【作者】 王静；

【导师】 李光玉；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2021， 博士

【摘要】 本文主要研究饲粮中添加白芍提取物对乌苏里貉生产性能及其有效成分芍药内酯苷(Albiflorin,ALB)抗肠炎机制。通过饲养试验及屠宰试验,研究饲粮中添加不同水平白芍提取物对育成期乌苏里貉生长性能、营养物质消化率、氮代谢、血清生化、小肠炎症及形态等指标的影响,以筛选出乌苏里貉育成期白芍提取物适宜添加水平。采集乌苏里貉直肠内容物,提取样本中细菌基因组DNA,进行16S V3-V4区基因测序,探究白芍提取物对乌苏里貉肠道微生物组成和多样性的影响。选用白芍提取物中的主要单萜苷类化合物ALB,评估ALB对甲氨蝶呤(Methotrexate,MTX)诱导的大鼠小肠炎的影响,并探讨其抗肠炎的机制。本研究主要由三部分组成:试验一:饲粮中添加白芍提取物对乌苏里貉生长性能、营养物质消化率及小肠形态的影响本试验旨在研究饲粮中添加白芍提取物对乌苏里貉生长性能、营养物质消化率、氮代谢、血清生化、小肠炎症及形态等指标的影响。将50只(120±5日龄)健康乌苏里貉公貉,随机分成5组,每组10个重复,每个重复1只。试验采用单因素随机试验设计,在基础饲粮中添加5个水平的白芍提取物,添加水平分别为0 g/kg、1 g/kg、2 g/kg、4 g/kg、8 g/kg。预饲期7 d,正试期40 d。结果表明:白芍1 g/kg组、白芍2 g/kg组平均日采食量显著高于其他各组(P<0.01),各组乌苏里貉末重、平均日增重、料重比差异不显著(P>0.05);白芍8 g/kg组干物质排出量显著高于其他各组(P<0.01),干物质消化率、粗蛋白质消化率显著低于其他各组(P<0.01),各组脂肪消化率无显著差异(P>0.05);白芍1 g/kg组、白芍2 g/kg组氮沉积均显著高于白芍8 g/kg组(P<0.05);随着饲粮中白芍提取物含量的逐渐增高,碱性磷酸酶活性先增高后降低,白芍2 g/kg组显著大于白芍0 g/kg组(P<0.05),各组乳酸脱氢酶、谷草转氨酶、谷丙转氨酶无显著差异(P>0.05);白芍4 g/kg组血清SOD活力显著大于其他各组(P<0.01),各组GSH-PX、MDA无显著差异(P>0.05);白芍2 g/kg组血清Ig A显著高于其他各组(P<0.05),各组Ig G、Ig M无显著差异(P>0.05);各组血清总蛋白、白蛋白、尿素、葡萄糖无显著差异(P>0.05);各组血清甘油三酯、胆固醇、低密度脂蛋白、高密度脂蛋白无显著差异(P>0.05);白芍1 g/kg组和白芍2 g/kg组小肠黏膜TNF-α的含量显著低于白芍0g/kg组(P<0.05),各组之间绒毛高度和隐窝深度无显著差异(P>0.05),与白芍0 g/kg组相比,白芍2 g/kg组绒毛高度与隐窝深度比值显著增加(P<0.05)。综上所述,在本试验条件下,饲粮中白芍提取物的添加水平为1～2 g/kg,有助于提高乌苏里貉的采食量和氮沉积,提高血清Ig A含量,降低小肠黏膜TNF-α的含量,提高小肠绒毛高度与隐窝深度的比值。试验二:饲粮中添加白芍提取物对乌苏里貉肠道微生物组成和多样性的影响本试验旨在研究饲粮中添加白芍提取物对乌苏里貉肠道微生物组成和多样性的影响。饲养试验如试验一,至第41 d屠宰试验时,随机选取白芍0 g/kg组、白芍2 g/kg组、白芍4 g/kg组和白芍8 g/kg组每组4只乌苏里貉,取其直肠内容物提取细菌组DNA进行16S V3～V4序列分析。结果表明:在门水平上,白芍0 g/kg组的优势菌门分别是厚壁菌门(42.33%±26.15%)、变形菌门(32.88%±20.07%)、梭杆菌门(9.78%±5.72%);白芍2 g/kg组的优势菌门分别是变形菌门(44.81%±21.63%)、厚壁菌门(30.83%±19.76%)、拟杆菌门(18.50%±21.62%);白芍4 g/kg组的优势菌门分别为厚壁菌门(73.78%±12.87%)、梭杆菌门(8.13%±8.42%)、变形菌门(5.76%±2.62%);白芍8 g/kg组的优势菌门分别为厚壁菌门(80.66%±20.40%)、变形菌门(19.22%±20.34%)、拟杆菌门(0.04%±0.03%)。相较于白芍0 g/kg组,白芍8 g/kg组的厚壁菌门的相对丰度显著增加(P<0.05)。在属水平上,白芍0 g/kg组的优势菌属是链球菌属(20.98%±13.14%)、脱硫弧菌属(14.17%±17.96%)、葡萄球菌属(9.82%±18.45%);白芍2 g/kg组的优势菌属是链球菌属(14.70%±23.08%)、伯杰氏菌属(8.68%±17.32%)、乳球菌属(6.37%±8.26%);白芍4 g/kg组的优势菌属是链球菌属(20.28%±23.15%)、葡萄球菌属(12.74%±22.15%)、魏斯氏菌属(8.18%±16.29%);白芍8 g/kg组的优势菌属是葡萄球菌属(29.79%±12.65%)、肠球菌属(11.31%±19.50%)、不动杆菌属(10.48%±17.57%)。Faith＿pd指数显示,白芍0 g/kg组与白芍8 g/kg组组间存在显著差异(P<0.05)。主坐标轴分析结果表明,基于Jaccard矩阵和Bray-Curtis矩阵算法,白芍8g/kg组与其他各组存在分离(P<0.05);组间差异分析结果表明,基于Jaccard矩阵和unweighted Uni Frac矩阵算法,白芍0 g/kg组与白芍8 g/kg组组间存在显著分离(P<0.05)。综上所述,乌苏里貉肠道优势菌门为厚壁菌门和变形菌门,白芍提取物能提高厚壁菌门和拟杆菌门的相对丰度。试验三:芍药内酯苷对甲氨蝶呤诱导的大鼠小肠炎的影响及机制研究本试验旨在研究白芍提取物中主要单萜成分芍药内酯苷对MTX诱导的大鼠小肠炎的影响,并探讨其可能的作用机制。将雄性SD大鼠随机分为4组:对照组,MTX组,MTX+ALB(20mg/kg)组,MTX+ALB(40 mg/kg)组。ALB 2组每天灌胃ALB 20 mg/kg或40 mg/kg,连续6d。对照组和MTX组大鼠灌胃等量生理盐水。ALB灌胃3d后,MTX组和ALB 2组大鼠腹腔注射MTX 7 mg/kg/d,连续3 d,同时对照组大鼠腹腔注射等量生理盐水。结果表明:ALB能显著增加MTX诱导的小肠炎大鼠的体重和肠道重量(P<0.05);ALB治疗后疾病活动性指数评分显著降低(P<0.01);在组织学检查中,ALB可显著减轻炎性细胞浸润,促进杯状细胞存活(P<0.01);此外,ALB还能降低MTX处理大鼠小肠组织中丙二醛、谷胱甘肽和超氧化物歧化酶的水平,并抑制活性氧的活性(P<0.05);在炎症相关因子检测中,ALB能显著降低MTX处理的肠组织中CD68+细胞的浸润(P<0.01),抑制髓过氧化物酶活性(P<0.01),抑制细胞间黏附分子-1和环氧合酶-2的表达(P<0.01)。ALB能有效抑制NLRP3的表达(P<0.01),抑制半胱氨酸天冬氨酸蛋白酶1p20和白细胞介素IL-1β和白细胞介素18的表达(P<0.01),并能阻断NF-κB p65的磷酸化和核转位(P<0.01)。综上所述,ALB通过抑制NF-κB/NLRP3信号通路而减轻MTX诱导的小肠炎。结论:综上所述,饲粮中白芍提取物的添加水平为1～2 g/kg,有助于提高乌苏里貉的采食量和氮沉积,提高血清Ig A含量,降低小肠黏膜TNF-α的含量,提高小肠绒毛高度与隐窝深度的比值,提高肠道厚壁菌门和拟杆菌门的相对丰度。白芍提取物的有效成分芍药内酯苷可抑制氧化应激和炎症反应,抑制NLRP3炎性小体激活,并调节NF-κB的表达,减轻大鼠小肠炎过程。即芍药内酯苷通过抑制NF-κB/NLRP3信号通路而减轻MTX诱导的大鼠小肠炎。更多还原

