日粮添加蛹虫草缓解脂多糖诱导的仔猪肝损伤和骨骼肌蛋白质降解

doi:10.3864/j.issn.0578-1752.2024.12.015

摘要/Abstract

摘要：

【目的】探讨日粮中添加蛹虫草对断奶仔猪生长和免疫性能的影响及调节断奶仔猪免疫应激的机制，为新型饲料添加剂筛选提供依据。【方法】选取相同日龄、初始体重相似的杜×长×大早期断奶仔猪144头并随机分为4组：NC组（基础日粮）、LPS组（LPS+基础日粮）、LPS+500CM组（LPS+基础日粮+500 mg·kg^-1 CM）、LPS+1 000 CM（LPS+基础日粮+1 000 mg·kg^-1 CM）。NC组和LPS组饲喂基础日粮，LPS+500CM组和LPS+1000CM组分别按照比例将蛹虫草粉与基础日粮充分混匀后饲喂。饲养结束后，每组随机挑选6头（公母各半）屠宰，屠宰前4h对LPS组、LPS+500CM组和LPS+1000CM组腹腔注射LPS，NC组注射等量生理盐水。屠宰后采集背最长肌、心脏、肝脏、脾脏、胰脏和肾脏进行检测。【结果】对体重及采食量统计分析，结果显示，日粮添加蛹虫草饲喂仔猪对仔猪平均日采食量无显著影响，而添加500 mg·kg^-1CM和1 000 mg·kg^-1CM的仔猪分别比对照组仔猪体重提高了8.58%（P<0.05）和9.08%（P<0.05）。相比于NC组，LPS组仔猪脾脏指数显著降低（P<0.05），而LPS+500CM组和LPS+1000CM组仔猪脾脏指数均显著高于LPS组（P<0.05）。肝功能生物标志物和总胆汁酸检测表明，LPS组仔猪肝脏ALT水平比NC组显著升高（P <0.05），AST、LDH、总胆汁酸水平有上升趋势。而饲料中添加有蛹虫草的LPS+500CM组和LPS+1000CM组的ALT、AST、LDH和总胆汁酸水平均比LPS组的指标显著降低。肝脏抗氧化指标和炎症因子表达检测显示，LPS组仔猪肝脏的MDA、LPO、T-AOC水平及炎症因子表达显著升高（P<0.05），而CAT、GSH-Px、T-SOD水平显著降低（P<0.01），而用蛹虫草饲喂仔猪可以显著缓解这些指标的升高或降低。LPS组骨骼肌粗蛋白和氨基酸含量显著减低（P<0.01），而日粮添加蛹虫草饲喂可以显著缓解LPS导致的蛋白和氨基酸含量减少（P<0.01）。与NC相比，LPS组炎症因子表达显著升高（P<0.05），而日粮添加蛹虫草饲喂后炎症因子表达比LPS组显著降低（P<0.05）。LPS组仔猪背最长肌中的MDA和LPO和水平显著高于NC组（P<0.01），而T-AOC、CAT、GSH-Px和T-SOD水平则显著降低（P<0.01），日粮添加蛹虫草可缓解这种异常的升高或降低。对Akt、t-mTOR、4EBP1、FOXO1磷酸化水平进行检测，发现LPS组相比于NC组具有降低的趋势，而MAFbx、MuRF1的蛋白表达水平显著升高（P<0.05）。日粮中添加蛹虫草饲喂后，仔猪背最长肌Akt、FOXO1磷酸化水平显著提高（P<0.05），而MAFbx、MuRF1蛋白表达水平显著降低（P<0.05）。【结论】日粮中添加蛹虫草可以提高断奶仔猪生长性能和免疫性能；蛹虫草可以抑制LPS导致的背最长肌粗蛋白和总氨基酸含量降低；蛹虫草可以抑制肝脏炎症和肝细胞过度增殖和凋亡，并提高了肝脏抗氧化能力；蛹虫草可以抑制断奶仔猪背最长肌炎症、氧化应激和蛋白质分解。总之，日粮中添加蛹虫草可以提高断奶仔猪生长和免疫性能，缓解LPS导致的肝损伤及蛋白质的降解，为筛选新型饲料添加剂及预防断奶仔猪免疫应激提供理论依据。

