肝脏作为脊椎动物体内重要的代谢与分泌器官，在营养代谢、物质供应、抵抗应激和解毒等生命活动中发挥着关键作用。近年研究表明，断奶过渡期是仔猪肝脏内质网应激的高发阶段。肝脏代谢旺盛，细胞内含有丰富内质网，对早期断奶引发的营养应激十分敏感，导致错误折叠与未折叠蛋白在腔内大量聚集，触发未折叠蛋白反应等应急机制。一定程度的内质网应激有助于内质网稳态的恢复，减少错误蛋白对细胞功能的损害。但应激持续存在或过于剧烈时，则会引起细胞凋亡与组织损伤。因而，维持内质网稳态是保障细胞存活的必要条件和肝脏功能正常发挥的重要基础。沉默信息调节因子（SIRT）家族是细胞内重要的去乙酰化酶，也是内质网应激的负调控蛋白，有望成为避免内质网应激持续激活的潜在干预靶点。紫檀芪是SIRT家族的一种多靶点激活剂，且具有改善细胞代谢与缓解组织损伤等作用。但紫檀芪对动物肝脏内质网应激的调节效果尚未明确，潜在的作用机理也有待揭示。为此，本试验首先在大样本量断奶仔猪上筛选紫檀芪的适宜用量；随后，采用腹腔注射衣霉素的方式建立断奶仔猪肝脏内质网应激模型，探究紫檀芪对内质网应激与肝脏损伤的保护作用，并借助修饰组学技术解析蛋白质乙酰化变化规律，明确发挥关键作用的SIRT；在此基础上，利用基因敲除技术，在模式动物上初步揭示紫檀芪改善肝脏内质网应激的潜在机制。

1  不同水平紫檀芪对断奶仔猪生长性能、肝脏损伤及内质网应激的影响

试验一旨在探究日粮添加不同水平紫檀芪对断奶仔猪生长性能、肝脏损伤及内质网应激的影响，并以经典的SIRT1天然激活剂白藜芦醇作为参照。选用300头21日龄杜×长×大三元断奶仔猪，随机分为5个处理组，每组6个重复，每重复10头仔猪。对照组饲喂基础日粮，白藜芦醇试验组饲喂含有250 mg/kg白藜芦醇的试验日粮，紫檀芪试验组分别饲喂含有100、250和500 mg/kg紫檀芪的试验日粮，试验期为21天。结果显示：添加250和500 mg/kg紫檀芪能够显著提高仔猪断奶后3周内饲料效率（P < 0.05），且250 mg/kg紫檀芪试验组平均日增重较对照组有升高趋势（P = 0.067）。添加250 mg/kg白藜芦醇部分降低了断奶仔猪肝脏细胞凋亡水平（P = 0.098），而添加250和500 mg/kg紫檀芪可显著减少断奶仔猪肝脏细胞凋亡比例（P < 0.05）。与对照组相比，白藜芦醇试验组肝脏活化转录因子6（ATF6）和葡萄糖调节蛋白78（GRP78）基因表达水平显著下调（P < 0.05）；250和500 mg/kg紫檀芪试验组肝脏蛋白激酶R样内质网激酶（PERK）、ATF6、GRP78及CCAAT增强子结合蛋白同源蛋白（CHOP）等内质网应激相关基因转录水平较对照组显著降低（P < 0.05），而SIRT1活性明显升高（P < 0.05）。此外，添加250 mg/kg紫檀芪显著提高了断奶仔猪肝脏钙联接蛋白（CANX）基因表达水平（P < 0.05）。综上，在提高断奶仔猪生长性能方面，紫檀芪的干预效果优于白藜芦醇，且紫檀芪减轻肝脏损伤及抑制肝脏内质网应激方面效果更优，其添加量以250 mg/kg为宜。

