辅酶Q10对子痫前期样孕鼠的肝脏保护作用及其机制的研究

发布时间：2018-06-28 07:00

本文选题：子痫前期 + 孕鼠　；参考：《福建医科大学》2014年硕士论文

【摘要】：第一部分子痫前期样孕鼠模型的建立及辅酶Q10疗效评价目的使用一氧化氮合成酶(nitric oxide synthase, NOS)抑制剂—左旋硝基精氨酸甲酯(L-nitro-arginine-methyl ester，L-NAME)建立子痫前期样孕鼠模型，从血压、24小时尿蛋白定量、肝功能、子代结局等方面评价辅酶Q10治疗子痫前期样孕鼠中的疗效。方法 1．子痫前期样孕鼠模型建立：妊娠第10-14天给予L-NAME125mg/kg/d皮下注射，妊娠第2天开始隔日无创测量血压，若较孕前血压升高30mmHg或以上则认为建模成功。 2．实验分组：选择SD(Sprague-Dawley)孕鼠30只，随机分为3组：正常妊娠组（n=10）及模型组（n=20，再分为辅酶Q10组与蒸馏水组，每组各10只孕鼠）。模型组：妊娠第10-14天予L-NAME125mg/kg/d皮下注射，正常妊娠组：妊娠第10-14天予同剂量生理盐水皮下注射；辅酶Q10组：妊娠第15-20天予辅酶Q1060mg/kg/d灌胃；蒸馏水组：妊娠第15-20天予同剂量蒸馏水灌胃。 3．血压、尿蛋白及肝功能测量：采用无创鼠尾血压检测方法，分别于孕前1周、妊娠第2天开始隔日测量血压；收集妊娠前、妊娠第10天、妊娠第15天、妊娠第20天24小时尿液进行尿蛋白定量分析；妊娠第21天留取心脏血，进行丙氨酸氨基转移酶（Alanine aminotransferase，ALT）及门冬氨酸氨基转移酶（Aspartateaminotransferase，AST）检测。 4．子代结局评价：妊娠第21天，剖宫术取出鼠胎、胎盘，计数正常鼠胎数，称取鼠胎及胎盘重量。结果 1．各组血压变化：妊娠前、妊娠第10天正常妊娠组及模型组（辅酶Q10组与蒸馏水组）孕鼠血压比较，差异均无统计学意义(P＞0.05)；妊娠第15天模型组血压较孕前均升高，差异有统计学意义(P＜0.05)，亦高于正常妊娠组，差异有统计学意义(P＜0.05)；妊娠第21天蒸馏水组孕鼠血压继续维持在较高水平，与妊娠第15天比较，差异无统计学意义；而辅酶Q10组妊娠第21天血压较妊娠第15天下降，且低于蒸馏水组，差异均有统计学意义(P＜0.05)，较正常妊娠组升高，差异有统计学意义(P＜0.05)。 2.各组24小时尿蛋白定量变化：比较三组孕鼠妊娠前、妊娠第10天24小时尿蛋白定量，差异无统计学意义；妊娠第15天，模型组24小时尿蛋白定量较妊娠前均升高，差异有统计学意义(P＜0.05)，亦高于正常妊娠组，差异有统计学意义(P＜0.05)；妊娠第20天，蒸馏水组尿蛋白高于正常妊娠组，辅酶Q10组尿蛋白低于蒸馏水组、高于正常妊娠组，分别比较，差异均有统计学意义(P＜0.05)。 3.各组肝功能变化：蒸馏水组ALT及AST均显著高于正常妊娠组，辅酶Q10组ALT及AST则较蒸馏水组显著降低，但仍较正常妊娠组升高，差异有统计学意义(P＜0.05)。 4.各组子代妊娠结局：蒸馏水组鼠胎数、鼠胎重量均低于正常妊娠组，差异均有统计学意义(P＜0.05)；辅酶Q10组鼠胎数、鼠胎重量均较蒸馏水组升高，差异均有统计学意义(P＜0.05)，低于正常妊娠组，差异均有统计学意义(P＜0.05)；模型组胎盘重量均低于正常妊娠组，差异均有统计学意义(P＜0.05)，辅酶Q10组胎盘重量高于蒸馏水组，差异均有统计学意义(P＜0.05)。结论妊娠第10-14天给予L-NAME125mg/kg/d皮下注射可以充分模拟子痫前期主要表现，成功建立子痫前期样孕鼠模型；予辅酶Q10治疗可以有效改善子痫前期样孕鼠病理生理表现，改善子代结局。第二部分辅酶Q10对子痫前期样孕鼠的肝脏保护作用及其机制的研究目的通过分析子痫前期样孕鼠肝脏氧化应激损伤水平及细胞凋亡水平的变化，探讨辅酶Q10在治疗子痫前期样孕鼠肝脏中的抗氧化应激损伤及抗细胞凋亡的作用机制。方法 1.实验分组：选择SD孕鼠50只，随机分为正常妊娠组（n=10）与模型组（n=40，均为子痫前期样孕鼠，再分为蒸馏水组、辅酶Q10组、硫酸镁+辅酶Q10组及硫酸镁组，每组各10只孕鼠）。妊娠第10-14天：模型组予左硝基精氨酸甲酯125mg/kg/d皮下注射，建立子痫前期样孕鼠模型；正常妊娠组予同剂量生理盐水皮下注射。妊娠第15-20天：蒸馏水组予蒸馏水1ml灌胃；辅酶Q10组予辅酶Q1060mg/kg/d灌胃；硫酸镁+辅酶Q10组予硫酸镁120mg/kg/d皮下注射+辅酶Q1060mg/kg/d灌胃；硫酸镁组予硫酸镁120mg/kg/d皮下注射+蒸馏水1ml灌胃。 2.