Pyroptosis is induced in radiation-induced intestinal injury
Mouse model of RIII was initially established via abdominal radiation (27Gy) (Figure 1A). Under radiation exposure, the height of the villi in the small intestine was greatly reduced and the depth of crypts were sharply decreased (Figure 1B-D). Additionally, the feces number and mass exhibited an evident downward trend, with concomitant hydration and a trend with no particles (Figure 1E-F). TUNEL assay revealed large numbers of brown positive cells in the intestinal villi and crypts, indicative of cell death (Figure 1G-H). These suggest severe intestinal injury after abdominal radiation at 27Gy.
Subsequently, the expression of pysroptosis-related proteins in intestinal tissue was detected. It was found that the expression of intestinal NLRP3, Caspase-1, Caspase-11 and GSDMD was increased after 24h of radiation exposure, with concomitant increasing cleavage of Caspase-1, Caspase-11 and GSDMD. Notably, the increase in villi was the most significant (Figure 1I). Further immunohistochemical assay for GSDMD confirmed the above findings (Figure 1J-K). Collectively the results identified the incidence of pyroptosis in the intestinal tissue following radiation, and the villous epithelium is involved predominantly.
Despite local radiation, total body radiation was performed at a dose of 12Gy (Figure S1A). The intestinal tissue was damaged remarkably, along with decreased length of villi and depth of crypts (Figure S1B-D). In the meantime, the GSDMD expression on villous epithelium was up-related as well (Figure S1E-F). All together concluded that radiation exposure could induce the incidence of pyroptosis in intestinal tissue.
GSDMD knock-out can alleviate the radiation-induced intestinal injury
It is established that GSDMD is the key executor involved in incidence of pyroptosis . To identify the association between pyroptosis and RIII, GSDMD knock-out was obtained in C57BL/6 mice and radiation was performed. As compared to wild-type, the mice of GSDMD knock-out had significantly prolonged survival after radiation (Figure 2A). Moreover, there was mitigatory intestinal injury, increasing length of villi and depth of crypt (Figure 2B-D) as well as elevating number and mass in feces (Figure 2E-G). In the TUNEL assay, radiation exposure led to reduced number of brown positive cells in mice with GSDMD deficiency (H-I). This indicated that GSDMD deficiency could decrease the radiation-induced intestinal injury, and the mediated pyroptosis is crucial in that process.
Radiation exposure induces the incidence of delayed pyroptosis and up-regulates GSDMD expression
We had proved that the intestinal villous epithelium was the predominant tissues with pyroptosis by radiation. Next, Mode-k cells, a type of intestinal epithelial cell line was used to further confirm the relationship between radiation and pyroptosis. With radiation, the cleavage of Caspase-1, Caspase-11 and GSDMD in Mode-k cells was noted (Figure 3A), which led to obvious cell swelling (Figure 3B), and subsequent increased release of LDH (Figure 3C), higher absorption of SYTOX Green (Figure 3D-E) and aggregation of GSDMD on cell membrane (Figure 3F). Concomitantly, Caspase-1, Caspase-11, GSDMD expression levels were highly up-regulated following radiation, especially the GSDMD expression (Figure 3A). This further proved that radiation could induce the pyroptosis in intestinal epithelial cells.
However, it was noting that radiation-induced pyroptosis occurred mainly after 12 h of radiation. It conventional cases, pyroptosis, such as the type induced by LPS, occurs acutely, and has a rapid onset, generally without significant up-regulation of pyroptosis-related proteins including GSDMD. To gain more insight into the phenomenon, a comparative study on pyroptosis induced by radiation and LPS+Nigericin (representing the canonical inflammasome activation) was performed. It was found that LPS+Nigericin led to significant cleavage of GSDMD within 1 h without evident increasing expression (Figure 3G-I). In addition, the release and absorption of LDH and SYTOX Green advanced prominently and acutely (Figure 3C-E), resulting in cell swelling earlier in time and more frequent (Figure 3B). Consistent finding was also noted in HIEC cells, a type of human intestinal epithelial cell lines (Figure S2).
This demonstrated that, the pyroptosis in intestinal epithelial cells induced by radiation is significantly different from the conventional type such as induced by LPS+Nigericin, which is characterized as delayed occurrence and with concomitant up-regulation of GSDMD expression.
