Sirtuin4 alleviates severe acute pancreatitis by regulating HIF-1α/HO-1 mediated ferroptosis

Abstract

Acute pancreatitis (AP) is a common emergency of the digestive system and serious cases can develop into severe acute pancreatitis (SAP), which ortality rates up to 30%. Sirtuin4 (SIRT4) is a member of the sirtuin family, and plays a key role in inflammation and oxidative stress. However, the potential role of SIRT4 in SAP has yet to be elucidated. In the present study, we found that the expression level of SIRT4 in human AP was downregulated by screening a public database, suggesting that SIRT4 may play a role in AP. Subsequently, we used L-arginine (L-Arg) to induce SAP in SIRT4 knockout (SIRT4_KO) and SIRT4 overexpression (AAV_SIRT4) mice. The results showed that the pancreatic tissue injury and related lung and kidney injury were serious in SIRT4_KO mice after SAP induction, but were significantly reduced in AAV_SIRT4 mice. More importantly, we found that the levels of antioxidant factors GSH and SOD were decreased in SIRT4_KO mice, and the production of oxidative products and lipid peroxidation markers was increased, suggesting that SIRT4 was involved in inflammation and oxidative stress during SAP. Further studies showed that the absence or overexpression of SIRT4 affected the expression level of Hypoxia-inducible factor-1α (HIF-1α) after SAP induction, and regulated the expression of ferroptosis related proteins by mediating HIF-1α/HO-1 pathway. Collectively, our study revealed that SIRT4 plays a protective role in SAP by regulating the HIF-1α/HO-1 pathway to inhibit ferroptosis.

Fig. 1. SIRT4 expression was down-regulated in SAP.

A The correlation between SIRT4 and AP was discovered by bioinformatics analysis. B SIRT4 mRNA expression was downregulated in AP patients (GEO dataset GSE194331). C SIRT4 is localized in mitochondria. D Animal SAP model and experimental protocol for drug (Fer-1, PX478) administration. E Serum amylase level and lipase activity. F Typical HE staining images of pancreatic tissue. Scale bar = 100 µm. G Protein expression level of SIRT4 in the pancreas. H Viability of AR42J cells. I The protein expression level of SIRT4 in AR42J cells. Sham group (saline), SAP group (4 g/kg L-Arg), NC group (PBS), L-Arg group (10 mg/ml L-Arg). E, G used GAPDH as the reference protein. *P < 0.05, ***P < 0.001. n = 6 per group.

Fig. 2. The pivotal role of SIRT4 in SAP mice.

A Mouse tail gene identification kit was used for gene identification, M was DNA Maker, T1 was SIRT4 heterozygous (SIRT4 +/-) mice, T2 was WT mice, T3 was water, T4-T9 was SIRT4_KO mice. B Protein levels of SIRT4 in the pancreas. C–D Serum amylase level and lipase activity. E–F Typical HE staining images of pancreatic tissue. Scale bar = 100 µm. G–H Statistics of pancreatic pathological damage. WT group (wild type mice), SIRT4_ KO group (SIRT4 knockout mice), WT+Sham group (WT+saline), SIRT4_KO+ Sham group (SIRT4_KO+saline), WT + SAP group (WT + 4 g/kg L-Arg), SIRT4_KO + SAP group (SIRT4_KO + 4 g/kg L-Arg), AAV_GFP group (empty virus+saline), AAV_SIRT4 group (SIRT4 overexpression+saline), AAV_GFP + SAP group (AAV_GFP + 4 g/kg L-Arg), AAV_SIRT4 + SAP group (AAV_SIRT4 + 4 g/kg L-Arg). B used GAPDH as the reference protein. **P < 0.01, ***P < 0.001, ns. not significant. n = 6 per group.

Fig. 3. SIRT4 deficiency aggravated SAP-related lung and kidney injury in mice.

A–B Representative histological HE staining images in lung of mice. Scale bar = 100 µm. C–D Statistics showing pathological injury in the lung of mice. E–F Representative histological HE staining images in kidney of mice. Scale bar = 50 µm. G–H Statistics showing pathological injury in the kidney of mice. *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant. n = 6 per group.

