Study on the mechanism of echinacoside in preventing and treating hypoxic pulmonary hypertension based on proteomic analyses

Abstract

Hypoxic pulmonary hypertension (HPH), a chronic condition affecting the cardiopulmonary system, has high mortality. Echinacoside (ECH) is a phenylethanoid glycoside, which is used to ameliorate pulmonary vascular remodeling and pulmonary vasoconstriction in rats. Accordingly, we aimed to explore the mechanism of ECH in preventing and treating HPH. Sprague Dawley rats were housed in a hypobaric hypoxia chamber for 28 days to obtain the HPH model. The experimental rats were randomly allocated into the following several groups: normoxia group, chronic hypoxia group, and ECH group. The therapeutic results of ECH (10, 20, and 40 mg/kg) showed that ECH reduced mPAP, Hb, Hct, and RVHI in HPH rats. Then this work employed label-free quantitative proteomic analysis, western blotting, and RT-PCR to investigate the mechanism by which ECH prevents HPH. The results found that in the chronic hypoxia group, the levels of ACSL1, COL6A1, COL4A2, COL1A1, and PC increased compared to the normoxia group. However, the opposite effect was observed in the chronic hypoxia group treated with ECH. The study indicates that the administration of ECH may slow the pathological progression of HPH by suppressing the inflammatory response, inhibiting smooth muscle cell proliferation, and minimizing the deposition of extracellular matrix.

Keywords: cell proliferation; echinacoside; hypoxic pulmonary hypertension; inflammation; proteomics.

FIGURE 1

Chemical structure of echinacoside.

FIGURE 2

Timeline of prevention of hypoxic pulmonary hypertension by ECH (A) and treatment of hypoxic pulmonary hypertension by ECH (B).

FIGURE 3

Venn diagram highlighting quantities of differential proteins between the chronic hypoxia group (M) and normoxia group (N) as well as between the chronic hypoxia group and ECH group (Y).

FIGURE 4

Effects of hypoxia and ECH on differential proteins expression. B, C, D, E, and F were the quantitative results of A. All data were expressed as mean ± standard deviation (SD), and each dot represents a biological replicate. For data with equal variances, the comparative analysis was conducted through one‐way ANOVA; the least significant difference (LSD) test was used for multiple comparisons of groups. For data with unequal variances, the rank sum test is used for comparative analysis; the Tamhane post‐hoc test was used for multiple comparisons of groups. *p < .05 versus Normoxia group, ^# p < .05 versus Chronic hypoxia group, n = 3 (biological replicates).

FIGURE 5

Changes of mRNA expression of differential proteins ACSL1 (A), COL6A1 (B), COL4A2 (C), COL1A1 (D), and PC (E) in three groups. All data were expressed as mean ± standard deviation (SD), and each dot represents a biological replicate. For data with equal variances, the comparative analysis was conducted through one‐way ANOVA; the least significant difference (LSD) test was used for multiple comparisons of groups. For data with unequal variances, the rank sum test is used for comparative analysis; the Tamhane post‐hoc test was used for multiple comparisons of groups. *p < .05 versus Normoxia group, ^# p < .05 versus Chronic hypoxia group, n = 3 (biological replicates).

FIGURE 6

Effect of ECH on mPAP (A), RVHI (B), Hb (C), Hct (D), and RBC (E). All data were expressed as mean ± standard deviation (SD), and each dot represents a biological replicate. For data with equal variances, the comparative analysis was conducted through one‐way ANOVA; the least significant difference (LSD) test was used for multiple comparisons of groups. For data with unequal variances, the rank sum test is used for comparative analysis; the Tamhane post‐hoc test was used for multiple comparisons of groups. *p < .05 versus normoxia, ^# p < .05 versus hypoxia,¹ p < .05 versus ECH (10 mg/kg), n = 10 (biological replicates).

FIGURE 7

Representative HE staining of the lung tissues in each group. (A) normoxia group; (B) chronic hypoxia group; and (C) ECH (40 mg/kg) group. The scale bar represents 100 μm. All data were expressed as mean ± standard deviation (SD), and each dot represents a biological replicate. For data with equal variances, the comparative analysis was conducted through one‐way ANOVA; the least significant difference (LSD) test was used for multiple comparisons of groups. For data with unequal variances, the rank sum test is used for comparative analysis; the Tamhane post‐hoc test was used for multiple comparisons of groups. *p < .05 versus normoxia group, ^# p < .05 versus chronic hypoxia group, n = 11 (biological replicates).