Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 3: 1513 - 1519

DOI: https://doi.org/10.12418/CJAN2023.143

RESEARCH PAPER

Dynamic Change of Apoptosis and Autophagy Signal Pathways in Intestinal Tract of Weaned Piglets after Lipopolysaccharide Challenge at Different Time Points

  • QIN Xu ,
  • ZHOU Mohan ,
  • YU Xintian ,
  • CHEN Shaokui ,
  • LIU Yulan ,
  • XIAO Kan , *
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  • Hubei Key Laboratory of Animal Nutrition and Feed Science, School of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan 430023, China
*associate professor, E-mail:

Received date: 2022-09-15

  Online published: 2023-03-16

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Abstract

The objective of this study was to investigate dynamic change of expression of genes related apoptosis and autophagy signal pathways in jejunum of weaned piglets after lipopolysaccharide (LPS) challenge at different time points. A total of 42 weaned piglets with body weight of (7.09±0.90) kg were randomly divided into 7 treatments according to different time points after LPS injection. There were 6 replicates in each treatment and 1 piglet in each replicate. After 14 days of preliminary feeding, the piglets were intraperitoneally injected of 100 μg/kg BW LPS. Piglets were slaughtered before (0 h) and 1, 2, 4, 8, 12 and 24 h after LPS injection, respectively. Jejunal samples were selected to measure the mRNA expression of apoptosis and autophagy signals. The results showed as follows: after LPS challenge, the mRNA relative expression level of Fas ligand (Fasl) significantly increased at 12 and 24 h (P<0.05). The mRNA relative expression levle of B-cell lymphoma XL proyein (Bcl-xl) significantly decreased at 2 h (P<0.05); the mRNA relative expression level of cystein-asparate protease 3 (Caspase 3) significantly increased at 2 h after LPS challenge (P<0.05); the mRNA relative expression level of Bcl-2 associated X protein (Bax) significantly increased at 12 h (P<0.01); the mRNA relative expression level of the mammalian target protein of rapamycin (mTOR) significantly decreased at 1 and 2 h (P<0.05); the mRNA relative expression level of microtubule-associated protein 1A/1B-light chain 3 (LC3) and Unc-51 kinase 1 (ULK1) significantly increased at 1 h (P<0.05); the mRNA relative expression level of autophagy-related gene 16 (Atg16) and autophagy adaptor protein 1 (SQSTM1) significantly increased at 4 h (P<0.01). In conclusion, LPS challenge activates apoptosis and autophagy signal pathways in jejunum of weaned piglets.

Key words： lipopolysaccharide; jejunum; apoptosis; autophagy; weaned piglets

Cite this article

QIN Xu , ZHOU Mohan , YU Xintian , CHEN Shaokui , LIU Yulan , XIAO Kan . Dynamic Change of Apoptosis and Autophagy Signal Pathways in Intestinal Tract of Weaned Piglets after Lipopolysaccharide Challenge at Different Time Points[J]. Chinese Journal of Animal Nutrition, 2023 , 35(3) : 1513 -1519 . DOI: 10.12418/CJAN2023.143

小肠不仅是食物消化吸收的主要部位,其完整性也是维持机体内外环境稳定的重要保障[1]。毒素、感染和应激等多种因素能够导致肠道损伤,造成肠上皮细胞的死亡[2]。脂多糖(LPS)是革兰氏阴性杆菌细胞壁的主要成分,它能引起机体肠道损伤[3]。肠道损伤常伴随细胞凋亡和自噬。凋亡是动物机体自主进行,并由基因控制的一种程序性细胞死亡方式[4]。凋亡主要由半胱天冬蛋白酶3(Caspase 3)和Bcl-2相关X蛋白(Bax)介导[5]。自噬是细胞通过降解自身衰老的细胞器及其他分子物质进行细胞自我吞噬的过程[6]。自噬过程主要由关键因子哺乳动物雷帕毒素靶蛋白(mTOR)、自噬相关基因16(Atg16)和自噬微管相关蛋白1轻链3(LC3)蛋白介导[7-8]。目前关于LPS刺激后肠道凋亡和自噬信号的变化鲜见研究报道。因此,本研究以LPS构建肠道损伤模型,探究LPS刺激后不同时间点空肠上皮细胞凋亡和自噬信号通路的动态变化,为营养素缓解仔猪肠道损伤提供研究靶点。

