【作者】 陈曦；

【导师】 方静； 王成；

【作者基本信息】 四川农业大学 ， 兽医硕士（专业学位）， 2023， 硕士

【摘要】 动物在运输应激状态下出现呼吸急促、发汗、失禁等症状,导致机体不同程度的脱水与电解质紊乱。肾脏是动物机体电解质代谢的重要场所,在维持动物机体电解质平衡的调节中发挥重要作用。当动物遭受运输应激时,肾脏通过肾素-血管紧张素-醛固酮（Renin-Angiotensin-Aldosterone System,RAAS）系统调控肾脏组织离子转运相关因子,从而调节Na⁺、Cl^-、K⁺等离子的吸收与排泄,以维持机体电解质平衡。巴尔吡尔是一种碱性离子添加物,目前研究发现其可以调节奶牛血清Na⁺浓度,缓解肉牛代谢性酸中毒等作用。然而,巴尔吡尔对运输应激所致的动物机体电解质紊乱的作用尚不清楚。因此,本研究选用大鼠作为研究对象,探究巴尔吡尔对运输应激大鼠电解质代谢与肾脏抗氧化指标的影响。本试验共设五个时间点,分别为运输前三天（D-3）、运输前（D0）、运输后（D0+）、运输后第三天（D3）、运输后第十天（D10）。将240只25日龄雄性SD大鼠随机分为6组:空白对照组（Ⅰ,60只,检测时间点为D-3、D0、D0+、D3、D10）、运输应激组（Ⅱ,36只,检测时间点为D0+、D3、D10）、巴尔吡尔运前添加组（Ⅲ,48只,检测时间点为D0、D0+、D3、D10）、维生素C运前添加组（Ⅳ,48只,检测时间点同Ⅲ组）、巴尔吡尔运后添加组（Ⅴ,24只,检测时间点为D3、D10）、维生素C运后添加组（Ⅵ,24只,检测时间点同Ⅴ组）。Ⅰ组大鼠不做运输处理,Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ组大鼠在运输当天（D0+）进行3.5 h的运输处理（公路运输,平均车速34km/h,全程116km）。按照试验设计,每个时间点不同试验组分别随机选12只大鼠采样,进行如下试验:试验一巴尔吡尔对运输应激大鼠肾功能相关指标及组织形态的影响:采用全自动生化仪检测大鼠血清BUN、Cre、CysC含量,HE染色观察大鼠肾脏组织形态。结果（1）肾脏组织形态:在D0,Ⅰ、Ⅲ、Ⅳ组大鼠肾脏组织未出现明显病理学变化;在D0+,与Ⅰ组大鼠相比,Ⅱ、Ⅲ、Ⅳ组大鼠肾脏均出现不同程度的肾间质小血管充血,Ⅱ组大鼠肾脏可见肾小管中有少量脱落的上皮细胞;在D3,Ⅱ-Ⅵ大鼠间质小血管充血明显,Ⅲ、Ⅴ组大鼠少数肾小管上皮细胞颗粒和空泡变性;在D10,Ⅱ-Ⅵ大鼠肾脏组织形态已恢复正常。（2）肾功能相关指标:在D0,与Ⅰ组相比,Ⅲ组大鼠血清BUN呈上升趋势,肾脏指数呈下降趋势。在D0+,与Ⅰ组相比,Ⅱ组大鼠肾脏指数呈升高趋势,血清Cre、BUN、CysC含量无显著差异。在D3,与Ⅰ组大鼠相比,Ⅱ组大鼠血清Cre、BUN、CysC含量显著下降,肾脏指数无显著差异;与Ⅱ组相比,Ⅲ-Ⅵ组大鼠血清Cre、BUN、CysC含量、肾脏指数无显著差异。在D10,与Ⅰ组相比,Ⅱ组大鼠血清Cre、BUN、CysC含量与肾脏指数已基本恢复。试验二巴尔吡尔对运输应激大鼠肾脏抗氧化能力的影响:用DHE染色法检测大鼠肾脏组织ROS水平,用比色法检测大鼠肾组织中抗氧化指标。结果（1）在D0,与Ⅰ组相比,Ⅲ大鼠肾脏ROS显著降低,SOD、CAT呈上升趋势,MDA呈下降趋势;Ⅳ组大鼠肾脏GSH-PX显著上升。（2）在D0+,与Ⅰ组相比,Ⅱ组大鼠肾脏ROS显著上升,GSH-PX、MDA呈上升趋势,SOD呈下降趋势。与Ⅱ组相比,Ⅲ大鼠肾脏SOD、GSH-PX、T-AOC呈上升趋势,MDA呈下降趋势。（3）在D3,与Ⅰ组相比,Ⅱ组大鼠肾脏ROS显著上升,GSH显著下降,T-AOC、SOD与GSH-PX呈下降趋势,CAT、MDA呈上升趋势。与Ⅱ组相比,Ⅲ-Ⅵ组大鼠肾脏GSH、GSH-PX显著上升,ROS显著下降;Ⅲ组大鼠肾脏T-AOC、SOD显著上升;Ⅴ组大鼠肾脏T-AOC显著上升,SOD、CAT呈上升趋势。（4）在D10,与Ⅰ组相比,Ⅱ组大鼠肾脏GSH显著上升,其余指标无显著变化。与Ⅱ组相比,Ⅴ大鼠肾脏ROS显著下降,肾脏T-AOC、GSH、CAT显著上升,SOD呈上升趋势。以上结果表明,运前、运后添加巴尔吡尔降低大鼠肾脏ROS水平,提高了肾脏抗氧化酶活性,有利于减轻运输应激导致的大鼠肾脏氧化应激。试验三巴尔吡尔对运输应激大鼠血清离子水平及激素的影响:采用全自动生化仪检测大鼠血清离子水平,用ELISA法测定大鼠血清肾素、AngⅡ、ALD水平。结果（1）血清离子水平:（1）在D0,与Ⅰ组相比,Ⅲ大鼠血清Na⁺水平显著上升（2）在D0+,与Ⅰ组相比,Ⅱ组大鼠血清Na⁺、Cl^-水平显著上升,Ca²⁺水平显著降低,K⁺水平呈下降趋势。与Ⅱ组相比,Ⅲ大鼠血清Na⁺、Ca²⁺水平显著上升,K⁺水平呈上升趋势,Cl^-水平呈下降趋势。（3）在D3,与Ⅰ组相比,Ⅱ组大鼠Cl^-水平显著上升,Mg²⁺水平显著下降,Na⁺、K⁺水平呈上升趋势。与Ⅱ组相比,Ⅲ大鼠血清Mg²⁺水平显著上升,K⁺水平呈上升趋势,Na⁺水平呈下降趋势。Ⅴ组大鼠血清Cl^-水平显著下降,K⁺水平呈下降趋势。（4）在D10,与Ⅰ组相比,Ⅱ组大鼠血清K⁺、Cl^-水平显著上升,Na⁺、Mg²⁺、Ca²⁺水平已基本恢复。与Ⅱ组相比,Ⅲ-Ⅵ组大鼠血清Na⁺水平显著上升,Cl^-水平显著下降,Mg²⁺、Ca²⁺水平无显著差异;Ⅲ、Ⅴ组大鼠血清K⁺水平显著下降。（2）血清激素水平:（1）在D0,与Ⅰ组相比,Ⅲ大鼠血清AngⅡ、ALD水平呈升高趋势（2）在D0+,与Ⅰ组相比,Ⅱ组大鼠血清肾素水平显著上升,AngⅡ、ALD水平呈上升趋势。与Ⅱ组相比,Ⅲ组大鼠血清肾素水平显著下降,AngⅡ水平呈上升趋势,ALD水平呈下降趋势。（3）在D3,与Ⅰ组相比,Ⅱ组大鼠血清肾素、AngⅡ、ALD水平呈上升趋势。与Ⅱ组相比,Ⅲ组大鼠血清肾素呈下降趋势,Ⅴ组大鼠血清肾素水平显著下降;Ⅲ、Ⅴ组大鼠血清AngⅡ水平呈下降趋势;Ⅲ-Ⅵ组大鼠血清ALD水平呈下降趋势。（4）在D10,与Ⅰ组相比,Ⅱ组大鼠血清激素水平已经基本恢复。与Ⅱ组相比,Ⅲ组大鼠血清激素水平无显著差异;Ⅴ组大鼠血清AngⅡ水平呈上升趋势,ALD水平呈下降趋势。以上结果表明,运前、运后添加巴尔吡尔在一定程度上减轻了运输应激导致的大鼠血清离子水平,并影响大鼠血清肾素、AngⅡ、ALD水平的变化。试验四巴尔吡尔对运输应激大鼠肾脏离子转运功能的影响:采用RT-qPCR与WB法检测肾脏离子转运蛋白（α-ENAC、AQP1、AQP2、ROMK、NCC）mRNA与蛋白表达水平,比色法检测大鼠肾脏NKA酶活性。