摘要
夏季高温高湿天气易诱发猪热应激,导致猪生产性能下降,对营养物质的吸收能力降低,给养猪业带来巨大损失。小肠是应激反应的敏感器官之一,又是营养物质消化吸收的主要场所。肠上皮细胞的刷状缘膜和基底膜上富含双糖酶和各类转运载体蛋白,在动物营养物质的消化吸收上起着重要作用。研究显示,藿香、苍术、黄柏等具有清热祛湿作用,同时还可改善动物小肠的消化吸收。本研究通过人工模拟夏季高温高湿环境诱发猪热应激,研究藿香、苍术提取物复合制剂对高温应激状态下猪小肠消化吸收功能的调控作用,建立小肠上皮细胞体外热应激模型,筛选抗热应激效果显著的提取物,探讨藿香、苍术提取物复合制剂改善猪小肠吸收功能的机制。
本研究包括以下五个试验部分：
1.藿香、苍术提取物复合制剂对高温应激猪生产性能及血清指标的影响
在人工气候仓模拟夏季高温高湿环境对中国小型实验猪(CEMP,农大Ⅰ系)进行为期10天的高温刺激(40℃,5h/d),研究不同配比的藿香、苍术、黄柏和石膏混合提取制备的复合制剂Ⅰ和Ⅱ在饲粮中添加后对高温应激猪生产性能和血清指标的影响。试验选用2月龄实验猪64头,分为常温组、高温组、提取物Ⅰ组和提取物Ⅱ组四个处理,每组16头猪。各组分别于试验的第1、3、6、10天,随机取4头猪称重,静脉采血后分离血清,测定血清SOD、GSH-Px活性,葡萄糖、MDA含量。结果表明,高温刺激可引起猪日采食量和日增重下降,血糖水平降低(P<0.05),血清SOD、GSH-Px活力升高；MDA含量升高(P<0.05)。提取物Ⅰ可降低高温应激猪血清MDA含量,提高日采食量(P<0.05),但对日增重没有明显改善(P>0.05)；提取物Ⅱ可显著提高高温应激猪的日增重和日采食量,降低其血清MDA含量(P<0.05)。以上结果提示在改善高温应激猪生产性能和血糖水平上,提取物Ⅱ效果优于提取物Ⅰ。
2.藿香、苍术提取物复合制剂对高温应激猪小肠脂质过氧化损伤的影响
研究了藿香、苍术提取物复合制剂Ⅰ和Ⅱ对高温应激猪小肠脂质过氧化损伤的保护作用。试验处理同试验1,在试验的第1、3、6、10天,从每组中取4头猪,快速放血处死,分离十二指肠、空肠和回肠,取部分肠组织制作电镜超薄切片,测定各段肠组织匀浆液SOD、GSH-Px活性及MDA含量。结果显示：(1)与常温组相比,高温组试猪小肠SOD、GSH-Px活性显著降低(P<0.05),MDA含量显著升高(P<0.05)；小肠上皮细胞微绒毛变细变短,线粒体内脊变短。(2)与高温组比较,提取物Ⅰ显著提高猪小肠组织SOD、GSH-Px活性；提取物Ⅱ可显著提高猪小肠组织SOD、GSH-Px活性,并降低小肠MDA含量,改善小肠上皮细胞微绒毛的吸收表面积(P<0.05)。这提示添加藿香、苍术提取物复合制剂可促进高温应激猪抗氧化酶活性的恢复,改善其受损的小肠上皮微绒毛结构,且提取物Ⅱ效果优于提取物Ⅰ。
3.藿香、苍术提取物复合制剂对高温应激猪小肠消化和吸收的影响
选择抗热应激作用显著的藿香、苍术提取物复合制剂Ⅱ,研究其对高温应激猪小肠消化吸收功能的影响。选用2月龄中国实验用小型猪48头,随机分成常温组、高温组和提取物组。各组试验猪分别于第1、3、6、10天采样,测定小肠木糖吸收水平,同时制备十二指肠、空肠和回肠刷状缘膜囊,测定蔗糖酶、乳糖酶、麦芽糖酶和碱性磷酸酶活性。结果表明：猪小肠各段的刷状缘二糖酶活性呈现明显差异,以空肠最高,其次是回肠和十二指肠。碱性磷酸酶活性在回肠最高,其次为空肠和十二指肠。高温应激猪十二指肠和空肠刷状缘三种二糖酶、碱性磷酸酶活性均在第1、3、6天比常温组猪显著下降(P<0.05),而后又逐渐恢复(P>0.05)；小肠木糖吸收水平在第3、6天显著下降(P<0.05),第10天回升(P>0.05)。添加藿香、苍术提取物复合制剂可显著上调高温应激猪小肠木糖吸收水平,提高小肠刷状缘二糖酶和碱性磷酸酶活性(P<0.05),使其在热应激的第6天提前恢复至正常水平。
4.藿香、苍术提取物复合制剂对高温应激猪小肠葡萄糖转运相关载体表达的影响
为探讨藿香、苍术提取物复合制剂对高温应激猪小肠营养吸收的调控作用,本试验研究了其对热应激第6天猪小肠葡萄糖转运相关载体表达的变化。测定小肠Na+/K+-ATP酶活性,采用ELISA法分析猪小肠SGLT1和GLUT2含量,荧光PCR技术检测小肠各段Na+/K+-ATP酶、SGLT1和GLUT2mRNA表达量。结果表明：高温应激猪小肠Na+/K+-ATP酶的活性降低(P<0.05),十二指肠和回肠GLUT2蛋白水平下降(P<0.05)；但小肠各段SGLT1和GLUT2mRNA表达量均无显著变化(P>0.05)。提取物组猪小肠Na+/K+-ATP酶的活性、Na+/K+-ATP酶mRNA表达量比高温组显著升高；小肠各段SGLT1和GLUT2蛋白水平,十二指肠和空肠SGLT1和GLUT2mRNA表达量比高温组和常温组都显著上升(P<0.05)。回肠SGLT1和GLUT2的mRNA表达量在三个处理组间均无显著差异(P>0.05)。以上结果提示,藿香、苍术提取物复合制剂可通过上调小肠葡萄糖转运相关蛋白的表达水平来改善高温应激猪葡萄糖吸收功能,但对小肠各段SGLT1和GLUT2mRNA表达的调控并不一致。