The radiation-induced GSDMD cleavage is dependent on the activation of Caspase-1 and Caspase-11
Former result had demonstrated concurrent activation in Caspase-1 and Caspase-11 (Figure 3A). In order to investigate the effect of Caspase-1 and Caspase-11 on GSDMD cleavage, 293T cell lines was then used, because 293T cell lines poorly express Caspase-1, Caspase-11 (Caspase-4 for human derived cells) and GSDMD as compared to Mode-k cell line (Figure 4A). Next, GSDMD was over-expressed in 293T cells, followed by radiation. After 8 h, no remarkable increase of GSDMD cleavage was observed (Figure 4B), which suggested that lack of Caspase-1 and Caspase-11 cannot led to radiation-induced GSDMD cleavage. Moreover, Caspase-1 or Caspase-11 expression in Mode-k cells was silenced by using targeted siRNA (Figure 4C). Compared to radiation alone group, Caspase-1 siRNA, and Caspase-11 siRNA both decreased absorption of SYTOX Green (Figure 4D-E) and reduced release of LDH (Figure 4F-G), of which Caspase-11 knock-down had the most significant effect (Figure 4H). A conclusion could be obtained that, the radiation-induced GSDMD cleavage is dependent on both activation of Caspase-1 and Caspase-11, in which Caspase-11 is predominate.
Radiation up-regulates the expression of GSDMD by P53 transcription
As aforementioned, radiation could not only induce GSDMD cleavage but also up-regulate GSDMD expression especially after 12 h of radiation exposure. This may also a vital cause of the delayed pyroptosis. In order to clarify the upstream regulatory molecules of GSDMD, we performed a predictive analysis of transcription factors combining JASPAR and PROMO database. Simultaneously, the RNA-seq of radiated and no-radiated Mode-k cells were also conducted to screen the differentially expressed genes (DEGs). Next, the intersection between predictive results of transcription factors and DEGs was made. As a result, five molecules including AR, RELA, SP1, TP53 and TWIST1 were identified as common transcription factors predicted from both two database (Figure 5A-B), among which AR, TP53 and TWIST1 were common genes from DEGs (Figure 5B). Gene ontology (GO) analysis and KEGG pathway analysis showed that the DEGs are mostly involved in the cellular processes of programmed cell death (Figure 5C left) and KEGG pathway of NOD-like receptor signaling pathway (related to pyroptosis) and apoptosis (Figure 5C right).
Subsequently, their expressions were detected by qPCR and results validated that AR, RELA, TP53 and TWIST1 were up-regulated upon radiation, within them TP53 had the most significant fold of change (Figure 5D). TP53 is the gene of P53, which is known for its anti-tumor activity. It is also one of the most common molecules which show aberrant expression after radiation. Upon radiation, P53 can be phosphorylated in early DNA injury, which is conducive to recruiting proteins including ART to promote DNA damage repair and concurrently making cell cycle arrest to provide enough time for DNA damage repair. Following that, P53 expression increases with concomitant increasing apoptotic proteins such as Bax, eventually inducing cell apoptosis . Combing the previous findings, we reasoned that the up-regulation of P53 might be the culprit for increasing GSDMD expression and incidence of delayed pyroptosis after radiation exposure.
In order to investigate the relationship between P53 and GSDMD, PTF-α, an inhibitor to P53 transcription was used. As compared with no IR group, radiation up-regulated the expression of GSDMD and P53 in a time-dependent manner. However, PTF-α eliminated this up-regulated trend in GSDMD, indicating that P53 can regulate GSDMD expression after radiation (Figure 5E). Further, the transcriptional effect of P53 on GSDMD was validated by dual-luciferase reporter gene assay. Results demonstrated profound activation of GSDMD promotor in presence of high P53 expression (Figure 5F), which was absent upon mutations in promotor (MUT1+MUT2, H20316) (Figure 5G). Chip-qPCR assay also confirmed that P53 indeed binds GSDMD promotor (Figure 5H).
Thereafter, the role of P53 in radiation-induced pyroptosis was also investigated. With PTF-α pretreatment, the release of LDH (Figure 5I) and absorption of SYTOX Green (Figure 5J-K) were decreased obviously after radiation. However, the application of PTF-α exhibited not remarkable effect on the absorption of SYTOX Green (Figure S3A-B) and release of LDH (Figure S3C) after LPS+Nigericin treatment.
Considering that DNA damage is the main biological effect upon radiation, we then investigated if DNA damage was related to transcription of P53 on GSDMD. Accordingly, the Etoposide, an inducer of DNA damage was applied in Mode-k cells.