结果（1）在D0,与Ⅰ组相比,Ⅲ组大鼠ROMK蛋白水平显著上升,Ⅲ组大鼠NCC mRNA和蛋白水平显著上升。（2）在D0+,与Ⅰ组相比,Ⅱ组大鼠肾脏α-ENAC mRNA和蛋白水平的显著上升,AQP2蛋白水平呈上升趋势,NKA活性显著下降。与Ⅱ组相比,Ⅲ组大鼠肾脏NKA活性显著上升,NCC mRNA和蛋白水平显著上升,α-ENAC mRNA和蛋白水平呈下降趋势。（3）在D3,与Ⅰ组相比,Ⅱ组大鼠肾脏NKA活性显著上升,α-ENAC、NCC mRNA和蛋白水平呈上升趋势,ROMK蛋白水平呈下降趋势。与Ⅱ组相比,Ⅲ组大鼠肾脏AQP2、α-ENAC、ROMK蛋白表达显著上升,NKA活性显著下降;Ⅴ组大鼠肾脏NKA活性、AQP2、NCC mRNA和蛋白表达显著上升,α-ENAC、ROMK mRNA和蛋白表达呈上升趋势。（4）在D10,与Ⅰ组相比,Ⅱ组大鼠肾脏AQP2、α-ENAC、ROMK、NCC mRNA和蛋白表达无显著差异,NKA活性呈上升趋势。与Ⅱ组相比,Ⅲ组大鼠肾脏NCC mRNA和蛋白表达显著上升,α-ENAC蛋白表达显著下降,NKA活性呈下降趋势;Ⅴ组大鼠AQP2 mRNA和蛋白表达显著上升。以上结果提示巴尔吡尔可能通过调控离子转运蛋白的表达,从而缓解运输应激所致的机体电解质紊乱。综上,运输应激导致大鼠肾脏ROS蓄积,抗氧化酶活性出现波动,组织形态改变,引起大鼠血清电解质紊乱,干扰大鼠肾脏蛋白转运相关蛋白水平。运前、运后添加巴尔吡均在一定程度上缓解了上述指标的变化,提示巴尔吡尔可以一定程度减轻运输应激所致的大鼠血清电解质紊乱与肾脏氧化应激。更多还原

【Abstract】 Animals suffer from shortness of breath,sweating and incontinence during transport stress,resulting in varying degrees of dehydration and electrolyte disturbances.The kidney is an important site of electrolyte metabolism in the animal body and plays an important role in maintaining its electrolyte balance.When animals are subjected to transport stress,the kidney regulates ion transport-related factors in renal tissues through the Renin-Angiotensin-Aldosterone System（RAAS）,which regulates the uptake and excretion of Na^+,Cl^-and K⁺ions to maintain the body’s electrolyte balance.IAMC is an alkaline water-soluble ionophore additive that has been found to regulate serum Na⁺concentrations in cows and alleviate metabolic acidosis in beef cattle.However,the effect of IAMC on electrolyte disturbances in animals due to transport stress is not known.Therefore,rats were selected as research objects in this study to explore the effects of Balpiril on electrolyte metabolism and renal antioxidant indexes in transport stressed rats.There were five time points in this experiment:three days before transportation（D-3）,before transportation（D0）,after transportation（D0+）,on the third day after transportation（D3）and on the tenth day after transportation（D10）.Two hundred and forty 25-day-old male SD rats were randomly divided into six groups:blank control group（I,60 rats,tested at time points D-3,D0,D0+,D3,D10）,transport stress group（II,36 rats,tested at time points D0+,D3,D10）,IAMC pre-transport addition group（III,48 rats,tested at time points D0,D0+,D3,D10）,vitamin C（Ⅳ,48 rats,the same time points as group III）,IAMC post-transport（V,24rats,the time points were D3 and D10）and vitamin C post-transport（VI,24 rats,the same time points as group V）.Rats in group I were not treated with transport,while rats in groups II,III,IV,V and VI were treated with transport stress for 3.5 h on the day of transport（D0+）（road transport,average speed 42 km/h,total distance 116 km,temperature 26°C,sunny on that day）.According to the experimental design,12 rats were randomly selected and sampled from different test groups