Acteoside alleviates UUO-induced inflammation and fibrosis by regulating the HMGN1/TLR4/TREM1 signaling pathway

Abstract

Purpose: Acteoside (Act), a phenylethanoid compound that was first isolated from mullein, has been widely used for the investigation of anti-inflammatory and anti-fibrotic effect. However, the mechanism of Act against unilateral ureteral obstruction (UUO)-mediated renal injury is largely unknown. Therefore, this study aimed to explore the effects of Act on UUO rats and possible mechanisms.

Methods: A total of 20 Sprague-Dawley (SD) rats were divided randomly into three groups (n ≥ 6): (i) sham-operated group (Sham); (ii) UUO group (UUO+Saline); and (iii) UUO + Act 40 mg/kg/day, (UUO+Act); Continuous gavage administration for 2 weeks postoperatively, while the rats in Sham and UUO+saline groups were given equal amounts of saline. All rats were sacrificed after 14 days, the urine and blood samples were collected for biochemical analysis, the renal tissues were collected for pathological staining and immunohistochemistry. Correlations between individual proteins were analyzed by Pearson correlation analysis.

Results: The results of renal function indexes and histopathological staining showed that Act could improve renal function by reducing serum creatinine, blood urea nitrogen and urine protein at the same time, Act could alleviate renal inflammation and fibrosis. In addition, the results of immunohistochemistry showed that Act could reduce the expression of inflammation and kidney injury-related proteins F4/80, Mcp-1, KIM-1 proteins, as well as the expression of fibrosis-related protein α-SMA and β-catenin. More importantly, Act can also reduce the expression of HMGN1, TLR4 and TREM-1 proteins.

Conclusion: These data demonstrate that Act can ameliorate UUO-induced renal inflammation and fibrosis in rats probably through triggering HMGN1/TLR4/TREM-1 pathway.

Keywords: Acteoside; HMGN1; Inflammation; Obstructive nephropathy; Renal fibrosis.

Figure 1. Act alleviated the renal function parameters and systemic inflammation in unilateral obstruction (UUO) rat models.

(A) Structural of Acteoside (Act); (B, C, D) Act decreases the levels of serum creatinine (Scr), blood urea nitrogen (BUN), urinary protein excretion rate in UUO groups. (E, F) The serum concentration of IL-6 and TNF- α in UUO rats were determined using enzyme-linked immunosorbent assay (ELISA) kits. All data are represented as mean ± SEM, n = 5 rats per group. ^∗p < 0.05, ^∗∗p < 0.001, ^∗∗∗p < 0.0001.

Figure 2. Act attenuates the renal inflammation and injury in unilateral obstruction (UUO) rat models.

(A) Kidney sections from all groups were stained with H&E for morphology changing, “ →” point to renal tubular cells necrosis fall off and filling with a tube-shaped protein; (B) immunohistochemical stain of Mcp-1, KIM-1 and F-4/80 in the kidney tissue of UUO rats (n = 5). Original magnification = 400 ×. Scale bars. 50 um. All data are represented as mean ± SD; (C) Renal injury score was analyzed based on H&E staining; (D) quantification of the levels of Mcp-1, KIM-1 and F-4/80 proteins. all values are presented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.001.

Figure 3. Act reduced the accumulation of collagen fibers and the expression of key proteins involved in UUO-induced renal fibrosis.

(A) Masson trichrome staining for collagen fibers (B) immunohistochemical stain of β-catenin and α-SMA in the kidney tissue of UUO rats (n = 5). Original magnification =400 ×. Scale bars. 50um. All data are represented as mean ± SEM; (C) Percentage of the fibrotic area by Masson’s trichrome staining. (D) Quantification of the levels of β-catenin and α-SMA proteins, all values are presented as mean ± SEM. ^∗∗p < 0.001, ^∗∗∗p < 0.0001.

Figure 4. HMGN1, TLR4, and TREM-1 protein levels in the kidney of different experimental groups.

(A) Immunohistochemical stain of HMGN1, TLR4 and TREM-1 in the kidney tissue of UUO rats (n = 5). Original magnification = 400 ×. Scale bars. 50 um. (B) Quantification of the levels of HMGN1, TLR4 and TREM-1 proteins. all values are presented as mean ± SEM. ^∗∗p < 0.001, ^∗∗∗p < 0.0001.

Figure 5. Correlation analysis of HMGN1 protein with the key proteins involved in UUO-induced renal inflammation and fibrosis.

(A) HMGN1 protein had a positive relation with F4/80+ cells. (B) HMGN1 protein was positively associated with Mcp-1 protein. (C) HMGN1 protein was positively related to KIM-1 protein. (D) HMGN1 protein was positively associated with α-SMA protein. (E) HMGN1 protein was positively associated with β-catenin protein. (F) TLR4 protein and HMGN1 protein had a positive relation. (G) TLR4 protein was positively associated with TREM-1 protein.