Chinese Journal of Animal Nutrition >

2026 , Vol. 38 >Issue 6: 4280 - 4290

DOI: https://doi.org/10.12418/CJAN2026.343

RESEARCH PAPER

Effects of Chitosan Oligosaccharide on Hepatic Function, Endoplasmic Reticulum Stress, Mitochondrial Mass and NOD-Like Receptor Protein 3 Inflammasome Activation in Heat-Stressed Broilers

  • WANG Haoxuan ,
  • GAO Jinren ,
  • ZHANG Jia ,
  • LAN Ruixia , *
Expand
  • College of Coastal Agriculture Sciences, Guangdong Ocean University, Zhanjiang 524088, China
* lecturer, E-mail:

Received date: 2025-11-25

  Online published: 2026-06-13

Fold

Abstract

The aim of this study was to investigate the effects of chitosan oligosaccharide (COS) on hepatic function, endoplasmic reticulum stress, mitochondrial mass and NOD-like receptor protein 3 (NLRP3) inflammasome activation in heat-stressed broilers. A total of one hundred of forty-four 28-day-old male Arbor Acres (AA) broilers with similar body weight [(1 469.07±117.26) g] were randomly divided into 3 groups, with 6 replicates per group and 8 broilers per replicate. Broilers in control (CON) group were housed in a room maintained at (24±1) ℃. Broilers in the heat stress (HS) group and COS group were housed in a room maintained at (34±1) ℃ from 08:00 to 18:00, while the temperature during the remaining time was maintained at (24±1) ℃. Broilers in the CON and HS groups were fed the basal diet, and those in the COS group were fed the basal diet supplemented with 200 mg/kg COS. The experiment lasted for 14 d. The results showed as follows: 1) compared with the CON and COS groups, hepatic alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutaminase (GLS) and glutamine synthase (GS) activities of the HS group were significantly increased (P<0.05). 2) Compared with the CON and COS groups, mRNA relative expression levels of hepatic progesterone X receptor (PXR), cytochrome P450 family 1 subfamily A member 2 (CYP1A2), cytochrome P450 family 2 subfamily D member 6 (CYP2D6) and cytochrome P450 family 3 subfamily A member 4 (CYP3A4) of the HS group were significantly up-regulated (P<0.05). 3) Compared with the CON and COS groups, mRNA relative expression levels of hepatic glucose regulated protein 78 (GRP78), protein kinase RNA-like ER kinase (PERK), X-box binding protein 1 (XBP1) and transcriptional factor C/EBP homologous protein (CHOP) of the HS group were significantly up-regulated (P<0.05). 4) Compared with the CON and COS groups, mRNA relative expression levels of hepatic peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) of the HS group were significantly down-regulated (P<0.05), while mRNA relative expression levels of mitochondrial dynamin-related protein 1 (DRP1), PTEN-induced kinase1 (PINK1) and E3 ubiquitin protein ligase (Parkin) of the HS group were significantly up-regulated (P<0.05). Compared with the CON group, mRNA relative expression level of hepatic selective autophagy adaptor protein p62 (p62) of the HS and COS group was significantly up-regulated (P<0.05). 5) Compared with the CON and COS groups, mRNA relative expression levels of hepatic NLRP3, cysteinyl aspartate specific proteinase 1 (Caspase 1) and interleukin-18 (IL-18) of the HS group were significantly up-regulated (P<0.05). In conclusion, dietary supplementation with 200 mg/kg COS can promote hepatic mitochondrial biogenesis in heat-stressed broilers, followed by inhibiting mitochondrial division, alleviating mitophagy, endoplasmic reticulum stress and inflammatory response, and finally alleviating hepatic damage and improving ammonia detoxification capacity in heat-stressed broilers.

Key words： chitosan oligosaccharide; heat-stressed broilers; hepatic function; endoplasmic reticulum stress; mitochondrial mass

Cite this article

WANG Haoxuan , GAO Jinren , ZHANG Jia , LAN Ruixia . Effects of Chitosan Oligosaccharide on Hepatic Function, Endoplasmic Reticulum Stress, Mitochondrial Mass and NOD-Like Receptor Protein 3 Inflammasome Activation in Heat-Stressed Broilers[J]. Chinese Journal of Animal Nutrition, 2026 , 38(6) : 4280 -4290 . DOI: 10.12418/CJAN2026.343

热应激会引发肉鸡代谢紊乱、组织器官氧化损伤及炎症反应,进而导致其生产性能下降[1]。肝脏是热应激的靶器官,也是机体能量代谢的枢纽器官,在糖类、脂质和蛋白质的代谢平衡及氨的解毒作用中有重要作用[2]。肝脏能量代谢异常,表现为肉鸡骨骼肌蛋白沉积下降,肝脏和腹部脂肪过度沉积,日增重下降[3-4]。本课题组前期研究表明,热应激诱导的氧化损伤和炎症反应导致肉鸡肝脏细胞损伤和凋亡,肝脏组织形态学结果显示有一定出血、胞浆疏松化、透明、空泡化严重且部分细胞有炎性浸润[5]。此外,TUNEL法检测肝细胞凋亡表明,热应激提高肉鸡肝细胞凋亡率,且伴随提高凋亡相关基因半胱天冬蛋白酶9(Caspase 9)和B细胞淋巴瘤-2相关X蛋白(B-cell lymphoma-2-associated X protein,Bax)mRNA相对表达量[5]。因此,缓解热应激肉鸡肝脏损伤的关键在于提高其抗氧化和抗炎能力。热应激诱导的肝脏损伤复杂多样,内质网和线粒体是热应激的靶细胞器,热应激引起的线粒体功能紊乱和内质网应激是导致肝脏损伤的关键因素,同时还会加剧氧化损伤和炎症反应[6-7]。线粒体是细胞的能量代谢中心,线粒体的质量控制,包括生物发生、分裂、融合及自噬的动态平衡,最终影响线粒体的形态、功能、活性氧(ROS)产生和细胞凋亡[8]。内质网对蛋白质的折叠和成熟至关重要,它能有效地感知外部刺激[9]。热应激条件下,内质网中未正确折叠蛋白质增多,通过葡萄糖调节蛋白78(GRP78)激活下游感应因子,蛋白激酶R样内质网激酶(PERK)、活化转录因子6(ATF6)和激活肌醇需要酶1(IRE1)导致内质网应激[10]。先前的研究已证实,线粒体功能障碍和内质网应激是导致肝脏损伤、炎症反应和氧化应激的关键因素[6-7]。因此,缓解热应激肉鸡肝脏线粒体功能障碍和内质网应激可能是缓解肝脏损伤的有效途径。壳寡糖(chitosan oligosaccharide,COS)具有抗炎、抗氧化、抗凋亡等多种生物学功能[11]。本课题组前期研究表明,COS可通过改善热应激肉鸡肝脏抗氧化能力和炎性反应缓解肝脏损伤[5]。此外,COS还能通过缓解线粒体自噬降低软骨细胞和骨肉瘤细胞的炎性损伤[12-13]。因此,本研究旨在探究COS对热应激肉鸡肝脏功能、内质网应激、线粒体质量及NOD样受体蛋白3(NLRP3)炎性小体活化的影响。