1 材料与方法

1.1 试验试剂

LPS:大肠杆菌血清型O55:B5(购于Sigma公司)。

1.2 试验设计

选取42头21日龄、体重为(7.09±0.90) kg的杜×长×大断奶仔猪(仔猪购自湖北奥登农牧科技有限公司),按注射LPS后时间点将仔猪随机分为7个处理,每个处理6个重复,每个重复1头猪。饲喂饲粮14 d后,腹膜皮下注射100 μg/kg体重(BW)的LPS,分别于注射前(0 h)和注射后1、2、4、8、12、24 h屠宰仔猪,取空肠黏膜样品。各处理饲喂相同的饲粮,参照NRC(1998)进行配制,其组成及营养水平见表1。
表1 饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of the diet (air-dry basis)%

项目 Items 含量 Content
原料 Ingredients
玉米 Corn 55.50
豆粕Soybean meal (CP 44%) 22.00
麦麸 Wheat bran 3.60
鱼粉 Fish meal 5.50
豆油 Soybean oil 5.00
大豆浓缩蛋白 Soy protein concentrate 2.50
代乳粉 Milk replacer 3.00
石粉 Limestone 0.70
磷酸氢钙 CaHPO4 1.00
食盐 NaCl 0.20
预混料 Premix1) 1.00
合计 Total 100.00
营养水平 Nutrient levels2)
消化能 DE/(MJ/kg) 14.00
粗蛋白质 CP 20.20
钙 Ca 0.90
总磷 TP 0.70
赖氨酸 Lys 1.35
蛋氨酸+半胱氨酸 Met+Cys 0.72
天冬氨酸+天冬酰胺 Asp+Asn 1.69

1)预混料为每千克饲粮提供The premix provided the following per kg of the diet:视黄醇乙酸酯 retinol acetate 2 700 μg,VD3 62.5 μg,DL-α-生育酚乙酸酯 DL-α-tocopheryl acetate 20 mg,VB2 4 mg,VB3 40 mg,胆酸氯化物 cholic acid chloride 400 mg,叶酸 folic acid 700 μg,VB5 15 mg,硫铵 ammonium sulfate 1.5 mg,VB12 18 μg,VB6 3 mg,VK3 3 mg,生物素 biotin 100 μg,Cu 25 mg(CuSO4·5H2O),Zn 80 mg(ZnSO4·7H2O),I 0.48 mg(KI),Mn 20 mg(MnSO4·5H2O),Fe 83 mg(FeSO4·H2O),Se 0.36 mg(Na2SeO3·5H2O)。

2)营养水平为计算值。Nutrient levels were calculated values.

1.3 样品采集及处理

将10 cm左右空肠段沿肠系膜纵向剪开,用4 ℃生理盐水轻轻冲洗肠内容物,用滤纸吸干水分,然后用载玻片轻轻刮取肠黏膜,分装并存于液氮中,最后转移至-80 ℃超低温冰箱保存待测。

1.4 mRNA相对表达量测定

1.4.1 主要试剂

RNA提取所用TRIzol试剂、反转录所用PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒以及实时荧光定量PCR所用TB GreenTM Premix Ex TaqTM Ⅱ试剂盒均由宝日医生物技术有限公司提供(TaKaRa)。

1.4.2 主要仪器

ABI 7500 Real-time PCR仪(Applied Biosystems),梯度升降温功能PCR仪(TaKaRa)。

1.4.3 RNA提取与反转录

研磨空肠样品,总RNA的提取和反转录参照陈逢[9]进行。

1.4.4 引物设计

根据已在NCBI上发表的猪的基因序列,利用Primer Premier 6.0设计实时荧光定量PCR引物,并由武汉擎科生物科技有限公司合成。引物序列见表2。
表2 引物序列