5.藿香、苍术挥发油对高温应激小肠上皮细胞葡萄糖吸收的影响
利用大鼠IEC-6细胞系,建立小肠上皮细胞体外热应激模型,研究组成提取物复合制剂的四种提取物(藿香挥发油、苍术挥发油、黄柏生物碱和石膏水提物)对高温应激状态下小肠上皮细胞增殖和葡萄糖吸收的影响。结果表明：41℃热应激2h可抑制大鼠肠道IEC-6细胞增殖作用,降低细胞葡萄糖吸收率和Na+/K+-ATP酶的活性；下调细胞Na+/K+-ATP酶mRNA表达量(P<0.05),但对细胞SGLT1、GLUT2的mRNA表达量无影响(P>0.05)。在常温和高温条件下,100μg/mL以上的藿香挥发油和50lμg/mL以上苍术挥发油、石膏水提物对大鼠IEC-6细胞系具有促增殖作用(P<0.05),而各浓度的黄柏生物碱均可抑制IEC-6细胞的增殖,且呈现剂量递增效应(P<0.05)；进一步的研究表明,100μg/mL藿香挥发油可促进葡萄糖的吸收,提高Na+/K+-ATP酶的活性,上调高温应激小肠上皮细胞SGLT1、GLUT2、Na+/K+-ATP酶mRNA表达(P<0.05),但在常温下这些基因的表达没有改变(P>0.05);50μg/mL苍术挥发油可上调常温下葡萄糖转运相关载体(SGLT1、GLUT2、Na+/K+-ATPase)的基因表达和高温下SGLT1的基因表达量,促进细胞对葡萄糖的吸收,提高Na+/K+-ATP酶的活性(P<0.05)。以上结果提示藿香挥发油和苍术挥发油可促进小肠上皮细胞葡萄糖吸收,且这种促进作用与葡萄糖转运相关载体的基因表达水平上调有关。
The heat stress in pig could happen under high temperature and high humidity environment in summer. It results in reduction of gastrointestinal nutrient digestion and absorption and accordingly the decrease of production performance, which bring great losing to the pig fostering. The mammalian small intestine is a central organ which is very sensitive to all stressors, which is also the major site of digesting and absorbing the nutrition. Hydrolases and transport proteins, which rich in the brush-border membrane vesicles (BBMVs) and basement membrane of small intestine, play a critical role on intestinal nutrient digestion and absorption. Agastache Rugosa, Rhizome Atractylodes, and Cortex Phellodendron have been reported to improve intestinal absorption on nutrition. Therefore, the current study was designed to investigate the effect of high temperature stress on growth performance and intestinal absorption of piglets under simulating high temperature and humidity environment in summer, and probe the regulation of extract compound preparation, which was extracted by extract compounds from Agastache Rugosa, Rhizome Atractylodes, Cortex Phellodendron and Gypsum Fibrosum on small intestinal digestion and absorption of heat stressed piglets. And then a model of heat stress in rat IEC-6cells was erected and used to select the effective extracts, which is helpful to illuminate the mechanism of the extract compounds on resisting high temperature stress.So the present studies including five parts as bellows.
