Inhibition of mtDNA-PRRs pathway-mediated sterile inflammation by astragalus polysaccharide protects against transport stress-induced cardiac injury in chicks

Abstract

Transport stress (TS) not only weakens poultry performance but also affects animal welfare. Additionally, TS can evoke cardiac damage by triggering sterile inflammation in chicks, but the underlying mechanism is not fully understood. Here, we aimed to elucidate how TS induces sterile inflammation and heart injury and to clarify the antagonism effect of astragalus polysaccharides (APS). We randomly divided 60 chicks (one-day-old female) into 5 groups (n = 12): Control_0h (Con_0h) group (chicks were slaughtered at initiation), Control group (stress-free control), TS group (simulated TS exposure for 8 h), TS plus water (TS+W) group, and TS plus APS (TS+APS) group. Before simulation transport, the chicks of TS+W and TS+APS groups were, respectively, dietary with 100 μL of water or APS (250 μg/mL). H&E staining was employed for cardiac histopathological observation. ELISA assay was used to measure oxidative stress marker levels (GSH, GPX, GST, and MDA). A commercial kit was used to isolate the mitochondrial portion, and qRT-PCR was employed to measure the mitochondrial DNA (mtDNA) levels. Furthermore, we evaluated the activity of mtDNA-mediated NF-κB, NLRP3 inflammasome, and cGAS-STING inflammatory pathways and the expression of downstream inflammatory factors by Western Blotting or qRT-PCR. Our findings revealed that APS notably relieved TS-induced myocardial histopathological lesions and infiltrations. Likewise, the decrease in proinflammatory factors (TNF-α, IL-1β, and IL-6) and IFN-β by APS further supported this result. Meanwhile, TS caused severe oxidative stress in the chick heart, as evidenced by decreased antioxidant enzymes and increased MDA. Importantly, APS prevented mtDNA stress and leakage by reducing oxidative stress. Interestingly, TS-induced mtDNA leakage caused a series of inflammation events via mtDNA-PRRs pathways, including TLR21-NF-κB, NLRP3 inflammasome, and cGAS-STING signaling. Encouragingly, all these adverse changes related to inflammation events induced by mtDNA-PRRs activation were all relieved by APS treatment. In summary, our findings provide the first evidence that inhibition of mtDNA-PRRs pathway-mediated sterile inflammation by APS could protect against TS-induced cardiac damage in chicks.

Keywords: NF-κB; animal welfare; cGAS-STING pathway; chick; mitochondrial stress.

Figure 1

The time flow chart for chick treatment and sample collection.

Figure 2

Astragalus polysaccharide alleviates TS-induced heart injury in chicks. (A) H&E staining of heart tissue sections. (B) Statistical analysis of the myocardial damage score. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA, followed by Turkey test. Different capital letters indicate P < 0.01, and different small letters indicate P < 0.05 and the same letters mean the difference is not significant (P > 0.05).

Figure 3

Astragalus polysaccharide improves TS-induced heart inflammation in chicks. (A–D) The mRNA levels of (A) IL-1β, (B) TNF-α, (C) IL-6, and (D) IFN-β. (E–I) The protein levels of (F) IL-1β, (G) TNF-α, (H) IL-6, and (I) IFN-β. detected by Western blotting analysis. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA, followed by Turkey test. Different capital letters indicate P < 0.01, and different small letters indicate P < 0.05 and the same letters mean the difference is not significant (P > 0.05).

Figure 4

Astragalus polysaccharide inhibits oxidative stress-mediated mtDNA leakage in the heart of chicks. (A–D) the contents of (A) GSH, (B) GPX, (C) GST, and (D) MDA. (E) The protein levels of SOD2. (F) Relative mtDNA level. (G) Relative cDNA level. (H) cDNA/ mtDNA ratio. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA, followed by Turkey test. Different capital letters indicate P < 0.01, and different small letters indicate P < 0.05 and the same letters mean the difference is not significant (P > 0.05).

Figure 5

Astragalus polysaccharide suppresses TS-activated TLR21-NF-κB pathway in the heart of chicks. (A) Heat map of relative mRNA levels TLRs. (B–D) The protein levels of (C) TLR2 and (D) TLR4. (E–G) The mRNA levels of (E) Myd88, (F) TRAF3, and (G) NF-κB. (H–M) The protein levels of (I) Myd88, (J) p-IκB-α, (K) TRAF3, (L) NF-κB, and (M) p-NF-κB. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA, followed by Turkey test. Different capital letters indicate P < 0.01, and different small letters indicate P < 0.05 and the same letters mean the difference is not significant (P > 0.05).

Figure 6

Astragalus polysaccharide restricts TS-induced NLRP3 inflammasome activation in the heart of chicks. (A, B) The mRNA levels of (A) NLRP3, and (B) Cas1. (C–E) The protein levels of (D) NLRP3, and (E) Cas1. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA, followed by Turkey test. Different capital letters indicate P < 0.01, and different small letters indicate P < 0.05 and the same letters mean the difference is not significant (P > 0.05).

Figure 7

Astragalus polysaccharide arrests TS-induced cGAS-STING pathway activation in the heart of chicks. (A–D) The mRNA levels of (A) cGAS, (B) STING, (C) TBK1, (D) IRF7. (E–L) The protein levels of (F) cGAS, (G) STING, (H) p-STING, (I) TBK1, (J) p-TBK1, (K) IRF7, and (L) p-IRF7. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA, followed by Turkey test. Different capital letters indicate P < 0.01, and different small letters indicate P < 0.05 and the same letters mean the difference is not significant (P > 0.05).

Figure 8

Correlation analysis between damage score and mtDNA-PRRs pathways. (A) PCA analysis of all results obtained in this study. (B, C) Pearson correlation coefficient scatterplot between the mtDNA and (B) myocardial damage score or (C) MDA level. (D) Correlation analysis between damage score or mtDNA and PRRs-related factors. * P < 0.05, * * P < 0.01.

Figure 9

A schematic diagram illustrating the proposed mechanism of inhibition of mtDNA-PRRs pathway-mediated sterile inflammation by APS protects against TS-induced cardiac damage in chicks.