRs. (A) The structural formula of Isoliensinine from PubChem (nih.gov) (B) The examination of pathological changes in renal tissue samples through H&E staining, with representative images taken at 400× magnification and a scale bar of 50 μm. The number of samples per group was n=6.

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  Figure 2. Isoliensinine treatment’s effects on the transcript expression profile of kidney tissues were analyzed by conducting RNA sequencing to identify differential transcripts (DETs) in each group. Comparing gene expression profiles using (A) hierarchical clustering plots and (B) volcano plots (|fold change| ≥ 2, P < 0.05). (C) Identification of integrated transcripts between the two comparisons with integrative analysis.

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  Figure 3. GO enrichment terms of renal tissues. GO Enrichment Analysis of DETs from (A) SHR vs WKY and (B) SHR+Isoliensinine vs SHR, presenting the top 30 enriched items in cellular composition, biological processes, and molecular function. (C) Venn diagram shows the overlap of cell composition, biological processes, and molecular functions between SHR and WKY groups and between SHR+ isoliensinine and WKY.

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  Figure 4. KEGG enrichment analysis of renal tissues: examining the impact of Isoliensinine on enriched signaling pathways in (A) SHR vs WKY and (B) SHR+Isoliensinine vs SHR, (C) with integrated analysis of the top 30 enriched pathways between both comparisons.

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  Figure 5. Isoliensinine reduced fibrosis in the renal interstitium and the levels of fibrosis markers in the kidney tissues of SHRs. (A) The collagen content in the kidneys of SHRs from each group was assessed using Masson trichrome staining. (B) The number of Masson trichrome-stained cells in the rats was calculated and IHC was performed to determine the positive areas. (C–F) IHC analysis was used to determine the protein expression of Collagen I and III. The number of collagen I and III-stained cells in the renal tissues was calculated and IHC was performed to detect the expressions of positive area. All micrographs were taken at 400x magnification with a scale bar of 50 µm. Results were presented as means ± standard deviations and statistical comparisons were made to the WKY (*P < 0.05 vs WKY) and SHR (#P < 0.05 vs SHR) groups. Representative images were depicted on left side and statistical graphs were shown on the right hand.

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  Figure 6. Inhibitory effect of Isoliensinie on TGF-β1/Smad2/3 signaling in-vivo using immunohistochemistry (IHC). (A and B) IHC was used to measure TGF-β1 protein expression in renal tissues and calculate the number of TGF-β1-positive cells. (C–F) IHC was also used to determine the expression of p-Smad2/Smad2 and p-Smad3/Smad3 and the proportion of positive regions in rat renal tissues. Micrographs were taken at 400× magnification with 50 µm scale bar and all results were presented as mean ± SD (n = 6 for each group). Significant differences were noted with *P < 0.05 vs WKY and #P < 0.05 vs SHR groups.