Intermedin通过抑制氧化应激减轻单侧输尿管梗阻大鼠肾脏间质纤维化

doi:10.3877/cma.j.issn.2095-3216.2017.01.006

目的

探讨intermedin(IMD)是否通过抑制氧化应激减轻单侧输尿管梗阻(UUO)大鼠肾脏间质纤维化。

方法

健康雄性Wistar大鼠，随机分为5组：假手术组、UUO模型组、UUO＋IMD组、UUO＋IMDshRNA组、UUO＋空质粒组，每组24只。转染组于质粒转染后7 d制做UUO模型，各组分别于术后1、3、7、14 d取6只大鼠：Masson染色观察肾脏纤维化程度；化学荧光法检测大鼠肾组织局部活性氧类(ROS)物质的表达；比色法检测超氧化物歧化酶(SOD)活性及脂质过氧化产物丙二醛(MDA)含量；real time RT-PCR检测肾组织血红素加氧酶-1(HO-1)的mRNA表达，免疫组化法检测其蛋白质表达。

结果

Masson染色结果显示，假手术组检测各时间点肾组织无明显纤维化表现；UUO各组随着梗阻时间的延长肾间质纤维化进行性加重(P<0.05)；UUO＋IMD组各时间点肾间质纤维化程度较相应UUO组明显减轻(P<0.05)；而UUO＋IMDshRNA组肾间质纤维化程度较相应UUO组更重(P<0.05)；UUO＋空质粒组与UUO组相比无明显区别。与假手术组相比，UUO组大鼠各检测时间点肾组织ROS和MDA含量明显增高(P<0.05)；UUO＋IMD组较相应时间点UUO组ROS和MDA含量明显降低；UUO＋IMDshRNA组较相应UUO组ROS和MDA含量更高(P<0.05)；UUO＋空质粒组则与相应UUO组相比无明显区别(P>0.05)。与假手术组相比，UUO组SOD活性、HO-1的mRNA和蛋白表达在第1d明显升高，以后逐渐下降，SOD活性第3天开始低于假手术组，而HO-1的mRNA和蛋白表达各检测时间点均高于假手术组(P<0.05)；UUO＋IMD组较相应时间点UUO组SOD活性和HO-1的表达更高(P<0.05)；UUO＋IMDshRNA组SOD活性和HO-1的表达较相应UUO组显著下降(P<0.05)；UUO＋空质粒组与UUO相比无明显区别(P>0.05)。

结论

IMD可以通过抑制氧化应激减轻UUO所致的大鼠肾脏纤维化。

关键词: 肾脏纤维化, Intermedin, 活性氧

Objective

To explore if intermedin (IMD) could reduce renal interstitial fibrosis through inhibiting reactive oxygen species (ROS) in rats with unilateral ureteral obstruction (UUO).

Methods

Healthy male Wistar rats were randomly divided into sham-operation group (n=24), UUO group (n=24), UUO+ IMD-pcDNA3.1 group (n=24), UUO+ IMDshRNA group (n=24), and UUO+ pcDNA3.1 group (n=24). Plasmid transfection was induced 7 days before UUO operation. Groups of 6 animals were killed at 1 d, 3 d, 7 d and 14 d after operation, respectively. The obstructed kidneys were harvested for further analysis. For analyzing the degree of tubulointerstitial collagen deposition, sections were stained with Masson trichrome. ROS content in the obstructed kidney was measured by chemiluminescence. Renal superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were evaluated by colorimetry. Expression of heme oxygenase-1 (HO-1) was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining, respectively.

Results

Masson trichrome showed no obvious fibrosis at each time point in kidneys of sham-operation rats. With the time of obstruction, the degree of renal interstitial fibrosis increased in the UUO group (P<0.05), but decreased in the UUO+ IMD group (P<0.05), and increased more in the UUO+ IMDshRNA group (P<0.05). Renal fibrosis showed no difference between the UUO group and UUO+ pcDNA3.1 group (P>0.05). At each time point, the ROS activity and MDA content increased obviously in the UUO group compared with the sham controls (P<0.05), but decreased in the UUO+ IMD group, and increased more in the UUO+ IMDshRNA group (P<0.05). The ROS activity and MDA content showed no difference between the UUO group and UUO+ pcDNA3.1 group (P>0.05). SOD activity and HO-1 expression increased significantly at d1 after UUO in the UUO group, and then gradually declined. The SOD activity was lower than that of the sham controls at d3, d7, d14 after UUO, while HO-1 expressions of mRNA and protein were higher than those of the sham controls at each time point (P<0.05). The SOD activity and expression of HO-1 were significantly higher in the UUO+ IMD group than in the UUO group (P<0.05), but markedly lower in the UUO+ IMDshRNA group (P<0.05). There were no significant differences in the SOD activity and HO-1 expression between the UUO group and UUO+ pcDNA3.1 group (P>0.05).

