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

Abstract

Purpose: This study aimed to investigate the molecular mechanisms of isoliensinine, a kind of bibenzyl isoquinoline alkaloid which isolated from a TCM named Lotus Plumule (Nelumbo nucifera Gaertn), in treating renal interstitial fibrosis (RIF) by using RNA sequencing, KEGG analysis and in vivo experimental approaches.

Methods: Spontaneous hypertension rats (SHRs) were randomly assigned into five groups, consisting of SHR, SHR+Isoliensinine-L (2.5 mg/kg/day), SHR+Isoliensinine-M (5 mg/kg/day), SHR+Isoliensinine-H (10 mg/kg/day), and SHR+Valsartan (10 mg/kg/day) groups (n = 6 for each group). A control group of Wistar Kyoto rats (n = 6) was also included. Rats were treated intragastrically with isoliensinine, valsartan, or double-distilled water of equal volume for 10 weeks. To examine the therapeutic impact on hypertensive renal injury, fibrosis, and its underlying mechanisms, multiple techniques were employed, including hematoxylin and eosin staining, Masson trichrome staining, RNA sequencing, gene ontology (GO) function and pathway enrichment analysis and immunohistochemistry.

Results: Resultantly, the use of isoliensinine at different concentrations or valsartan showed significant improvement in renal pathological injury in SHRs. RNA sequencing and KEGG analysis uncovered 583 differentially expressed transcripts and pathways enriched in collagen formation and ECM-receptor interaction after treatment with isoliensinine. There was also a reduction in the increase of collagen and upregulation of collagen I & III, TGF-β1, p-Smad2, and p-Smad3 in the renal tissue of SHRs. Thus, isoliensinine ameliorated renal injury and collagen deposition in hypertensive rats, and inhibiting the activation of the TGF-β1/Smad2/3 pathway might be one of the underlying mechanisms.

Conclusion: This study showed that treatment with isoliensinine effectively reduced the renal injury and fibrosis in SHRs. In addition, isoliensinine inhibited the TGF-β1/Smad2/3 signaling in-vivo. These findings provided strong evidence for the therapeutic benefits of isoliensinine in combating renal injury and fibrosis.

Keywords: RNA sequencing; TGF-β1/Smad2/3 pathway; collagen deposition; hypertension; isoliensinine; renal injury.

Graphical abstract

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.

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.

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.

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.

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.

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.