1. Effects of extract compounds from Atractylodes Rhizome, Agastache Rugosa on growth performance and serum indexes in piglets under high temperature stress
The swine was put into the man-made granary to stimulate the weather in summer to heat stress (40℃,5h/d) in order to study the effect of extracts compound preparation I and II, which was extracted from Rhizome Atractylodes, Agastache Rugosa, Cortex Phellodendron and Gypsum Fibrosum, on the growth performance and serum indexes of swine under heat stress. Total sixty-four2-month-old Chinese experimental piglets (CEMP, agriculture university I series) were blocked according to weight, sex and litter origin, and then randomly allotted to three groups and treated as follows:Normal temperature control group (NTG), High temperature control group (HTG); Extract compound preparation group I (ETGI) and Extracts compound preparation group Ⅱ (ETGII)(n=16per group). On the day1,3,6,10of trial, four piglets were selected from each group, weighed and collected blood separately, and serum glucose levels, MDA levels and SOD, GSH-Px activity were analyzed by commercial kit. The result shows that the high temperature stress decreased ADFI and ADG, induced the decline of serum glucose levels, and increased the activity of SOD and GSH-Px in piglets. Extract Ⅰ improved the ADFI and ADG of heat stressed piglets (P>0.05), and decreased serum MDA contents (P<0.05);extract Ⅱ improved porcine growth performance, increased the activity of SOD and GSH-Px in serum, and induced the decline of MDA contents under high temperature stress (P<0.05). The current results indicated that extract Ⅱ plays a more effective role in the preventive and curative of heat stress in swine compared with that of extract Ⅰ.
2. Effects of extract compounds from Atractylodes Rhizome, Agastache Rugosa on small intestinal lipid peroxidation in piglets under high temperature stress
To determine the effect of supplemental extracts compound preparation Ⅰ and Ⅱ on small intestinal lipid peroxidate in piglets under high temperature stress. In each group, four piglets were collected duodenum, jejunum, ileum samples after treatment on the day1,3,6, and10respectively, one part were used to make intestinal electron microscopic section, and others were determined SOD、GSH-PX activity、MDA contents in small intestine by kit. The result showed:(1) Compared with the NTG, jejunum SOD、GSH-Px activity in the HTG were significant decreased on day6and10(p<0.05), MDA content was increased on day1and3(p<0.05), and the activities of SOD and GSH-Px in ileum were decreased remarkably on day10(p<0.05).(2) Compared with the HTG, extract Ⅱ decreased MDA content in jejunum on day1and3, and improved intestinal cell membrane structure (p<0.05). These results indicated that extract Ⅱ plays an important role on improving the antioxidant status and cell membrane structure of small intestine in piglets than extract Ⅰ under high temperature stress.