at each time point.The following tests were conducted:Experiment 1:Effect of IAMC on indicators related to renal function and histomorphology in transport-stressed rats:The contents of BUN,Cre and CysC in serum of rats were detected by automatic biochemical analyzer,and the morphology of kidney tissue was observed by HE staining.Results（1）Morphology of renal tissue:No obvious pathological changes were observed in D0,Ⅰ,ⅢandⅣgroups.In D0+,compared with rats in groupⅠ,the kidney of rats in groupsⅡ,ⅢandⅣshowed different degrees of renal interstitial small vessel congestion,and a small number of epithelial cells were shed in the renal tubules of rats in groupⅡ.In D3,Ⅱ-Ⅵrats,the interstitial small vessel congestion was obvious,and a few renal tubular epithelial cell granules and vacuoles were denaturated inⅢandⅤrats.The morphology of kidney tissue in D10 ratsⅡ-Ⅵwas restored to normal.（2）Related indexes of renal function:in D0,compared with groupⅠ,serum BUN of groupⅢshowed an increasing trend and renal index showed a decreasing trend.In D0+,compared with groupⅠ,the kidney index of rats in groupⅡshowed an increased trend,and the contents of serum Cre,BUN and CysC had no significant difference.In D3,compared with groupⅠ,the contents of serum Cre,BUN and CysC in groupⅡwere significantly decreased,but the renal index had no significant difference.Compared with groupⅡ,the contents of serum Cre,BUN,CysC and renal index of rats in groupⅢ-Ⅵhad no significant differences.In D10,compared with groupⅠ,the contents of serum Cre,BUN,CysC and renal index of rats in groupⅡhad basically recovered.Experiment 2:Effect of IAMC on the antioxidant capacity of the kidney in transport-stressed rats:The levels of ROS in rat kidney tissues were detected by DHE staining and the antioxidant indexes in rat kidney tissues were detected by colorimetric method.Results（1）Compared with groupⅠ,ROS of rats in D0,SOD and CAT of rats inⅢshowed an increasing trend,while MDA showed a decreasing trend.Kidney GSH-PX in groupⅣwas significantly increased.（2）In D0+,compared with groupⅠ,ROS,GSH-PX and MDA in groupⅡwere significantly increased,while SOD was decreased.Compared with groupⅡ,SOD,GSH-PX and T-AOC in kidney of ratsⅢshowed an increasing trend,while MDA showed a decreasing trend.（3）In D3,compared with groupⅠ,ROS,GSH,T-AOC,SOD and GSH-Px were significantly increased in groupⅡ,while CAT and MDA were increased.Compared with groupⅡ,kidney GSH and Gsh-Px in groupsⅢ-Ⅵwere significantly increased,while ROS was significantly decreased;The kidney T-AOC and SOD of groupⅢwere increased significantly.In groupⅤ,kidney T-AOC increased significantly,SOD and CAT increased.