Mechanistic Insights into PM2.5-Induced Cardiac Fibrosis Involving ERS/TXNIP/NLRP3-Driven Pyroptosis in Multiple Cell Types

Figures

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  Figure 1. PM2.5 exposure causes damage to J774A.1 cells. J774A.1 cells were treated with different dose concentrations of PM2.5 and positive control (LPS + ATP) for 24 h. (a) Changes in cell morphology were observed in PM2.5 and positive control. Scale bar: 20 μm (white, lower right). (b) The cell viability changes were detected by MTT assay. (c) The LHD kit was used to detect LDH exposure in the supernatant of treated J774A.1 cells to assess cell viability in each group. (d) Calcein/PI staining assay was performed to detect the cell membrane integrity and metabolic activity of the cells to accurately assess the effect of PM2.5 and positive control treatments on J774A.1 cell viability. Scale bar: 100 μm (white, lower right). The data shown as mean ± SD (n = 3). ns, P ≥ .05; *P < .05.

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  Figure 2. PM2.5 exposure causes an increase in ROS levels in J774A.1 cells, resulting in oxidative stress and inflammation. J774A.1 cells were treated with different dose concentrations of PM2.5 and LPS (100 ng/ml) + ATP (3 mM) for 24 h. (a) DCFH-DA fluorescent probe assay to detect ROS levels in J774A.1 cells. Scale bar: 50 μm (white, lower right). (b) JC-1 fluorescent probe detects the level of cellular mitochondrial membrane potential, reflecting the early events of ROS action on cells. Scale bar: 50 μm (white, lower right). (c) MDA, T-SOD, and GSH-Px kits were used to detect the level of oxidative stress occurring in the cells. The Western blot analysis of (d) Sod1 and Nrf-2 in the processed groups of J774A.1 cells, along with the Western blot analysis of (e) NF-κB, IL-6, and IL-1β. The data shown as mean ± SD (n = 3). ns, P ≥ .05; *P < .05.

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  Figure 3. PM2.5 exposure causes J774A.1 cell ERS activation, activates TXNIP, and leads to pyroptosis and apoptosis. J774A.1 cells were treated with different concentrations of PM2.5 and LPS (100 ng/ml) + ATP (3 mM) for 24 h. (a) Western blot analysis of the expression levels of PERK, p-PERK, CHOP, and Bip proteins in the ERS response in J774A.1 cells and (b) the expression levels of TXNIP protein and pyroptotic markers NLRP3, Caspase-1, and GSDMD-N proteins. (c) The Annexin V/PI staining method for detecting the level of cell apoptosis. Scale bar: 100 μm (white, lower right). (d) Western blot analysis of CRT, Caspase-3, and Caspase-9 in each group of cells. The data shown as mean ± SD (n = 3). ns, P ≥ .05; *P < .05.

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  Figure 4. PM2.5 exposure causes cytokine release from J774A.1 cells to induce MCF cell activation. The supernatants from macrophages treated with varying doses of PM2.5 in combination with LPS (100 ng/mL) and ATP (3 mM) were collected, and cytokine levels were analyzed. These supernatants were subsequently used for indirect co-treatment experiments with cardiac fibroblasts. (a) Flowchart for establishing an indirect co-culture model of macrophages and cardiac fibroblasts. (b) The levels of IL-1β, IL-18, and TGF-β1 in the supernatant of macrophages. After treatment with the collected macrophage supernatant for 24 h, investigate (c) changes in the morphology of cardiac fibroblasts (CFs) (scale bar: 20 μm) and the (d) mRNA expression levels of COL-III, COL-I, E-cadherin, α-SMA, TGF-β1, and FN. (e) The expression levels of COL-III, COL-I, E-cadherin, α-SMA, and TGF-β1 proteins. Changes in (f) α-SMA and (g) E-cadherin fluorescence intensity. Scale bar: 50 μm (white, lower right). The data shown as mean ± SD (n = 3). ns, P ≥ .05; *P < .05.

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  Figure 5. PM2.5 exposure causes damage to H9c2 cells. The H9c2 cells were treated with different concentrations of PM2.5 for 24 h, and the extent of cardiomyocyte damage was assessed. (a) The morphological changes of H9c2 cells after treatment with different concentrations of PM2.5 and the control group. Scale bar: 50 μm (white, lower right). (b) The changes in LDH release levels in H9c2 cells following exposure to PM2.5. (c) The effect of different doses of PM2.5 exposure on cellular ROS levels. Scale bar: 100 μm (white, lower right). (d) The changes in the levels of MDA, T-SOD, and GSH-Px in each group of H9c2 cells after exposure to PM2.5. (e) The changes in the expression levels of Sod1, Nrf-2, NF-κB, IL-6, and IL-1β proteins in H9c2 cells after exposure to different doses of PM2.5 compared to the control group. (f) The change in mitochondrial membrane potential of H9c2 cells induced by PM2.5. Scale bar: 50 μm (white, lower right). The data shown as mean ± SD (n = 3). ns, P ≥ .05; *P < .05.

