褐点石斑鱼天然免疫增强剂筛选及黄芪对其免疫功能影响研究
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摘要
本论文以褐点石斑鱼(Epinephelus fuscoguttatus)为研究对象,通过氧呼吸暴发活性检测和吞噬活性检测,从8种中药,即淫羊藿、杜仲、黄芪、红参、复方1(生脉玉屏风散)、复方2(十全大补汤)以及2种泰药,即姜黄和穿心莲,筛选出5种对褐点石斑鱼具有较强免疫刺激效果的中药,即红参、黄芪、香菇、生脉玉屏风散及姜黄。以筛选中药黄芪按0%、0.5%、1.0%和1.5%添加到褐点石斑鱼的饲料中,饲喂28天后,测定褐点石斑鱼血样中的红细胞数目、白细胞数目、溶菌酶活性、氧呼吸暴发活性(氧负离子产生强弱)、吞噬活性共5项免疫指标,利用荧光定量PCR技术分析6个器官(鳃、头肾、肝脏、脾脏、心脏、肠系膜)中的MHC1-α、IL-2、Mx、IFN和TNF共5个免疫功能基因的表达水平差异,同时,对部分饲喂28天后的试验动物用创伤弧菌进行人工感染,测定感染后24h的上述5项血液学免疫指标和鳃、头肾、肝脏、脾脏、心脏、肠系膜中的上述5个免疫功能基因的表达水平差异。结果如下：
1.天然免疫增强剂的筛选：利用水相萃取法和酒精萃取法从8种中药和2种泰药中提取有效成分,采用氮蓝四唑(NBT)还原法检测褐点石斑鱼白细胞氧呼吸暴发活性和吞噬活性,开展褐点石斑鱼天然免疫增强剂的筛选工作。白细胞氧呼吸暴发活性试验结果表明,当药物水相萃取物浓度为10mg/mL时,复方1组免疫效果最好；当药物水相萃取物浓度为1mg/mL时,淫羊藿组作用效果最好；当药物水相萃取物浓度为0.1mg/mL时,所有实验组的作用效果均不明显。当药物酒精萃取物浓度为10mg/mL时,香菇组作用效果最好；当药物酒精萃取物浓度为5mg/mL时,所有实验组的作用效果均不明显；当药物酒精萃取物浓度为2.5mg/mL时,黄芪组、香菇组、红参组和复方1组的作用效果最为突出；当药物酒精萃取物浓度1.25mg/mL时,黄芪组、红参组、复方1组和姜黄组作用效果最为突出；当药物酒精萃取物浓度为0.625mg/mL时,黄芪组、杜仲组和姜黄组作用效果最为突出。白细胞吞噬活性检测结果表明,红参组、香菇组、黄芪组、复方1组和姜黄组5个试验组的白细胞吞噬活性极显著高于对照组(P<0.01)。根据上述结果,确定了5种能明显提高褐点石斑鱼非特异性免疫力的天然免疫增强剂,即红参、香菇、黄芪、生脉玉屏风散及姜黄。
2.黄芪对褐点石斑鱼部分血液学指标的影响：添加黄芪的饲料饲喂28d时,1.5%黄芪试验组红细胞数目显著低于对照组,1.5%黄芪试验组白细胞数目显著低于对照组,0.5%黄芪试验组溶菌酶活性极显著高于对照组,1.0%黄芪试验组溶菌酶活性显著高于对照组,1.5%黄芪试验组氧呼吸暴发活性检测中STI值极显著高于对照组,1.5%黄芪试验组吞噬活性极显著高于对照组,1.5%黄芪试验组和0.5%黄芪试验组吞噬指数显著高于对照组；创伤弧菌感染24h,样品中各实验组的红细胞数目和白细胞数目与对照组差异不显著,0.5%黄芪试验组溶菌酶活性与对照组差异极显著,1.0%黄芪试验组溶菌酶活性与对照组差异显著,样品中各实验组的氧呼吸暴发活性和吞噬活性与对照组差异不显著；感染试验中,1.5%黄芪试验组14天免疫保护率为86.7%,显著高于对照组。
3.黄芪对褐点石斑鱼免疫基因表达差异分析：添加黄芪的饲料饲喂28d时,试验动物脾脏和鳃中各免疫基因在添加1.5%黄芪试验组中表达量最高,其中脾脏中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因、TNF基因和IFN基因表达量分别是对照组的140.40倍、39.90倍、69.70倍、115.89倍和36.76倍；鳃中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因、TNF基因和IFN基因表达量分别是对照组的5.03倍、3674.50倍、69.70倍、8.38倍和270.60倍。上述试验动物以创伤弧菌感染24h后,脾脏中Mx基因、IL-2基因、MHC I-α基因、TNF基因在添加1.5%黄芪试验组表达量最高,脾脏中IFN基因表达在量添加0.5%黄芪试验组最高,鳃中各基因在添加1.5%黄芪试验组表达量最高,其中,脾脏中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因和TNF基因表达量分别是对照组的22.11倍、206.5倍、5.90倍和68.99倍,脾脏中添加0.5%黄芪试验组IFN基因表达量是对照组的19.74倍；鳃中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因、TNF基因和IFN基因表达量分别是对照组的1.17倍、12.15倍、1.11倍、1.49倍和1.15倍。
综上所述,本文初步筛选了对褐点石斑鱼具有较强免疫增强作用的4种天然药物,从血液学水平和分子水平确定了不同浓度黄芪对褐点石斑鱼的免疫功能的影响,为进一步开发海水鱼类新型、安全的免疫增强剂及及今后阐明鱼类对免疫增强剂的免疫应答机制奠定了良好基础,具有一定的理论水平和良好的应用前景。
In this study, five herbs (Radix astragali, Lentinan, Radix ginseng rubra, Shengmai Yu Ping Feng San and Turmeric, respectively) with strong immune stimulating effects on brown spotted grouper (Epinephelus fuscoguttatus), were selected from a total of eight Chinese herbs and two Thai herbs using superoxide anion production and phagocytic activity detection methods. The eight Chinese herbs include Epimedium herb (Epimedium sagittatum), Eucommia bark (Eucommia ulmoides), Radix astragali (Astragalus