促肝细胞生长素通过调节核呼吸因子1和线粒体转录因子A表达对大鼠肝脏缺血再灌注损伤保护作用的研究

doi:10.3877/cma.j.issn.1674-0793.2020.06.003

目的

研究促肝细胞生长素（PHGF）是否通过上调核呼吸因子1（NRF-1）和线粒体转录因子A（TFAM）表达对大鼠肝脏缺血再灌注损伤（HIRI）起到保护作用。

方法

首先将备选干扰序列导入大鼠肝星状细胞HSC-T6中，通过检测NRF-1、TFAM蛋白及mRNA表达，筛选出干扰效果最强序列；然后将108只SD大鼠依据再灌注时间不同（1、3、7 d）随机分为3组，每组36只。每个时间点大鼠又分为实验组和对照组，每组18只，实验组术前4 d分别尾静脉注射相应慢病毒颗粒（1×10⁸个/只），术后每只大鼠腹腔注射PHGF 15 μg/d，对照组术后每只大鼠每日腹腔注射等量0.9%氯化钠溶液。实验组和对照组分别设置3个组，每组6只，分别为阴性沉默组、靶向沉默NRF-1组、靶向沉默TFAM组，检测每个时间点大鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、总胆红素（TBIL）水平及肝组织NRF-1、TFAM mRNA表达水平，苏木精-伊红染色再灌注7 d时大鼠肝脏病理学改变。

结果

从备选序列中选出干扰TFAM、NRF-1表达效果最强序列分别为T2、N3，使用PHGF对HIRI模型RNAi大鼠处理发现，与阴性沉默组比较，降低NRF-1、TFAM mRNA表达水平可导致ALT、AST、TBIL水平明显升高（P<0.05）。使用PHGF处理的实验组大鼠较对照组大鼠ALT、AST、TBIL水平明显降低，以第7天差异最为明显，且NRF-1、TFAM mRNA明显升高（P<0.05）。

结论

PHGF通过上调NRF-1和TFAM表达对大鼠HIRI起到保护作用。

关键词: 促肝细胞生长素, 缺血再灌注损伤, 核呼吸因子1, 线粒体转录因子A

Objective

To investigate whether rats’ hepatic ischemia-reperfusion injury (HIRI) can be promoted by hepatocyte growth factor (PHGF) by up-regulating the expression of nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM).

Methods

First, the candidate interference sequences were introduced into HSC-T6 of rat hepatic satellate cells, and the expression of NRF-1, TFAM proteins and mRNA was detected to select the strongest interference sequences. Second, 108 SD rats were randomly divided into three groups according to different reperfusion time (1,3,7 d), with 36 rats in each group. At each time point, the rats were divided into two groups with 18 rats in each group, which were the experimental group and the control group (in the experimental group, the corresponding lentiviral particles were injected into the tail vein, 4 days before the operation). The experimental group and the control group were set into 3 groups, 6 rats in each group, respectively a negative silencing group, a targeted silencing NRF-1 group, and a targeted silencing TFAM group. Rat serum alanine aminotransferase (ALT) was detected at each time point, aspartate aminotransferase (AST), total bilirubin (TBIL) content, and expression levels of NRF-1 and TFAM mRNA in liver tissue, pathological changes in rat liver after 7 days of HE staining.

Results

T2 and N3 were selected from the candidate sequences to interfere with the expression of TFAM and NRF-1. Treatment of HIRI model RNAi rats with PHGF showed that compared with the negative silencing group, reducing the levels of NRF-1 and TFAM mRNA would result in significantly higher levels of ALT, AST, and TBIL (P<0.05). After treatment with PHGF, the contents of ALT, AST, and TBIL were significantly lower than those treated with normal saline, and the differences were most obvious on the 7th day, and the increase of NRF-1 and TFAM mRNA increased significantly (P<0.05).

Conclusion

PHGF can protect HIRI by up-regulating the expression of NRF-1 and TFAM.

