【作者】 李莹；

【导师】 刘芳萍；

【作者基本信息】 东北农业大学 ， 兽医博士（专业学位）， 2019， 博士

【摘要】 脂多糖(LPS)是革兰氏阴性菌细胞壁的主要成分之一,肠道内的阴性菌溶解后可释放大量的LPS进入血液或由门静脉进入肝脏,对雏鸡的肝脏和脾脏等组织造成损伤,破坏雏鸡的免疫系统,影响雏鸡的生长性能和免疫功能,甚至导致雏鸡死亡,给家禽养殖业造成严重的经济损失。二氢杨梅素(DHM)是一种类黄酮化合物,其结构明确,存在于多种植物中,具有清除氧自由基、抗氧化和抗炎等多种生理活性,在畜牧业发展中拥有广阔的应用前景。本实验以海兰白雏鸡为研究对象,在雏鸡饲料中添加不同剂量的DHM,研究其对雏鸡生长性能和免疫功能的影响及对LPS诱导雏鸡肝脾损伤的保护作用,为DHM在兽医临床上的广泛应用提供理论基础和科学依据。试验内容如下:(1)研究DHM对雏鸡生长性能和免疫功能的影响:1日龄雏鸡适应性饲喂7 d,于雏鸡7日龄时进行新城疫疫苗免疫,然后将雏鸡分为五组,在饲料中添加不同剂量(0.025%、0.05%、0.1%、0.2%)的DHM,连续饲喂35 d。每周称量雏鸡空腹体重,计算平均日增重(ADG)和平均料重比(F/G);称量体重后心脏采血分离血清,剖取心脏、肝脏、肾脏、脾脏、胸腺和法氏囊进行称重,计算脏器指数。采用生化自动分析仪检测血清总蛋白(TP)、白蛋白(ALB)、总胆固醇(TC)、甘油三酯(TG)、葡萄糖(GLU)、直接胆红素(DBIL)、钙(Ca)和磷(P)的含量和谷草转氨酶(AST)、谷丙转氨酶(ALT)、乳酸脱氢酶(LDH)的活性;ELISA法检测血清中IgG、IL-2、IFN-γ含量;血凝抑制法检测血清中新城疫抗体效价;将肝脏的部分组织进行匀浆,采用试剂盒检测肝脏匀浆中总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性、总抗氧化能力(T-AOC)及丙二醛(MDA)含量。(2)研究DHM对雏鸡肝脏和脾脏中TLR2、TLR4及炎性因子mRNA表达的影响:将7日龄雏鸡分为五组,在饲料中添加不同剂量(0.025%、0.05%、0.1%、0.2%)的DHM,连续饲喂35 d。于雏鸡43日龄时,颈动脉放血处死雏鸡,分离肝脏和脾脏组织,用于提取总RNA,RT-PCR法检测雏鸡肝脏和脾脏中TLR2、TLR4及炎性因子(IL-1β、IL-6、IL-8、IL-10和TNF-α)mRNA的表达。(3)研究DHM对LPS诱导雏鸡肝脾损伤的保护作用:将7日龄雏鸡分为五组,在饲料中添加不同剂量(0.025%、0.05%、0.1%、0.2%)的DHM,连续饲喂14 d。于雏鸡22日龄时,模型组和DHM添加组腹腔注射LPS(60 mg/kg),对照组腹腔注射等量的生理盐水,注射12 h后收集血液和肝脾组织,检测血清中ALT、AST和LDH活性及DBIL、TC和TG的含量;肝脏和脾脏的部分组织用于提取总RNA和蛋白,RT-PCR法检测雏鸡肝脏和脾脏中TLR2、TLR4、MyD88、NF-κB p65及炎性因子(IL-1β、IL-6、IL-8、IL-10和TNF-α)mRNA的表达,western blot法检测肝脾组织中NF-κB p65蛋白的磷酸化水平;肝脏和脾脏的部分组织用于制作石蜡切片和透射电镜切片,观察肝脾组织形态和超微形态。试验结果如下:(1)DHM对雏鸡生长性能影响结果:与对照组相比,0.05%DHM显著增加28日龄、35日龄和42日龄的雏鸡体重(p<0.05或p<0.01),增加7-21日龄雏鸡的ADG并降低22-42日龄雏鸡的F/G;在0.05%DHM添加组中,35日龄雏鸡血清中TC和TG含量显著高于对照组(p<0.05),不同日龄雏鸡血清中GLU含量显著高于对照组(p<0.05或p<0.01),14日龄、21日龄和28日龄雏鸡血清中LDH活性和14日龄雏鸡血清中DBIL含量显著低于对照组(p<0.05或p<0.01)。(2)DHM对雏鸡免疫功能影响结果:在0.05%DHM添加组中,14日龄雏鸡的脾脏指数、胸腺指数和法氏囊指数及42日龄雏鸡的脾脏指数显著高于对照组(p<0.05或p<0.01);与对照组相比,0.025%、0.05%和0.1%DHM分别明显提高了21日龄雏鸡的脾脏指数、胸腺指数和法氏囊指数(p<0.05或p<0.01);在0.025%、0.05%和0.1%DHM添加组中,14日龄雏鸡血清中IL-2、IFN-γ和IgG含量极显著高于对照组(p<0.01),0.05%DHM添加组中,21日龄雏鸡血清中IL-2、IFN-γ和IgG含量极显著高于对照组(p<0.01);0.05%、0.1%和0.2%DHM添加组中,14日龄、28日龄和42日龄雏鸡血清中的新城疫抗体效价均明显高于对照组(p<0.05或p<0.01);在0.025%和0.05%DHM添加组中,21日龄和42日龄雏鸡肝脏内的GSH-Px和A-TOC活性显著高于对照组(p<0.05或p<0.01),0.05%DHM提高21和42日龄雏鸡肝脏内SOD活性,与对照组相比差异显著(p<0.05)。(3)RT-PCR结果显示:与对照组相比,0.05%和0.1%DHM可明显促进肝脏中TLR4、IL-1β、IL-6、IL-8和IL-10的mRNA表达,明显抑制肝脏中TLR2和TNF-α的mRNA表达(p<0.05或p<0.01);与对照组相比,0.05%和0.1%DHM可显著促进脾脏中TLR2、TLR4和TNF-α的mRNA表达,显著抑制脾脏中IL-1β、IL-6、IL-8和IL-10的mRNA表达(p<0.05或p<0.01)。(4)腹腔注射LPS后,雏鸡血清中AST、ALT和LDH活性及DBIL、TG含量显著高于对照组(p<0.05或p<0.01),0.05%DHM可极显著降低雏鸡血清中AST和ALT及LDH活性及DBIL和TG含量(p<0.01)。(5)腹腔注射LPS后,与对照组相比,TLR2的mRNA表达在肝脏和脾脏中均显著升高(p<0.05或p<0.01),TLR4的mRNA表达在脾脏中极显著降低(p<0.01),MyD88和NF-κB p65的mRNA表达在肝脏中极显著降低而在脾脏中极显著升高(p<0.01),其中NF-κB p65蛋白的磷酸化水平在肝脏中无显著变化,而在脾脏中极显著降低(p<0.01);IL-1β和IL-8的mRNA的表达在肝脏和脾脏中升高显著(p<0.05或p<0.01),IL-6的mRNA表达在肝脏中极显著降低(p<0.01),IL-10的mRNA表达在肝脏和脾脏中均极显著降低(p<0.01),TNF-α的mRNA表达在肝脏中升高极显著(p<0.01)。