氧化应激损伤水平及抗氧化酶检测：检测肝脏组织丙二醛（malondialdehyde，MDA）含量、过氧化物歧化酶（superoxide dismutase，SOD）及谷胱甘肽过氧化物酶（glutathione peroxidase, GSH-PX）活性。 3.细胞凋亡检测：提取肝脏组织蛋白， Western-blot法检测肝脏组织活化Caspase-3、Bcl-2及Bax蛋白表达水平。结果 1.各组血压变化：孕前正常妊娠组及模型组（蒸馏水组、辅酶Q10组、硫酸镁+辅酶Q10组及硫酸镁组）孕鼠血压比较，差异无统计学意义（P＞0.05）；妊娠第15天模型组孕鼠血压较孕前均升高，差异有统计学意义（P＜0.05），也高于正常妊娠组，差异有统计学意义（P＜0.05）；妊娠第21天蒸馏水组孕鼠血压继续维持在高水平，与妊娠第15天比较，差异无统计学意义（P＞0.05）；硫酸镁+辅酶Q10组、硫酸镁组、辅酶Q10组妊娠第21天血压较妊娠第15天下降，且低于蒸馏水组，差异均有统计学意义（P＜0.05）；辅酶Q10组较正常妊娠组仍升高，差异有统计学意义（P＜0.05）。 2.各组24小时尿蛋白定量变化：比较各组孕鼠妊娠前、妊娠第10天24小时尿蛋白定量，差异无统计学意义；妊娠第15天，模型组（蒸馏水组、辅酶Q10组、硫酸镁+辅酶Q10组及硫酸镁组）24小时尿蛋白定量较孕前均升高，差异有统计学意义（P＜0.05），也高于正常妊娠组，差异有统计学意义（P＜0.05）。妊娠第20天，蒸馏水组尿蛋白高于正常妊娠组，差异有统计学意义（P＜0.05）；硫酸镁+辅酶Q10组、硫酸镁组、辅酶Q10组低于蒸馏水组，差异有统计学意义（P＜0.05），仍高于正常妊娠组，差异有统计学意义（P＜0.05）。 3.各组肝功能变化：蒸馏水组ALT及AST均高于正常妊娠组，差异有统计学意义（P＜0.05）；硫酸镁+辅酶Q10组、硫酸镁组、辅酶Q10组ALT及AST较蒸馏水组降低，较正常妊娠组升高，差异均有统计学意义（P＜0.05）。 4.各组子代妊娠结局：蒸馏水组鼠胎数、鼠胎重量均显著低于正常妊娠组，硫酸镁+辅酶Q10组、硫酸镁组、辅酶Q10组鼠胎数、鼠胎重量均较蒸馏水组升高，差异有统计学意义（P＜0.05），三组之间比较，差异无统计学意义（P＞0.05）；仍低于正常妊娠组，差异有统计学意义（P＜0.05）。模型组胎盘重量均低于正常妊娠组，差异有统计学意义（P＜0.05），硫酸镁+辅酶Q10组、硫酸镁组及辅酶Q10组均高于蒸馏水组，差异有统计学意义（P＜0.05）；三组之间比较，差异无统计学意义（P＞0.05）。 5.各组肝脏GSH-PX及SOD活性水平：模型组肝脏GSH-PX及SOD活性水平均低于正常妊娠组，两两比较，差异均有统计学意义（P＜0.05）。辅酶Q10组与蒸馏水组、硫酸镁组比较均升高，分别比较，差异均有统计学意义（P＜0.05）。硫酸镁+辅酶Q10组与蒸馏水组比较升高，差异有统计学意义（P＜0.05）；与辅酶Q10组比较轻度升高，但差异无统计学意义（P＞0.05）；与硫酸镁组比较亦升高，差异有统计学意义（P＜0.05）。硫酸镁组与蒸馏水组比较升高，差异有统计学意义（P＜0.05）。 6.各组肝脏MDA水平：正常妊娠组肝脏MDA水平为（5.49±0.32）nmol/mg protein，模型组肝脏MDA水平均高于正常妊娠组，两两比较，差异均有统计学意义（P＜0.05）。蒸馏水组、辅酶Q10组、硫酸镁+辅酶Q10组及硫酸镁组分别为（9.38±0.51）nmol/mg protein、（6.80±0.32）nmol/mg protein、（6.86±0.45）nmol/mg protein及（7.73±0.28）nmol/mg protein，除辅酶Q10组与硫酸镁+辅酶Q10组比较，差异无统计学意义（P＞0.05），其余各组间两两比较，差异均有统计学意义（P＜0.05）。 7.各组肝脏组织活化Caspase-3、Bcl-2及Bax蛋白水平：蒸馏水组孕鼠肝脏组织活化Caspase-3及Bax蛋白较正常妊娠组、硫酸镁+辅酶Q10组、硫酸镁组、辅酶Q10组增多，Bcl-2较正常妊娠组减少，差异均有统计学意义（P＜0.05），模型组经过硫酸镁、辅酶Q10治疗后与蒸馏水组比较，活化Caspase-3及Bax蛋白表达减少（P＜0.05），Bcl-2蛋白表达增多，差异均有统计学意义（P＜0.05）。结论子痫前期样孕鼠肝脏组织氧化应激水平、细胞凋亡水平均升高；辅酶Q10可以有效改善子痫前期样孕鼠肝功能，减少肝脏细胞凋亡，具有保护肝脏的作用，其机制可能是通过抗氧化应激损伤和调节Bcl-2/Bax平衡从而抑制细胞凋亡。
[Abstract]:Establishment of Preeclampsia Model and Evaluation of Coenzyme Q10 in the First Part of Preeclampsia