2  紫檀芪对内质网应激断奶仔猪肝脏损伤和代谢异常的影响

试验二旨在探究紫檀芪对内质网应激仔猪肝脏损伤和代谢异常的保护作用。选用54头21日龄断奶仔猪，随机分为3组，每组6重复，每重复3头仔猪。正常对照（NC）组和内质网应激（TC）组饲喂基础日粮，紫檀芪缓解（TP）组饲喂含有250 mg/kg紫檀芪的试验日粮。在仔猪第35日龄，TC和TP组腹腔注射衣霉素溶液，NC组腹腔注射等体积的无菌生理盐水，24 h后每重复中随机挑选1头仔猪进行屠宰取样。结果显示：衣霉素攻毒导致断奶仔猪体重显著下降（P < 0.05），紫檀芪具有改善内质网应激断奶仔猪体重增速放缓的趋势（P = 0.059）。与NC组相比，TC组肝脏损伤现象较为明显，表现为血浆丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）活性显著升高（P < 0.05），肝脏细胞排列紊乱、空泡化增加、线粒体嵴断裂和内质网肿胀等不良现象。相较于基础日粮，紫檀芪试验日粮可显著降低内质网应激断奶仔猪血浆ALT活性（P < 0.05），维持肝脏细胞形态结构完整性，减少线粒体嵴断裂，缓解内质网扩张现象。衣霉素攻毒导致断奶仔猪肝脏GRP78和CHOP蛋白表达以及肌醇需求酶α（IRE1α）相对磷酸化水平显著升高（P < 0.05），紫檀芪干预则可有效抑制GRP78和CHOP表达并降低IRE1α磷酸化水平（P < 0.05）。转录组分析显示，紫檀芪可通过激活过氧化物酶体增殖物激活受体（PPAR）信号途径并抑制炎症和凋亡相关通路。重要的是，紫檀芪干预主要改变了PPAR、叉头框蛋白O（FOXO）和5’-磷酸腺苷活化蛋白激酶（AMPK）通路以及过氧化物还原酶（PRDX）家族蛋白乙酰化水平，并多以去乙酰化调节作用为主，这可能解释了紫檀芪缓解内质网应激断奶仔猪肝脏损伤的潜在机理。上述结果表明，紫檀芪可能通过激活某个或某些去乙酰化酶，缓解内质网应激引起的仔猪肝脏损伤。

3  紫檀芪对SIRT3敲除内质网应激小鼠肝脏损伤的影响

试验三旨在验证紫檀芪对肝脏内质网应激及组织损伤的保护作用是否依赖于SIRT3。选取5周龄C57BL/6J野生型和SIRT3敲除型雄性小鼠各18只，随机分为野生型对照（WCS）组、野生型攻毒（WCT）组、野生型缓解（WPT）组、敲除型对照（KCS）组、敲除型攻毒（KCT）组和敲除型缓解（KPT）组。在5~8周龄阶段，WCS、WCT、KCS和KCT组饲喂基础日粮，WPT和KPT组饲喂含有400 mg/kg紫檀芪的试验日粮。饲喂结束时，WCT、WPT、KCT和KPT组腹腔注射衣霉素，WCS和KCS组腹腔注射等体积的无菌生理盐水，24 h后对小鼠进行麻醉和样本采集。结果显示：紫檀芪显著抑制衣霉素诱导的野生型小鼠血浆ALT及AST活性升高（P < 0.05），并降低肝脏细胞凋亡率（P < 0.05），改善肝脏细胞空泡化及脂质异常积聚等现象。然而，SIRT3敲除后紫檀芪的上述保护效应消失（P > 0.05）。同时，紫檀芪可显著下调野生型小鼠肝脏内质网应激标志物GRP78和CHOP的蛋白表达（P < 0.05），但该调控作用在SIRT3敲除模型中失效（P > 0.05）。此外，紫檀芪显著降低野生型小鼠肝脏PRDX3乙酰化水平（P < 0.05），而SIRT3缺失阻断了其对PRDX3乙酰化的调控能力（P > 0.05）。以上结果表明，SIRT3在紫檀芪缓解内质网应激动物肝脏损伤过程中发挥重要作用。

综上所述：在断奶仔猪日粮中添加紫檀芪具有提高生长性能、减轻肝脏损伤及抑制肝脏内质网应激的积极作用，当添加量为250 mg/kg时效果较优。紫檀芪处理能够有效缓解内质网应激断奶仔猪肝脏细胞凋亡增多、未折叠蛋白反应加剧、糖脂代谢异常等不良现象，且对肝脏蛋白质具有明显的去乙酰化调节作用。在模式动物上，敲除SIRT3会明显削弱紫檀芪对肝脏内质网应激及组织损伤的保护作用，表明SIRT3是介导紫檀芪上述积极功效的关键因子。

