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

Abstract

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

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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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

Dietary Addition of Cordyceps Militaris Can Alleviate Lipopolysaccharide- Induced Liver Damage and Skeletal Muscle Protein Degradation in Early Weaning Piglets

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项目 Item	NC	LPS	LPS+500 M	LPS+1000 CM
心脏指数Cardiac index	5.71±0.06aA	5.36±0.08aA	5.33±0.33aA	5.67±0.14aA
肝脏指数Liver index	25.45±0.71aA	27.82±0.73aA	27.23±1.84aA	29.25±1.38aA
脾脏指数Spleen index	2.51±0.05aA	2.13±0.05bA	2.39±0.27aA	2.61±0.15aA
胰脏指数Pancreatic index	2.46±0.16aA	2.45±0.13aA	2.12±0.16aA	1.95±0.21aA
肾脏指数Kidney index	6.04±0.28aA	6.90±0.05bA	6.23±0.31bA	6.54±0.16abA

项目 Item	NC	LPS	LPS+500CM	LPS+1000CM	P值P value
项目 Item	NC	LPS	LPS+500CM	LPS+1000CM	P₁	P₂	P₃
粗蛋白Crude protein	19.52±0.03	17.69±0.23	18.59±0.10	18.39±0.22	<0.01	0.022	0.089

项目Item	NC	LPS	LPS+500 CM	LPS+1000 CM
Asp	0.5±0.05aA	0.14±0.03cB	0.39±0.04bA	0.54±0.09aA
Glu	2.66±0.1aA	1.88±0.04cB	2.35±0.16abAB	1.99±0.1bcB
Ser	1.55±0.07aA	1.5±0.02aAB	1.53±0.09aAB	1.31±0.08bB
Gly	1.64±0.09aA	1.37±0.03aA	1.56±0.11aA	1.63±0.12aA
His	1.4±0.05aA	1.58±0.19aA	1.38±0.26aA	1.61±0.1aA
Arg	1.43±0.12aA	1.38±0.09aA	1.51±0.12aA	1.46±0.18aA
Thr	0.95±0.01aA	0.37±0.04cC	0.95±0.06aA	0.76±0.04bB
Ala	1.54±0.19aA	1.56±0.27aA	1.51±0.22aA	1.57±0.15aA
Pro	1.59±0.16aA	1.48±0.24aA	1.38±0.14aA	1.19±0.09aA
Tyr	1.11±0aA	0.48±0.01cC	0.97±0.07bAB	0.89±0.03bB
Val	1.12±0.02aA	0.41±0.02cC	1.01±0.07aAB	0.87±0.05bB
Met	0.96±0.01aA	0.4±0.02cC	0.74±0.06bB	0.69±0.02bB
Cys	0.12±0.01aA	0.07±0cC	0.1±0.01bAB	0.09±0bBC
Ile	1.22±0aA	0.64±0.03cC	1.09±0.06bAB	1±0.02bB
Leu	1.59±0.03aA	0.57±0.03cC	1.3±0.12bB	1.23±0.05bB
Phe	1.76±0.03aA	0.83±0.03dC	1.26±0.11bB	1.09±0.05cB
Lys	1.52±0.02aA	0.7±0.03cC	1.35±0.1aAB	1.1±0.08bB
EAA	10.99±0.09aA	6.51±0.38cC	9.65±0.35bB	9.04±0.2bB
FAA	9.21±0.21aA	6.26±0.15cC	8.04±0.27bAB	7.71±0.16bB
TAA	22.65±0.15aA	15.36±0.49dC	20.38±0.63bB	19.01±0.35cB

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[3]	YING ShiJia, DAI ZiChun, GUO JiaJia, SHI ZhenDan. Time Course Effect of Lipopolysaccharide on Toll-Like Receptors Expression in the Goose Follicular Stroma [J]. Scientia Agricultura Sinica, 2017, 50(6): 1147-1156.
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项目 Item	NC	500CM	1000CM	P值 P value
项目 Item	NC	500CM	1000CM	P₁	P₂	P₃
初始体重 Initial weight	8.26±0.26	8.89±0.35	8.45±0.21	0.112	0.635	0.261
第一周 First week	12.46±0.50	13.44±0.41	12.72±0.32	0.100	0.658	0.224
第二周 Second week	16.32±0.53	17.61±0.55	15.77±0.29	0.059	0.416	0.008
第三周 Third week	18.15±0.55	19.36±0.66	19.36±0.71	0.313	0.188	0.754
第四周 Forth week	20.04±0.50	21.76±0.77	21.86±0.34	0.035	0.026	0.897