Conclusion

IMD attenuated renal fibrosis induced in the UUO rats through inhibiting oxidative stress.

Key words: Renal fibrosis, Intermedin, Reactive oxygen species

表1 UUO术后不同时间各组大鼠细胞外基质比较[纤维化面积(%), ±s]

组别	1 d	3 d	7 d	14 d
假手术	1.29±0.33	1.31±0.41	1.29±0.35	1.27±0.37
UUO	2.47±0.67^a	5.13±1.38^a	13.78±3.16^a	20.03±5.05^a
UUO＋IMD	2.05±0.49^b	3.81±1.03^b	10.15±3.05^b	16.48±4.12^b
UUO＋IMDshRNA	2.53±0.65^b	7.89±2.22^b	18.84±4.81^b	25.80±6.56^b
UUO＋空质粒	2.39±0.66	5.66±1.51	13.27±3.04	20.49±5.27

表1 UUO术后不同时间各组大鼠细胞外基质比较[纤维化面积(%), ±s]

组别	1 d	3 d	7 d	14 d
假手术	1.29±0.33	1.31±0.41	1.29±0.35	1.27±0.37
UUO	2.47±0.67^a	5.13±1.38^a	13.78±3.16^a	20.03±5.05^a
UUO＋IMD	2.05±0.49^b	3.81±1.03^b	10.15±3.05^b	16.48±4.12^b
UUO＋IMDshRNA	2.53±0.65^b	7.89±2.22^b	18.84±4.81^b	25.80±6.56^b
UUO＋空质粒	2.39±0.66	5.66±1.51	13.27±3.04	20.49±5.27

图1 UUO术后不同时间各组大鼠肾组织细胞外基质比较(Masson染色×200倍)

表2 UUO术后不同时间各组大鼠肾组织ROS的含量(×10⁴/mg, ±s)

组别	1 d	3 d	7 d	14 d
假手术	23.93±5.37	23.12±5.02	26.26±7.79	22.83±6.57
UUO	70.46±16.07^a	88.67±23.11^a	105.56±27.13^a	125.57±31.75^a
UUO＋IMD	49.58±14.78^b	66.77±18.21^b	87.72±22.82^b	110.82±27.86^b
UUO＋IMDshRNA	84.70±22.65^b	110.57±28.49^b	124.95±32.86^b	165.16±41.60^b
UUO＋空质粒	72.32±23.11	88.70±26.31	103.32±31.01	130.22±34.53

表2 UUO术后不同时间各组大鼠肾组织ROS的含量(×10⁴/mg, ±s)

组别	1 d	3 d	7 d	14 d
假手术	23.93±5.37	23.12±5.02	26.26±7.79	22.83±6.57
UUO	70.46±16.07^a	88.67±23.11^a	105.56±27.13^a	125.57±31.75^a
UUO＋IMD	49.58±14.78^b	66.77±18.21^b	87.72±22.82^b	110.82±27.86^b
UUO＋IMDshRNA	84.70±22.65^b	110.57±28.49^b	124.95±32.86^b	165.16±41.60^b
UUO＋空质粒	72.32±23.11	88.70±26.31	103.32±31.01	130.22±34.53

表3 UUO术后不同时间各组大鼠肾组织MDA的含量(nmol/mg, ±s)

组别	1 d	3 d	7 d	14 d
假手术	2.23±0.67	2.61±0.59	2.89±0.77	2.95±0.48
UUO	5.73±1.55^a	7.81±2.09^a	9.92±2.52^a	13.57±3.73^a
UUO＋IMD	4.20±1.58^b	5.06±1.59^b	7.21±2.26^b	10.36±3.09^b
UUO＋IMDshRNA	8.95±1.67^b	10.64±2.69^b	12.94±3.38^b	16.90±4.28^b
UUO＋空质粒	5.73±1.37	7.55±2.66	9.77±2.43	13.63±3.68

表3 UUO术后不同时间各组大鼠肾组织MDA的含量(nmol/mg, ±s)