Purpose

Using nitric oxide synthase ( NOS ) inhibitor , L - nitro - arginine methyl ester ( L - NAME ) to establish a rat model of pre - eclampsia , the effect of coenzyme Q10 in the treatment of pre - eclampsia was evaluated from the aspects of blood pressure , 24 - hour urinary protein , liver function and progeny outcome .

method

1 . The model of pre - eclampsia was established : L - NAME125mg / kg / day subcutaneous injection was administered on the 10th - 14th day of pregnancy , and the blood pressure was not measured on the 2nd day of gestation . If the blood pressure increased by 30 mmHg or above , the model was considered successful .

2 . Experimental group : Thirty - three Sprague - Dawley rats were randomly divided into three groups : normal pregnancy group ( n = 10 ) and model group ( n = 20 , subdivided into coenzyme Q10 group and distilled water group , 10 pregnant rats in each group ) . Model group : subcutaneous injection of L - NAME125mg / kg / day at 10 - 14 days of gestation , normal pregnancy group : subcutaneous injection with same dose of physiological saline at 10 - 14 days of pregnancy ;
Coenzyme Q10 group : coenzyme Q1060 mg / kg / day was administered to the stomach at the 15th to 20th days of pregnancy ;
Distilled water group : 15 - 20 days gestation , the same dose of distilled water was administered to the stomach .

3 . Measurement of blood pressure , urinary protein and liver function : The blood pressure was measured at 1 week before pregnancy and the blood pressure was measured on the 2nd day of gestation , respectively .
Urine protein was analyzed quantitatively in urine before pregnancy , gestation day 10 , gestation day 15 , gestation day 20 24 hours ;
Heart blood was collected on Day 21 of gestation and alanine aminotransferase ( ALT ) and aspartate aminotransferase ( AST ) were detected .

4 . Evaluation of the outcome of the progeny : 21 days of pregnancy , the uterus was taken out to remove the fetal placenta and the placenta , and the number of normal fetuses was counted , and the weight of the fetus and the placenta was weighed .