Key words: Promoting hepatocyte growth factor, Ischemia reperfusion injury, Nuclear respiratory factor 1, Mitochondrial transcription factor A

表1 各组细胞靶基因mRNA相对表达量（±s，n=6）

组别	TFAM mRNA	组别	NRF-1 mRNA
T1	0.453±0.013^ab	N1	0.833±0.033^ac
T2	0.303±0.025^a	N2	0.775±0.025^ac
T3	0.725±0.052^ab	N3	0.542±0.041^a
LIPO	1.009±0.071^b	LIPO	1.002±0.043^c
CON	0.994±0.023^b	CON	0.942±0.026^c

表1 各组细胞靶基因mRNA相对表达量（±s，n=6）

组别	TFAM mRNA	组别	NRF-1 mRNA
T1	0.453±0.013^ab	N1	0.833±0.033^ac
T2	0.303±0.025^a	N2	0.775±0.025^ac
T3	0.725±0.052^ab	N3	0.542±0.041^a
LIPO	1.009±0.071^b	LIPO	1.002±0.043^c
CON	0.994±0.023^b	CON	0.942±0.026^c

图1 不同序列降低线粒体转录因子A（TFAM）蛋白表达水平与T1、T3序列比较，*P<0.05

图2 不同序列降低核呼吸因子1（NRF-1）蛋白表达水平与N1、N2序列比较，*P<0.05

图3 各组大鼠不同再灌注时间血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、总胆红素（TBIL）水平测定　A为阴性沉默组；B为靶向沉默TFAM组；C为靶向沉默NRF-1组；1为术后注射促肝细胞生长素（PHGF）；2为术后注射0.9%氯化钠溶液

表2 术后各组大鼠目的基因相对表达水平变化情况(±s，n=6)

mRNA	组别	术后第1天	术后第3天	术后第7天	变化幅度(%)
TFAM	A1组	0.552±0.021	0.715±0.019	1.138±0.034	106.2±8.3^b
	A2组	0.544±0.032	0.603±0.026	0.980±0.038	80.3±6.8
	B1组	0.415±0.024^a	0.538±0.019	0.784±0.025	89.0±6.3^b
	B2组	0.373±0.027^a	0.461±0.017	0.607±0.017	63.0±5.7
NRF-1	A1组	0.518±0.030	0.721±0.026	1.196±0.011	130.8±8.1^b
	A2组	0.494±0.024	0.614±0.031	1.013±0.031	104.9±6.5
	C1组	0.400±0.020^a	0.570±0.018	0.810±0.021	102.4±5.9^b
	C2组	0.384±0.020^a	0.501±0.021	0.696±0.019	81.3±5.1

表2 术后各组大鼠目的基因相对表达水平变化情况(±s，n=6)

mRNA	组别	术后第1天	术后第3天	术后第7天	变化幅度(%)
TFAM	A1组	0.552±0.021	0.715±0.019	1.138±0.034	106.2±8.3^b
	A2组	0.544±0.032	0.603±0.026	0.980±0.038	80.3±6.8
	B1组	0.415±0.024^a	0.538±0.019	0.784±0.025	89.0±6.3^b
	B2组	0.373±0.027^a	0.461±0.017	0.607±0.017	63.0±5.7
NRF-1	A1组	0.518±0.030	0.721±0.026	1.196±0.011	130.8±8.1^b
	A2组	0.494±0.024	0.614±0.031	1.013±0.031	104.9±6.5
	C1组	0.400±0.020^a	0.570±0.018	0.810±0.021	102.4±5.9^b
	C2组	0.384±0.020^a	0.501±0.021	0.696±0.019	81.3±5.1

图4 再灌注7 d时各组大鼠肝脏组织苏木精-伊红染色结果（×200）　A为阴性沉默组，注射PHGF；B为阴性沉默组，注射0.9%氯化钠溶液；C为靶向沉默TFAM组，注射PHGF；D为靶向沉默TFAM组，注射0.9%氯化钠溶液；E为靶向沉默NRF-1组，注射PHGF；F为靶向沉默NRF-1组，注射0.9%氯化钠溶液

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Protective effect of promoting hepatocyte growth factor in rats’ hepatic ischemia-reperfusion injury byregulating the expression of NRF-1 /TFAM

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Protective effect of promoting hepatocyte growth factor in rats’ hepatic ischemia-reperfusion injury byregulating the expression of NRF-1 /TFAM