二甲双胍下调水通道蛋白4表达改善大鼠颅脑创伤早期脑水肿的研究

doi:10.3877/cma.j.issn.2095-123X.2022.04.006

目的

探讨二甲双胍对大鼠液压冲击伤后脑组织水通道蛋白4（AQP4）表达的影响及其与颅脑创伤（TBI）后脑水肿的关系。

方法

96只雄性SD大鼠采用随机数字表法分为4组，每组24只。假手术对照组（SO组）行颅骨开窗，不进行液压冲击；单纯脑损伤组（BI组）制备大鼠侧位中度液压冲击脑损伤模型；生理盐水对照组（NS组）在液压冲击伤后30 min腹腔注射0.9%氯化钠注射液；二甲双胍治疗组（MT组）大鼠液压冲击伤后30 min腹腔注射100 mg/（kg·d）的二甲双胍。4组大鼠分别于24、48和72 h进行神经功能评分，并测定脑组织含水量及AQP4蛋白和mRNA表达。

结果

BI组、NS组及MT组大鼠在24、48和72 h的神经功能评分均低于SO组，差异具有统计学意义（P<0.05）。BI组大鼠在24、48和72 h脑含水量、AQP4蛋白和AQP4 mRNA表达均高于SO组、MT组，差异均具有统计学意义（P<0.05），与NS组比较差异无统计学意义（P>0.05）。MT组和SO组24 h时的脑含水量、AQP4蛋白和AQP4 mRNA表达比较，差异无统计学意义（P>0.05），48、72 h时均高于SO组，差异具有统计学意义（P<0.05）。

结论

二甲双胍干预可明显降低TBI后脑组织AQP4蛋白和AQP4 mRNA的表达，减轻早期脑水肿，改善大鼠神经功能障碍。

关键词: 颅脑创伤, 脑水肿, 二甲双胍, 水通道蛋白4

Objective

To investigate the effect of metformin on aquaporin 4 (AQP4) expression in brain tissue after hydraulic percussion injury in rats and its relationship with brain edema, and to provide a theoretical basis for metformin treatment of brain edema after traumatic brain injury (TBI).

Methods

Ninety-six male SD were divided into 4 groups with 24 rats in each group by random number table method. The sham operation control group (SO group) only underwent skull fenestration without hydraulic impact; In the simple brain injury group (BI group), the lateral moderate hydraulic impact brain injury model was established; The normal saline control group (NS group) was intraperitoneally injected with 0.9% sodium chloride injection 30 min after hydraulic shock injury; The rats in the metformin treatment group (MT group) were injected with 100 mg/(kg·d) metformin intraperitoneally 30 min after hydraulic shock injury. The neurological function was scored at 24, 48 and 72 h, and the water content of brain tissue and the expression of AQP4 protein and mRNA were measured.

Results

The neurological function scores of rats in BI group, NS group and MT group at 24, 48 and 72 h were lower than that in SO group, and the differences were statistically significant (P<0.05). The water content of brain tissue, AQP4 protein and AQP4 mRNA expression of rats in BI group at 24, 48 and 72 h were higher than those in SO group and MT group, and the differences were statistically significant (P<0.05), while there was no significant difference compared with NS group (P>0.05). There was no significant difference in brain water content, AQP4 protein and AQP4 mRNA expression between MT group and SO group at 24 h (P>0.05), but at 48 and 72 h, the differences were higher than those of SO group (P<0.05).

Conclusion

Metformin intervention markedly reduces AQP4 protein and AQP4 mRNA expression in brain tissue after TBI, reduces brain edema, and improves neurological dysfunstion in rats.

Key words: Traumatic brain injury, Brain edema, Metformin, Aquaporin 4

表1 4组大鼠不同时间点的神经功能评分比较（分，Mean±SD）

组别	只数	24 h	48 h	72 h
SO组	8	15.00±0.00	15.00±0.00	15.00±0.00
BI组	8	6.50±0.93^c	6.75±0.89^c	8.50±0.93^abc
NS组	8	6.50±0.76^c	7.00±1.07^c	8.63±1.06^abc
MT组	8	7.88±1.25^cde	9.50±1.31^acde	10.63±1.69^abcde
F值		10.251	3.453	15.257
P值		<0.001	0.010	<0.001

