急性热应激对山羊血液生化指标及血淋巴细胞热休克蛋白70家族基因表达的影响

Effects of Acute Heat Stress on Blood Biochemistry Indices and Expression of HSP70 Family Genes in Blood Lymphocytes in Goats

旨在研究急性热应激对山羊抗氧化能力、免疫功能和血淋巴细胞热休克蛋白70(HSP70)家族基因表达的影响。本试验选取5只健康、体况接近的(12±0.5)月龄波尔山羊×关中奶山羊杂交F1母羊,饲养于环控舱内(温度维持20℃,相对湿度60%),适应5d。第6天利用环控舱对5只试验羊进行38℃急性热应激处理12h,采集热应激前(0h,20℃)和热应激后2、4、8和12h试验羊血样。分别利用比色法测定血清抗氧化指标(总抗氧化能力、超氧化物歧化酶、谷胱甘肽过氧化物酶活性和丙二醛含量),ELISA法测定血清免疫指标(免疫球蛋白和细胞因子含量),实时荧光定量PCR法测定血淋巴细胞HSP70家族基因(HSPA1A、HSPA6和HSPA8)mRNA的表达量。结果显示:1)热应激时间对血清抗氧化指标有显著影响。与热应激前0h相比,血清T-AOC(P<0.05)、SOD(P<0.05)和GSH-Px(P<0.01)活性均在热应激8h后显著下降,MDA含量在热应激4h后显著增加(P<0.05)。2)热应激时间对血清免疫指标有显著影响。与热应激前0h相比,血清TNF-α、IL-1β、IFN-γ和IL-2含量分别在热应激4、8、8和4h后显著增加(P<0.05);IL-4(P<0.01)、IgG(P<0.01)、IgM(P<0.01)和IgA(P<0.05)含量分别在热应激12、4、4和4h后显著下降。3)热应激时间显著提高血淋巴细胞中HSP70家族基因(HSPA1A、HSPA6和HSPA8)的表达量。HSPA1AmRNA表达量呈先升高后下降的趋势,在热应激4h时达到峰值,各检测时间点均显著高于应激前水平(P<0.01);HSPA6mRNA表达量在热应激2h时显著升高(P<0.01),4h后恢复到应激前水平(P>0.05);HSPA8mRNA表达量在热应激4(P<0.05)、8(P<0.01)、12h(P<0.01)时显著高于应激前水平。在本试验条件下,38℃急性热应激能够抑制山羊的免疫和抗氧化功能;提高血淋巴细胞HSPA1A、HSPA6和HSPA8基因的表达量,其中HSPA1A对热应激温度和时间更敏感,可作为山羊热应激早期的分子标志物。

This experiment was conducted to explore the effects of acute heat stress on antioxidant capacity,immune function and expression of HSP70 family genes in blood lymphocytes in goats. Five (12±0.5)-month-old healthy female goats (F 1 of Boer goat × Guanzhong dairy goat) were selected and raised separately in cage in an environment controlled chamber (temperature: 20 ℃,relative humidity:60%),adaptation period lasted for 5 days. At the 6th day,five goats were treated with acute heat stress at 38 ℃ for 12 h. Blood samples were collected before heat stress (0 h,20 ℃) and after 2,4,8 and 12 h of heat stress. Serum antioxidant indexes (total antioxidant capacity,superoxide dismutase,glutathione peroxidase activity and malondialdehyde content), serum immune indexes (serum immunoglobulin and cytokine contents) and the expression of HSP70 family member genes ( HSPA 1A, HSPA 6 and HSPA 8) in blood lymphocytes were measured by colorimetric method,ELISA and RT-PCR,respectively. The results showed as follows: 1) Heat stress time had significant effects on serum antioxidant indexes. Compared with 0 h before heat stress,serum T-AOC ( P <0.05),SOD ( P <0.05) and GSH-Px ( P <0.01) activities decreased significantly after 8 h of heat stress,and MDA content increased significantly after 4 h of heat stress ( P <0.05). 2) Heat stress time had also significant influence on serum immune indexes. The contents of serum TNF-α and IL-2 increased significantly after 4 h of heat stress ( P <0.05), while the contents of IL-1β and IFN-γ increased significantly after 8 h of heat stress ( P <0.05). The contents of IgG ( P <0.01),IgM ( P <0.01) and IgA ( P <0.05) decreased significantly after 4 h of heat stress,while the content of IL-4 decreased significantly after 12 h of heat stress( P <0.01). 3) Heat stress time significantly increased the expression levels of HSP70 family genes ( HSPA 1A, HSPA 6 and HSPA 8) in blood lymphocytes. The mRNA expression of HSPA 1A rose firstly,then declined,and then it reached the peak at 4 h after heat stress. In addition,the mRNA expression of HSPA 1A was significantly higher at 2,4,8 and 12 h after heat stress than at 0 h ( P <0.01). The mRNA expression of HSPA 6 increased significantly at 2 h after heat stress ( P <0.01),and returned to pre-stress level after 4 h ( P >0.05). The mRNA expression of HSPA 8 was significantly higher at 4 ( P <0.05),8 ( P <0.01),12 h ( P <0.01) after heat stress than that at 0 h. In this experiment,acute heat stress at 38 ℃ could inhibit the immune and antioxidant functions and increased the mRNA expression of HSPA 1A, HSPA 6 and HSPA 8 genes in blood lymphocytes. HSPA 1A was more sensitive to temperature and time of heat stress,and could be used as a molecular marker of early heat stress in goats.