1 材料与方法

1.1 试验材料

本试验所使用的COS纯度为93.6%,平均分子质量为1 768 Da,脱乙酰度≥90%,由青岛颂田生物技术有限公司提供。

1.2 试验设计

本试验方案经广东海洋大学滨海农业学院动物福利委员会批准(批准编号为SYXK-2018-0147)。
选取28日龄体重[(1 469.07±117.26) g]相近的雄性爱拔益加(AA)肉鸡144只,随机分为3组,每组6个重复,每个重复8只鸡。对照(CON)组肉鸡饲养在温度为(24±1) ℃的鸡舍;热应激(HS)组和COS组肉鸡饲养在08:00—18:00时间段的温度为(34±1) ℃,其余时间段的温度为(24±1) ℃的鸡舍。CON组和HS组饲喂基础饲粮,COS组在基础饲粮中添加200 mg/kg的COS。试验期为14 d。基础饲粮的配制方法与其饲粮组成及营养水平均参见本课题组前期报道[5]。试验期间肉鸡自由采食和饮水,按肉鸡饲养管理和免疫程序进行饲养管理和免疫接种。

1.3 样品采集

试验结束当日,禁食8 h后,每个重复随机选取1只鸡放血屠宰,取3份约1 g的肝脏样品,液氮速冻后,-80 ℃保存,用于检测肝脏功能相关酶活性及相关基因mRNA的表达。

1.4 检测指标及方法

1.4.1 肝脏功能相关指标测定

按购于南京建成生物工程研究所的试剂盒说明书测定肝脏中谷丙转氨酶(ALT)、谷草转氨酶(AST)、谷氨酰胺酶(GLS)和谷氨酰胺合成酶(GS)活性。

1.4.2 荧光定量PCR检测相关基因mRNA相对表达量

肝脏组织中的RNA提取、cDNA合成及荧光定量PCR方法参照本课题组前期试验的方法[14],以β-肌动蛋白(β-actin)为内参基因,采用2-ΔΔCt法计算目的基因mRNA相对表达量[15]。引物序列见表1。
表1 引物序列

Table 1 Primer sequences

基因名称 Gene names 引物序列 Primer sequences (5'— 3')
β-肌动蛋白
β-actin		 F:ATCCGGACCCTCCATTGTC
R:AGCCATGCCAATCTCGTCTT
芳香烃受体
AHR		 F:TTCAGGAAAGCAGAACAGCAA
R:TCACAACTAATACGAAGCCAT
孕烷X受体
PXR		 F:CCCTCAAGAGCTACATCGACCA
R:TGTTCTCCATCTTCAGCGTCT
细胞色素P450家族1亚家族A成员2
CYP1A2		 F:CTATGACAAGAACAGCATCCGAGACT
R:CCCCAAAGATGTCATTCACC
细胞色素P450家族1亚家族B成员1
CYP1B1		 F:TGAAGAAACGGGAACTGGAAG
R:CAGCCTTATCAAGCAACTCCA
细胞色素P450家族2亚家族D成员6
CYP2D6		 F:GAACCCTGCTTACATCCGAGA
R:CATGAACAGGAACGCCCAT
细胞色素P450家族3亚家族A成员4
CYP3A4		 F:TCATAGTGTTGTTCCCCTT
R:GGTATCCTTCTTCCCGTTC
细胞色素P450家族3亚家族A成员7
CYP3A7		 F:GACTCCATGAACAACCCCAA
R:AAATCTACTCTGCCCGTGTG
核呼吸因子1
NRF1		 F:CAGTATAGCACACCTGGTACCCTC
R:CTCCGATGCCTGCGTTGTCT
过氧化物酶体增殖物激活受体-γ共激活因子-1α
PGC-1α		 F:GATTCTTCACCTGGGTGG CA
R:TCAGCCCGAATTTCCTGGTC
线粒体转录因子A
TFAM		 F:GTGAAAGCCTGGCGAAACTG
R:CACAGCTCAGGTTACACCGT
线粒体融合蛋白1
MFN1		 F:CCTGCTGCAACTCCAGAGAACAC
R:TCACTCCGCCAACAACGATGATG
线粒体融合蛋白2
MFN2		 F:AGCTGGCTGCGTACATCAATGAG
R:GCCTTGCCAACACTTCACTAATGC
视神经萎缩症蛋白1
OPA1		 F:TGTATGTGATGAGATAGCCTGG
R:CCAGCTCCACTGTACAAGACA
线粒体动力学相关蛋白1
DRP1		 F:CACGAGACAAGGCTGCTGCTG
R:TTCCTGCTGATACCTCCTCTGC
线粒体裂变因子
MFF		 F:GGCTCCTCAGAATGCTGACCTTG
R:CACTACAATCCGCTCTGGAACCTG
PTEN诱导激酶1
PINK1		 F:TGCAGTTGTTGGAAGGTGTG
R:CAGCCAGCAGAATCGAACTCTTA
E3泛素连接酶
Parkin		 F:GTCCAGCAAAGCATCGTTCA
R:CAACGATGGAAGGATGCTGG
微管相关蛋白1轻链3Ⅰ
LC3Ⅰ		 F:TTACACCCATATCAGATTCTTG
R:ATTCCAACCTGTCCCTCA
微管相关蛋白1轻链3Ⅱ
LC3Ⅱ		 F:AGTGAAGTGTAGCAGGATGA
R:AAGCCTTGTGAACGAGAT
选择性自噬接头蛋白p62
p62		 F:GACCCAGCCAAGACTACCAT
R:CAGAGGCATGTAGTTTCGGC
葡萄糖调节蛋白78
GRP78		 F:TCCTGCTCCTCGTGGTGTCC
R:CTCCTCTGGTGTTAGCCGATTCTG
蛋白激酶R样内质网激酶
PERK		 F:GTGGATGAGCAGGAGGCAATGATG
R:ATCCTTAACCAGCCATGCAGAAGC
真核翻译起始因子2α
EIF2α		 F:GCTGCGAGTCAGTAATGGGTATAA
R:CTGCCAGGAAACTTGCCACA
活化转录因子4
ATF4		 F:AATTGGCTCGCTGTGGACAG
R:CGGTGGCTTCCAGATGTTCC
活化转录因子6
ATF6		 F:GATTGTGGGCGTCACTTCTCG
R:TGGGATGCCAATGTTAGCCTG
肌醇需要酶1
IRE1		 F:TGAGGGCAATGAGAAATAAGAAGC
R:TGTAGGAGCAGGTGAGGGAAGC
X盒结合蛋白1
XBP1		 F:GCGAGTCTACGGATGTGAAGGA
R:TGTGGAGGTTGTCAGGAATGGT
C/EBP同源蛋白
CHOP		 F:CAGGAAGAAGAGCTGGCCCCACT
R:TGCTGTGCTCGCCGTGCTGT
NOD样受体蛋白3
NLRP3		 F:CCGCTACACCAACCTGACC
R:CACCTGGCCGTCTTTGCT
半胱天冬蛋白酶1
Caspase 1		 F:CTCTGACAGCACCTTCCT
R:CCGTGGTCCCATTACTCT
白细胞介素-18
IL-18		 F:AAGCGTGGCAGCTTTTGAAG
R:CTGAAGGTGCGGTGGTTTTG