Table 2 Primer sequences

基因
Genes		 引物序列
Primer sequences (5'—3')		 登录号
Accession number		 产物大小
Product size/bp
Fas 配体
Fasl		 F:TCCTCTTCCTCTTGCCATAGA
R:CGAAGCCATTCCGAGTCTG		 BH168716.1 148
半胱天冬蛋白酶3
Caspase 3		 F:ACCCAAACTTTTCATAATTCA
R:ACCAGGTGCTGTAGAATATGC		 NM_214131.1 145
半胱天冬蛋白酶9
Caspase 9		 F:AACCAGTAGACAAGCAACAA
R:TGAATCCTCCAGAACCAATG		 XM_003127618.4 209
Bcl-2相关X蛋白
Bax		 F:CTACCAAGAAGTTGAGCGAGTG
R:CCAGTTGAAGTTGCCGTCAG		 XM_003127290.5 158
B淋巴细胞瘤XL蛋白
Bcl-xl		 F:GCAACCCATCCTGGCACCT
R:TCAAACTCATCGCCCGCCT		 XM_021077292.1 136
哺乳动物雷帕毒素靶蛋白
mTOR		 F:TGCGTCAAGAAGGAGAAGGAA
R:AGGCTGCTCGGATGATGTC		 XM_003127584.6 121
自噬微管相关蛋白1轻链3
LC3		 F:CGGAATCTCATCAGACATAGACAA
R:GTAGGATAGCGTAGGCGAATAAG		 EU979279.1 218
Unc-51激酶1
ULK1		 F:AGCATCGGCACCATCGT
R:AGCGTCTTGTTCTTCTCGTAG		 XM_021072525.1 101
自噬相关基因12
Atg12		 F:GCCTTCCTAGCGACTCTCA
R:CGGCAGGTTCTTCTGTTCC		 NM_001190282.1 231
自噬相关基因16
Atg16		 F:CGAACAGGATGATGACATTGAAG
R:GGAGCAGGAACGGAAGAGA		 XM_021074592.1 123
自噬接头蛋白1
SQSTM1		 F:GATGGCGATGTCGTATGTGAA
R:CTTGCTGTGCTCCTTGTGAA		 XM_005654936.2 244
β-肌动蛋白
β-actin		 F:TGCGGGACATCAAGGAGAAG
R:AGTTGAAGGTGGTCTCGTGG		 XM_021086047.1 216

1.4.5 实时荧光定量PCR

以空肠的cDNA为模板进行PCR扩增,采用的20 μL反应体系是由10.0 μL SYBR® Premix Ex TaqTM(2×)、0.4 μL ROX reference dye Ⅱ(10×)、2.0 μL cDNA、6.8 μL RNase free dH2O、0.4 μL上游引物(10 μmol/L)、0.4 μL下游引物(10 μmol/L)组成。使用ABI 7500 Real-time PCR仪进行扩增。循环参数:首先在95 ℃ 30 s,随后进行95 ℃ 5 s,60 ℃ 34 s,40个循环。测定凋亡和自噬相关目的基因的mRNA相对表达量。以β-肌动蛋白(β-actin)为内参,采用2-ΔΔCt法[10]计算各目的基因的mRNA相对表达量。

1.5 统计分析

用Excel 2010软件归一化处理试验数据,采用SPSS 22.0统计软件的独立样本t检验,分别对LPS注射后不同时间点与0 h进行组间比较,结果以平均值±均值标准误表示,P≤0.05为差异显著标准。