关键词: 蛹虫草, 早期断奶仔猪, 脂多糖, 免疫应激, 肝损伤, 蛋白质代谢

Abstract:

【Objective】 This study aims to explore the effect of cordyceps militaris on the growth and immune performance of early weaned piglets and the mechanism of regulating immune stress. 【Method】A total of Duroc × Landrace × large 144 early-weaned piglets of the same age and similar initial weight were randomly divided into 4 groups: NC (basal diet), LPS (LPS+ basal diet), LPS+500CM (LPS+ basal diet+ 500 mg·kg^-1 CM) and LPS+1000CM (LPS+ basal diet+ 1 000 mg·kg^-1 CM). Group NC and group LPS were fed with basal diet, while group LPS+500CM and group LPS+1000CM were fed by fully mixed basal diet with cordyceps militaris (CM) powder according to the proportion. After feeding, 6 piglets in each group (half male and half female) were randomly selected and slaughtered. Group LPS, group LPS+500CM and group LPS+1000CM were intraperitoneally injected with LPS according to body weight 4 hours before slaughter, and group NC was injected with the same amount of normal saline. After slaughtering, the longissimus dorsi, heart, liver, spleen, pancreas and kidney were collected for the next detection. 【Result】Statistical analysis of body weight and feed intake showed that adding Cordyceps militaris to the diet of piglets had no notable effect on the average daily feed intake of piglets, while the weight of piglets supplemented with 500 mg·kg^-1 CM and 1 000 mg·kg^-1CM was respectively higher than that of the control group. 8.58% (P<0.05) and 9.08% (P<0.05). Compared with the NC group, the spleen index of piglets in the LPS group was significantly lower (P<0.05), while the spleen index of the piglets in the LPS+500CM group and LPS+1000CM group was significantly higher than that in the LPS group (P<0.05). The detection of liver function biomarkers and total bile acids showed that the liver ALT level of piglets in the LPS group was significantly higher than that of the NC group (P<0.05), and the levels of AST, LDH, and total bile acids showed an upward trend. The ALT, AST, LDH and total bile acid levels of the LPS+500CM group and LPS+1000CM group with Cordyceps militaris added to the feed were significantly lower than those of the LPS group. The detection of liver antioxidant indicators and inflammatory factor expression showed that the levels of MDA, LPO, T-AOC and the expression of inflammatory factors in the liver of piglets in the LPS group were significantly increased (P<0.05), while the levels of CAT, GSH-Px and T-SOD were significantly higher. There was a significant decrease (P<0.01), and feeding piglets with Cordyceps militaris could significantly alleviate the increase or decrease in these indicators. The crude protein and amino acid contents of skeletal muscle in the LPS group were significantly reduced (P<0.01), and adding Cordyceps militaris to the diet could significantly alleviate the decrease in protein and amino acid contents caused by LPS (P<0.01). Compared with NC, the expression of inflammatory factors in the LPS group was significantly higher (P<0.05), while the expression of inflammatory factors after feeding Cordyceps militaris in the diet was significantly lower than that in the LPS group (P<0.05). The levels of MDA and LPO in the longissimus dorsi muscle of piglets in the LPS group were significantly higher than those in the NC group (P<0.01), while the levels of T-AOC, CAT, GSH-Px and T-SOD were significantly lower (P<0.01), adding Cordyceps militaris to the diet can alleviate this abnormal increase or decrease. The phosphorylation levels of Akt, t-mTOR, 4EBP1, and FOXO1 were detected and it was found that the LPS group had a decreasing trend compared with the NC group, while the protein expression levels of MAFbx and MuRF1 were significantly increased (P<0.05). After Cordyceps militaris was added to the diet, the phosphorylation levels of Akt and FOXO1 in the longissimus dorsi muscle of piglets were significantly increased (P<0.05), while the protein expression levels of MAFbx and MuRF1 were significantly decreased (P<0.05)【Conclusion】 The results show that adding CM to the diet can improve the growth and immune performance of weaned piglets; CM can inhibit the decrease in crude protein and total amino acid content of longissimus dorsi caused by LPS; CM can inhibit liver inflammation and excessive proliferation and apoptosis and hepatocytes and improve liver antioxidant capacity; CM can inhibit longissimus dorsi inflammation, oxidative stress and protein degradation in weaned piglets. Overall, adding CM to the deit can improve the growth and immune performance of weaned piglets, alleviate liver damage and protein degradation cause by LPS, and provide a theoretical basis for screening new feed additives and preventing immune stress in weaned piglets.