1.5 数据统计分析

试验数据使用SAS 9.1.3软件进行单因素方差分析,采用Duncan氏法进行组间多重比较,结果数据以“平均值±标准误”表示,P<0.05表示差异显著。

2 结果

2.1 COS对热应激肉鸡肝脏功能的影响

由表2所示,HS组肝脏中ALT、AST、GLS和GS活性均显著高于CON组(P<0.05);COS组肝脏中ALT、AST、GLS和GS活性均显著低于HS组(P<0.05),而与CON组差异均不显著(P>0.05)。
表2 壳寡糖对热应激肉鸡肝脏功能的影响

Table 2 Effects of COS on hepatic function in heat-stressed broilers

项目
Items		 组别 Groups P值
P-value
对照 CON 热应激 HS 壳寡糖 COS
谷丙转氨酶 ALT/(U/g prot) 24.34±3.31b 34.55±3.45a 25.93±3.02b <0.001
谷草转氨酶 AST/(U/g prot) 34.08±4.01b 44.33±4.56a 37.82±3.18b 0.009
谷氨酰胺酶 GLS/[nmol/(min·g)] 928.89±166.66b 1 379.64±111.06a 832.43±45.88b <0.001
谷氨酰胺合成酶 GS/[μmol/(h·g)] 21.76±5.17b 29.82±1.58a 22.06±1.39b 0.003

同行数据肩标不同字母表示差异显著(P<0.05),无字母或相同字母表示差异不显著(P>0.05)。下表同。

In the same row, values with different letter superscripts indicated significant difference (P<0.05), with no letter or the same letter superscripts indicated no significant difference (P>0.05). The same as below.

2.2 COS对热应激肉鸡肝脏核受体和细胞色素酶相关基因表达的影响

如表3所示,与CON组相比,HS组肝脏孕烷X受体(PXR)、细胞色素P450家族1亚家族A成员2(CYP1A2)、细胞色素P450家族2亚家族D成员6(CYP2D6)和细胞色素P450家族3亚家族A成员4(CYP3A4) mRNA的相对表达量显著上调(P<0.05)。与HS组相比,COS组肝脏PXR、CYP1A2、CYP2D6和CYP3A4 mRNA的相对表达量显著下调(P<0.05)。
表3 壳寡糖对热应激肉鸡肝脏核受体和细胞色素酶相关基因表达的影响

Table 3 Effects of COS on hepatic nuclear receptor and cytochrome enzyme related gene expression in heat-stressed broilers

项目
Items		 组别 Groups P值
P-value
对照 CON 热应激 HS 壳寡糖 COS
芳香烃受体 AHR 1.00±0.40 1.69±0.59 1.26±0.36 0.057
孕烷X受体 PXR 1.00±0.70b 2.95±0.72a 1.37±0.40b <0.001
细胞色素P450家族1亚家族A成员2 CYP1A2 1.00±0.69b 3.39±0.62a 1.59±1.20b 0.006
细胞色素P450家族1亚家族B成员1 CYP1B1 1.00±0.59 1.34±0.47 1.07±0.54 0.466
细胞色素P450家族2亚家族D成员6 CYP2D6 1.00±0.52b 1.78±0.25a 1.07±0.53b 0.041
细胞色素P450家族3亚家族A成员4 CYP3A4 1.00±0.29b 1.78±0.21a 1.07±0.45b 0.004
细胞色素P450家族3亚家族A成员7 CYP3A7 1.00±0.38 1.22±0.57 1.01±0.32 0.695