2 结果

2.1 LPS刺激后不同时间点断奶仔猪空肠凋亡相关基因的mRNA表达

如表3所示,与注射前相比,空肠中Fasl的mRNA相对表达量在LPS刺激后12和24 h均显著升高(P<0.05);Caspase 9的mRNA相对表达量在12 h有升高的趋势(P=0.071);Bcl-xl的mRNA相对表达量在2 h显著下降(P<0.05);Bax的mRNA相对表达量在12 h极显著升高(P<0.01);Caspase 3的mRNA相对表达量在2 h显著升高(P<0.05)。
表3 LPS刺激后不同时间点断奶仔猪空肠凋亡关键基因mRNA表达变化

Table 3 Change of LPS stimulation challenge on mRNA expression of key genes in apoptosis at different time points in jejunum of weaned piglets

项目
Items		 0 h 1 h 2 h 4 h 8 h 12 h 24 h P值 P-value
0 h vs
1 h		 0 h vs
2 h		 0 h vs
4 h		 0 h vs
8 h		 0 h vs
12 h		 0 h vs
24 h
Fas配体 Fasl 1.00±0.04 0.98±0.23 0.42±0.07 0.12±0.01 0.70±0.11 4.56±0.96 1.34±0.17 0.921 <0.001 <0.001 0.038 <0.001 0.019
半胱天冬蛋白3
Caspase 3		 1.00±0.09 0.95±0.16 1.45±0.20 2.72±0.54 1.24±0.27 1.55±0.32 1.00±0.11 0.805 0.074 0.002 0.335 0.073 0.978
半胱天冬蛋白9
Caspase 9		 1.00±0.11 0.50±0.03 0.12±0.01 0.23±0.03 0.85±0.14 1.32±0.12 1.03±0.21 0.001 <0.001 <0.001 0.412 0.071 0.918
Bcl-2相关X蛋白
Bax		 1.00±0.07 0.68±0.05 0.52±0.09 0.85±0.07 1.38±0.16 2.43±0.27 1.21±0.24 0.003 0.001 0.161 0.072 <0.001 0.282
B淋巴细胞瘤XL蛋白
Bcl-xl		 1.00±0.04 1.31±0.18 0.70±0.05 1.23±0.09 1.73±0.22 2.91±0.37 1.25±0.23 0.067 0.001 0.054 0.002 <0.001 0.350

2.2 LPS刺激后不同时间点断奶仔猪空肠自噬相关基因的mRNA表达

如表4所示,与注射前相比,mTOR的mRNA相对表达量在LPS刺激后1和2 h显著下降(P<0.05);Atg12的mRNA相对表达量在1和2 h显著下降(P<0.05);ULK1的mRNA相对表达量在1 h显著升高(P<0.05);LC3的mRNA相对表达量在1 h显著升高(P<0.05);Atg16和SQSTM1的mRNA相对表达量均在4 h极显著升高(P<0.01)。
表4 LPS刺激后不同时间点断奶仔猪空肠自噬关键基因mRNA表达变化

Table 4 Change of LPS stimulation challenge on mRNA expression of key genes in autophagy at different time points in jejunum of weaned piglets

项目
Items		 0 h 1 h 2 h 4 h 8 h 12 h 24 h P值 P-value
0 h vs
1 h		 0 h vs
2 h		 0 h vs
4 h		 0 h vs
8 h		 0 h vs
12 h		 0 h vs
24 h
哺乳动物雷帕毒素靶蛋白
mTOR		 1.00±0.13 0.55±0.05 0.34±0.02 1.01±0.21 1.28±0.21 1.59±0.17 0.92±0.21 0.020 0.002 0.972 0.291 0.021 0.751
自噬微管相关蛋白1轻链3
LC3		 1.00±0.26 3.08±0.59 0.02±0.01 0.45±0.09 0.64±0.29 1.47±0.61 0.93±0.43 0.015 0.010 0.120 0.379 0.439 0.887
Unc-51激酶1
ULK1		 1.00±0.05 1.49±0.18 0.48±0.10 0.36±0.06 0.23±0.04 0.24±0.02 0.70±0.08 0.007 0.002 <0.001 <0.001 <0.001 0.027
自噬相关基因12
Atg12		 1.00±0.05 0.68±0.06 0.59±0.05 1.02±0.15 0.98±0.15 1.10±0.15 0.78±0.14 0.002 <0.001 0.904 0.896 0.556 0.213
自噬相关基因16
Atg16		 1.00±0.10 0.95±0.04 1.93±0.41 3.10±0.53 2.11±0.64 1.61±0.49 0.44±0.14 0.690 0.074 <0.001 0.055 0.164 0.016
自噬接头蛋白1
SQSTM1		 1.00±0.05 1.10±0.15 0.98±0.15 1.55±0.03 0.92±0.19 0.91±0.11 0.88±0.02 0.487 0.886 <0.001 0.687 0.460 0.186