Key words: cordyceps militaris, early-weaned piglets, lipopolysaccharide, immune stress, liver injury, protein metabolism

蔡睿杰, 褚艺心, 史新娥, 靳建军, 杨公社. 日粮添加蛹虫草缓解脂多糖诱导的仔猪肝损伤和骨骼肌蛋白质降解[J]. 中国农业科学, 2024, 57(12): 2467-2482.

CAI RuiJie, CHU YiXin, SHI XinE, JIN JianJun, YANG GongShe. Dietary Addition of Cordyceps Militaris Can Alleviate Lipopolysaccharide- Induced Liver Damage and Skeletal Muscle Protein Degradation in Early Weaning Piglets[J]. Scientia Agricultura Sinica, 2024, 57(12): 2467-2482.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.12.015

https://www.chinaagrisci.com/CN/Y2024/V57/I12/2467

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饲料成分 Ingredient	含量 Content (%)	营养物质 Nutrient component	对照组 Control	蛹虫草组1 500 CM	蛹虫草组2 1000 CM
玉米Corn	47.62	水分Moisture (%)	11.34	11.64	11.53
挤压玉米Extruded corn	32.30	干物质Dry matter (%)	88.66	88.36	88.47
豆粕Soybean meal	5.00	粗蛋白Crude protein (%)	17.71	17.52	17.54
膨化大豆 Extruded soybean	4.00	粗纤维Crude fiber (%)	2.09	1.98	1.98
豆油Soybean oil	1.50	灰分Crude ash (%)	4.40	4.37	4.36
乳清粉Whey mist	0.64	钙Calcium (%)	0.82	0.82	0.82
鱼粉Fish meal	3.50	磷phosphorus (%)	0.63	0.64	0.63
蔗糖Saccharose	3.00	表观可消化磷	0.18	0.18	0.18
维生素和矿物质预混料 Vitaminsand minerals Premix^a	0.60	Available phosphorus (%)
氯化钠NaCl	0.40	消化能DE (kcal·kg^-1)	3631.84	3619.00	3623.32
赖氨酸硫酸盐Lysine sulfate (70%)	0.42	净能NE (kcal·kg^-1)	2624.98	2606.67	2612.18
苏氨酸Threonine (98%)	0.11
蛋氨酸Methionine (98%)	0.15
色氨酸Tryptophan (98%)	0.02
磷酸氢钙Calcium monophosphate	0.64
植酸酶Phytase	0.10
合计Total	100

营养成分 Nutrition content	含量 Content (%)	活性物质 active substance composition	含量 Content
粗蛋白CP	15.97	虫草多糖Cordyceps polysaccharide(%)	4.96
粗脂肪CF	2.79	虫草酸Cordycepic acid(%)	3.75
灰分Ash	3.18	总甾醇Total sterols(%)	0.60
钙Ca	0.15	虫草素Cordycepin(mg·kg^-1)	1654.56
磷P	0.61	腺苷Adenosine(mg·kg^-1)	440.88
		肌苷Inosine(mg·kg^-1)	83.27