2.3 COS对热应激肉鸡肝脏内质网应激相关基因表达的影响

如表4所示,与CON组相比,HS组肝脏GRP78、PERK、X盒结合蛋白1(XBP1)和C/EBP同源蛋白(CHOP)mRNA的相对表达量显著上调(P<0.05)。与HS组相比,COS组肝脏GRP78、PERK、XBP1和CHOP mRNA的相对表达量显著下调(P<0.05)。
表4 壳寡糖对热应激肉鸡肝脏内质网应激相关基因表达的影响

Table 4 Effects of COS on hepatic endoplasmic reticulum stress related gene expression in heat-stressed broilers

项目
Items		 组别 Groups P值
P-value
对照 CON 热应激 HS 壳寡糖 COS
葡萄糖调节蛋白78 GRP78 1.00±0.54b 1.63±0.79a 0.90±0.38b 0.046
蛋白激酶R样内质网激酶 PERK 1.00±0.40b 2.05±0.85a 1.03±0.37b 0.027
真核翻译起始因子2α EIF2α 1.00±0.50 1.39±0.59 0.74±0.18 0.110
活化转录因子4 ATF4 1.00±0.31 1.19±0.41 1.01±0.34 0.640
活化转录因子6 ATF6 1.00±0.51 1.35±0.55 1.04±0.35 0.351
肌醇需要酶1 IRE1 1.00±0.24 1.53±0.53 1.10±0.50 0.053
X盒结合蛋白1 XBP1 1.00±0.35b 1.68±0.47a 1.08±0.40b 0.007
C/EBP同源蛋白 CHOP 1.00±0.49b 1.97±0.59a 1.28±0.36b 0.025

2.4 COS对热应激肉鸡肝脏线粒体质量相关基因表达的影响

如表5所示,与CON组相比,HS组肝脏过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC-1α)和线粒体转录因子A(TFAM)mRNA的相对表达量显著下调(P<0.05),并且肝脏线粒体动力学相关蛋白1(DRP1)、PTEN诱导激酶1(PINK1)、E3泛素连接酶(Parkin)和选择性自噬接头蛋白p62(p62) mRNA的相对表达量显著上调(P<0.05)。与HS组相比,COS组肝脏PGC-1α和TFAM mRNA的相对表达量显著上调(P<0.05),并且肝脏DRP1、PINK1和Parkin mRNA的相对表达量显著下调(P<0.05)。
表5 壳寡糖对热应激肉鸡肝脏线粒体质量相关基因表达的影响

Table 5 Effects of COS on hepatic mitochondrial mass related gene expression in heat-stressed broilers

项目
Items		 组别 Groups P值
P-value
对照 CON 热应激 HS 壳寡糖 COS
核呼吸因子1 NRF1 1.00±0.38 0.54±0.16 1.02±0.41 0.107
过氧化物酶体增殖物激活受体-γ共激活因子-1α
PGC-1α		 1.00±0.19a 0.56±0.16b 1.36±0.46a 0.004
线粒体转录因子A TFAM 1.00±0.32a 0.50±0.16b 0.88±0.24a 0.033
线粒体融合蛋白1 MFN1 1.00±0.36 1.11±0.46 0.67±0.06 0.178
线粒体融合蛋白2 MFN2 1.00±0.17 1.04±0.33 0.74±0.27 0.186
视神经萎缩症蛋白1 OPA1 1.00±0.40 1.78±0.61 1.32±0.58 0.115
线粒体动力学相关蛋白1 DRP1 1.00±0.20b 1.89±0.69a 0.96±0.30b 0.013
线粒体裂变因子 MFF 1.00±0.23 0.76±0.35 0.80±0.32 0.390
PTEN诱导激酶1 PINK1 1.00±0.37b 1.79±0.55a 1.10±0.37b 0.028
E3泛素连接酶 Parkin 1.00±0.34b 1.56±0.49a 1.07±0.28b 0.046
微管相关蛋白1轻链3Ⅰ LC3Ⅰ 1.00±0.41 1.66±0.45 1.21±0.37 0.098
微管相关蛋白1轻链3Ⅱ LC3Ⅱ 1.00±0.26 1.36±0.36 1.30±0.31 0.182
选择性自噬接头蛋白p62 p62 1.00±0.42b 1.93±0.66a 1.82±0.60a 0.023

2.5 COS对热应激肉鸡肝脏NLRP3炎性小体相关基因表达的影响

如表6所示,与CON组相比,HS组肝脏NLRP3、半胱天冬蛋白酶1(Caspase 1)和白细胞介素-18(IL-18) mRNA的相对表达量显著上调(P<0.05)。与HS组相比,COS组肝脏NLRP3、Caspase 1和IL-18 mRNA的相对表达量显著下调(P<0.05)。
表6 壳寡糖对热应激肉鸡肝脏NLRP3炎性小体相关基因表达的影响

Table 6 Effects of COS on hepatic NLRP3 inflammasome related gene expression in heat-stressed broilers

项目
Items		 组别 Groups P值
P-value
对照 CON 热应激 HS 壳寡糖 COS
NOD样受体蛋白3 NLRP3 1.00±0.40b 2.68±0.71a 1.57±0.32b 0.001
半胱天冬蛋白酶1 Caspase 1 1.00±0.32b 1.62±0.31a 0.94±0.39b 0.008
白细胞介素-18 IL-18 1.00±0.25b 2.03±0.61a 1.12±0.51b 0.005