3. Effects of extract compounds from Atractylodes Rhizome, Agastache Rugosa on small intestinal digestion and absorption in piglets under high temperature stress
To study the effects of extracts compound preparation Ⅱ on small intestinal digestion and absorption of piglets during high temperature stress. Forty-eight Chinese experimental piglets with2-month-old were blocked according to weight, sex and litter origin, and then randomly allotted to three groups and treated as follows:Normal temperature control group (NTG); High temperature control group (HTG); The extract compound preparation group (ETG)(n=16per group). In each group, duodenum, jejunum, ileum samples were collected respectively from four piglets on day1,3,6and10of heat stress; and then small intestinal brush border membrane vesicle (BBMV) was prepared. Sucrase, lactase, maltase and AKP activity of BBMV were determined by kit, and at the same time small intestinal xylose absorption of piglets were investigated. The result showed: the highest activity of disaccharidases and AKP was detected in jejunum, followed by duodenum and ileum. Disaccharidases and AKP activity of BBMV in duodenum and jejunum in the HTG were decreased significantly (P<0.05) on day1,3,6comparing to the NTG, then trend to normal level on day10(P>0.05); high temperature treatment induced the decreased of intestinal xylose absorption on day3,6. Extracts compound preparation up-regulated significantly disaccharidase activity in BBMV of small intestine during heat stress, and recovered intestinal absorption to normal level ahead on day6after heat stress.
4. Effects of extract compounds from Atractylodes Rhizome, Agastache Rugosa on small intestinal glucose transport correctional carrier expression in piglets under high temperature stress
To study the effect of extracts compound preparation on small intestinal absoption in heat stressed piglets, the activity of Na+/K+-ATPase and expressional levels of sodium-glucose co-transporter1(SGLT1), glucose transporter2(GLUT2) were determined by chemical assay and ELISA kit, and a method of real time PCR was applied to determine SGLT1and GLUT2mRNA expression in small intestine on day6. The result showed that heat stress induced a decrease of the activity of Na+/K+-ATPase in small intestine, and GLUT2contents in duodenal and ileal of the HTG were decreased compared with the NTG (P<0.05). The expression of SGLT1and GLUT2mRNA in small intestine with HTG did not change compared to the NTG (P<0.05). The extract compound preparation increased intestinal Na+/K+-ATPase activity and its gene expression, and improved the SGLT1/GLUT2protein levels in small intestine of heat stressed piglets. Only duodenal and jejunal expression of SGLT1and GLUT2genes expression in the ETG were increased when it compared with the NTG and HTG. However, no difference was showed on SGLT1 and GLUT2mRNA expression of ileum among three groups. These results indicated that extracts compound preparation improved significantly the absorption of glucose in small intestine during heat stress, but the extract affected SGLT1and GLUT2mRNA expression in small intestine of piglets in a spatio-temporal specific manner.
5. Effects of Atractylodes Rhizome essential oil, Agastache Rugosa essential oil on small intestinal epithelial cells under high temperature stress
We established the small intestinal epithelial cells model of heat stress by using the rat IEC-6cells, and investigated the effects of heat stress on the intestinal epithelail cells proliferation and glucose absorption as well as the regulation of four extracts which composed the extract compounds Ⅱ. Results showed that high temperature stress treatment (41℃,2h) decreased the proliferation of IEC-6cells. Four extracts altered the cell proliferation in a dose-dependent manner, and Agastache Rugosa essential oil(100μg/mL,200μg/mL), Rhizome Atractylodes essential oil and Gypsum Fibrosum water extract (50μg/mL,100μg/mL,200μg/mL)increased the proliferation of IEC-6cells(P<0.05), but Cortex Phellodendron alkaloid with100μg/mL,50μg/mL,100μg/mL,200μg/mL decreased the proliferation of IEC-6cells under both normal and high temperature(P>0.05) Moreover,100μg/mL Agastache Rugosa essential oil treatment increased glucose absorption and Na+/K+-ATPase activity in small intestinal epithelial cell under both normal and high temperature(37℃and41℃, P<0.05), and up-regulated the expression of SGLT1, GLUT2and Na+/K+-ATPase gene in IEC-6cells under heat stress (41℃, P<0.05) but without changing their expression under normal temperature (37℃, P>0.05). However, Rhizome Atractylodes essential oil with50μg/mL improved glucose absorption and the activity of Na+/K+-ATPase, and up-regulated the expression of SGLT1and Na+/K+-ATPase gene in IEC-6cells under normal temperature(37℃, P<0.05), only SGLT1gene expression in cells were increased by Rhizome Atractylodes essential oil treatment under heat stress (41℃,P<0.05)
引文
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