中草药复方制剂对副溶血弧菌致南美白对虾肝胰腺坏死的保护作用

Protective effect of Chinese herbal compound preparations on hepatopancreas necrosis of Penaeus vannamei induced by Vibrio parahaemolyticus

为了研究中药复方制剂三黄连合剂、柴茵护肝合剂、辣蓼黄酮提取物对副溶血弧菌致肝胰腺损伤的南美白对虾的保护作用,试验先测定副溶血弧菌临床分离株HJ05感染南美白对虾的半数致死浓度(LC_(50));然后利用该浓度感染南美白对虾,观察南美白对虾的临床症状、检测肝胰腺生化指标[丙氨酸氨基转移酶(ALT)、谷胱甘肽过氧化物酶(GSH-Px)、碱性磷酸酶(AKP)、总超氧化物歧化酶(SOD)、酸性磷酸酶(ACP)]和组织病理变化,建立南美白对虾肝胰腺坏死综合征(HPNS)模型;最后将249尾南美白对虾随机分为8组,分别为对照组、模型组、三黄连合剂药物组、三黄连合剂预防组、柴茵护肝合剂药物组、柴茵护肝合剂预防组、辣蓼黄酮药物组、辣蓼黄酮预防组,分析3种中药制剂对患HPNS南美白对虾死亡率和组织病理损伤的影响,利用荧光定量PCR技术研究3种中草药制剂对患HPNS南美白对虾免疫相关因子[酚氧化酶(po)、β-1,3-葡聚糖结合蛋白(lgbp)和过氧化物酶(pe)基因]表达量的影响。结果表明:HJ05株副溶血弧菌48 h的LC_(50)是4.79×10^(5) cfu/mL,南美白对虾感染HJ05株副溶血弧菌后食欲减退,采食量下降,肝胰腺轮廓不清晰,肝胰腺上皮细胞坏死脱落,腺管萎缩塌陷;肝胰腺ALT、GSH-Px活性显著低于对照组(P<0.05),AKP和SOD活性极显著低于对照组(P<0.01),ACP活性显著高于对照组(P<0.05)。感染前15 d饲喂三黄连合剂、柴茵护肝合剂、辣蓼黄酮提取物可明显降低HPNS对虾死亡数量,缓解肝胰腺的组织病理损伤,48 h的保护率分别是48.95%、68.90%、76.67%;三黄连合剂、柴茵护肝合剂、辣蓼黄酮药物组南美白对虾肝胰腺免疫相关因子po、lgbp和pe基因mRNA相对表达量均显著或极显著高于对照组(P<0.05或P<0.01),三黄连合剂预防组lgbp和pe基因mRNA相对表达量显著或极显著高于模型组(P<0.05或P<0.01),柴茵护肝合剂预防组lgbp和po基因mRNA相对表达量极显著高于模型组(P<0.01),辣蓼黄酮预防组po基因mRNA相对表达量极显著高于模型组(P<0.01)。说明HJ05株副溶血弧菌感染南美白对虾可诱发南美白对虾HPNS;三黄连合剂、柴茵护肝合剂、辣蓼黄酮提取物可提高机体非特异性免疫,且对HJ05株副溶血弧菌感染所致肝胰腺损伤的南美白对虾有较好的保护作用,其中辣蓼黄酮提取物的保护效果最好;推测3种中草药制剂可能是通过提高南美白对虾肝胰腺相关免疫因子的表达和抑制副溶血弧菌生长以抵抗副溶血弧菌感染。

In order to study the protective effect of Chinese herbal compound preparations,Sanhuanglian mixture,Chaiyin Hugan mixture and Polygonum flavonoid extract on hepatopancreas damage of Penaeus vannamei induced by Vibrio parahaemolyticus,the experiment firstly determined the median lethal concentration(LC_(50))of Vibrio parahaemolyticus HJ05 clinical isolate infected Penaeus vannamei;using this concentration to infect Penaeus vannamei;the clinical symptoms of Penaeus vannamei were observed,and the biochemical indexes of hepatopancreas[alanine aminotransferase(ALT),glutathione peroxidase(GSH-Px),alkaline phosphatase(AKP),total superoxide dismutase(SOD),acid phosphatase(ACP)]and pathological changes were detected for the establishment of a model of hepatopancreas necrosis syndrome(HPNS)in Penaeus vannamei.249 Penaeus vannamei were randomly divided into 8 groups,namely control group,model group,Sanhuanglian mixture drug group,Sanhuanglian mixture prevention group,Chaiyin Hugan mixture drug group,Chaiyin Hugan mixture prevention group,Polygonum flavonoid drug group,and Polygonum flavonoid prevention group.The effect of three Chinese herbal preparations on the mortality and pathological damage of Penaeus vannamei with HPNS was determined,and quantitative PCR was used to detect the effect of three Chinese herbal preparations on the expression of immune-related factors in Penaeus vannamei with HPNS[phenol oxidase(po),β-1,3-glucan-binding protein(lgbp)and peroxidase(pe)genes].The results showed that the LC_(50) of HJ05 strain Vibrio parahaemolyticus at 48 h was 4.79×10^(5) cfu/mL.After the infection of HJ05 strain of Vibrio parahaemolyticus,the appetite of Penaeus vannamei decreased;the feed intake decreased;the outline of the hepatopancreas was unclear;the hepatopancreatic gland epithelial cells were necrotic and shed,and the glandular ducts atrophied and collapsed.The activities of ALT and GSH-Px in the hepatopancreas were significantly lower than those in the control group(P<0.05);the activities of AKP and SOD were significantly lower than those in the control group(P<0.01),and the activity of ACP were significantly higher than those in the control group(P<0.05).Feeding Sanhuanglian mixture,Chaiyin Hugan mixture,and Polygonum flavonoid extract 15 days before infection could significantly reduce the death number of HPNS Penaeus vannamei and alleviate the histopathological damage of hepatopancreas;the protection rates at 48 h were 48.95%,68.90%,and 76.67%,respectively.The relative expression levels of the Penaeus vannamei hepatopancreas immune-related factors po,lgbp and pe gene mRNAs in the Sanhuanglian mixture drug group,Chaiyin Hugan mixture drug group,and Polygonum flavonoid drug group were significantly or extremely significantly higher than those in the control group(P<0.05 or P<0.01).The expression levels of lgbp and pe genes mRNA relative expression in the Sanhuanglian mixture prevention group were significantly or extremely significantly higher than those in the model group(P<0.05 or P<0.01);the mRNA relative expression of lgbp and po genes in the Chaiyin Hugan mixture prevention group were significantly higher than those in the model group(P<0.01);the expression level of po gene mRNA relative expression in the Polygonum flavonoid prevention group was significantly higher than that in the model group(P<0.01).The results suggested that HJ05 strain of Vibrio parahaemolyticus could induce HPNS in Penaeus vannamei.Sanhuanglian mixture,Chaiyin Hugan mixture,and Polygonum flavonoid extract could improve the body’s non-specific immunity,and have a good protective effect on the hepatopancreas damage caused by HJ05 strain of Vibrio parahaemolyticus.Among them,Polygonum flavonoid extract had the best protective effect.It was speculated that the three Chinese herbal preparations may resist the infection of Vibrio parahaemolyticus by increasing the expression of hepatopancreas-related immune factors and inhibiting the growth of Vibrio parahaemolyticus.