4-羟基壬烯醛通过MAPK信号促进猪小肠上皮细胞凋亡、降低肠屏障功能的机理研究

The intestinal barrier function is critical for the growth, development and health in animals including pigs. To maintain appropriate intestinal function, the balance between cell proliferation, differentiation and apoptosis of intestinal epithelial cell, one of the predominant cell types in intestine, is critical for the intestinal structure of small intestine. As the first line of defense, intestinal epithelial cells are exposed to endogenous and exogenous stimuli such as pathogenic bacteria, toxin and antigens which might influence the proliferation or apoptosis by regulating genes involved in related signaling. 4-hydroxynonenal, an end product of lipid oxidation, has recently been reported to function as a molecular signal and is associated with multiple diseases. In our previous study, we found that the intestinal permeability and the expression of tight junction protein were lowered in weanling piglet as compared with that of suckling piglets. Also the food intakes and growth performance were also decreased. To investigate the underlying mechanisms, we found that the plasma and the intestinal 4-hydroxynonenal (4-HNE) were increased compared with that of controls. Importantly, we found that glutamine supplementation abolished weanling stress induced barrier dysfunction and tight junction protein expression alteration in in vitro and in vivo studies. Also the plasma 4-HNE were decreased accompanied the beneficial effects of glutamine. These results suggest a critical role of 4-HNE in intestinal barrier function. As 4-HNE is an end product of oxidative stress and associated with oxidative damage in multiple metabolic diseases, it is plausible to hypothesis that oxidative damage exists in intestinal environment and contributes to the barrier function dysfunction. However, as far as we know, there is very few data to support this viewpoint. The aim of this study is to test the hypothesis that 4-HNE induces apoptosis in intestinal epithelial cells and contributes to the barrier function disruption. In this study, weanling piglets or pig intestinal epithelial cells were left untreated as control or were subjected to 4-HNE, NAC or 4-HNE+NAC. Apoptosis and barrier function were determined by using Real-time PCR, Western blot analysis (especially the MAPK family proteins), ussing chamber technology, trans-epithelial electric resistance (TEER) analysis, and immunohistochemistry analysis. siRNA mediated knockdown experiments or over-expression of the critical target genes were conducted for further functional analysis to reveal the underlying molecular mechanism. Results from this study will elucidate the effect of MAPK signaling activated by 4-HNE on apoptosis in intestinal epithelial cells as well in the maintenance of integrity of barrier. Also, NAC, a well-known antioxidant will be applied to validate whether it can protect 4-HNE induced cell death and barrier function disruption in piglets and porcine intestinal epithelial cells.

小肠屏障功能对猪的生长、发育和健康有重要影响。机体内外环境中的各种应激，是影响仔猪屏障功能的重要因素。应激条件下，猪体内脂质氧化产物 4-羟基壬烯醛（4-HNE）含量明显升高，4-HNE 能否通过MAPK家族蛋白，诱导肠上皮细胞凋亡，进而造成猪肠屏障功能紊乱，目前还不清楚。本研究拟采用体内、体外试验相结合的方法，应用免疫组化、肠通透性检测、Western blot、流式细胞技术、活性氧检测、Real-time PCR、siRNA干扰、基因过表达等技术手段，研究4-HNE通过激活MAPK信号蛋白（p38MAPK、JNK、ERK1/2）诱导肠上皮细胞凋亡的机制及其与肠屏障功能的关系, 并对抗氧化剂（NAC）抑制细胞凋亡，改善肠屏障功能的可能机制进行研究。拟开展工作对阐明氧化应激引起的肠上皮细胞凋亡在肠屏障功能中的作用，以及通过应用抗氧化剂减少氧化应激对健康造成的不良影响具有重要研究意义。

小肠屏障功能对营养物质的转运吸收以及猪的生长、发育和健康有重要影响。断奶应激影响仔猪的肠屏障功能和饲料利用效率，增加了养殖成本。因此改善断奶应激对仔猪肠屏障功能的不良影响是提高饲料利用效率，降低养殖成本的重要措施。本项目通过体内试验，发现断奶仔猪空肠组织和血液中脂质过氧化产物 4-羟基壬烯醛（4-HNE）含量明显升高，肠道通透性增加，紧密连接蛋白表达下降，氧自由基含量及细胞凋亡明显增加，空肠组织中应激相关蛋白表达明显增加。体外研究发现，4-HNE能够激活应激相关信号蛋白p38MAPK、JNK以及ERK1/2磷酸化，引起肠上皮细胞的凋亡和单层细胞电阻的下降，通透性明显增加，出现Caspase-3依赖性的细胞凋亡。补充氮乙酰半胱氨酸（NAC）或谷氨酰胺后，明显降低了细胞凋亡的发生，并改善了肠上皮的通透性, 提高了肠上皮细胞中还原型谷胱苷肽（GSH）的含量，增加了氧自由基清除能力，从而抑制4-HNE引起ERK1/2磷酸化和细胞凋亡，ERK1/2抑制剂也能够缓解4-HNE诱导的细胞凋亡，提示ERK1/2是参与凋亡的关键蛋白。进一步的研究发现，氮乙酰半胱氨酸（NAC）主要通过调节丝裂原活化蛋白激酶磷酸化酶（MKP-1）来发挥其生物学功能。在揭示其凋亡机制的基础上，我们进一步研究了补充谷氨酰胺对猪空肠上皮细胞凋亡和肠屏障功能的影响。发现谷氨酰胺能增加肝脏中4-HNE的代谢，促进GSH的生成，从而降低4-HNE引起的细胞凋亡。谷氨酰胺的保护作用与其调节猪肠上皮细胞内质网蛋白质的翻译、折叠和修饰密切相关。