3 讨论

3.1 COS对热应激肉鸡肝脏功能的影响

肝脏是热应激的靶器官,也是肉鸡营养代谢的核心器官,在能量代谢及氨的解毒作用中有重要作用[2]。本课题组前期研究发现,热应激导致肉鸡血液ALT活性升高,肝细胞出血、肿胀、空泡化及出现炎症浸润,表明热应激导致肝脏损伤[5]。肝脏是氨的解毒器官,ALT和AST是机体重要的2种转氨酶。本研究发现,热应激显著增加肝脏中ALT和AST活性,表明热应激增强肝脏氨基酸的分解代谢。氨基酸脱氨基作用生成的氨,需转运到肝脏进行无毒化处理。由于家禽缺少氨甲酰磷酸合成酶Ⅰ,不能将氨合成尿素排出,只能通过GS合成无毒的谷氨酰胺(Gln)或通过嘌呤核苷酸代谢生产尿酸排出。本研究发现,热应激显著提高肝脏GS和GLS活性。GS和GLS活性变化可能与肝脏损伤相关,降低肝脏对氨的解毒能力。肝脏损伤导致GS活性下降,此外GLS催化Gln水解生成谷氨酸(Glu)和氨,Glu激活肝星状细胞,促进肝脏组织的修复,同时导致血氨水平升高[16-17]。马冰冰[18]报道,热应激降低肝脏GS活性,提高ALT、AST和GLS活性及血氨含量。我们前期研究发现,COS通过缓解热应激肉鸡氧化应激和炎症反应缓解肝脏损伤,降低热应激肉鸡血清ALT和AST活性及肝细胞凋亡[5]。本研究发现,COS降低热应激肉鸡肝脏ALT、AST、GLS和GS活性,表明COS缓解肝脏损伤可能通过缓解肝脏星状细胞的激活,进而使GLS活性下降。

3.2 COS对热应激肉鸡肝脏核受体和细胞色素酶相关基因表达的影响

核受体芳香烃受体(AHR)和PXR参与调控Ⅰ相代谢酶基因的转录,其中AHR主要调控细胞色素P450家族1亚家族(CYP1)的转录,而PXR参与调控细胞色素P450家族2亚家族(CYP2)和细胞色素P450家族3亚家族(CYP3)的转录[19]。本研究发现,热应激上调肝脏PXR、CYP1A2、CYP2D6和CYP3A4 mRNA的相对表达量,表明热应激诱导肝脏核受体PXR的应答反应,上调CYP1A2、CYP2D6和CYP3A4的转录,激活Ⅰ相代谢反应,加重肝脏氧化损伤。Ye等[20]报道,黄曲霉毒素暴露诱导的肉鸡肝脏氧化损伤,同样表现为激活Ⅰ相代谢反应,上调肝脏细胞色素P450家族1亚家族A成员1(CYP1A1)和CYP2A6 mRNA的相对表达量。COS下调热应激肉鸡肝脏PXR、CYP1A2、CYP2D6和CYP3A4 mRNA的相对表达量,表明COS通过抑制Ⅰ相代谢反应来缓解热应激诱导的肝脏损伤[21]。

3.3 COS对热应激肉鸡肝脏内质网应激相关基因表达的影响

热应激可诱导内质网应激,并启动未折叠蛋白质反应,继而激活PERK、ATF6和IRE1信号通路,以维持内质网的稳态,但过度的内质网应激将引发氧化损伤和炎症反应[6,10]。研究表明,热应激诱导肉鸡肝脏出现内质网应激时,常伴随PERK、活化转录因子4(ATF4)、真核翻译起始因子2α(EIF2α)、IRE1、XBP1、ATF6、GRP78、CHOP mRNA的相对表达量的上调[6-7,10]。本研究中,热应激肉鸡肝脏GRP78、PERK、XBP1和CHOP mRNA的相对表达量显著上调。持续的内质网应激激活CHOP并诱导肝细胞凋亡,表明热应激肉鸡肝脏引发了内质网应激,诱发肝脏损伤,降低肝脏解毒能力。这与本课题组先前报道一致,热应激提高肝细胞凋亡率,上调肝脏Caspase 9和Bax mRNA的相对表达量[5]。COS的抗氧化功能在缓解内质网应激中有重要作用[22-23]。肝脏富含内质网,同时内质网是热应激的靶细胞器,易受ROS攻击发生内质网应激,诱发肝细胞凋亡和肝脏损伤[6-7]。本研究中,COS显著下调热应激肉鸡肝脏GRP78、PERK、XBP1和CHOP mRNA的相对表达量。本课题组前期研究报道,COS可通过调节热应激肉鸡肝脏的氧化损伤和炎症反应,缓解肝细胞凋亡和损伤[5],表明COS可能通过缓解热应激肉鸡肝脏氧化损伤,进而缓解内质网应激,最终缓解肝脏损伤。

3.4 COS对热应激肉鸡肝脏线粒体质量相关基因表达的影响

线粒体质量主要与线粒体生物发生、融合和分裂相关[24]。核呼吸因子1(NRF1)、PGC-1α和TFAM与线粒体的生物发生密切相关,本质上是为了提升线粒体的数量和质量[25]。线粒体融合蛋白1(MFN1)、线粒体融合蛋白2(MFN2)和视神经萎缩症蛋白1(OPA1)调节线粒体的融合,DRP1和线粒体裂变因子(MFF)调节线粒体分裂[26]。若线粒体的生物发生受阻、融合和分裂不平衡,将引起线粒体形态结构改变和功能障碍[27]。线粒体是热应激的靶细胞器,热应激暴露导致线粒体断裂、DRP1的mRNA的相对表达量增加,MFN1、MFN2和OPA1 mRNA的相对表达量减少[28-29]。戚秋蓉[30]报道,热应激肉鸡肝脏的线粒体发生肿胀、嵴溶解及膜破裂,肝脏DRP1的mRNA和蛋白的相对表达量出现上调,而MFN1、MFN2和OPA1 mRNA和蛋白的相对表达量出现下调。杨泰[31]报道,热应激肉鸡空肠和回肠NRF1、PGC-1α和TFAM mRNA相对表达量出现下调。本研究结果同样表明,热应激肉鸡肝脏PGC-1α和TFAM mRNA相对表达量下调,并且DRP1 mRNA相对表达量上调,表明热应激导致肉鸡肝细胞线粒体功能障碍。COS能否参与调节线粒体功能还未见相关报道。Liu等[32]报道,木聚糖能够上调脂多糖(LPS)刺激的断奶仔猪空肠MFN1 mRNA相对表达量,下调DRP1 mRNA相对表达量,表明寡糖可参与调节线粒体功能。此外,藻酸寡糖和巴戟天寡糖也被证实参与调节线粒体功能[33-34]。本研究结果表明,COS上调热应激肉鸡肝脏PGC-1α和TFAM mRNA的相对表达量,并显著下调肝脏DRP1 mRNA的相对表达量,表明COS可能通过促进热应激肉鸡肝脏线粒体生物发生,抑制线粒体分裂,减少受损线粒体的数量,以维持线粒体质量和功能。
线粒体自噬可选择性清除受损线粒体以维持细胞正常功能,与线粒体生物发生、分裂和融合密切相关[8]。热应激损伤线粒体功能,膜电位下降,通过激活PINK1/Parkin信号通路,由p62协助与微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)结合,最终诱导线粒体自噬[35]。Shi等[36-37]报道,热应激可激活肉鸡心肌和肝细胞自噬,上调PINK1和Parkin蛋白的相对表达量。本研究结果表明,热应激上调肝脏PINK1、Parkin和p62 mRNA的相对表达量,表明热应激激活了肝脏线粒体的自噬。对软骨细胞和骨肉瘤细胞的研究发现,COS通过调节线粒体自噬缓解炎性损伤[12-13]。本研究结果同样表明,COS下调热应激肉鸡肝脏PINK1和Parkin mRNA的相对表达量。但值得注意的是,COS缓解热应激肉鸡肝脏线粒体自噬,是COS的直接作用,还是由于COS促进热应激肉鸡肝脏线粒体生物发生,抑制线粒体分裂,减少受损线粒体的数量,进而缓解线粒体自噬,亦或是二者的协同作用,还需做进一步研究。