与模型组相比,0.05%或0.1%DHM可极显著降低肝脏中TLR2、IL-1β、IL-8和TNF-α的mRNA表达(p<0.01),显著升高肝脏中TLR4、MyD88、IL-6和IL-10的mRNA表达(p<0.05或p<0.01),DHM的添加对肝脏中NF-κB p65的mRNA表达及其磷酸化水平无显著影响;0.025%DHM可显著降低脾脏中TLR2的mRNA表达(p<0.05),极显著升高脾脏中IL-10的mRNA表达(p<0.01),0.05%DHM可显著升高脾脏中TLR4、IL-6和IL-10的mRNA表达(p<0.01),0.05%和0.1%DHM可明显降低脾脏中MyD88、IL-1β和IL-8的mRNA表达(p<0.05或p<0.01);在DHM(0.05%和0.1%)添加组中,脾脏中NF-κB p65的mRNA显著降低(p<0.01),而NF-κB p65蛋白的磷酸化水平极显著升高(p<0.01)。(6)形态学检查结果显示,雏鸡腹腔注射LPS后,肝脏组织中细胞肿胀,肝索结构紊乱且有炎性细胞浸润,脾脏组织中白髓和红髓界限模糊;超微形态学结果显示,肝细胞的内质网中有大量空泡形成,而脾脏细胞中的内质网中有少量空泡形成。在雏鸡饲料中添加0.05%和0.1%DHM可减轻肝脏和脾脏的损伤情况,缓解LPS对雏鸡肝脏和脾脏组织造成的损伤。实验结果表明,在海兰白雏鸡饲料中添加DHM,可促进雏鸡体重的增加,改善雏鸡的生化指标,增加血清中抗体和细胞因子含量,调节雏鸡肝脏和脾脏中TLR2、TLR4和炎性因子(IL-1β、IL-6、IL-8、IL-10和TNF-α)mRNA的表达;DHM可通过调节炎症反应中TLR2、TLR4、MyD88、NF-κB p65和炎性因子mRNA在雏鸡肝脏和脾脏中的表达,以及肝脾组织中NF-κB p65蛋白的磷酸化水平,改善雏鸡肝脾组织的损伤程度。综上可知,0.05%DHM可提高雏鸡的生长性能和免疫功能,缓解LPS对雏鸡造成的肝脾损伤。更多还原

【Abstract】 Lipopolysaccharide(LPS)is one of the major components of the cell walls of gram-negative bacteria.Bacterium of negative of gut dissolves can release a large number of LPS into the blood or into the liver via portal vein.LPS can damage the liver and spleen tissue and destroy the immune system of chicken,and affect the growth performance and immune function,and even cause death,resulting in serious economic losses.Dihydromyricetin(DHM)is a flavonoid compound.DHM exists in many kinds of plants and its structure is clear.DHM has a variety of physiological activities such as scavenging oxygen free radicals,antioxidant and anti-inflammatory and has a high application prospect in the production of animal husbandry.In this experiment,different dose DHM were added into the blue sea chicks feed to study the effect of DHM on the growth performance and immune function,and the protective effect of DHM on liver and spleen injury induced by LPS,which provides the theoretical and scientific basis for the wide application of DHM in veterinary clinic.The experiments are designed as followed:(1)Study the effect of DHM on growth performance and immune function of chicks: The 1-day-old chicks were given adaptive feeding for 7 days,and the chickens were immunized with Newcastle disease vaccine at the age of 7 days.Then the chicks were divided into five groups and different dose DHM(0.025%,0.05%,0.1%,0.2%)was added to the feed for 35 days.Then the chickens were weighed on an empty stomach weekly to calculate the average daily weight gain(ADG)and average feed weight ratio(F/G).After weighing the chicks,blood samples were collected from heart to separate the serum,and the organs(heart,liver,kidney,spleen,thymus and bursa)were isolated and weighted to calculate the organ index.The content of serum total protein(TP),albumin(ALB),total