The liver is a crucial metabolic and secretory organ in vertebrates, playing a pivotal role in nutrient metabolism, biosynthetic supply, stress resistance, and detoxification. Recent studies have identified the early post-weaning period as a critical phase characterized by heightened endoplasmic reticulum stress susceptibility in liver. Due to its high metabolic activity and abundant endoplasmic reticulum networks, the liver is highly sensitive to nutritional stress induced by early weaning, leading to the accumulation of misfolded and unfolded proteins in the endoplasmic reticulum lumen, thereby triggering the unfolded protein response. Moderate endoplasmic reticulum stress facilitates the restoration of endoplasmic reticulum homeostasis and mitigates cellular injury caused by aberrant proteins. However, sustained or excessive endoplasmic reticulum stress can induce apoptosis and tissue injury. Therefore, maintaining endoplasmic reticulum homeostasis is essential for cell survival and proper hepatic function. The sirtuin (SIRT) family, encompassing NAD⁺-dependent deacetylases, serves as a negative regulator of endoplasmic reticulum stress and represents a potential therapeutic target to prevent sustained endoplasmic reticulum stress activation. Pterostilbene, a multi-target activator of SIRT, has been shown to improve cellular metabolism and alleviate tissue injury. Nevertheless, its regulatory effects on hepatic endoplasmic reticulum stress in animals remain unclear, and the underlying mechanisms require further elucidation. To address this, our study first screened the optimal dosage of pterostilbene in a large cohort of weaned piglets. Subsequently, we established an endoplasmic reticulum stress model in piglets via intraperitoneal tunicamycin injection to investigate the protective effects of pterostilbene against liver injury and endoplasmic reticulum stress. Using advanced proteomic techniques, we analyzed protein acetylation dynamics to identify key SIRT involved in this process. Furthermore, through gene knockout technology in model animals, we preliminarily elucidated the potential mechanisms by which pterostilbene ameliorates hepatic endoplasmic reticulum stress.

1  Effects of different levels of pterostilbene on growth performance, liver injury, and endoplasmic reticulum stress in weaned piglets

The Experiment 1 aimed to investigate the effects of dietary supplementation with different levels of pterostilbene on growth performance, liver injury, and endoplasmic reticulum stress in weaned piglets, with resveratrol, a classical natural activator of SIRT1, as the reference. A total of 300 weaned piglets (21-day-old; Duroc × Landrace × Large White) were randomly divided into 5 treatment groups, with 6 replicates per group and 10 piglets per replicate. The control group was fed a basal diet, while the resveratrol group was fed a diet supplemented with 250 mg/kg resveratrol. The pterostilbene groups were fed diets supplemented with 100, 250, and 500 mg/kg pterostilbene. The experiment lasted for 21 days. The results showed that supplementation with 250 and 500 mg/kg pterostilbene significantly improved the feed efficiency in piglets during the 3-week post-weaning period (P < 0.05), and the 250 mg/kg pterostilbene group showed a tendency to increase average daily weight gain compared with the control group (P = 0.067). Supplementation with 250 mg/kg resveratrol partially reduced liver cell apoptosis in weaned piglets (P = 0.098), whereas 250 and 500 mg/kg pterostilbene significantly reduced the levels of apoptotic liver cells (P < 0.05). Compared with the control group, the resveratrol group showed significantly lower expression levels of activating transcription factor 6 (ATF6) and glucose-regulated protein 78 (GRP78) in the liver (P < 0.05). The 250 and 500 mg/kg pterostilbene groups exhibited significantly reduced transcription levels of endoplasmic reticulum stress-related genes, including protein kinase R-like ER kinase (PERK), ATF6, GRP78, and C/EBP-homologous protein (CHOP) compared with the control group (P < 0.05), while SIRT1 activity was significantly increased (P < 0.05). Additionally, supplementation with 250 mg/kg pterostilbene significantly increased calnexin expression levels in the liver (P < 0.05). In conclusion, pterostilbene was more effective than resveratrol in improving growth performance, alleviating liver injury, and regulating endoplasmic reticulum stress in weaned piglets, with an optimal level of 250 mg/kg.

2  Effects of pterostilbene on endoplasmic reticulum stress-induced liver injury and metabolic disorders in weaned piglets

