The Pyroptosis Level is Significantly Increased with The Severity of The Acute Lung Injury Patients.
Firstly, we sought to determine whether the plasmatic N-GSDMD and pyroptosis related inflammatory cytokines IL-18 and IL-1β levels differed between ALI groups and Control group. The plasmatic N-GSDMD(Fig. 1A), IL-18 (Fig. 1B)and IL-1β (Fig. 1C)levels were higher in the ALI (p < 0.0001) as compared to the Control group, and the levels were increased accompany with the severity of the ALI. Meanwhile, the infection cytokines IL-6 expression in plasma were augmented in the ALI groups (p < 0.0001) compared to Control group (Fig. 1D).
LPS Increases GSDMD Expression and Deficiency of Gsdmd Restores LPS-induced Inflammation in Acute Lung Injury
To determine the status of pyroptosis and effects of GSDMD on LPS-induced ALI, we established LPS-induced ALI model in mouse models. Lung tissue was histologically examined with H&E staining. Consistent with published findings, hemorrhage, pulmonary edema, alveolar wall thickening, and neutrophil infiltration were increased in the WT treated with LPS group compared with the Control group[19], and then GSDMD deficiency can attenuate LPS-induced ALI (Fig. 2A). The lung W/D weight ratio(Fig. 2B), and number of total cells, macrophages, neutrophils in BALF(Fig. 2C), as well as the expression of IL-6 and TNF-α (Fig. 2D) in plasma were significantly increased in the WT-LPS groups compared with the other groups,which was effectively decreased by Gsdmd-knockout.
Gsdmd Knockout Ameliorated Acute Lung Injury Related Intestinal Mucosal impairment
Through our research, intestinal barrier damage was observed in ALI. But it remains unclear whether GSDMD-dependent pyroptosis plays a role in ALI-induced intestinal barrier, we next investigated pyroptosis level in intestine mucosal epithelium. Intestine mucosal tissue was histologically examined with H&E staining. Intestinal damage was characterized by a reduction in villus height, villus epithelial lifting/loss, lamina propria swelling and a large amount of neutrophil infiltration into the mucosa. Intestinal mucosal impairment level was significantly increased in the WT-LPS groups compared with the other groups, which was effectively decreased by Gsdmd-knockout (Fig. 3A). Consistently, the expression of ZO-1 and Occludin, two markers of intestinal mucosal barrier, significantly increased in WT-LPS groups, compared to controls, and Gsdmd deficiency decreased the expression of ZO-1 and Occludin (Fig. 3B). We speculated intestinal and alveolar-capillary permeability by measuring DAO and D-lactic acid in serum, we found DAO and D-lactic acid expression were significantly elevated in the WT-LPS group, and attenuated by Gsdmd Knockout (Fig. 3C-D).
Gsdmd is Required for LPS-induced Pyroptosis in ALI and Related Intestinal Mucosal impairment.
To explore the effects of GSDMD on LPS-induced pyroptosis in the lung of ALI and ALI-induced intestinal mucosal dysfunction, we found that the pyroptosis inflammatory factors, IL-1β and IL-18 were increased in the serum of WT-LPS group, and Gsdmd deficiency reversed the high expression (Fig. 4A). Meanwhile, similar result was obtained using Wb in the lung tissue (Fig. 4B&C). Then, we got the similar results in intestinal epithelium, IL-18, IL-1β and N-GSDMD protein was also notably increased in the WT-LPS group and was substantially inhibited by Gsdmd Knockout (Fig. 4D).
Disulfiram Protected Mice from Acute Lung Injury Induced by LPS
To investigate the treatment effect of disulfiram on lung inflammation in ALI, we detected the severity of ALI among the Gsdmd−/− groups and WT groups treat with disulfiram. Disulfiram was administered at a dose of 25, 50, or 100mg/kg at 0h, 12h before LPS administration, mice were anesthetized at 24h after LPS challenge. The lung inflammation and cytokines of TNF-α and IL-6 levels were markedly down-regulated at dose of 50mg/kg in the WT mice.
We demonstrated that disulfiram could significantly alleviated LPS-induced lung inflammation in ALI (Fig. 5A). The lung W/D weight ratio (Fig. 5B), the number of total cells, macrophages, neutrophils in BALF(Fig. 5C), and serum TNF-α and IL-6 were reduced by disulfiram treatment (Fig. 5D). The data demonstrated that disulfiram’ treatment on ALI was effective.
Disulfiram Alleviated ALI Related Intestinal Mucosal impairment.
To determine the function of disulfiram on ALI-induced intestinal mucosal dysfunction, histological analyses showed that ALI-induced intestinal inflammation was restored by disulfiram administration compared with the control group in WT mice (Fig. 6A). Meantime, we assessed intestinal epithelial barrier permeability by examining DAO and D-lactic acid in serum, WT ALI mice exhibited higher concentrations of serum DAO and D-lactic acid than those in control mice, and disulfiram significantly decreased the expression of DAO and D-lactic acid (Fig. 6B).
Compared with controls, the expression of ZO-1 and Occludin in the intestinal mucosa of ALI mice were up-regulated, while that of ZO-1 and Occludin decreased by disulfiram (Fig. 6C). Thus, disulfiram may play an important role on the treatment of intestinal epithelial barrier dysfunction induced by ALI.
Disulfiram Reduced the Level of Pyroptosis-induced by LPS-induced ALI.
To explore the effects of disulfiram on ALI-induced pyroptosis in the lung and intestinal epithelium, we found that the pyroptosis inflammatory factors, IL-1β and IL-18 were increased in the serum of WT-LPS group, and disulfiram reversed the high expression (Fig. 7A). Similar result was obtained using Wb and immunofluorescence histochemistry in the lung tissue (Fig. 7B, C). Meanwhile, we tested the pyroptosis level of in ALI-induced intestinal epithelium. The expressions of IL-18, IL-1β and N-GSDMD in intestine mucosal epithelium tissue were significantly increased in the LPS group, which was effectively decreased by disulfiram treatment (Fig. 7D).