3 讨论

正常情况下,肠道可以抵御机体代谢物和有害病菌等进入体内,但在机体受到毒素等刺激时,肠道就会发生损伤。肠道损伤常伴随着细胞的凋亡和自噬。细胞中的凋亡和自噬密切相关,二者过程中的关键调节蛋白能够通过直接或间接作用达到对自噬或者凋亡的双重调节[11]。本试验采用LPS刺激仔猪,探究LPS对仔猪空肠上皮细胞凋亡和自噬信号通路的影响。
在细胞凋亡信号通路中,Bcl-2家族蛋白具有调控细胞凋亡的作用[12],其中的抗凋亡蛋白Bcl-xl能够维持线粒体膜的完整性,而促凋亡蛋白Bax在外界刺激信号作用下被激活,细胞色素C从线粒体释放到胞质,随后结合凋亡蛋白酶相关激活因子,通过活化Caspase 9前体,导致下游效应因子Caspase 3活化,Caspase 3通过切割参与细胞骨架调节蛋白等底物最终使细胞发生凋亡[13⇓-15]。胞外信号同样能够通过死亡因子Fasl介导的死亡受体通路诱导细胞发生凋亡[16]。本试验中,LPS刺激后2 h Caspase 3的mRNA相对表达量升高,Bax的mRNA相对表达量在12 h升高,Bcl-xl的mRNA相对表达量在2 h降低,这与其他研究结果[17-18]相符。宋勇等[19]研究表明,给大鼠腹腔注射LPS后肺组织细胞Fasl的mRNA和蛋白表达量均有显著的上升,这与本试验结果一致,表明LPS刺激会使仔猪空肠上皮细胞发生凋亡。
细胞自噬信号通路中,mTOR是自噬启动阶段的关键负调控因子[20],细胞发生自噬时,首先ULK1及其复合物与mTOR之间相互作用启动自噬,自噬前体形成后,Atg12和LC3等物质会被募集参与吞噬泡的延伸和扩张[21],自噬前体经过此调控过程形成自噬体然后与溶酶体结合,最后发挥降解细胞器等物质的作用,在自噬发生阶段中,SQSTM1能够将LC3标记的自噬小体与底物结合,促进自噬的发生[22⇓-24]。本试验结果表明,LPS刺激后1和2 h mTOR的mRNA相对表达量均下降,LC3和ULK1的mRNA相对表达量在1 h均升高,这与既往研究结果[25⇓-27]均一致。本试验中,LPS刺激后4 h SQSTM1的mRNA相对表达量升高,这与陈晶等[28]研究结果相符。还有研究表明,LPS诱导大鼠滑膜细胞RSC-364可使自噬蛋白Atg12升高[29],但本试验LPS刺激并没有使Atg12的mRNA相对表达量升高,这可能与其他自噬基因的显著表达激活机体其他信号通路导致的反馈调控有关。综上所述,试验结果表明LPS刺激会使仔猪空肠上皮细胞发生自噬。

4 结论

在LPS刺激断奶仔猪后,空肠上皮细胞凋亡和自噬相关因子的mRNA表达量改变,信号通路被激活,说明LPS刺激可诱导空肠上皮细胞发生凋亡和自噬。

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