membranaceus), Lentinan (Lentinus edodes), Radix ginseng rubra (Panax ginseng), Five leaf Gynostemma herb (Gynostemma pentaphyllum), compound1(Shengmai Yu Ping Feng San), and compound2(Shi Quan Da Bu Tang). The two Thai herbs were Turmeric (Curcuma longa) and Fahtalaijons (Andrographis paniculata). Different proportion (0%,0.5%,1.0%and1.5%) of Radix astragali, were added to brown spotted grouper feed for28d. The immune parameters, RBC, WBC, lysozyme activity, superoxide union production and phagocytic activity, were selected to test the immunity of the fed fish. At the same time, gene expression differences of five immune genes, MHC1-α, IL-2, Mx, IFN and TNF, in five organs (gill, kidney, heart, spleen, liver and intestine) were analyzed with fluorescent quantitative PCR. Furthermore, brown spotted grouper were infected with Vibrio vulnificus after fed with the herb feed for28days, and the immunity parameters and immunity-related gene expression differences were analyzed after24h of infection.
1. Selection of natural immunostimulant:Active ingredients were extracted from eight Chinese medicine and two Thai herbs using ethyl aqueous extraction and alcohol extraction methods, and screening of natural immunostimulants for brown spotted grouper was carried out by detecting of superoxide anion production with tetrazolium (NBT) reduction and phagocytic activity of its white blood cell. The results showed that, at aqueous extraction concentrations10mg/mL and1mg/mL, the groups with best effect were group compound1and Epimedium herb, respectively, while at aqueous extraction concentration O.lmg/mL, none of experimental groups showed any significant effect. At alcohol extraction concentration10mg/mL, Lentinan group showed the best effect, with the alcohol extraction concentration of5mg/mL, all experimental groups showed no significant effect, at alcohol extraction concentrations of2.5mg/mL, group Radix astragali, Lentinan, Radix ginseng rubra and compound1got the most prominent effect, at alcohol extraction concentrations of1.25mg/mL, group Radix astragali, Radix ginseng rubra, compound1and Turmeric got the best prominent effect, at alcohol extraction concentration of0.625mg/mL, group Radix astragali, Eucommia bark, and Turmeric showed the optimal effect. White blood cell phagocytic activity test showedthatRadix astragali, Lentinan, Radix ginseng rubra and compound1had significant immune enhancement effect on brown spotted grouper (P<0.01). Based on above experimental results, four herbs (Radix astragali, Lentinan, Radix ginseng rubra, Shengmai Yu Ping Feng San and Turmeric respectively) were seleted as effective natural immunostimulants for brown spotted grouper from above experiments.
