Primers sequences used in this study.

The medicinal application of pomegranate peel extract enriched with polyphenols (PPE) as a therapeutic strategy for managing inflammatory bowel diseases (IBD) is still limited. Integrating pomegranate peel extract (PPE) into an effective nanocarrier system could enhance its mechanistic actions, potentially aiding in the remission of colitis. Therefore, this approach aimed to enhance PPE’s stability and bioavailability and investigate mitigating impact of pomegranate peel extract-loaded nanoparticles (PPE-NPs) in a colitis model. Colonic injury was induced by 5% dextran sulfate sodium (DSS) and efficacy of disease progression after oral administration of PPE-NPs for 14 days was assessed by evaluating clinical signs severity, antioxidant and inflammatory markers, expressions of endoplasmic reticulum associated genes and histopathological and immunostaining analysis in colonic tissues. Clinical signs and disease activity index were effectively reduced, and the levels of fecal calprotectin were decreased in groups treated with PPE-NPs compared to DSS group. The colitic group showed a significant increase (P < 0.05) in C-reactive protein (CRP) and myeloperoxidase (MPO) and nitric oxide (NO) (35.60, 163.30 and 280 nmol/g tissue respectively) and higher expression (P < 0.05) IL-17, TNF-α, and IL-1β (increased up to 2.99, 4.36 and 4.90 respectively unlike PPE-NPsIII that recorded reduced levels of CRP, MPO and NO (8,96, 78.30 and 123 nmol/g tissue respectively) and much lower (P < 0.05) levels of IL-17, TNF-α, and IL-1β expression (decreased to 1.23, 1.69 and 1.64, respectively). The most improvement of colon damage PPE-NPsIII group was also associated with the reduction MDA level (P < 0.05) (decreased to 21.60 vs 90.65 in DSS non treated group). The highest glutathione peroxidase, superoxide dismutase and catalase activities were noted in PPE-NPsIII received group (42.60, 50.30 and 62.70 U/mg). Notably, prominent free radical scavenging activities were noticed in group received 150 mg/kg of PPE-NPs as supported by higher scavenging of 1,1-diphenyl-2-picrylhydrazyl (9.85 mg/g) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid tested radicals (19.98 mg/g). Balancing between endoplasmic reticulum stressors (ERS), inflammation and autophagy was prominently noted in group treated with 150 mg/kg of PPE-NPs. These findings were supported by subsiding the excessive expression of ERS related genes (CHOP, JUNK, ATF6, BIP, and Elf-2) and immunostaining expression regulation of key markers regulating autophagy (Beclin-2) in this group. The histopathological changes in the colon were less severe in the PPE-NPs received groups (especially at the level of 150 mg/kg) compared to DSS group. Collectively, these findings suggest that the nanoencapsulation of PPE enhances its effectiveness in promoting recovery of colonic tissue damage and achieving remission of colitis.富含多酚的石榴皮提取物（pomegranate peel extract, PPE）用于炎症性肠病（inflammatory bowel diseases, IBD）的治疗策略，其临床应用仍较为有限。将石榴皮提取物（PPE）整合至高效纳米载体系统中，可增强其药理作用机制，有望辅助结肠炎的缓解。因此，本研究旨在提升PPE的稳定性与生物利用度，并探究负载石榴皮提取物的纳米颗粒（pomegranate peel extract-loaded nanoparticles, PPE-NPs）对结肠炎模型的缓解作用。本研究采用5%葡聚糖硫酸钠（dextran sulfate sodium, DSS）诱导结肠损伤，通过评估结肠组织的临床症状严重程度、抗氧化与炎症标志物水平、内质网相关基因表达，以及组织病理学与免疫染色分析，来评价连续14天口服PPE-NPs后疾病进程的改善效果。与DSS模型组相比，PPE-NPs干预组的临床症状与疾病活动指数显著降低，粪便钙卫蛋白水平也有所下降。结肠炎模型组的C反应蛋白（C-reactive protein, CRP）、髓过氧化物酶（myeloperoxidase, MPO）与一氧化氮（nitric oxide, NO）水平分别升至35.60、163.30与280 nmol/g组织，白细胞介素17（Interleukin-17, IL-17）、肿瘤坏死因子α（Tumor Necrosis Factor-α, TNF-α）及白细胞介素1β（Interleukin-1β, IL-1β）的表达量分别上调至2.99、4.36与4.90（P<0.05）；而PPE-NPsIII组的CRP、MPO与NO水平分别降至8.96、78.30与123 nmol/g组织，IL-17、TNF-α及IL-1β的表达量也显著下调至1.23、1.69与1.64（P<0.05）。PPE-NPsIII组的结肠损伤改善效果最为显著，同时伴随丙二醛（malondialdehyde, MDA）水平的显著降低（P<0.05），从未经处理的DSS组的90.65降至21.60。该组的谷胱甘肽过氧化物酶、超氧化物歧化酶与过氧化氢酶活性也达到最高，分别为42.60、50.30与62.70 U/mg。值得注意的是，150 mg/kg剂量的PPE-NPs干预组表现出显著的自由基清除活性：对1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl, DPPH）自由基的清除能力达9.85 mg/g，对2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸（2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS）自由基的清除能力达19.98 mg/g。150 mg/kg剂量的PPE-NPs干预组可有效平衡内质网应激（endoplasmic reticulum stress, ERS）、炎症与自噬过程，具体表现为下调内质网应激相关基因（CHOP、c-Jun氨基末端激酶（JNK）、ATF6、BIP与Elf-2）的过度表达，并调控自噬关键标志物Beclin-2的免疫染色表达。与DSS模型组相比，PPE-NPs干预组（尤其是150 mg/kg剂量组）的结肠组织病理学改变显著减轻。综上，本研究结果表明，对PPE进行纳米包封可增强其修复结肠组织损伤、缓解结肠炎的治疗效果。