In our preliminary study, we found that SiO₂ induced ER stress in A549 cells [7] and MLE-12 cells [8]. As a follow-up, this study was aimed at further investigating whether ER stress was also manifested in silica-induced macrophages. As shown in Figure 1A, the fluorescence intensity of phospho-PKR-like ER kinase (p-PERK) was significantly enhanced in a dose-dependent manner by supplementation of the macrophages with 100, 200, and 250 µg/mL SiO₂. Furthermore, exposure of the macrophages to SiO₂ induced an increase in the expression of phospho-inositol-requiring enzyme 1α (p-IRE-1α) (Figure 1B). We also found that SiO₂ significantly increased intracellular ROS production (Figure 1C). Western blotting analysis indicated that the expressions of p-PERK, p-IRE-1α, and phospho-eukaryotic initiation factor 2 alpha (p-eIF-2α) were upregulated in a dose-dependent manner by supplementation of the macrophages with 100, 200, and 250 µg/mL SiO₂ (Figure 1D). These results suggested that silica induced ER stress in macrophages.

Since glycolytic reprogramming and ER stress are critical for the functional and phenotypic transformation of macrophages, we further investigated whether the targeted inhibition of LDHA had an effect on the regulation of glycolysis and ER stress. Oxamate alleviated the silica-induced increase in the expression of p-PERK and p-IRE-1α as well as the increase in ROS production (Figure 2A–C). Similarly, oxamate treatment reduced the extent of ER stress by inhibiting the silica-induced upregulation of p-PERK and p-IRE-1α, but not by inhibiting p-eIF-2α (Figure 2D). To investigate the effect of oxamate on metabolic changes in macrophages, we assessed the changes in the extracellular acidification rate (ECAR), lactate concentration, and the mitochondrial oxygen consumption rate (OCR) after treatment of the silicotic macrophages with oxamate. After exposure of the macrophages to silica, we detected increases in both the ECAR and lactate concentration but a decrease in the OCR, indicating the transition from oxidative phosphorylation to aerobic glycolysis. However, these effects were reversed upon treatment of the silica-exposed macrophages with oxamate (Figure 3).

To determine the effect of lactate on the expression of ER stress-related factors in NR8383 cells, we stimulated NR8383 cells with different concentrations of lactate. As shown in Figure 4A,B, the expression of p-PERK and p-IRE-1α gradually increased with the increase in the lactate concentration. It was previously reported that exogenous lactate stimulation resulted in an increase in intercellular ROS production in skin fibroblasts [19]. Our study corroborated this finding, as lactate was also found to stimulate the production of ROS in NR8383 cells (Figure 4C). Supplementation of exogenous lactate triggered an increase in the expression of p-PERK, p-IRE-1α, and p-eIF-2α in NR8383 cells in a dose-dependent manner (Figure 4D). Taken together, these results indicated that lactate played a key role in the activation of ER stress in macrophages.

Next, we used oxamate to inhibit LDH and prevent the conversion of pyruvate to lactate, reducing the production of lactate. We found that treatment with oxamate inhibited the enhanced expression of p-PERK and p-IRE-1α and ROS production induced by exogenous lactate (Figure 5A–C). In addition, treatment of the silica-exposed macrophages with oxamate inhibited the activation of ER stress by targeting p-PERK and p-IRE-1α rather than p-eIF-2α (Figure 5D). Next, we sought to verify whether oxamate regulated lactate-induced metabolic changes. Treatment of the macrophages with oxamate resulted in lower levels of ECAR, a reduced production of lactate, and higher levels of OCR due to the inhibition of LDHA (Figure 6). These findings indicated that oxamate alleviated the lactate-induced increase in glycolytic metabolism and ER stress by inhibiting LDHA.

Since we observed that oxamate inhibited glycolysis and ameliorated ER stress in macrophages, we considered whether oxamate could mitigate fibrotic responses to silica in vivo. To test this hypothesis, we administered different doses of oxamate to mice exposed to silica. Treatment of the silicotic mice with 100 mg/kg or 500 mg/kg oxamate per day for 4 weeks led to a significant reduction in the expression of LDHA and attenuation of fibrotic remodeling in the silica-exposed lungs of the mice, as assessed by HE. and Sirius Red histological staining of the lung tissue (Figure 7A–C). Consistent with our in vitro observations, treatment of the mice exposed to silica with 100 mg/kg or 500 mg/kg oxamate led to the reduced expression of p-PERK and p-IRE-1α but had no effect on p-eIF-2α; in addition, the effects of the 500 mg/kg oxamate dosage on the expression of the two proteins were more significant than the 100 mg/kg dosage (Figure 7D–F). Taken together, these data supported that oxamate played an anti-fibrosis role in vivo by inhibiting glycolysis and ER stress in macrophages.