Endogenous tRNA‐derived small RNA (tRF3‐Thr‐AGT) inhibits ZBP1/NLRP3 pathway‐mediated cell pyroptosis to attenuate acute pancreatitis (AP)

Figures

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  FIGURE 1. Association of tRF3‐Thr‐AGT levels with AP progression. (A) The generation process of tsRNAs. (B) The tsRNA sequencing results were shown in heat map. (C) The structure and sequence information for tRF3‐Thr‐AGT. Real‐Time qPCR was performed to examine the expression levels of tRF3‐Thr‐AGT in (D) AR42J cells (each experiment repeated for three times), (E) rat pancreatic tissues (each group had 6 rats, and individual experiment was repeated for three times). *p < 0.05

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  FIGURE 2. Cell pyroptosis was closely associated with AP development. (A) MTT assay and (B) trypan blue staining assay were, respectively, performed to examine cell proliferation and viability in AR42J cells (each experiment was repeated for three times). (C) The expression levels of NLRP3, ASC and Gasdermin D were determined by Western Blot analysis (each experiment was repeated for three times). (D) The expression levels and localization of caspase‐1 in AR42J cells were determined by immunofluorescence staining assay. (E, F) The mRNA and (G, H) protein levels of IL‐1β and IL‐18 were measured in AR42J cells and its supernatants (each experiment was repeated for three times). *p < 0.05

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  FIGURE 3. Cell pyroptosis and inflammation occurred during AP pathogenesis in vivo. (A) The rat pancreatic tissues were collected, and the expression levels of NLRP3, ASC, and Gasdermin D were examined by Western Blot analysis (each experiment was repeated for three times). The levels of the pro‐inflammatory cytokines in (B–F) rat serum (each group contained 3 rats). *p < 0.05

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  FIGURE 4. Upregulation of tRF3‐Thr‐AGT attenuated STC‐induced cell death and pyroptosis in AR42J cells. (A) MTT assay and (B) trypan blue staining assay were, respectively, used to determine cell proliferation and viability (each experiment was repeated for three times). The pyroptosis associated signatures were, respectively, examined by using (C) Western Blot analysis and (D) immunofluorescence staining assay. IL‐1β and IL‐18 expressions in (E, F) AR42J cells and (G, H) supernatants were measured by Real‐Time qPCR and ELISA. *p < 0.05

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  FIGURE 5. NLRP3‐deficiency reversed STC‐induced cell pyroptosis and inflammation in AR42J cells. Knock‐down of NLRP3 rescued cell (A) proliferation and (B) viability in STC‐treated AR42J cells (each experiment repeated for three times). (C, D) The generation and (E, F) secretion of IL‐1β and IL‐18 were suppressed by NLRP3‐deficiency in STC‐treated AR42J cells and its supernatants (each experiment repeated for three times). *p < 0.05

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  FIGURE 6. The regulatory mechanisms of tRF3‐Thr‐AGT and ZBP1. (A) The potential downstream targets for tRF3‐Thr‐AGT were predicted by using the bioinformatics analysis. (B) The binding sites between tRF3‐Thr‐AGT and 3′UTR of ZBP1 mRNA were predicted, which were validated by using the following (C) dual‐luciferase reporter gene system assay and (D) RNA pull‐down assay (each experiment repeated for three times). tRF3‐Thr‐AGT negatively regulated ZBP1 at both (E) mRNA and (F) protein levels (each experiment repeated for three times). (G, H) ZBP1 was upregulated in the AP rats pancreatic tissues, in contrast with the normal rats (each group had 3 rats, and individual experiment was repeated for three times). *p < 0.05

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  FIGURE 7. Overexpression of tRF3‐Thr‐AGT suppressed STC‐induced cell pyroptosis by downregulating ZBP1. (A, B) The expression levels of NLRP3, ASC and Gasdermin D were determined by Western Blot analysis (each experiment was repeated for three times). (C) Immunofluorescence staining assay was performed to determine the expression levels and localization of caspase‐1 in AR42J cells. *p < 0.05

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  FIGURE 8. tRF3‐Thr‐AGT suppressed STC‐induced cell death and inflammation via modulating the ZBP1/NLRP3 pathway. (A, B) Cell proliferation and viability were measured by MTT assay and trypan blue staining assay (each experiment was repeated for three times). (C–F) The mRNA and protein levels of IL‐1β and IL‐18 in AT42J cells and its supernatants were measured by using Real‐Time qPCR and ELISA (each group had 6 rats, and individual experiment was repeated for three times). *p < 0.05