Astragalus polysaccharide alleviates transport stress-induced heart injury in newly hatched chicks via ERS-UPR-autophagy dependent pathway

Abstract

Transport stress (TS) not only affects animal welfare but also eventually leads to higher morbidity and mortality. Moreover, TS could induce heart injury in animals, but the possible mechanism has yet to be fully explored. Astragalus polysaccharide (APS) is a main active component of Radix Astragali, which has an extensive anti-stress effect. However, the effect of APS on TS-induced heart injury has not yet been elucidated. In this study, a chick model of simulated TS was used. 240 newly hatched chicks were arranged into 4 groups: Control (Con), Transport group (T), Transport + water group (TW), and Transport + APS group (TA). Before transport, the chicks of the TW and TA groups were treated with deionized water and APS (0.25 mg/mL, 100 µL) by oral drops respectively. The histopathological analysis of myocardial tissue was assessed by hematoxylin and eosin staining. qRT-PCR and Western Blotting assays were employed to measure the expression of genes and proteins. Semiquantitative PCR was performed for the X box-binding protein-1 (XBP-1) mRNA splicing assay. The results indicated that APS significantly reduced TS-induced myocardial histopathological changes. Meanwhile, TS induced endoplasmic reticulum stress (ERS), evidenced by an activation of the unfolded protein response (UPR) signaling pathway and up-regulation of ERS-markers (P < 0.05). Moreover, TS markedly triggered autophagy induction by activating AMP-activated protein kinase (AMPK), reflected by augmented LC3-II/LC3-I, AMPK phosphorylation and autophagy-related genes (ATGs) expression (P < 0.05). Importantly, our study manifested that treatment of APS could reduce TS-induced ERS and AMPK-activated autophagy, accordingly alleviating heart injury of transported chicks. In summary, these findings indicate that TS induces heart injury in chicks via an ERS-UPR-autophagy-dependent pathway, and APS as an effective therapeutic method to alleviate it.

Keywords: Astragalus polysaccharide; endoplasmic reticulum stress; heart injury; newly hatched chick; transport stress.

Figure 1

Effect of TS and APS on myocardial histopathological changes in transported-chicks. (A) Histopathology with hematoxylin and eosin staining of the heart section in different groups and different time points (Con, T, TW, and TA). (B) Myocardial damage score. Slight granule denaturation (→, 1 point), vacuolar degeneration (↑, 2 points), Capillary congestion (∆, 3 points), necrosis (←, 4 points). Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01. At the same time point, the same letters mean the difference is not significant (P > 0.05), different small letters mean a significant difference (P < 0.05), and different capital letters for the extremely significant difference (P < 0.01).

Figure 2

Effect of TS and APS on the transcript levels of ERS-UPR-related genes in transported-chicks. (A) GRP78, glucose regulated protein78kD. (B) ATF6, activating transcription factor 6. (C) PERK, protein kinase RNA‑like ER kinase. (D) eIF2α, eukaryotic initiation factor 2. (E) ATF4, activating transcription factor 4. (F) IRE1, inositol-requiring enzyme 1α. (G) Heat map of relative mRNA levels of ERS-UPR-related genes. (H) PCA of ERS-UPR-related factor levels. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01. At the same time point, the same letters mean the difference is not significant (P > 0.05), different small letters mean a significant difference (P < 0.05) and different capital letters for the extremely significant difference (P < 0.01).

Figure 3

Effect of TS and APS on protein levels of ERS markers in transported-chicks. (A) The protein levels of ERS-UPR-related factors detected by Western blotting analysis; GAPDH was served as loading control for the total fractions respectively. (B) Luminance value analysis of XBP-1s/XBP-1u. (C) AFT4/GAPDH. (D) ATF6/GAPDH. (E) XBP-1s/XBP-1u. (F) Correlation analysis between ERS markers and damage score. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01. At the same time point, the same letters mean the difference is not significant (P > 0.05), different small letters mean a significant difference (P < 0.05) and different capital letters for the extremely significant difference (P < 0.01).

Figure 4

Effect of TS and APS on gene expression of AMPKs and ATGs in transported-chicks. (A) AMPK-associated subunits mRNA expression. (B) Beclin1. (C) LC3. (D) mTOR. (E) P62. (F) ATG7. (I) Heat map of relative mRNA levels ATGs. (J) PCA of the ERS-UPR-related factor levels. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01. At the same time point, the same letters mean the difference is not significant (P > 0.05), different small letters mean a significant difference (P < 0.05) and different capital letters for the extremely significant difference (P < 0.01).

Figure 5

Effect of TS and APS on protein levels of autophagy signaling pathway in transported-chicks. (A) AMPK/GAPDH. (B) p-AMPK/GAPDH. (C, D) The protein levels of autophagy-related factors determined by Western blotting analysis; GAPDH was served as loading control for the total fractions respectively. (E) Beclin1/GAPDH. (F) LC3II/I ratio. (G) Correlation analysis between autophagy-related factors and damage score. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01. At the same time point, the same letters mean the difference is not significant (P > 0.05), different small letters mean a significant difference (P < 0.05) and different capital letters for the extremely significant difference (P < 0.01).

Figure 6

Correlation analysis between ERS-UPR and autophagy signal pathway.

Figure 7

Schematic diagram illustrating the proposed mechanism of APS alleviates TS-induced heart injury mechanism in chicks via an ERS-UPR-autophagy dependent pathway.