cholesterol(TC),triglyceride(TG),glucose(GLU),direct bilirubin(DBIL),calcium(Ca),phosphorus(P),as well as the activity of aspertate aminotransferase(AST),alanine aminotransferase(ALT)and lactic dehydrogenase(LDH)were detected by automatic biochemical analyser.The concentration of serum Ig G,IL-2,IFN-γ were detected by ELISA.The antibody titer of Newcastle disease in serum were tested by hemagglutination inhibition(RPHI).The activities of total superoxide dismutase(T-SOD),glutathione peroxidase(GSH-Px),total antioxidant capacity(T-AOC)and malondialdehyde(MDA)in liver homogenate were tested with the kit.(2)Study the effect of DHM on the m RNA expression of TLR2,TLR4 and inflammatory factor in the liver and spleen of chicks: the 7-day-old chicks were divided into five groups,and dose DHM(0.025%,0.05%,0.1%,0.2%)was added to the feed for 35 days.At the age of 43 days,the chickens were killed by carotid artery bleeding and the liver and spleen tissues were separated for extraction of total RNA.And then m RNA expression of TLR2,TLR4 and inflammatory factors(IL-1β,IL-6,IL-8,IL-10 and TNF-α)in the chicks liver and spleen were detected with RT-PCR.(3)Study the protective effect of DHM on liver and spleen injury induced by LPS in chicks: the 7-day-old chicks were divided into five groups,dose DHM(0.025%,0.05%,0.1%,0.2%)was added to the feed for 14 days.At the age of 43 days,the chicks in the model group and DHM added group were intraperitoneally injected with LPS(60 mg/kg),and the chicks in the control group were intraperitoneally injected with equivalent normal saline.Intraperitoneal injection after 12 hours,blood was collected for testing ALT and AST activity and LDH and DBIL content and the liver and spleen tissue were collectedthe for extraction of total RNA and protein,making paraffin section and ultrafine slices.The m RNA expression of TLR2,TLR4,My D88,NF-κB p65 and inflammatory factors(IL-1β,IL-6,IL-8,IL-10 and TNF-α)in the liver and spleen were detected with RT-PCR.Western blot was performed to determine the phosphorylation level of NF-κB p65 protein in liver and spleen tissues.The test results are as follows:(1)The effect of DHM on the growth performance of chicks: compared with the control group,0.05% DHM significantly increased the weight of chicks at 28,35 and 42 days(p<0.05 or p<0.01),increased the ADG of chicks at 7-21 days and decreased the F/G of chicks at 22-42 days.In the 0.05% DHM group,the level of TC and TG in serum of 35 days chicks were significantly higher than that of control group(p<0.05).The serum GLU content of different day-old chicks were significantly higher than that in control group(p<0.05 or p<0.01).The LDH levels in serum of 14,21 and 28 days chicks and the DBIL content in serum of 14 days chicks were lower than that in control group and the difference were significant(p<0.05 or p< 0.01).