The Experiment 2 aimed to investigate the protective effects of pterostilbene on liver injury and metabolic disorders induced by endoplasmic reticulum stress in piglets. A total of 54 weaned piglets (21-day-old) were randomly divided into 3 groups, with 6 replicates per group and 3 piglets per replicate. The normal control (NC) group and the endoplasmic reticulum stress (TC) group were fed a basal diet, while the pterostilbene-supplemented (TP) group received a diet containing 250 mg/kg pterostilbene. On day 35, the TC and TP groups were intraperitoneally injected with tunicamycin solution, while the NC group was injected with an equal volume of sterile saline. Twenty-four hours later, one piglet per replicate was randomly selected for slaughter and sample collection. The results showed that tunicamycin challenge significantly decreased body weight of the weaned piglets (P < 0.05), while pterostilbene intervention tended to mitigate the weight loss caused by tunicamycin exposure (P = 0.059). Compared to the NC group, the TC group exhibited more severe liver injury, characterized by significantly increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities (P < 0.05), liver cell disorganization, increased vacuolization, mitochondrial cristae rupture, and endoplasmic reticulum swelling. Compared to the basic diet, the pterostilbene-supplemented diet significantly reduced plasma ALT activity in the endoplasmic reticulum stress-induced weaned piglets (P < 0.05), protected the liver cell morphology, reduced mitochondrial cristae rupture, and alleviated endoplasmic reticulum swelling. Tunicamycin challenge significantly increased the protein expression of GRP78 and CHOP, as well as the relative phosphorylation level of inositol-requiring enzyme 1 alpha (IRE1α) (P < 0.05), whereas pterostilbene intervention effectively suppressed GRP78 and CHOP expression and reduced IRE1α phosphorylation (P < 0.05). Transcriptomic analysis indicated that pterostilbene activated the peroxisome proliferator-activated receptor (PPAR) signaling pathway and inhibited inflammation-related and apoptosis-related pathways. Importantly, pterostilbene intervention primarily altered the PPAR, forkhead box protein O (FOXO), and adenosine monophosphate-activated protein kinase (AMPK) pathways, as well as the acetylation levels of peroxiredoxin (PRDX) family proteins, predominantly through deacetylation regulation. This may explain the potential mechanism by which pterostilbene alleviates liver injury induced by endoplasmic reticulum stress in weaned piglets. In conclusion, these results suggest that pterostilbene may alleviate liver injury in weaned piglets caused by endoplasmic reticulum stress through the activation of one or more SIRT.

3  Effects of pterostilbene on hepatic injury in SIRT3 knockout mice under endoplasmic reticulum stress.

The Experiment 3 aimed to investigate whether the protective effects of pterostilbene on hepatic endoplasmic reticulum stress and tissue injury are dependent on SIRT3. A total of 18 five-week-old male C57BL/6J wild-type mice and 18 SIRT3 knockout mice were randomly divided into six experimental groups: wild-type control (WCS), wild-type tunicamycin-challenged (WCT), wild-type pterostilbene-treated (WPT), knockout control (KCS), knockout tunicamycin-challenged (KCT), and knockout pterostilbene-treated (KPT). From weeks 5 to 8, the WCS, WCT, KCS, and KCT groups were fed a basal diet, whereas the WPT and KPT groups received a diet supplemented with 400 mg/kg pterostilbene. At the end of the feeding period, the WCT, WPT, KCT, and KPT groups were intraperitoneally injected with tunicamycin, while the WCS and KCS groups received an equal volume of sterile saline. Twenty-four hours later, mice were anesthetized for sample collection. The results showed that pterostilbene significantly inhibited tunicamycin-induced increases in plasma ALT and AST activities (P < 0.05) and reduced hepatic apoptosis (P < 0.05) in wild-type mice, concomitant with ameliorated hepatocyte vacuolation and abnormal lipid accumulation. However, these protective effects were abolished in SIRT3 knockout mice (P > 0.05). Furthermore, pterostilbene markedly downregulated the protein expression of endoplasmic reticulum stress markers (GRP78 and CHOP) in wild-type mice (P < 0.05), but these beneficial effects were not observed in SIRT3 knockout mice (P > 0.05). Additionally, pterostilbene markedly reduced hepatic PRDX3 acetylation levels in wild-type mice (P < 0.05), while SIRT3 knockout blocked the regulatory effect of pterostilbene on PRDX3 acetylation (P > 0.05). These findings highlight the critical role of SIRT3 in mediating the protective effects of pterostilbene against endoplasmic reticulum stress-induced liver injury.

Summary of findings: Dietary supplementation of pterostilbene in weaned piglets significantly improved growth performance, alleviated liver injury, and suppressed hepatic endoplasmic reticulum stress. The optimal dosage for these beneficial effects was determined to be 250 mg/kg. Pterostilbene treatment effectively mitigated endoplasmic reticulum stress-induced adverse effects, including increased hepatocyte apoptosis, enhanced unfolded protein response, abnormal glucose and lipid metabolism. Additionally, it significantly regulated the deacetylation of hepatic proteins. In the murine model, knockout of SIRT3 significantly attenuated the protective effects of pterostilbene against endoplasmic reticulum stress and liver injury, confirming that SIRT3 is the key mediator of the beneficial effects of pterostilbene.