Fig. 4. SIRT4 regulated inflammatory response and oxidative stress during SAP in mice.

A–B Statistics for the levels of inflammatory factors (IL-6, IL-1β, TNF-α and MCP-1). C–D Statistics for the levels of oxidative stress (GSH, H₂O₂, SOD and MDA) in the pancreas of mice. E–F Representative immunohistochemical staining of CD11b, MPO and Ly6G in the pancreas of mice. Scale bar = 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant. n = 6 per group.

Fig. 5. SIRT4 mediated oxidative stress in SAP in AR42J cells.

A Western blot showing the SIRT4 knock down efficiency. B Transfection efficiency of SIRT4 overexpressed using the Flag plasmid in AR42J cells. C Viability of AR42J cells. D–E The levels of oxidative stress (GSH, SOD and MDA) in AR42J cells. F–G Representative images showing ROS in the AR42J cells. Scale bar = 100 µm. CON group (PBS), SIRT4 KD group (SIRT4 knockdown), CON + L-Arg group (10 mg/ml L-Arg), SIRT4 KD + L-Arg group (SIRT4 KD + 10 mg/ml L-Arg), SIRT4 OE group (SIRT4 overexpression), SIRT4 OE + L-Arg group (SIRT4 OE + 10 mg/ml L-Arg). A used GAPDH as the reference protein. *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant. n = 6 per group.

Fig. 6. SIRT4 regulated ferroptosis in vivo and in vitro.

A–B Representative IHC staining of 4-HNE in the pancreas of mice. Scale bar = 100 µm. C–D Western blot of different types of ferroptosis-related proteins (ACSL4, GPX4, SLC7A11) in pancreas of mice. E–F The protein expression level of ferroptosis-related proteins (ACSL4, GPX4, SLC7A11) in AR42J cells. A–F used GAPDH as the reference protein. *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant. n = 6 per group.

Fig. 7. SIRT4 mitigated SAP by suppressing ferroptosis.

A Fer-1 decreased the serum amylase level and lipase activity of SAP mice induced by L-Arg. B Representative HE staining images of pancreas. Scale bar = 100 µm. C The levels of oxidative stress (GSH, H₂O₂, SOD, MDA) in the pancreas of mice. D Representative IHC staining of CD11b, MPO, Ly6G and 4-HNE in the pancreas of mice in different groups. Scale bar = 100 µm. E The protein expression level of ferroptosis-related proteins (ACSL4, GPX4, SLC7A11) in the pancreas of mice in different groups. WT + SAP group, SIRT4_KO + SAP group, SIRT4_KO+Fer-1 + SAP group. E used GAPDH as the reference protein. *P < 0.05, **P < 0.01, ***P < 0.001. n = 6 per group.

Fig. 8. The regulatory effect of SIRT4 on ferroptosis in SAP depends on the HIF-1α/HO-1 pathway.

A–B The protein expression levels of HIF-1α, HO-1 and NQO-1 in the pancreas of mice. C Serum amylase level and lipase activity of mice in different groups after using HIF-1α inhibitor PX478 (100 mg/kg). D Representative HE staining images of mice pancreas in different groups after using HIF-1α inhibitor PX478 (100 mg/kg). Scale bar = 100 µm. E The levels of oxidative stress factors (GSH, H₂O₂, SOD, MDA) in the pancreas of mice in different groups after using HIF-1α inhibitor PX478 (100 mg/kg). F Representative IHC staining of CD11b, MPO, Ly6G and 4-HNE in the pancreas of mice in different groups after using HIF-1α inhibitor PX478(100 mg/kg). Scale bar = 100 µm. G The protein expression level of HIF-1α, HO-1, NQO-1, ACSL, GPX4 and SLC7A11 in the pancreas of mice in different groups after using HIF-1α inhibitor PX478 (100 mg/kg). WT + SAP group, SIRT4_KO + SAP group, SIRT4_KO + PX478 + SAP group. A–G used GAPDH as the reference protein. *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant. n = 6 per group.