(2)The effect of DHM on the immune function of chickens: in the 0.05% DHM group,the spleen index,thymus index and bursa index of chickens at 14 days and the spleen index of chickens at 42 days were higher than those in the control group with significant differences(p<0.05 or p<0.01).Compared with the control group,0.025%,0.05% and 0.1% DHM increased the spleen index,thymus index and bursa index of 21 days chicks respectively with significant differences(p<0.05 or p<0.01).In the 0.025%,0.05% and 0.1% DHM group,the serum levels of IL-2,IFN-γ and Ig G in chicks at 14 days were significantly higher than those in the control group(p<0.01).In the 0.05% DHM group,the serum levels of IL-2,IFN-γ and Ig G in chicks at 21 days were significantly higher than those in the control group(p<0.01).The Newcastle disease antibody titers of 14,28 and 42 days chicks in the 0.05%,0.1% and 0.2% DHM group were significantly higher than those in the control group(p<0.05 or p<0.01).In the 0.025% and 0.05% DHM group,GSH-Px activity and A-AOC in the liver of 21 and 42-day-old chicks were significantly higher than those of the control group(p<0.05 or p<0.01),while 0.05% DHM increased SOD activity in the liver of 21 and 42-day-old chicks,which was significantly different from that of the control group(p<0.05).(3)RT-PCR results: compared to the control group,0.05% and 0.1% DHM promoted the m RNA expression of TLR4,IL-1β,IL-6,IL-8,IL-10 and inhibited the m RNA expression of TLR2,TNF-α in the liver(p<0.01).DHM(0.05% and 0.1%)can promote the m RNA expression of TLR2,TLR4,TNF-α and inhibit m RNA expression of IL-1β,IL-6,IL-8 and IL-10 in the spleen(p<0.01).(4)After intraperitoneal injection of LPS,the activity of AST,ALT and LDH as well as the content of DBIL and TG in chicks serum were significantly higher than those in the control group(p<0.05 or p<0.01).0.05% DHM significantly reduced the activities of LDH,AST and ALT,and the levels of DBIL and TG in the serum of chicks(p<0.01).(5)After intraperitoneal injection of LPS,compare with control group,TLR2 m RNA expressions was significantly increased in the liver and spleen(p<0.05 or p<0.01).Compare with control group,TLR4 m RNA expression was not significantly changed in the liver(p<0.01).The m RNA expression of My D88 and NF-κB p65 were sgnificantly lower in liver than that of control group and siginficantly higher in spleen than that of control group(p<0.01).Moreover,the phosphorylation level of NF-κB p65 was not significantly changed in the liver and decreased significantly in the spleen(p<0.01).Compared to control group,the m RNA expression of