表1 4组大鼠不同时间点的神经功能评分比较（分，Mean±SD）

组别	只数	24 h	48 h	72 h
SO组	8	15.00±0.00	15.00±0.00	15.00±0.00
BI组	8	6.50±0.93^c	6.75±0.89^c	8.50±0.93^abc
NS组	8	6.50±0.76^c	7.00±1.07^c	8.63±1.06^abc
MT组	8	7.88±1.25^cde	9.50±1.31^acde	10.63±1.69^abcde
F值		10.251	3.453	15.257
P值		<0.001	0.010	<0.001

表2 4组大鼠不同时间点的脑含水量比较（%，Mean±SD）

组别	只数	24 h	48 h	72 h
SO组	8	77.80±1.10	77.80±1.10	77.80±1.10
BI组	8	80.62±1.41^b	82.46±0.93^ab	82.43±1.52^ab
NS组	8	80.56±1.24^b	82.56±0.45^ab	82.61±1.26^ab
MT组	8	77.51±1.18^bcd	79.57±1.49^abcd	80.93±1.70^abcd
F值		12.453	14.538	5.235
P值		<0.001	<0.001	0.010

表2 4组大鼠不同时间点的脑含水量比较（%，Mean±SD）

组别	只数	24 h	48 h	72 h
SO组	8	77.80±1.10	77.80±1.10	77.80±1.10
BI组	8	80.62±1.41^b	82.46±0.93^ab	82.43±1.52^ab
NS组	8	80.56±1.24^b	82.56±0.45^ab	82.61±1.26^ab
MT组	8	77.51±1.18^bcd	79.57±1.49^abcd	80.93±1.70^abcd
F值		12.453	14.538	5.235
P值		<0.001	<0.001	0.010

表3 4组大鼠不同时间点AQP4蛋白相对水平比较（Mean±SD）

组别	只数	24 h	48 h	72 h
SO组	8	0.26±0.02	0.26±0.02	0.26±0.02
BI组	8	0.37±0.06^c	0.56±0.06^c	0.81±0.08^abc
NS组	8	0.37±0.08^c	0.56±0.08^c	0.77±0.09^abc
MT组	8	0.29±0.03^bcde	0.48±0.09^acde	0.66±0.11^abcde
F值		2.356	5.354	3.254
P值		<0.001	0.002	<0.001

表3 4组大鼠不同时间点AQP4蛋白相对水平比较（Mean±SD）

组别	只数	24 h	48 h	72 h
SO组	8	0.26±0.02	0.26±0.02	0.26±0.02
BI组	8	0.37±0.06^c	0.56±0.06^c	0.81±0.08^abc
NS组	8	0.37±0.08^c	0.56±0.08^c	0.77±0.09^abc
MT组	8	0.29±0.03^bcde	0.48±0.09^acde	0.66±0.11^abcde
F值		2.356	5.354	3.254
P值		<0.001	0.002	<0.001

图1 BI组和MT组大鼠AQP4蛋白表达部位的免疫组化资料（×400）黑色箭头：大脑皮质深部；白色箭头：血管周围细胞膜

图2 4组大鼠不同时间点免疫组化AQP4蛋白表达趋势图

图3 BI组和MT组大鼠AQP4蛋白Western blot检测

表4 4组大鼠AQP4 mRNA表达水平比较

组别	只数	24 h	48 h	72 h
SO组	8	0.53±0.07	0.53±0.07	0.53±0.07
BI组	8	0.60±0.03^c	0.68±0.06^c	0.76±0.05^abc
NS组	8	0.61±0.04^c	0.68±0.06^c	0.77±0.06^abc
MT组	8	0.53±0.06^cde	0.60±0.03^acde	0.69±0.05^abcde
F值		10.021	3.254	6.231
P值		<0.001	0.020	<0.001

表4 4组大鼠AQP4 mRNA表达水平比较

组别	只数	24 h	48 h	72 h
SO组	8	0.53±0.07	0.53±0.07	0.53±0.07
BI组	8	0.60±0.03^c	0.68±0.06^c	0.76±0.05^abc
NS组	8	0.61±0.04^c	0.68±0.06^c	0.77±0.06^abc
MT组	8	0.53±0.06^cde	0.60±0.03^acde	0.69±0.05^abcde
F值		10.021	3.254	6.231
P值		<0.001	0.020	<0.001

图4 BI组和MT组大鼠AQP4 mRNA表达RT-PCR检测结果

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Metformin ameliorates traumatic brain edema in rats by downregulating aquaporin 4 expression

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Metformin ameliorates traumatic brain edema in rats by downregulating aquaporin 4 expression