2. Effect of Radix astragali on the immune hematology in brown spotted grouper. RBC, WBC, lysozyme activity, STI value and phagocytic activity were tested after the fish were fed with0.5%,1.0%or1.5%Radix astragali for28days. Results indicated that at0.5%Radix astragali concentration, lysozyme activity showed a highly significant effect (p<0.01), while phagocytic activity had a significant effect (p<0.05). At1.0%Radix astragali concentration, only lysozyme activity showed a significant effect (p<0.05). At1.5%Radix astragali concentration, RBC, WBC and phagocytic activity were significant different (p<0.05), and STI were highly significant different between the test and control groups (p<0.01). Samples in the test fish injected by Vibrio vulnificus for24h, WBC, RBC and phagocytic activity in all experimental groups were no significant difference with the control group. However, lysozyme activity showed significant and highly significant differences in1.0%and0.5%Radix astragali concentration groups, respectively (p<0.05, p<0.01). In infection experiment,1.5%Radix astragali concentration group had an immune protection rate of86.7%, which was significantly higher than the control group.
3. Gene expression analysis of brown spotted grouper: before and after Radix astragali feeding the test group with Radix astragali for28days, immune genes of spleen and gill in test group at1.5%Radix astragali concentration showed the highest expression. In spleen, genes Mx, IL-2, MHC I-α, TNF and IFN expression were140.40times,39.90times,69.70times,115.89times and36.76times as the control group, respectively. In gill, genes Mx, IL-2, MHC I-α, TNF and IFN expression were5.03times,3674.50times,69.70times,8.38times and270.60times as the control group, respectively. After infecting the test group fish with Vibrio vulnificus for24h, immune genes Mx, IL-2, MHC I-a and TNF in group1.5%Radix astragali concentration got the highest expression in spleen, and immune gene IFN in group0.5%Radix astragali concentration got the highest expression. In gill, all immune genes expression in group1.5%Radix astragali concentration was the highest. In spleen, genes Mx, IL-2, MHC I-α, and TNF expression in group1.5%Radix astragali adding were respectively22.11times,206.50times,5.90times and68.99times as the control group. In spleen, gene IFN expression in group0.5%Radix astragali concentration was19.74times as the control group. In gill, genes Mx, IL-2, MHC I-α, TNF and IFN expression in group1.5%Radix astragali concentration were1.17,12.15,1.11,1.49and1.15as the control group, respectively.
In summary, four nature herbs with strong immune enhancing effects were preliminarily screened for brown spotted grouper. The immune enhancing effect of Radix astragali on the brown grouper was mainly studied at hematological and gene expression level. It laid a good foundation for the new and safeimmune enhancer selection in salt water fish. It also has broad theoretical and applicational prospect.
