热应激对从江香猪十二指肠黏膜结构、HIF-1及其相关蛋白表达的影响

doi:10.11843/j.issn.0366-6964.2024.10.040

摘要/Abstract

摘要：

旨在研究热应激(heat stress, HS)对从江香猪十二指肠黏膜屏障结构的影响，同时跟踪检测低氧诱导因子-1(hypoxia inducible factor-1, HIF-1)的表达特征及调控机制。试验选取24头育肥期从江香猪，随机分为6组，除对照组(Con)，其余5组进行不同时间热应激处理(6、12、24、48、72 h)，试验过程中检测从江香猪直肠温度和呼吸频率，试验结束时采集各组从江香猪十二指肠。苏木精-伊红染色(HE)和阿利新蓝-过碘酸雪夫反应(AB-PAS)观察各组十二指肠组织结构和杯状细胞数量变化；TUNEL染色检测十二指肠细胞凋亡率；实时荧光定量(qRT-PCR)、蛋白免疫印迹(Western blot)和免疫组织化学分别检测紧密连接相关蛋白、HIF-1及其上游调控因子HSP90和PHD-2的表达变化。结果显示，与对照组相比，热应激可使从江香猪直肠温度和呼吸频率明显升高，肠绒毛高度下降，隐窝深度增加，绒腺比降低，闭合蛋白(Occludin)和紧密连接蛋白1(ZO-1)表达水平呈时间依赖性下降，杯状细胞数量增多。TUNEL检测结果显示，HS处理72 h后，十二指肠上皮细胞凋亡率显著上升(P < 0.001)。免疫组织化学、qRT-PCR和Western blot对HIF-1α、HSP90和PHD-2的表达进行检测，结果显示，相较于对照组，HS处理72 h后十二指肠黏膜上皮HIF-1α mRNA和蛋白表达量随HS时间延长而增加；HSP90阳性细胞在对照组和热应激组中均集中于黏膜上皮，其表达趋势与HIF-1α一致；PHD-2表达特征与HSP90相反，其表达强度在HS组中明显减弱，且随HS时间增加，PHD-2 mRNA和蛋白水平均呈时间依赖性下降。以上结果说明，HS可引起十二指肠上皮细胞凋亡，损伤十二指肠黏膜屏障；HIF-1的表达升高与十二指肠细胞凋亡密切相关，且可能同时受HSP90和PHD-2调控。

关键词: 热应激, 从江香猪, 肠黏膜屏障, 低氧诱导因子1(HIF-1), 表达调控

Abstract:

The aim of this study was to investigate the effects of heat stress (HS) on the duodenal mucosal barrier structure and the character of hypoxia inducible factor 1 (HIF-1) expression and regulation in Congjiang Xiang pigs. Twenty-four fattening pigs were divided into 6 groups randomly, the control group (Con) kept under normal conditions, and the other 5 groups were treated with heat stress for 6, 12, 24, 48, 72 h), respectively. Rectal temperature and respiratory rate were measured during treatment, and the duodenum of each group was collected at the end of the experiment. The change of duodenal tissue structure and goblet cell number in each group were observed with hematoxylin-eosin staining (HE) and alcian blue-periodate Schiff reaction (AB-PAS). The apoptosis cell of duodenal was detected by TUNEL staining. Real-time fluorescence quantification (qRT-PCR), Western blot and immunohistochemistry were used to detect the expression changes of tight junction related proteins, HIF-1 and its upstream regulatory factors respectively. The results showed that, compared with the control group, HS could induce rectal temperature and respiratory rate increase, and the villus height kept decreasing with extension of HS time, while the crypt depth increased synchronously, the ratio of villus height/gland depth and the expression of Occludin and ZO-1 decreased significantly. On the contrary, the number of goblet cells kept increasing. TUNEL showed the apoptosis rate of group HS 72 h was significantly higher than that of control group (P < 0.001).The positive expression of HIF-1α was mainly observed in small intestinal epithelial cells, and which expression enhanced at HS 72 h, the trend was as same as which HIF-1α mRNA and protein expression. The character of HSP90 expression were basically consistent with HIF-1α while the profile of PHD-2 was obviously the opposite. These results indicated that HS could induce apoptosis of small intestinal epithelial cells and damage duodenal mucosal barrier. The increase of HIF-1 expression is closely related to the apoptosis of small intestine cells, and may be regulated by both HSP90 and PHD-2.

Key words: heat stress, Congjiang Xiang pig, intestinal mucosal barrier, hypoxia inducible factor 1(HIF-1), expression regulation

中图分类号:

S828.89

刘勇庆, 张刚, 熊艳玲, 孙忠鑫, 高凡, 刘婷, 李慧. 热应激对从江香猪十二指肠黏膜结构、HIF-1及其相关蛋白表达的影响[J]. 畜牧兽医学报, 2024, 55(10): 4690-4699.

Yongqing LIU, Gang ZHANG, Yanling XIONG, Zhongxin SUN, Fan GAO, Ting LIU, Hui LI. Effects of Heat Stress on Duodenal Mucosal Structure, HIF-1 and Its Related Protein Expression in Congjiang Xiang Pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4690-4699.

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基因名称 Gene name	引物序列(5′→3′) Primer sequence(5′→3′)	产物大小/bp Product size	退火温度/℃ Annealing temperature	登录号 Accesion No.
PHD-2	F：TCATGAGCAGCATGGACGAC	117	63	XM_021073581.1
	R：CGTAACCTGTTCCGTTGCC
HIF-1α	F: TCCTTCGATCAGTTGTCACCATTGG	89	64	NM_001123124.1
	R: GGCATTGGAGTGGGCTGGAATAC
HSP90	F: CAGAGGCGGACAAGAACGACAAG	116	63	NM_213973.2
	R: GATCCTGTTGGCGTGCGTCTG
β-actin	F: TCTGGCACCACACCTTCT	114	60	XM_021086047.1
	R: TGATCTGGGTCATCTTCTCAC

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