【Abstract】 The purpose of this study was to investigate the effects of dietary Paeoniae radix alba extract on the production performance of raccoon dog and the anti-enteritis mechanism of Albiflorin(ALB).Through feeding experiment and slaughter experiment,the effects of different supplemental levels of Paeoniae radix alba extract on growth performance,nutrient digestibility,nitrogen metabolism,serum biochemistry,small intestinal inflammation and morphology of raccoon dog were studied,so as to screen out the appropriate supplemental level of Paeoniae radix alba extract for raccoon dog during growing period.Bacterial genomic DNA was extracted from the samples which collected from the rectal contents of raccoon dog,then the 16 S V3～V4 genes were sequenced to investigate the effects of dietary Paeoniae radix alba extract on intestinal microbial composition and diversity of raccoon dog.The effects of ALB,the main monoterpene glycoside from Paeoniae radix alba extract,on Methotrexate(MTX)-induced enteritis in rats were evaluated,and its anti-enteritis mechanism was discussed.This study is mainly composed of three parts:Part 1: Effects of dietary Paeoniae radix alba extract on growth performance,nutrient digestibility and small intestine morphology of raccoon dog.This experiment was conducted to investigate the effects of Paeoniae radix alba extract on growth performance,nutrient digestibility,nitrogen metabolism,serum biochemistry,intestinal inflammation and morphology of raccoon dog.Fifty healthy male raccoon dogs(120 ± 5 day-old)were randomly divided into 5 groups with 10 replicates in each group and 1 in each replicate.A single factor randomized trial was designed in the experiment.Five levels of Paeoniae radix alba extract were added to the basic diet,and the addition levels were 0 g/kg,1 g/kg,2 g/kg,4 g/kg,8 g/kg respectively.The prefeeding period lasted for 7 days and the trial period lasted for 40 days.The results showed that: The average daily feed intake in group PRA1 and PRA2 was significantly higher than that in other groups(P<0.01),there were no significant differences in final weight,average daily gain and feed/gain among all groups(P>0.05);The dry matter excretion in group PRA8 was significantly higher than that in other groups(P<0.01)while the dry matter digestibility and protein digestibility in group PRA8 were significantly lower than those in other groups(P<0.01).There was no significant difference in fat digestibility among all groups(P>0.05);Nitrogen deposition in group PRA1 and PRA2 was significantly higher than that in group PRA8(P<0.05);With the increasing of dietary Paeoniae radix alba extract content,alkaline phosphatase activity was firstly increased and then decreased,and the activity of PRA2 was significantly higher than that of PRA0(P<0.05),but there were no significant differences in lactate dehydrogenase,aspartate aminotransferase and alanine aminotransferase among all groups(P>0.05);The activity of serum SOD in PRA4 was significantly higher than that in other groups(P<0.01),and there was no significant difference in GSH-Px and MDA in each group(P>0.05);The content