（4）In D10,compared with groupⅠ,kidney GSH in groupⅡwas significantly increased,while other indexes had no significant changes.Compared with groupⅡ,kidney ROS of ratsⅤdecreased significantly,kidney T-AOC,GSH,CAT increased significantly,and SOD increased.The above results indicate that the supplementation of balpiril before and after transport reduces the ROS level of the kidney,improves the activity of antioxidant enzymes in the kidney,and is beneficial to alleviate the oxidative stress of the kidney caused by transport stress.Experiment 3:Effects of IAMC on serum ion levels and hormones in rats under transport stress:The serum ion levels of rats were detected by automatic biochemical instrument,and the serum renin,AngⅡand ALD levels were determined by ELISA.Results（1）Serum ion levels:（1）In D0,compared with groupⅠ,serum Na⁺level of ratsⅢwas significantly increased;（2）In D0+,compared with groupⅠ,serum Na⁺and Cl^-levels of ratsⅡwere significantly increased,Ca²⁺level was significantly decreased,and K+level showed a downward trend.Compared with groupⅡ,the serum Na+and Ca²⁺levels of ratsⅢwere significantly increased,the serum K⁺level showed an increasing trend,and the serum Cl^-level showed a decreasing trend.（3）In D3,compared with groupⅠ,the level of Cl^-and Mg²⁺in groupⅡwas significantly increased,while the level of Na⁺and K⁺was significantly decreased.Compared with groupⅡ,the serum Mg²⁺level of ratsⅢwas significantly increased,the serum K⁺level was increased,and the serum Na⁺level was decreased.The serum Cl^-level of rats in groupⅤwas significantly decreased,and the K⁺level showed a downward trend.（4）In D10,compared with groupⅠ,serum K⁺and Cl^-levels of rats in groupⅡwere significantly increased,and the levels of Na⁺,Mg²⁺and Ca²⁺had basically recovered.Compared with groupⅡ,the serum Na⁺level of rats in groupsⅢ-Ⅵwas significantly increased,the Cl^-level was significantly decreased,and the levels of Mg²⁺and Ca²⁺were not significantly different.The serum K⁺level of rats in groupsⅢandⅤwas significantly decreased.（2）Serum hormone levels:（1）In D0,compared with groupⅠ,serum AngⅡand ALD levels of ratsⅢshowed an increasing trend;（2）In D0+,compared with groupⅠ,serum renin level of ratsⅡwas significantly increased,and AngⅡand ALD levels showed an increasing trend.Compared with groupⅡ,the serum renin level of rats in groupⅢwas significantly decreased,AngⅡlevel was increased,and ALD level was decreased.（3）Compared with groupⅠ,the serum levels of renin,AngⅡand ALD in groupⅡshowed an increasing trend in D3.Compared with groupⅡ,the serum renin level of rats in groupⅢwas decreased,and that in groupⅤwas significantly decreased.The serum AngⅡlevel in groupsⅢandⅤshowed a decreasing trend;The serum ALD level of rats in groupⅢ-Ⅵshowed a decreasing trend.