1.天然免疫增强剂的筛选：利用水相萃取法和酒精萃取法从8种中药和2种泰药中提取有效成分,采用氮蓝四唑(NBT)还原法检测褐点石斑鱼白细胞氧呼吸暴发活性和吞噬活性,开展褐点石斑鱼天然免疫增强剂的筛选工作。白细胞氧呼吸暴发活性试验结果表明,当药物水相萃取物浓度为10mg/mL时,复方1组免疫效果最好；当药物水相萃取物浓度为1mg/mL时,淫羊藿组作用效果最好；当药物水相萃取物浓度为0.1mg/mL时,所有实验组的作用效果均不明显。当药物酒精萃取物浓度为10mg/mL时,香菇组作用效果最好；当药物酒精萃取物浓度为5mg/mL时,所有实验组的作用效果均不明显；当药物酒精萃取物浓度为2.5mg/mL时,黄芪组、香菇组、红参组和复方1组的作用效果最为突出；当药物酒精萃取物浓度1.25mg/mL时,黄芪组、红参组、复方1组和姜黄组作用效果最为突出；当药物酒精萃取物浓度为0.625mg/mL时,黄芪组、杜仲组和姜黄组作用效果最为突出。白细胞吞噬活性检测结果表明,红参组、香菇组、黄芪组、复方1组和姜黄组5个试验组的白细胞吞噬活性极显著高于对照组(P<0.01)。根据上述结果,确定了5种能明显提高褐点石斑鱼非特异性免疫力的天然免疫增强剂,即红参、香菇、黄芪、生脉玉屏风散及姜黄。
2.黄芪对褐点石斑鱼部分血液学指标的影响：添加黄芪的饲料饲喂28d时,1.5%黄芪试验组红细胞数目显著低于对照组,1.5%黄芪试验组白细胞数目显著低于对照组,0.5%黄芪试验组溶菌酶活性极显著高于对照组,1.0%黄芪试验组溶菌酶活性显著高于对照组,1.5%黄芪试验组氧呼吸暴发活性检测中STI值极显著高于对照组,1.5%黄芪试验组吞噬活性极显著高于对照组,1.5%黄芪试验组和0.5%黄芪试验组吞噬指数显著高于对照组；创伤弧菌感染24h,样品中各实验组的红细胞数目和白细胞数目与对照组差异不显著,0.5%黄芪试验组溶菌酶活性与对照组差异极显著,1.0%黄芪试验组溶菌酶活性与对照组差异显著,样品中各实验组的氧呼吸暴发活性和吞噬活性与对照组差异不显著；感染试验中,1.5%黄芪试验组14天免疫保护率为86.7%,显著高于对照组。
3.黄芪对褐点石斑鱼免疫基因表达差异分析：添加黄芪的饲料饲喂28d时,试验动物脾脏和鳃中各免疫基因在添加1.5%黄芪试验组中表达量最高,其中脾脏中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因、TNF基因和IFN基因表达量分别是对照组的140.40倍、39.90倍、69.70倍、115.89倍和36.76倍；鳃中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因、TNF基因和IFN基因表达量分别是对照组的5.03倍、3674.50倍、69.70倍、8.38倍和270.60倍。上述试验动物以创伤弧菌感染24h后,脾脏中Mx基因、IL-2基因、MHC I-α基因、TNF基因在添加1.5%黄芪试验组表达量最高,脾脏中IFN基因表达在量添加0.5%黄芪试验组最高,鳃中各基因在添加1.5%黄芪试验组表达量最高,其中,脾脏中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因和TNF基因表达量分别是对照组的22.11倍、206.5倍、5.90倍和68.99倍,脾脏中添加0.5%黄芪试验组IFN基因表达量是对照组的19.74倍；鳃中添加1.5%黄芪试验组Mx基因、IL-2基因、MHC I-α基因、TNF基因和IFN基因表达量分别是对照组的1.17倍、12.15倍、1.11倍、1.49倍和1.15倍。
综上所述,本文初步筛选了对褐点石斑鱼具有较强免疫增强作用的4种天然药物,从血液学水平和分子水平确定了不同浓度黄芪对褐点石斑鱼的免疫功能的影响,为进一步开发海水鱼类新型、安全的免疫增强剂及及今后阐明鱼类对免疫增强剂的免疫应答机制奠定了良好基础,具有一定的理论水平和良好的应用前景。
In this study, five herbs (Radix astragali, Lentinan, Radix ginseng rubra, Shengmai Yu Ping Feng San and Turmeric, respectively) with strong immune stimulating effects on brown spotted grouper (Epinephelus fuscoguttatus), were selected from a total of eight Chinese herbs and two Thai herbs using superoxide anion production and phagocytic activity detection methods. The eight Chinese herbs include Epimedium herb (Epimedium sagittatum), Eucommia bark (Eucommia ulmoides), Radix astragali (Astragalus membranaceus), Lentinan (Lentinus edodes), Radix ginseng rubra (Panax ginseng), Five leaf Gynostemma herb (Gynostemma pentaphyllum), compound1(Shengmai Yu Ping Feng San), and compound2(Shi Quan Da Bu Tang). The two Thai herbs were Turmeric (Curcuma longa) and Fahtalaijons (Andrographis paniculata). Different proportion (0%,0.5%,1.0%and1.5%) of Radix astragali, were added to brown spotted grouper feed for28d. The immune parameters, RBC, WBC, lysozyme activity, superoxide union production and phagocytic activity, were selected to test the immunity of the fed fish. At the same time, gene expression differences of five immune genes, MHC1-α, IL-2, Mx, IFN and TNF, in five organs (gill, kidney, heart, spleen, liver and intestine) were analyzed with fluorescent quantitative PCR. Furthermore, brown spotted grouper were infected with Vibrio vulnificus after fed with the herb feed for28days, and the immunity parameters and immunity-related gene expression differences were analyzed after24h of infection.