Comet assay indicated that Etoposide induced comet tails comparable to radiation (Figure S4A), and simultaneously up-regulated the expression of γ-H2AX proteins, marker of DNA damage (Figure S4B), indicating an evident DNA damage upon Etoposide treatment. Next, using PTF-α, the expression of γ-H2AX had not obvious change, however GSDMD up-regulation induced by Etoposide was significantly suppressed (Figure S4B-C), meanwhile the release of LDH was also reduced (Figure S4D).
Above results demonstrated that radiation-induced DNA damage up-regulates P53 which subsequently transcribes GSDMD to finally bring about pyroptosis.
Radiation-induced pyroptosis promotes apoptosis in intestinal epithelial cells
P53 is a well-known pro-apoptotic molecule that can directly transcribe apoptosis-related genes, such as Bax, as a transcription factor. On the other hand, we had proved that P53 could transcribe GSDMD and thereby induce the occurrence of pyroptosis after radiation exposure. In the following part, the association between P53-induced apoptosis and pyroptosis after radiation was investigated. At first, GSDMD was knocked out in Mode-k cells (Figure 6A-B) and was confirmed by decreasing pyroptosis as demonstrated by reductions in release of LDH (Figure 6C) and absorption of SYTOX Green (Figure 6D, F). After knocking out GSDMD, the apoptosis induced by radiation was further detected. As a result, the expression of Bax was decreased remarkably (Figure 6A-B), meanwhile flow cytometry assay also demonstrated a reduced apoptosis in Mode-k cells with GSDMD knock-out (Figure 6E-G). Both two findings indicated that the GSDMD-mediated pyroptosis facilitated cell apoptosis. Reversely, the effect of apoptosis on pyroptosis was studied. By using siRNA-Bax, the expression of Bax after radiation was decreased, while there was no notable change in GSDMD expression and cleavage (Figure 6H-J). In the flow cytometry, application of Peptide V5, a Bax inhibitor, did not show significant effect on the release of LDH induced by radiation (Figure 6K-L), suggesting that the radiation-induced apoptosis exhibited less effect on pyroptosis.
Previous research revealed that GSDMD expression on cell membrane could increase Ca2+ influx, and the Ca2+ overload was regarded as an important promoter for cell apoptosis. It triggered speculation that the GSDMD-mediated pyroptosis might promote cell apoptosis by increasing Ca2+ influx. Here, Ca2+ probe was used to evaluate intracellular Ca2+ content at different time points after radiation. At 4 h after radiation, the fluorescence intensity of Ca2+ in Mode-k cells gradually increased, which was higher and more sharply than that in cells with GSDMD knock-out (Figure 7M-O). This demonstrated that, the up-regulation and activation of GSDMD-mediated pyroptosis after radiation could accelerate cell apoptosis mainly via increasing Ca2+ influx in late stage.
Disulfiram, a GSDMD inhibitor alleviates radiation-induced intestinal injury
Since the GSDMD-mediated pyroptosis could promote the occurrence and development of radiation-induced intestinal injury, suppressing GSDMD, therefore, might be potentially protection for radiation-induced intestinal injury. Here we applied a new GSDMD inhibitor called disulfiram. It is a classical alcohol deterrent. In 2021, Jun Jacob Hu et al  firstly reported the suppressive action of disulfiram for pyroptosis, which was achieved by inhibiting membrane pore formation activated by N-GSDMD. Our study found that disulfiram could significantly block the aggregation of GSDMD on cell membrane under radiation (Figure 3F) and decrease the absorption of SYTOX Green (Figure 7A-B) as well as the release of LDH (Figure 7C), indicative of decreasing pyroptosis. In the meantime, the cell apoptosis was concurrently inhibited remarkably (Figure 7D-E), which further demonstrated the relationship between pyroptosis and apoptosis. In vivo experiment was performed to identify the role of disulfiram in radiation-induced intestinal injury. It was found that application of disulfiram prolonged the survival of mice exposed to radiation (Figure 7F), remitted intestinal injury (Figure 7G-I), and simultaneously decreasing intestinal epithelial death (Figure 7J-K). In all, disulfiram could suppress the GSDMD-mediated pyroptosis thereby decreasing radiation-induced intestinal injury. Disulfiram, therefore, is a potential new preventive agent for radiation-induced intestinal injury.