Results

1 . Blood pressure changes in each group : Before pregnancy , there was no significant difference in blood pressure between normal pregnant group and model group ( coenzyme Q10 group and distilled water group ) in the normal pregnancy group and the model group ( P > 0.05 ) .
The blood pressure of the 15th day of pregnancy was higher than that in normal pregnancy group ( P < 0.05 ) , and the difference was statistically significant ( P < 0.05 ) .
The blood pressure of pregnant rats in distilled water group was maintained at a high level in the 21st day of gestation , and the difference was not statistically significant compared with the 15th day of pregnancy .
Compared with the control group , the blood pressure of the coenzyme Q10 group was lower than that in the group of distilled water ( P < 0.05 ) , and the difference was statistically significant ( P < 0.05 ) .

2 . The quantitative changes of urinary protein 24 hours in each group : there was no significant difference in the amount of urinary protein 24 hours after pregnancy in three groups .
In the 15th day of gestation , the 24 - hour urinary protein of the model group was higher than that in the normal pregnancy group ( P < 0.05 ) , which was higher than that in the normal pregnancy group ( P < 0.05 ) .
The urinary protein in the distilled water group was higher than that in the normal pregnancy group on the 20th day of gestation , and the urinary protein in the coenzyme Q10 group was lower than that in the normal pregnancy group , which was higher than that in the normal pregnancy group , and the difference was statistically significant ( P < 0.05 ) .

3 . The changes of liver function in each group : ALT and AST in the distilled water group were significantly higher than those in the normal pregnancy group , but the ALT and AST in the coenzyme Q10 group were significantly lower than that in the distilled water group , but still higher in the normal pregnancy group ( P < 0.05 ) .

4 . The pregnant outcome of each group : the number of mice in the distilled water group and the weight of the fetus were lower than that of the normal pregnancy group ( P < 0.05 ) .
The number of Coenzyme Q10 group and the weight of fetus were higher than that in the distilled water group ( P < 0 . 05 ) , and the difference was statistically significant ( P < 0 . 05 ) .
The placental weight of the model group was lower than that in normal pregnancy group ( P < 0.05 ) , and the placental weight of coenzyme Q10 group was higher than that in distilled water group ( P < 0.05 ) .

Conclusion

Subcutaneous injection of L - NAME125mg / kg / day on the 10th to 14th days of pregnancy can fully simulate the main manifestations of the pre - eclampsia , and successfully establish a pre - eclampsia - like pregnant rat model ;
Coenzyme Q10 treatment can effectively improve the pathological physiology of pregnant mice in the early stage of eclampsia and improve the outcome of offspring .

Study on the Protective Effect of the Second Part of Coenzyme Q10 on the Liver of Preeclampsia - like Pregnant Rats and Its Mechanism

Purpose

The effects of coenzyme Q10 on oxidative stress injury and cell apoptosis in the liver of pregnant rats with pre - eclampsia were investigated by analyzing the changes of oxidative stress injury level and cell apoptosis level in pre - eclampsia - like pregnant rats .

method

1 . Experimental group : 50 SD pregnant rats were randomly divided into normal pregnancy group ( n = 10 ) and model group ( n = 40 , all were pre - eclampsia - like pregnant rats , then divided into distilled water group , coenzyme Q10 group , magnesium sulfate + coenzyme Q10 group and magnesium sulfate group , 10 pregnant rats in each group ) . 10 - 14 days of gestation : the model group was injected subcutaneously with left nitro - arginine methyl ester 125mg / kg / day to establish a pre - eclampsia - like pregnant rat model ;
The normal pregnancy group was administered subcutaneously with the same dose of physiological saline . Days 15 - 20 of gestation : 1 ml of distilled water was administered to the distilled water .
Coenzyme Q10 group was administered with coenzyme Q 101 mg / kg / d ;
Magnesium sulfate + coenzyme Q10 group was administered subcutaneously with 120 mg / kg / day magnesium sulfate + coenzyme Q1060 mg / kg / day ;
Magnesium sulfate group was given 120 mg / kg / day magnesium sulfate and 1 ml of distilled water .

2 . Oxidative stress injury level and anti - oxidation enzyme detection : detect the content of malondialdehyde ( MDA ) , superoxide dismutase ( SOD ) and glutathione peroxidase ( GSH - PX ) activity in liver .

3 . Apoptosis was detected by Western - blot . Caspase - 3 , Bcl - 2 and Bax protein expression levels were detected by Western - blot .