of serum Ig A in group PRA2 was significantly higher than that in other groups(P<0.05),and there was no significant difference in Ig G and Ig M in each group(P>0.05);There was no significant difference in serum total protein,albumin,urea and glucose in each group(P>0.05);There was no significant difference in serum triglyceride,cholesterol,low density lipoprotein and high density lipoprotein(P>0.05);The content of TNF-α in intestinal mucosa in group PRA1 and group PRA2 was significantly lower than that in group PRA0(P<0.05);There was no significant difference in villus height and crypt depth among the groups(P>0.05);The ratio of villus height to crypt depth was significantly increased in group PRA2 compared with group PRA0(P<0.05).In conclusion,under the conditions of this experiment,when the dietary Paeoniae radix alba extract supplemental level is 1～2g/kg,it can improve the feed intake and nitrogen deposition,increase the serum Ig A content,decrease the content of TNF-α in small intestinal mucosa,and increase the ratio of villus height to crypt depth of raccoon dog.Part 2: Effects of dietary Paeoniae radix alba extract on the composition and diversity of intestinal microorganisms in raccoon dog.The purpose of this experiment was to investigate the effects of dietary Paeoniae radix alba extract on the composition and diversity of intestinal microorganisms in raccoon dog.The feeding experiment was as sample as experiment 1.On the 41 st day in the slaughtering experiment,4 raccoon dogs in each group were randomly selected from group PRA0,PRA2,PRA4 and PRA8,and the DNA of bacterial group in their rectal contents was extracted for 16 S V3～V4 sequence analysis.The results showed that:at the phylum level,the dominant bacteria in group PRA0 were Firmicutes(42.33% ± 26.15%),Proteobacteria(32.88%±20.07%),Fusobacteria(9.78%±5.72%);The dominant bacteria in group PRA2 were Proteobacteria(44.81%±21.63%),Firmicutes(30.83%±19.76%),Bacteroidetes(18.50%±21.62%);The dominant bacteria in group PRA4 were Firmicutes(73.78%±12.87%),Fusobacteria(8.13% ± 8.42%),Proteobacteria(5.76% ± 2.62%);The dominant bacteria in group PRA8 were Firmicutes(80.66%±20.40%),Proteobacteria(19.22%±20.34%),Bacteroidetes(0.04%±0.03%).The relative abundance of Firmicutes in group PRA8 was significantly higher than that in group PRA0(P<0.05).At the genus level,the dominant bacteria in group PRA0 were Streptococcus(20.98%±13.14%),Desulfovibrio(14.17%±17.96%),Staphylococcaceae＿Staphylococcus(9.82%±18.45%);The dominant bacteria in group PRA2 were Streptococcus(14.70% ± 23.08%),Bergeyella(8.68% ±17.32%),Lactococcus(6.37%±8.26%);The dominant bacteria in group PRA4 were Streptococcus(20.28% ± 23.15%),Staphylococcaceae＿Staphylococcus(12.74% ± 22.15%),Weissella(8.18% ±16.29%);The dominant bacteria in group PRA8 were Staphylococcaceae＿Staphylococcus(29.79%±12.65%),Enterococcus(11.31%±19.50%),Acinetobacter(10.48%±17.57%).Faith＿pd index showed that there were significant difference between group PRA0 and group PRA8(P<0.05).The results of PCo A analysis showed that there were differences between group PRA8 and other groups based on Jaccard distance and Bray-Curtis distance(P<0.05);The results of difference analysis between groups