（4）In D10,compared with groupⅠ,the serum hormone level of rats in groupⅡhad basically recovered.Compared with groupⅡ,the serum hormone level of groupⅢhad no significant difference.The serum AngⅡlevel of rats in groupⅤshowed an increasing trend,while the serum ALD level showed a decreasing trend.The above results indicated that adding balpiril before and after transportation could reduce the serum ion level caused by transportation stress in rats to some extent,and affect the changes of serum renin,AngⅡand ALD levels in rats.Experiment 4:Effect of IAMC on renal ion transport function in transport-stressed rats:The mRNA and protein expression levels of renal ion transport proteins（α-ENAC,AQP1,AQP2,ROMK,NCC）were detected by RT-q PCR with WB method,and NKA enzyme activity in rat kidney was detected by colorimetric method.Results（1）Compared with groupⅠ,ROMK protein level in groupⅢand NCC mRNA and protein level in groupⅢwere significantly increased in D0.（2）In D0+,compared with groupⅠ,the levels ofα-ENAC mRNA and protein in the kidney of rats in groupⅡwere increased significantly,the levels of AQP2 protein were increased,and the activity of NKA was decreased significantly.Compared with groupⅡ,NKA activity,NCC mRNA and protein levels were significantly increased in groupⅢ,whileα-ENAC mRNA and protein levels were decreased.（3）In D3,compared with groupⅠ,NKA activity in kidney of rats in groupⅡwas significantly increased,mRNA and protein levels ofα-ENAC and NCC were increased,while ROMK protein levels were decreased.Compared with groupⅡ,the protein expressions of AQP2,α-ENAC and ROMK in kidney of groupⅢwere significantly increased,while the activity of NKA was significantly decreased.In groupⅤ,NKA activity,AQP2,NCC mRNA and protein expressions were significantly increased,whileα-ENAC,ROMK mRNA and protein expressions were increased.（4）In D10,compared with groupⅠ,mRNA and protein expressions of AQP2,α-ENAC,ROMK and NCC in groupⅡshowed no significant differences,and NKA activity showed an upward trend.Compared with groupⅡ,the expression of NCC mRNA and protein in kidney of groupⅢwas significantly increased,the expression ofα-ENAC protein was significantly decreased,and the activity of NKA was decreased.The mRNA and protein expressions of AQP2 in groupⅤwere significantly increased.These results suggest that Balpiril may regulate the expression of ion transporters to relieve the electrolyte disorder caused by transport stress.In conclusion,transport stress leads to ROS accumulation,antioxidant enzyme activity fluctuations,tissue morphological changes,serum electrolyte disturbance,and interference with protein transport-related protein levels in rat kidneys.Adding IAMC before and after transportation alleviated the changes of the above indexes to a certain extent,suggesting that IAMC can alleviate the serum electrolyte disturbance and renal oxidative stress caused by transportation stress in rats to a certain extent.更多还原