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  Figure 6. PM2.5 exposure causes H9c2 cells ERS activates, increases the expression level of TXNIP, and leads to pyroptosis and apoptosis. Treatment of H9c2 cells with different doses of PM2.5 for 24 h. (a) The expression levels of ER stress-related proteins (PERK, p-PERK, CHOP, and Bip), TXNIP, and pyroptosis-related proteins (NLRP3, Caspase-1, and GSDMD-N) in cardiomyocytes were assessed using Western blotting. (b) The Annexin V/PI double staining method was used to detect changes in the level of apoptosis in cardiomyocytes. Scale bar: 200 μm (white, lower right). Additionally, panel (c) assessed the changes in the protein expression levels of CRT, Caspase-3, and Caspase-9 in cardiomyocytes. The data shown as mean ± SD (n = 3). ns, P ≥ .05; *P < .05.

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  Figure 7. ERS and TXNIP play a key role in pyroptosis and apoptosis in J774A.1 cells induced by PM2.5 exposure, and also contribute to J774A.1-MCF crosstalk. Add the classic inhibitors of ERS and TXNIP, 4-PBA and SRI-37330, and treat macrophages for 24 h. Assess macrophage injury and changes in signaling pathways (a–e). (a) A time-flow diagram for the treatment of macrophages, cardiomyocytes, and the establishment of an indirect co-treatment model of macrophages and cardiac fibroblasts with PM2.5, 4-PBA, and SRI-37330. (b) The changes in ROS levels in macrophages after treatment. Scale bar: 50 μm (white, lower right). (c) The changes in the protein expression levels of NF-κB. (d) The expression levels of ERS-related proteins (PERK, p-PERK, CHOP, and Bip), TXNIP, and pyroptosis-related proteins (NLRP3, Caspase-1, and GSDMD-N) were analyzed for changes. (e) The changes in the expression levels of CRT, Caspase-3, and Caspase-9 proteins. The culture supernatant of macrophages exposed to PM2.5, treated with 4-PBA and SRI-37330, was collected and used to treat cardiac fibroblasts for 24 h. The role of ERS and TXNIP in the activation of cardiac fibroblasts induced by these treatments was then…

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  Figure 8. ERS and TXNIP play a key role in pyroptosis and apoptosis in H9c2 cells caused by PM2.5 exposure. The classic inhibitors of ERS and TXNIP, 4-PBA and SRI-37330, were added to H9c2 cardiomyocytes, which were treated for 24 h. The following parameters were assessed: (a) changes in ROS levels across different groups (scale bar: 50 μm), (b) alterations in NF-κB protein expression, and (c) variations in the expression levels of ERS-related proteins (PERK, p-PERK, CHOP, and Bip), TXNIP, and pyroptosis-associated proteins (NLRP3, Caspase-1, and GSDMD-N). And (d) the changes in the expression levels of CRT, Caspase-3, and Caspase-9 proteins. The data shown as mean ± SD (n = 3). ns, P ≥ .05; *P < .05.

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  Figure 9. The schematic illustration of PM2.5 induces cardiac fibrosis via ERS/TXNIP/NLRP3 pathway in cells crosstalk. PM2.5 exposure induces ERS in macrophage cell line J774A.1 and cardiomyocyte cell line H9c2, and promotes pyroptosis through the activation of TXNIP. Pyroptosis triggers the release of cytokines such as IL-18, IL-1β, and TGF-β1 by macrophages. The release of these cytokines can activate cardiac fibroblasts, leading to the upregulation of fibrosis markers such as α-SMA, COL-I, and COL-III, which indicates the differentiation of fibroblasts into myofibroblasts, thereby promoting the progression of cardiac fibrosis. PM2.5 promotes the transcriptional upregulation of Pro-Caspase-1, ASC, and NLRP3 in the nucleus through the TLR/MyD88/NF-κB pathway, as well as the expression of Pro-IL-18 and Pro-IL-1β. On the other hand, after exposure to PM2.5, the stimulation of cells induces a high-level change in ROS. Excessive ROS triggers the occurrence of ERS, leading to the dissociation of Bip from the endoplasmic reticulum, which in turn activates the phosphorylation of PERK. This activation results in the phosphorylation of eIF2α, subsequently causing an upregulation of CHOP…