IL-1β and IL-8 was significantly increased in the liver and spleen(p<0.05 or p<0.01),while the m RNA expression of IL-6 was siginficantly decreased in the liver(p<0.01).The m RNA expression of IL-10 in the liver and spleen was sinificantly lower than that in the control group(p<0.01),while the m RNA expression of TNF-α in liver were significantly higher than that in the control group(p<0.01).There was no significant change in the phosphorylation level of NF-κB p65 in the liver,but it was significantly decreased in the spleen(p<0.01).Compared with model group,0.05% and 0.1% DHM significantly reduced the m RNA expression of TLR2,IL-1β,IL-8 and TNF-α,(p<0.01),and significantly increased the m RNA expression of TLR4,My D88、IL-6 and IL-10 in the liver(p<0.05 or p<0.01).DHM had no significant effect on the m RNA expression of NF-κB p65 and the phosphorylation level of NF-κB p65 protein in the liver.Meanwhile,0.025% DHM can inhibit TLR2 expression and promote IL-10 expression in the spleen with significantly difference(p<0.05 or p<0.01).0.05% DHM could significantly increase the expression of TLR4,IL-6 and IL-10 expression(p<0.01).0.1% DHM reduced the m RNA expression of My D88,IL-1β and IL-8 in the spleen with significantly difference(p<0.05 or p<0.01).In the group of DHM(0.05% and 0.1%),the m RNA of NF-κB p65 was significantly reduced(p<0.01),and the phosphorylation level of NF-κB p65 protein was significantly increased in spleen(p<0.01).(6)Morphological examination results showed that after intritonesl injection of LPS,hepatocytes were swollen,hepatic cords were disorganized with the inflmma tory cell infiltration,and the boundaries between white pulp and red pulp in the spleen tissue were blurred.The ultrastructural results showed a large number of vacuoles were formed in the endoplasmic reticulum of liver cells,while a small number of vacuoles were formed in the endoplasmic reticulum of spleen cells.Adding 0.05% and 0.1% DHM can alleviate the damage of liver and spleen cells of chicks.The results showed that adding DHM into the feed of helan white chicks could promote the weight gain of chicks,improve the biochemical indexes of serum,increase the content of antibodies and cytokines in serum,and regulate the m RNA expression of TLR2,TLR4 and inflammatory factors(IL-1β,IL-6,IL-8,IL-10 and TNF-α)in the liver and spleen of chicks.DHM can improve the degree of injury of chick liver and spleen by regulating the m RNA expression of TLR2,TLR4,My D88,NF-κB p65 and inflammatory factor,and the phosphorylation level of NF-κB p65 protein in the liver and spleen tissue.In conclusion,0.05% DHM can improve the growth performance and immune function of chicks,alleviate the liver and spleen damage caused by LPS.更多还原