1. Selection of natural immunostimulant:Active ingredients were extracted from eight Chinese medicine and two Thai herbs using ethyl aqueous extraction and alcohol extraction methods, and screening of natural immunostimulants for brown spotted grouper was carried out by detecting of superoxide anion production with tetrazolium (NBT) reduction and phagocytic activity of its white blood cell. The results showed that, at aqueous extraction concentrations10mg/mL and1mg/mL, the groups with best effect were group compound1and Epimedium herb, respectively, while at aqueous extraction concentration O.lmg/mL, none of experimental groups showed any significant effect. At alcohol extraction concentration10mg/mL, Lentinan group showed the best effect, with the alcohol extraction concentration of5mg/mL, all experimental groups showed no significant effect, at alcohol extraction concentrations of2.5mg/mL, group Radix astragali, Lentinan, Radix ginseng rubra and compound1got the most prominent effect, at alcohol extraction concentrations of1.25mg/mL, group Radix astragali, Radix ginseng rubra, compound1and Turmeric got the best prominent effect, at alcohol extraction concentration of0.625mg/mL, group Radix astragali, Eucommia bark, and Turmeric showed the optimal effect. White blood cell phagocytic activity test showedthatRadix astragali, Lentinan, Radix ginseng rubra and compound1had significant immune enhancement effect on brown spotted grouper (P<0.01). Based on above experimental results, four herbs (Radix astragali, Lentinan, Radix ginseng rubra, Shengmai Yu Ping Feng San and Turmeric respectively) were seleted as effective natural immunostimulants for brown spotted grouper from above experiments.
2. Effect of Radix astragali on the immune hematology in brown spotted grouper. RBC, WBC, lysozyme activity, STI value and phagocytic activity were tested after the fish were fed with0.5%,1.0%or1.5%Radix astragali for28days. Results indicated that at0.5%Radix astragali concentration, lysozyme activity showed a highly significant effect (p<0.01), while phagocytic activity had a significant effect (p<0.05). At1.0%Radix astragali concentration, only lysozyme activity showed a significant effect (p<0.05). At1.5%Radix astragali concentration, RBC, WBC and phagocytic activity were significant different (p<0.05), and STI were highly significant different between the test and control groups (p<0.01). Samples in the test fish injected by Vibrio vulnificus for24h, WBC, RBC and phagocytic activity in all experimental groups were no significant difference with the control group. However, lysozyme activity showed significant and highly significant differences in1.0%and0.5%Radix astragali concentration groups, respectively (p<0.05, p<0.01). In infection experiment,1.5%Radix astragali concentration group had an immune protection rate of86.7%, which was significantly higher than the control group.
3. Gene expression analysis of brown spotted grouper: before and after Radix astragali feeding the test group with Radix astragali for28days, immune genes of spleen and gill in test group at1.5%Radix astragali concentration showed the highest expression. In spleen, genes Mx, IL-2, MHC I-α, TNF and IFN expression were140.40times,39.90times,69.70times,115.89times and36.76times as the control group, respectively. In gill, genes Mx, IL-2, MHC I-α, TNF and IFN expression were5.03times,3674.50times,69.70times,8.38times and270.60times as the control group, respectively. After infecting the test group fish with Vibrio vulnificus for24h, immune genes Mx, IL-2, MHC I-a and TNF in group1.5%Radix astragali concentration got the highest expression in spleen, and immune gene IFN in group0.5%Radix astragali concentration got the highest expression. In gill, all immune genes expression in group1.5%Radix astragali concentration was the highest. In spleen, genes Mx, IL-2, MHC I-α, and TNF expression in group1.5%Radix astragali adding were respectively22.11times,206.50times,5.90times and68.99times as the control group. In spleen, gene IFN expression in group0.5%Radix astragali concentration was19.74times as the control group. In gill, genes Mx, IL-2, MHC I-α, TNF and IFN expression in group1.5%Radix astragali concentration were1.17,12.15,1.11,1.49and1.15as the control group, respectively.
In summary, four nature herbs with strong immune enhancing effects were preliminarily screened for brown spotted grouper. The immune enhancing effect of Radix astragali on the brown grouper was mainly studied at hematological and gene expression level. It laid a good foundation for the new and safeimmune enhancer selection in salt water fish. It also has broad theoretical and applicational prospect.
引文
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