3.5 COS对热应激肉鸡肝脏NLRP3炎性小体相关基因表达的影响

热应激能通过激活自噬和内质网应激诱导机体炎症发生[38],热应激能够上调肉鸡肝脏核因子-κB p65亚基(NF-κB p65)、白细胞介素-1β(IL-1β)、NLRP3、Caspase 1和IL-18 mRNA的相对表达量[5,39-40]。本研究结果表明,热应激可上调肉鸡肝脏NLRP3、Caspase 1和IL-18 mRNA的相对表达量。线粒体自噬通过清除受损的线粒体和内质网来抑制NLRP3炎性小体的激活,表明热应激诱导了线粒体功能失调和内质网应激,促进了炎症反应[41]。Liu等[39]报道,热应激会激活肉鸡肝脏NLRP3炎性小体的活化,上调NLRP3和Caspase 1蛋白的相对表达量。体外试验也证实,热应激会激活肝细胞NLRP3炎性小体的活化,上调NLRP3蛋白的相对表达量以及Caspase 1活性和IL-1β含量[42]。COS具有抗炎功能,可通过抑制Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路和NLRP3炎性小体的活化,下调相关促炎症因子的表达[5,43]。Hu等[43]报道,COS能够下调巨噬细胞白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、NLRP3、Caspase 1和IL-18 mRNA的相对表达量,从而缓解炎症反应。本试验结果同样表明,COS下调热应激肉鸡肝脏NLRP3、Caspase 1和IL-18 mRNA的相对表达量,表明COS可抑制NLRP3炎性小体的活化。
内质网与线粒体通过线粒体相关内质网膜相互连接构成内质网-线粒体系统,参与调控炎症反应、内质网应激、细胞凋亡、自噬以及线粒体动力等生物学过程[44]。内质网-线粒体系统上的多种蛋白质决定了其功能的多样性,如MFN2[45]。MFN2位于线粒体外膜,并在内质网-线粒体系统中含量丰富,具有调节线粒体融合及线粒体与内质网之间的距离的双重作用。敲除MFN2基因会导致线粒体和内质网的形态异常,增加内质网-线粒体系统的间距,并影响钙离子(Ca2+)向线粒体的转移[46]。先前有研究报道,热应激导致的心肌细胞损伤与线粒体钙过载相关[47]。本试验中,未检测肝脏Ca2+的浓度,也未进行体外细胞试验验证机制,COS改善热应激肉鸡肝脏线粒体功能、缓解内质网应激和炎症反应是否与调节Ca2+向线粒体的转移相关还需做进一步研究证实。

4 结论

本试验条件下,饲粮中添加200 mg/kg的COS可以通过促进肝脏线粒体生物发生,抑制线粒体分裂,缓解线粒体自噬、内质网应激和炎症反应,最终缓解热应激肉鸡肝脏损伤并提高肝脏氨解毒能力。

References

Publishing order | Descend order by publishing year | Descend order by cited within
[1]
KHAN R U, NAZ S, ULLAH H, et al. Physiological dynamics in broiler chickens under heat stress and possible mitigation strategies[J]. Animal Biotechnology, 2023, 34(2):438-447.

DOI

[2]
ZAEFARIAN F, ABDOLLAHI M R, COWIESON A, et al. Avian liver:the forgotten organ[J]. Animals, 2019, 9(2):63.

DOI

[3]
MA B B, ZHANG L, LI J L, et al. Heat stress alters muscle protein and amino acid metabolism and accelerates liver gluconeogenesis for energy supply in broilers[J]. Poultry Science, 2021, 100(1):215-223.

DOI PMID

[4]
LAN R X, LUO H W, WU F, et al. Chitosan oligosaccharides alleviate heat-stress-induced lipid metabolism disorders by suppressing the oxidative stress and inflammatory response in the liver of broilers[J]. Antioxidants, 2023, 12(8):1497.

DOI

[5]
王浩轩, 高金稔, 张迦, 等. 壳寡糖缓解热应激肉鸡肝脏损伤的作用研究[J]. 动物营养学报, 2025, 37(11):7534-7546.

DOI

WANG H X, GAO J R, ZHANG J, et al. Study on effects of chitosan oligosaccharides in alleviating liver injury in heat-stressed broilers[J]. Chinese Journal of Animal Nutrition, 2025, 37(11):7534-7546. (in Chinese)

DOI

[6]
MA B B, XING T, LI J L, et al. Chronic heat stress causes liver damage via endoplasmic reticulum stress-induced apoptosis in broilers[J]. Poultry Science, 2022, 101(10):102063.

