Echinacoside inhibited cardiomyocyte pyroptosis and improved heart function of HF rats induced by isoproterenol via suppressing NADPH/ROS/ER stress

Abstract

Prevalence of heart failure (HF) continues to rise over time and is a global difficult problem; new drug targets are urgently needed. In recent years, pyroptosis is confirmed to promote cardiac remodelling and HF. Echinacoside (ECH) is a natural phenylethanoid glycoside and is the major active component of traditional Chinese medicine Cistanches Herba, which is reported to possess powerful anti-oxidation and anti-inflammatory effects. In addition, we previously reported that ECH reversed cardiac remodelling and improved heart function, but the effect of ECH on pyroptosis has not been studied. So, we investigated the effects of ECH on cardiomyocyte pyroptosis and the underlying mechanisms. In vivo, we established HF rat models induced by isoproterenol (ISO) and pre-treated with ECH. Indexes of heart function, pyroptotic marker proteins, ROS levels, and the expressions of NOX2, NOX4 and ER stress were measured. In vitro, primary cardiomyocytes of neonatal rats were treated with ISO and ECH; ASC speckles and caspase-1 mediated pyroptosis in cardiomyocytes were detected. Hoechst/PI staining was also used to evaluate pyroptosis. ROS levels, pyroptotic marker proteins, NOX2, NOX4 and ER stress levels were all tested. In vivo, we found that ECH effectively inhibited pyroptosis, down-regulated NOX2 and NOX4, decreased ROS levels, suppressed ER stress and improved heart function. In vitro, ECH reduced cardiomyocyte pyroptosis and suppressed NADPH/ROS/ER stress. We concluded that ECH inhibited cardiomyocyte pyroptosis and improved heart function via suppressing NADPH/ROS/ER stress.

Keywords: echinacoside; endoplasmic reticulum stress; heart failure; heart function; pyroptosis.

FIGURE 1

ECH effectively inhibited cardiomyocyte pyroptosis and improved heart function of HF rats induced by ISO. (A) Representative echocardiographic image of Ctrl (n = 6), ISO (n = 6) and ECH (n = 6) treated rat. (B–E) ECH effectively decreased LVIDd, LVIDs and increased LVEF, LVFS. (F) There is no significant difference in HR among three groups. (G) Representative bands of Western blot of cleaved caspase‐1, caspase‐1, NLRP3, ASC, GSDMD‐N, IL‐1β and IL‐18. (H–L) Statistical histogram of relative protein expression/GAPDH and the ratio of cleaved caspase‐1/caspase‐1. All *p < 0.05 versus Ctrl, **p < 0.01 versus Ctrl; #p < 0.05 versus ISO, ##p < 0.01 versus ISO. Error bars represent SD.

FIGURE 2

ECH significantly suppressed NOX2, NOX4, p‐SAPK/JNK, ROS and ER stress in left ventricular tissue of HF rats induced by ISO. (A, B) Representative immunohistochemical image of NOX2 and NOX4 in Ctrl, ISO and ECH‐treated rats. (C, D) Statistical histogram of mean optical density (OD) of NOX2 and NOX4 in rats. (E) ROS level in left ventricular tissue of Ctrl, ISO and ECH‐treated rats. (F) Representative bands of Western blot of ATF6, IRE1α, p‐IRE1α, PERK and p‐PERK in Ctrl, ISO and ECH‐treated rats. (G, H) Representative immunohistochemical image of GRP78 and CHOP in Ctrl, ISO and ECH‐treated rats. (I, J) Statistical histogram of OD of GRP78 and CHOP. (K–Q) Statistical histogram of relative ATF6, p‐IRE1α, p‐PERK expression/GAPDH and the ratios of p‐IRE1α/IRE1α, p‐PERK/PERK. (R) Representative bands of Western blot of p‐SAPK/JNK and statistical histogram. All *p < 0.05 versus Ctrl, **p < 0.01 versus Ctrl; #p < 0.05 versus ISO, ##p < 0.01 versus ISO. Error bars represent SD.

FIGURE 3

ECH reduced caspase‐1 mediated pyroptosis and ASC speckles in primary cardiomyocytes of neonatal rat induced by ISO. (A) Representative flow cytometry image of caspase‐1 mediated pyroptosis in Ctrl, ISO and ECH‐treated cardiomyocytes. (B) Representative immunofluorescence staining image of ASC in Ctrl, ISO and ECH‐treated cardiomyocytes. (C) Statistical histogram of cardiomyocyte pyroptotic rate mediated by caspase‐1. (D) Statistical histogram of ASC speckles positive rate of cardiomyocytes. All *p < 0.05 versus Ctrl, **p < 0.01 versus Ctrl; #p < 0.05 versus ISO, ##p < 0.01 versus ISO. Error bars represent SD.

FIGURE 4

ECH reduced cardiomyocyte pyroptosis in primary cardiomyocytes of neonatal rat induced by ISO. (A) Representative Hoechst/PI staining image of cardiomyocytes treated with Ctrl, ISO, ECH. (B) Statistical histogram of PI‐positive rate of cardiomyocytes. (C) Representative bands of Western blot of cleaved caspase‐1, caspase‐1, NLRP3, ASC, GSDMD‐N, IL‐1β and IL‐18 in cardiomyocytes treated with Ctrl, ISO, ECH. (D–H) Statistical histogram of relative cleaved caspase‐1, caspase‐1, NLRP3, ASC, GSDMD‐N, IL‐1β, IL‐18 expression/GAPDH and the ratio of cleaved caspase‐1/caspase‐1. All *p < 0.05 versus Ctrl, **p < 0.01 versus Ctrl; #p < 0.05 versus ISO, ##p < 0.01 versus ISO. Error bars represent SD.

FIGURE 5

ECH down‐regulated p‐SAPK/JNK, NOX2, NOX4, ROS and ER stress in primary cardiomyocytes of neonatal rat induced by ISO. (A) Representative immunohistochemical image of NOX2, NOX4 and pSAPK/JNK in Ctrl, ISO and ECH‐treated cardiomyocytes. (B, C) Statistical histogram of NOX2, NOX4 and pSAPK/JNK relative expression/GAPDH. (D) Representative flow cytometry image of ROS level. (E) Intracellular ROS level in Ctrl, ISO and ECH‐treated cardiomyocytes. (F) Representative bands of Western blot of GRP78, ATF6, IRE1α, p‐IRE1α, PERK, p‐PERK and CHOP in Ctrl, ISO and ECH‐treated cardiomyocytes. (G–N) Statistical histogram of relative GRP78, ATF6, p‐IRE1α, p‐PERK, CHOP expression/GAPDH and the ratios of pIRE1α/ IRE1α, p‐PERK/PERK. All *p < 0.05 versus Ctrl, **p < 0.01 versus Ctrl; #p < 0.05 versus ISO, ##p < 0.01 versus ISO. Error bars represent SD.