项目 Item		指标 Index			检验方法 Testing method
项目 Item		特级 Special	一级 Level 1	二级 Level 2	检验方法 Testing method
水分 Moisture (g/100g)	≤	12.0			GB 5009.3
灰分 Ash content (g/100g)	≤	10.0			GB 5009.4
虫草素 Cordycepin (mg·kg^-1)		≥10000	5000—9999	1000—4999	NY/T 2116
腺苷 Adenosine (mg·kg^-1)	≤	550			NY/T 2116
粗多糖 Crude polysaccharide (g/100g)	≤	2.5			NY/T 1676
杂质 Impurities (%)	≤	1.0			GB/T 12533
二氧化碳残留量（以SO₂计） Carbon dioxide residual amount (calculated as S O₂，g·kg^-1)	≤	0.05			GB 5009.34

基因Gene	引物序列 Primer sequence (5'-3')
TLR4	F: 5'-TTAGTTCTCACCTTCTCCTG-3'
TLR4	R: 5'-GTTCATTCCTCACCCAGTCTTC-3'
MyD88	F: 5'-GATGGTAGCGGTTGTCTCTGAT-3'
MyD88	R: 5'-GATGCTGGGGAACTCTTTCTTC-3'
TRAF6	F: 5'-CAAGAGAATACCCAGTCGCACA-3'
TRAF6	R: 5'-ATCCGAGACAAAGGGGAAGAA-3'
IRAK1	F: 5'-CAAGGCAGGTCAGGTTTCGT-3'
IRAK1	R: 5'-TTCGTGGGGCGTGTAGTGT-3'
NOD1	F: 5'-CTGTCGTCAACACCGATCCA-3'
NOD1	R: 5'-CCAGTTGGTGACGCAGCTT-3'
NOD2	F: 5'-GAGCGCATCCTCTTAACTTTCG-3'
NOD2	R: 5'-ACGCTCGTGATCCGTGAAC-3'
RIPK2	F: 5'-CAGTGTCCAGTAAATCGCAGTTG-3'
RIPK2	R: 5'-CAGGCTTCCGTCATCTGGTT-3'
NF-κB	F: 5'-AGTACCCTGAGGCTATAACTCGC-3'
NF-κB	R: 5'-TCCGCAATGGAGGAGAAGTC-3'
TNF-α	F: 5'-TCCAATGGCAGAGTGGGTATG-3'
TNF-α	R: 5'-AGCTGGTTGTCTTTCAGCTTCAC-3'
Bax	F: 5'-GACGCTGGACTTCCTTCGAG-3'
Bax	R: 5'-GTGGCCCGAGAGAGGTTTATT-3'
Bcl-2	F: 5'-GCTACTTACTGCCAAAGGGA-3'
Bcl-2	R: 5'-TTCAGGCGGAGCTGTAAGAG-3'
Caspase3	F: 5'-GGAATGGCATGTCGATCTGGT-3'
Caspase3	R: 5'-ACTGTCCGTCTCAATCCCAC -3'
Caspase8	F: 5'-TCTGCGGACTGGATGTGATT-3'
Caspase8	R: 5'-TCTGAGGTTGCTGGTCACAC-3'
Caspase9	F: 5'-AATGCCGATTTGGCTTACGT-3'
Caspase9	R: 5'-CATTTGCTTGGCAGTCAGGTT-3'
PCNA	F: 5'-ATGCCTTCTGGTGAATTTGC-3'
PCNA	R: 5'-TCCATTTCCGAGTTCTCCAC-3'

项目 Item	NC	500CM	1000CM	P值 P value
项目 Item	NC	500CM	1000CM	P₁	P₂	P₃
第一周 First week	0.57±0.13	0.63±0.17	0.52±0.14	0.450	0.442	0.191
第二周 Second week	0.86±0.18	0.86±0.13	0.80±0.12	0.973	0.458	0.396
第三周 Third week	0.75±0.16	0.72±0.15	0.73±0.13	0.688	0.717	0.882
第四周 Forth week	0.93±0.15	0.89±0.11	0.87±0.15	0.641	0.513	0.772