DOI

[7]
JING J Z, ZENG H J, SHAO Q J, et al. Selenomethionine alleviates environmental heat stress induced hepatic lipid accumulation and glycogen infiltration of broilers via maintaining mitochondrial and endoplasmic reticulum homeostasis[J]. Redox Biology, 2023, 67:102912.

DOI

[8]
LENIN R R, KOH Y H, ZHANG Z T, et al. Dysfunctional autophagy,proteostasis,and mitochondria as a prelude to age-related macular degeneration[J]. International Journal of Molecular Sciences, 2023, 24(10):8763.

DOI

[9]
SCHWARZ D S, BLOWER M D. The endoplasmic reticulum:structure,function and response to cellular signaling[J]. Cellular and Molecular Life Sciences, 2016, 73(1):79-94.

DOI

[10]
WANG J Y, JING J Z, GONG Z Y, et al. Different dietary sources of selenium alleviate hepatic lipid metabolism disorder of heat-stressed broilers by relieving endoplasmic reticulum stress[J]. International Journal of Molecular Sciences, 2023, 24(20):15443.

DOI

[11]
LI Q Y, SHI W R, HUANG Y L. Comparison of the protective effects of chitosan oligosaccharides and chitin oligosaccharide on apoptosis,inflammation and oxidative stress[J]. Experimental and Therapeutic Medicine, 2024, 28(2):310.

DOI

[12]
PAN Z, CHENG D D, WEI X J, et al. Chitooligosaccharides inhibit tumor progression and induce autophagy through the activation of the p53/mTOR pathway in osteosarcoma[J]. Carbohydrate Polymers, 2021, 258:117596.

DOI

[13]
CAO R Q, YU H M, LONG H B, et al. Low deacetylation degree chitosan oligosaccharide protects against IL-1β induced inflammation and enhances autophagy activity in human chondrocytes[J]. Journal of Biomaterials Science (Polymer Edition), 2022, 33(4):517-531.

[14]
LAN R X, LIU F, HE Z B, et al. Immunolocalization of GnRHRI,gonadotropin receptors,PGR,and PGRMCI during follicular development in the rabbit ovary[J]. Theriogenology, 2014, 81(8):1139-1147.

DOI

[15]
LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 2001, 25(4):402-408.

DOI

[16]
FRIEG B, GÖRG B, GOHLKE H, et al. Glutamine synthetase as a central element in hepatic glutamine and ammonia metabolism:novel aspects[J]. Biological Chemistry, 2021, 402(9):1063-1072.

DOI

[17]
EL-ASHMAWY N E, AL-ASHMAWY G M, FAKHER H E, et al. The role of WNT/β-catenin signaling pathway and glutamine metabolism in the pathogenesis of CCl4-induced liver fibrosis:repositioning of niclosamide and concerns about lithium[J]. Cytokine, 2020, 136:155250.

DOI

[18]
马冰冰. 慢性热应激影响肉鸡氮代谢机制及牛磺酸缓解作用研究[D].博士学位论文. 南京: 南京农业大学, 2022:47-57.

MA B B. Mechanism of chronic heat stress affecting nitrogen metabolism and alleviation effect of taurine in broilers[D].Ph.D. Thesis. Nanjing: Nanjing Agricultural University, 2022:47-57. (in Chinese)

[19]
RAKATELI L, HUCHZERMEIER R, VAN DER VORST E P C. AhR,PXR and CAR:from xenobiotic receptors to metabolic sensors[J]. Cells, 2023, 12(23):2752.

DOI

[20]
YE X Q, YANG Y Y, YAO Q H, et al. The ameliorative role of phlorotannin on aflatoxin B1-induced liver oxidative stress and mitochondrial injury is related to the activation of Nrf2 and Nrf1 signaling pathways in broilers[J]. Journal of Animal Science and Biotechnology, 2025, 16(1):75.

DOI

[21]
YAO H T, LUO M N, LI C C. Chitosan oligosaccharides reduce acetaminophen-induced hepatotoxicity by suppressing CYP-mediated bioactivation[J]. Journal of Functional Foods, 2015, 12:262-270.

DOI

[22]
EI Z Z, HUTAMEKALIN P, PROMMEENATE P, et al. Chitooligosaccharide prevents vascular endothelial cell apoptosis by attenuation of endoplasmic reticulum stress via suppression of oxidative stress through Nrf2-SOD1 up-regulation[J]. Pharmaceutical Biology, 2022, 60(1):2155-2166.

DOI PMID

[23]
ZHAI X C, YUAN S J, YANG X, et al. Chitosan oligosaccharides induce apoptosis in human renal carcinoma via reactive-oxygen-species-dependent endoplasmic reticulum stress[J]. Journal of Agricultural and Food Chemistry, 2019, 67(6):1691-1701.

DOI PMID

[24]
MARZETTI E, CSISZAR A, DUTTA D, et al. Role of mitochondrial dysfunction and altered autophagy in cardiovascular aging and disease:from mechanisms to therapeutics[J]. American Journal of Physiology-Heart and Circulatory Physiology, 2013, 305(4):H459-H476.

DOI

[25]
SCARPULLA R C. Transcriptional paradigms in mammalian mitochondrial biogenesis and function[J]. Physiological Reviews, 2008, 88(2):611-638.

DOI PMID

[26]
FRIEDMAN J R, NUNNARI J. Mitochondrial form and function[J]. Nature, 2014, 505(7483):335-343.

DOI

[27]
BI J B, ZHANG J, REN Y F, et al. Irisin alleviates liver ischemia-reperfusion injury by inhibiting excessive mitochondrial fission,promoting mitochondrial biogenesis and decreasing oxidative stress[J]. Redox Biology, 2019, 20:296-306.

DOI

[28]
YU T Z, FERDJALLAH I, ELENBERG F, et al. Mitochondrial fission contributes to heat-induced oxidative stress in skeletal muscle but not hyperthermia in mice[J]. Life Sciences, 2018, 200:6-14.

