Background

Pancreatic inflammation plays a key role in diabetes pathogenesis and progression. Urolithin A (UA), an intestinal flora metabolite of pomegranate, has anti-diabetic, anti-inflammatory and kidney protection effects among others. However, its effects on pancreatic inflammation and the potential mechanisms have not been clearly established.

Purpose

This study aimed at investigating the molecular mechanisms of UA anti-pancreatic inflammation under a diabetic environment.

Methods

Diabetes induction in male C57BL/6 mice was achieved by a high fat diet and intraperitoneal streptozotocin injections. Then, diabetic mice were orally administered with UA for 8 weeks. In vitro, endoplasmic reticulum stress and MIN6 pancreatic β cell inflammation were induced using 25 mM glucose and 0.5 mM palmitic acid. The effects of UA were evaluated by immunohistochemistry, Western blot, and enzyme linked immunosorbent assays. Finally, the underlying mechanisms were elucidated using an autophagy inhibitor (chloroquine, CQ) and an AMPK inhibitor (dorsomorphin dihydrochloride).

Results

UA significantly inhibited IL-1β secretion and TXNIP/NLRP3 expression in the pancreas of diabetic mice and in MIN6 pancreatic cells. UA downregulated the ER stress protein, p-PERK, and promoted AMPK phosphorylation. UA activated autophagy to inhibit TXNIP/NLRP3 IL-1β inflammatory signal, an effect that was reversed by CQ. Dorsomorphin 2HCL, reversed the autophagy-activation and anti-inflammatory effects of UA. Verapamil, clinically applied as an antiarrhythmic drug, is a TXNIP inhibitor for prevention of beta cell loss and diabetes development, but limited by its cardiac toxicity. In this study, verapamil (as positive control) inhibited NLRP3 /IL-1β signaling in MIN6 cells. Inhibitory effects of UA on TXNIP and IL-1β were weaker than those of verapamil (both at 50 μM, p < 0.05, p < 0.01). Conversely, inhibitory effects of UA on p62 were stronger, relative to those of verapamil (p < 0.05), and there were no differences in AMPK activation and LC3 enhancement effects between UA and verapamil.

Conclusion

UA is a potential anti-pancreatic inflammation agent that activates AMPK and autophagy to inhibit endoplasmic reticulum stress associated TXNIP/NLRP3/IL-1β signal pathway.

中文翻译：

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尿石素 A 通过调节 AMPK 和自噬抑制胰岛 β 细胞中糖脂毒性诱导的内质网应激和 TXNIP/NLRP3/IL-1β 炎症信号

背景

胰腺炎症在糖尿病的发病机制和进展中起关键作用。尿石素 A (UA) 是石榴的一种肠道菌群代谢物，具有抗糖尿病、抗炎和肾脏保护等作用。然而，其对胰腺炎症的影响及其潜在机制尚未明确。

目的

本研究旨在探讨糖尿病环境下UA抗胰腺炎症的分子机制。

方法

雄性 C57BL/6 小鼠的糖尿病诱导是通过高脂肪饮食和腹腔内链脲佐菌素注射实现的。然后，糖尿病小鼠口服 UA 8 周。在体外，使用 25 mM 葡萄糖和 0.5 mM 棕榈酸诱导内质网应激和 MIN6 胰腺 β 细胞炎症。通过免疫组织化学、蛋白质印迹和酶联免疫吸附试验评估 UA 的作用。最后，使用自噬抑制剂（氯喹，CQ）和 AMPK 抑制剂（dorsomorphin dihydrochloride）阐明了潜在机制。

结果

UA 显着抑制糖尿病小鼠胰腺和 MIN6 胰腺细胞中 IL-1β 的分泌和 TXNIP/NLRP3 的表达。UA 下调内质网应激蛋白 p-PERK，并促进 AMPK 磷酸化。UA 激活自噬以抑制 TXNIP/NLRP3 IL-1β 炎症信号，这一作用被 CQ 逆转。Dorsomorphin 2HCL，逆转了 UA 的自噬激活和抗炎作用。临床上用作抗心律失常药物的维拉帕米是一种 TXNIP 抑制剂，用于预防 β 细胞丢失和糖尿病的发展，但受到其心脏毒性的限制。在这项研究中，维拉帕米（作为阳性对照）抑制了 MIN6 细胞中的 NLRP3 /IL-1β 信号传导。UA 对 TXNIP 和 IL-1β 的抑制作用弱于维拉帕米（均为 50 μM，p  < 0.05，p < 0.01)。相反，UA对p62的抑制作用强于维拉帕米（p  < 0.05），UA和维拉帕米的AMPK激活和LC3增强作用没有差异。

结论

UA 是一种潜在的抗胰腺炎症剂，可激活 AMPK 和自噬以抑制与 TXNIP/NLRP3/IL-1β 信号通路相关的内质网应激。