showed that there was significant separation between group PRA8 and group PRA0 based on Jaccard distance and unweighted Uni Frac distance(P<0.05).To sum up,the dominant bacteria in intestinal of raccoon dog were Firmicutes and Proteobacteria.Paeoniae radix alba extract can significantly increase the richness of Firmicutes and Bacteroidetes.Part 3: Effect of Albiflorin on methotrexate-induced small enteritis in rats and its mechanism.The purpose of this study was to explore the effect of Albiflorin,the main monoterpene component in the Paeoniae radix alba extract,on enteritis induced by MTX in rats and to explore its possible mechanism.Male SD rats were randomly divided into four groups: group control,group methotrexate(MTX),group MTX ALB(20 mg/kg)and group MTX ALB(40 mg/kg).The two groups of ALB was given ALB 20 mg/kg or 40 mg/kg daily for 6 days.The rats in the group control and group MTX were perfused with the same amount of normal saline.After 3 days of intragastric administration of ALB,the rats in group of MTX and the two groups of ALB were intraperitoneally injected with MTX 7 mg/kg/d for 3 days.At the same time,the rats in the group control were injected with the same amount of normal saline.The results showed that: ALB significantly increased body weight and intestinal weight of rats with MTX-induced enteritis(P<0.05).The disease activity index(DAI)score was also decreased after ALB administration(P<0.01).In histological examination,ALB significantly attenuated inflammatory cells infiltration and promoted survival of goblet cells(P<0.01).Additionally,ALB reduced malondialdehyde,glutathione and superoxide dismutase levels and suppressed reactive oxygen species activity in the intestinal tissues of MTX-treated rats(P<0.05).In the detection of inflammatoryassociated factors,we observed that ALB significantly decreased CD68+ cells infiltration(P<0.01),inhibited Myeloperoxidase activity(P<0.01),and suppressed intercellular cell adhesion molecule-1 and cyclooxygenase-2 expression in MTX-treated intestinal tissues(P<0.01).Moreover,inflammasome NLR family pyrin domain containing 3(NLRP3)was found to be increased in MTX-exposed intestine,while ALB effectively inhibited NLRP3 expression(P<0.01),as well as the caspase-1 p20 and interleukin(IL)-1β and 18(P<0.01).Finally,NF-κB p65 phosphorylation and nuclear translocation was also demonstrated to be blocked upon ALB treatment(P<0.01).In conclusion,ALB alleviated MTXinduced enteritis via inhibiting the NF-κB/NLRP3 signaling pathway.Conclusion: to sum up,when the dietary Paeoniae radix alba extract is 1～2 g/kg,it is helpful to increase the feed intake and nitrogen deposition of raccoon dog,increase the content of serum Ig A,decrease the content of TNF-α in intestinal mucosa,increase the ratio of villus height to crypt depth,and improve the richness of intestinal Firmicutes and Bacteroidetes.Albiflorin,the active component of Paeoniae radix alba extract,can inhibit oxidative stress and inflammation,inhibit the activation of NLRP3 inflammatory bodies,regulate the expression of NF-κB,and alleviate the process of enteritis in rats.That is,Albiflorin attenuates methotrexate-induced enteritis by inhibiting NF-κB/NLRP3 signal pathway.更多还原