Astragalus Polysaccharide Nanoparticles Alleviate Sepsis-Induced Myocardial Injury by Targeting the HSP90AA1/NLRP3 Signaling Pathway

Figures

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  Figure 1. Network pharmacology predicted the potential therapeutic effects of APS on sepsis-induced myocardial injury via the NOD-like receptor signaling pathway. (A) To search for the overlapped targets of APS against septic myocardial injury, the Venny figure was utilized. (B) Core target proteins from the PPI network were identified using the MCODE algorithm. (C) KEGG enrichment analysis was performed using the DAVID database to explore signaling pathways. The red box highlights the strong association between sepsis-induced myocardial injury and the NOD-like receptor signaling pathway. (D) The drug-component-target-pathwaynetwork was visualized with the Cytoscape software. The red box indicates the higher interaction with the core components of APS and HSP90AA1.

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  Figure 2. To further examine the interaction between APS and HSP90AAl using molecular docking and Microscale thermophoresis (MST). (A) Molecular docking was utilized to confirm the interaction between the HSP90AA1 and the core targets of APS polysaccharides. (B) The interaction between APS and HSP90AA1 was further verified APS had a high affinity to bind to HSP90AA1 by from MST assay.

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  Figure 3. The components of APS and characterization of APS-CS/TPP. (A) The ingredients of APS was characterized via HPLC. The A, B, and C peaks correspond to rhamnose (Rha), glucose (Glc), galactose (Gal) respectively which correspond to the key components of APS. (B) The characteristic absorption peaks of APS and APS-CS/TPP using Infrared spectroscopy showed a unique absorption peak at 618.3 cm−1 in the spectrum of the APS-CS/TPP meaning the successful preparation of the APS-CS/TPP. (C) The size distribution of the APS-CS/TPP was relatively uniform, with an average of 186.35±0.31 nm. (D) The zeta potential values of APS-CS/TPP were positively charged with a value of +47.89±0.11 mV.

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  Figure 4. Effects of APS-CS/TPP on septic myocardial injury in vivo. (A) A schematic diagram of the experimental group and protocol. There were three groups: sham (n=22), model control group (CLP group, n=30), Astragalus polysaccharides nanoparticles group (CLP+APS -CS/TPP group, n=30) which were received gavage administration of 200mg/kg APS-CS/TPP once daily for 3 consecutive days prior to establish the CLP model. (B) The 72 h survival analysis of mice induced by CLP in each group. (C) Representative echocardiography images of the short axis using an animal specific transthoracic echocardiography at 8 h post-CLP in mice. (D) Statistical graph of LVEF, LVFS, SV as well as CO.*P<0.05,**P<0.01, compared with the CLP group. (E) Representative hematoxylin and eosin (H&E) stained sections of myocardial tissue from mice 48 h after cecal ligation and puncture (CLP) surgery. The black arrows indicate infiltrating inflammatory cells in the myocardial tissue. (F) Bar graphs of the levels of cTnI, CK-MB, and LDH in each group at 48 h post-CLP in mice.*P<0.05,**P<0.01, compared with the CLP group.

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  Figure 5. APS-CS/TPP inhibited the HSP90AA1/NLRP3 signaling pathway. (A) Representative images and (B–E) quantitative analysis of Western blots of HSP90AA1, NLRP3, Caspase-1, and IL-1B.*P<0.05,**P<0.01. Values are means± SD. The results represent three independent experiments.

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  Figure 6. APS-CS/TPP suppressed inflammatory responses in LPS-induced H9c2 cells. (A) Cell viability was determined using the CCK-8 assay. (B and C) CK-MB and cTNI levels in LPS-induced H9c2 cells were measured by ELISA. (D) Morphological changes in H9c2 cardiomyocytes across groups were observed using Swiss staining. The red arrows indicate varying degrees of damage to the integrity of the cardiomyocyte cell membrane and cytoplasmic volume in each group. (E–G) The levels of TNF-α, IL-6, and IL-1β in LPS-induced H9c2 cells were determined by ELISA. The mean and standard deviation (n = 3 per group) represent the data. *P < 0.05, **P < 0.01, compared to the LPS group and the APS-CS/TPP group.

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  Figure 7. APS-CS/TPP attenuated protein expression in the HSP90AA1/NLRP3 signaling pathway in LPS-induced H9c2 cells. (A and B) Western blotting was used to visualize the successful inhibition of the HSP90AA1 activation by Tas-116 in the experimental groups. (C) Bands of NLRP3, Caspase-1, IL-1β, and β-actin were analyzed to verify whether the suppression of HSP90AA1 activation by APS-CS/TPP was dependent on the NLRP3 pathway. (D–F) The Western blot data was quantified using densitometric analysis and normalized to β-actin. *P<0.05,**P<0.01, compared to the LPS group and the APS-CS/TPP group.

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  Figure 8. APS-CS/TPP intervene in septic myocardial injury, involving the HSP90AA1/NLRP3 signaling pathway. (A) Schematic diagram of the HSP90AA1/NLRP3 signaling pathway. The purple box designates the Hsp90AA1/NLRP3 signaling pathway, and the red box indicates the inflammatory effects ultimately produced by this pathway. (B) APS-CS/TPP suppressed the release of inflammatory cytokines while reducing the protein levels of HSP90AA1, NLRP3, caspase-1, and IL-1β.