Results

1 . Blood pressure changes in each group : the blood pressure of pregnant rats in the normal pregnant group and the model group ( distilled water group , coenzyme Q10 group , magnesium sulfate + coenzyme Q10 group and magnesium sulfate group ) had no statistical significance ( P > 0.05 ) ;
The blood pressure of pregnant rats was higher than that in normal pregnancy group ( P < 0.05 ) , and the difference was statistically significant ( P < 0.05 ) .
The blood pressure of pregnant rats in distilled water group was maintained at a high level in the 21st day of gestation , and the difference was not statistically significant ( P > 0.05 ) compared with the 15th day of pregnancy .
The blood pressure of magnesium sulfate + coenzyme Q10 group , magnesium sulfate group and coenzyme Q10 group decreased on the 15th day of gestation and lower than that in distilled water group ( P < 0.05 ) .
There was significant difference between coenzyme Q10 group and normal pregnancy group ( P < 0.05 ) .

2 . Changes of urinary protein in 24 hours in each group : compared with that of pregnant rats in each group , there was no significant difference in the amount of urine protein in 24 hours of gestation at the 10th day of pregnancy ;
In the 15th day of gestation , the 24 - hour urinary protein of the model group ( distilled water group , coenzyme Q10 group , magnesium sulfate + coenzyme Q10 group and magnesium sulfate group ) was higher than that in normal pregnancy group ( P & lt ; 0.05 ) , and the difference was statistically significant ( P < 0.05 ) . The urinary protein of distilled water group was higher than that of normal pregnancy group ( P < 0.05 ) .
Magnesium sulfate + coenzyme Q10 group , magnesium sulfate group and coenzyme Q10 group were lower than that in distilled water group ( P < 0 . 05 ) , but the difference was statistically significant ( P < 0 . 05 ) .

3 . Changes of liver function in each group : ALT and AST in distilled water group were higher than those in normal pregnancy group ( P < 0.05 ) .
ALT and AST in the magnesium sulfate + coenzyme Q10 group , the magnesium sulfate group and the coenzyme Q10 group were lower than those in the distilled water group , and the difference was statistically significant ( P < 0.05 ) .

4 . The pregnant outcome of each group : the number of rats in the distilled water group and the weight of the fetus were significantly lower than that in the normal pregnancy group , the magnesium sulfate + coenzyme Q10 group , the magnesium sulfate group , the coenzyme Q10 group and the mouse embryo , the weight of the rats was higher than that in the distilled water group , the difference was statistically significant ( P < 0.05 ) , and the difference was not statistically significant ( P > 0.05 ) ;
The placental weight of the model group was lower than that in the normal pregnancy group ( P < 0 . 05 ) , the difference was statistically significant ( P < 0 . 05 ) , the magnesium sulfate + coenzyme Q10 group , the magnesium sulfate group and the coenzyme Q10 group were all higher than that of the distilled water group , the difference was statistically significant ( P < 0.05 ) ;
There was no significant difference between the three groups ( P > 0.05 ) .

5 . The levels of GSH - PX and SOD in the liver of each group were lower than those in normal pregnancy group ( P < 0 . 05 ) . Compared with the group of distilled water and magnesium sulfate ( P < 0.05 ) , there was significant difference between the coenzyme Q10 group and the distilled water group ( P < 0.05 ) .
Compared with coenzyme Q10 group , there was no significant difference ( P > 0.05 ) .
Compared with the magnesium sulfate group , the difference was statistically significant ( P < 0.05 ) . There was significant difference between the magnesium sulfate group and the distilled water group ( P < 0.05 ) .

6 . The level of MDA in liver of normal pregnancy group was ( 5.49 卤 0.32 ) nmol / mg protein , and MDA level in the model group was higher than that in normal pregnancy group ( 9.38 卤 0.51 ) nmol / mg protein , ( 6.80 卤 0.32 ) nmol / mg protein , ( 6.86 卤 0.45 ) nmol / mg protein and ( 7.73 卤 0.28 ) nmol / mg protein , respectively .

7 . The levels of Caspase - 3 , Bcl - 2 and Bax protein in liver tissues of each group were higher than those in normal pregnancy group , magnesium sulfate + coenzyme Q10 group , magnesium sulfate group , coenzyme Q10 group , and Bcl - 2 group . The expression of Caspase - 3 and Bax protein decreased ( P < 0.05 ) .

Conclusion

The level of oxidative stress and the level of apoptosis in the liver tissues of pregnant rats with pre - eclampsia were increased .
Coenzyme Q10 can effectively improve the liver function of the pre - eclampsia - like pregnant mice , reduce the apoptosis of the liver cells , and have the function of protecting the liver , and the mechanism may be to inhibit apoptosis by resisting oxidative stress injury and regulating the Bcl - 2 / Bax balance .
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.244

【参考文献】