DOI PMID

[29]
WANG R, SHI Z G, LI J L, et al. Protective effect of manganese on apoptosis and mitochondrial function of heat-stressed primary chick embryonic myocardial cells[J]. Biological Trace Element Research, 2022, 200(10):4419-4429.

DOI

[30]
戚秋蓉. 染料木素通过调控Mfn2与PLIN1互作缓解热应激引起的肉鸡肝脏脂质沉积[D].硕士学位论文. 南昌: 江西农业大学, 2024:25-27.

QI Q R. Genistein reduces liver lipid deposition in broilers under heat stress by regulating the interaction between Mfn2 and PLIN1[D].Master’s Thesis. Nanchang: Jiangxi Agricultural University, 2024:25-27. (in Chinese)

[31]
杨泰. 鞣花酸对热应激肉鸡肠道损伤保护效应及机制研究[D].博士学位论文. 长沙: 湖南农业大学, 2022:70.

YANG T. Study on the protective effect and mechanism of ellagic acid on intestinal damage in heat-stressed broiler chickens[D]. Ph.D.Thesis. Changsha: Hunan Agricultural University, 2022:70. (in Chinese)

[32]
LIU G M, SUN W X, ZHANG R N, et al. Dietary xylo-oligosaccharides alleviates LPS-induced intestinal injury via endoplasmic reticulum-mitochondrial system pathway in piglets[J]. Journal of Animal Science, 2024, 102:skae238.

DOI

[33]
FENG W J, LIU J Y, WANG S, et al. Alginate oligosaccharide alleviates D-galactose-induced cardiac ageing via regulating myocardial mitochondria function and integrity in mice[J]. Journal of Cellular and Molecular Medicine, 2021, 25(15):7157-7168.

DOI PMID

[34]
WANG X L, JIANG H, ZHANG N, et al. Anti-diabetic activities of agaropectin-derived oligosaccharides from Gloiopeltis furcata via regulation of mitochondrial function[J]. Carbohydrate Polymers, 2020, 229:115482.

DOI

[35]
YIN K L, LEE J, LIU Z L, et al. Mitophagy protein PINK1 suppresses colon tumor growth by metabolic reprogramming via p53 activation and reducing acetyl-CoA production[J]. Cell Death and Differentiation, 2021, 28(8):2421-2435.

DOI PMID

[36]
SHI J C, LI C Y, LIANG Q J, et al. HSP90-regulated mitophagy can alleviate heat stress damage by inhibiting pyroptosis in the hepatocytes of Wenchang chickens[J]. International Journal of Biological Macromolecules, 2024, 280(Pt 4):135979.

DOI

[37]
SHI J C, JI Z P, YAO X, et al. HSP90 enhances mitophagy to improve the resistance of car-diomyocytes to heat stress in Wenchang chickens[J]. International Journal of Molecular Sciences, 2024, 25(21):11695.

DOI

[38]
罗烨, 刘慧敏, 瞿明仁, 等. 热应激诱导炎症反应及细胞自噬的调控机制[J]. 动物营养学报, 2024, 36(10):6191-6200.

DOI

LUO Y, LIU H M, QU M R, et al. Regulatory mechanism of heat stress-induced inflammatory response and autophagy[J]. Chinese Journal of Animal Nutrition, 2024, 36(10):6191-6200. (in Chinese)

DOI

[39]
LIU Y L, DING K N, SHEN X L, et al. Chronic heat stress promotes liver inflammation in broilers via enhancing NF-κB and NLRP3 signaling pathway[J]. BMC Veterinary Research, 2022, 18(1):289.

DOI

[40]
欧阳经鑫, 李秋粉, 周华, 等. 饲粮添加色氨酸对热应激肉鸡肝脏、肠道抗氧化能力和炎症反应的影响[J]. 中国兽医学报, 2022, 42(6):1256-1262,1269.

OUYANG J X, LI Q F, ZHOU H, et al. Effects of dietary tryptophan supplementation on antioxidant capacity and inflammatory response in liver and intestine of broilers subjected to heat stress[J]. Chinese Journal of Veterinary Science, 2022, 42(6):1256-1262,1269. (in Chinese)

[41]
ZHOU R B, YAZDI A S, MENU P, et al. A role for mitochondria in NLRP3 inflammasome activation[J]. Nature, 2011, 469(7329):221-225.

DOI

[42]
DU D H, LV W T, JING X X, et al. Dietary supplementation of camel whey protein attenuates heat stress-induced liver injury by inhibiting NLRP3 inflammasome activation through the HMGB1/RAGE signalling pathway[J]. Journal of Functional Foods, 2021, 84:104584.

DOI

[43]
HU H M, XIA H, ZOU X J, et al. N-acetyl-chitooligosaccharide attenuates inflammatory responses by suppression of NF-κB signaling,MAPK and NLRP3 inflammasome in macrophages[J]. Journal of Functional Foods, 2021, 78:104364.

DOI

[44]
TUBBS E, RIEUSSET J. Metabolic signaling functions of ER-mitochondria contact sites:role in metabolic diseases[J]. Journal of Molecular Endocrinology, 2017, 58(2):R87-R106.

DOI

[45]
MAO H, CHEN W, CHEN L X, et al. Potential role of mitochondria-associated endoplasmic reticulum membrane proteins in diseases[J]. Biochemical Pharmacology, 2022, 199:115011.

DOI

[46]
HAN S, ZHAO F P, HSIA J, et al. The role of Mfn2 in the structure and function of endoplasmic reticulum-mitochondrial tethering in vivo[J]. Journal of Cell Science, 2021, 134(13):jcs253443.

DOI

[47]
YAO X, ZHU J, LI L, et al. Hsp90 protected chicken primary myocardial cells from heat-stress injury by inhibiting oxidative stress and calcium overload in mitochondria[J]. Biochemical Pharmacology, 2023, 209:115434.

DOI

Options
Outlines

/

〈 〉

京ICP备17050989号-2
Copyright © Chinese Journal of Animal Nutrition, All Rights Reserved.
Tel: 010－62817823　E-mail: cjan@vip.163.com
Powered by Beijing Magtech Co., Ltd.