组别	1 d	3 d	7 d	14 d
假手术	2.23±0.67	2.61±0.59	2.89±0.77	2.95±0.48
UUO	5.73±1.55^a	7.81±2.09^a	9.92±2.52^a	13.57±3.73^a
UUO＋IMD	4.20±1.58^b	5.06±1.59^b	7.21±2.26^b	10.36±3.09^b
UUO＋IMDshRNA	8.95±1.67^b	10.64±2.69^b	12.94±3.38^b	16.90±4.28^b
UUO＋空质粒	5.73±1.37	7.55±2.66	9.77±2.43	13.63±3.68

表4 UUO术后不同时间各组大鼠肾组织SOD活性(U/g，±s)

组别	1 d	3 d	7 d	14 d
假手术	694.20±172.15	689.64±146.64	691.55±157.39	693.02±161.44
UUO	755.24±186.11^a	637.54±157.05^a	358.11±99.04^a	260.26±64.64^a
UUO＋IMD	1263.87±266.06^b	771.66±168.00^b	482.41±128.19^b	348.53±77.28^b
UUO＋IMDshRNA	634.12±157.74^b	425.24±117.64^b	305.14±78.68^b	227.73±59.79^b
UUO＋空质粒	736.31±188.00^b	633.07±150.18	369.50±95.06	261.93±59.91

表4 UUO术后不同时间各组大鼠肾组织SOD活性(U/g，±s)

组别	1 d	3 d	7 d	14 d
假手术	694.20±172.15	689.64±146.64	691.55±157.39	693.02±161.44
UUO	755.24±186.11^a	637.54±157.05^a	358.11±99.04^a	260.26±64.64^a
UUO＋IMD	1263.87±266.06^b	771.66±168.00^b	482.41±128.19^b	348.53±77.28^b
UUO＋IMDshRNA	634.12±157.74^b	425.24±117.64^b	305.14±78.68^b	227.73±59.79^b
UUO＋空质粒	736.31±188.00^b	633.07±150.18	369.50±95.06	261.93±59.91

表5 UUO术后不同时间各组大鼠肾组织HO-1 mRNA的相对表达量(±s)

组别	1 d	3 d	7 d	14 d
假手术	0.092±0.035	0.073±0.026	0.089±0.028	0.082±0.028
UUO	3.477±1.058^a	2.675±0.954^a	1.859±0.455^a	1.076±0.312^a
UUO＋IMD	4.549±1.687^b	3.518±1.155^b	2.619±0.636^b	1.711±0.626^b
UUO＋IMDshRNA	2.312±0.650^b	1.535±0.479^b	0.804±0.113^b	0.474±0.117^b
UUO＋空质粒	3.593±1.038	2.645±0.709	1.765±0.472^b	1.101±0.279

表5 UUO术后不同时间各组大鼠肾组织HO-1 mRNA的相对表达量(±s)

组别	1 d	3 d	7 d	14 d
假手术	0.092±0.035	0.073±0.026	0.089±0.028	0.082±0.028
UUO	3.477±1.058^a	2.675±0.954^a	1.859±0.455^a	1.076±0.312^a
UUO＋IMD	4.549±1.687^b	3.518±1.155^b	2.619±0.636^b	1.711±0.626^b
UUO＋IMDshRNA	2.312±0.650^b	1.535±0.479^b	0.804±0.113^b	0.474±0.117^b
UUO＋空质粒	3.593±1.038	2.645±0.709	1.765±0.472^b	1.101±0.279

表6 UUO术后不同时间各组大鼠肾组织HO-1蛋白表达的比较(光密度值，±s)

组别	1 d	3 d	7 d	14 d
假手术	0.55±0.15	0.48±0.13	0.58±0.13	0.59±0.14
UUO	5.32±1.46^a	3.93±1.06^a	2.32±0.59^a	1.68±0.57^a
UUO＋IMD	6.75±1.61^b	4.97±1.08^b	3.86±0.83^b	2.75±0.69^b
UUO＋IMDshRNA	4.11±1.15^b	3.06±0.95^b	2.02±0.62^b	0.73±0.21^b
UUO＋空质粒	5.31±1.36	3.97±1.11	2.31±0.62	1.67±0.64

表6 UUO术后不同时间各组大鼠肾组织HO-1蛋白表达的比较(光密度值，±s)

组别	1 d	3 d	7 d	14 d
假手术	0.55±0.15	0.48±0.13	0.58±0.13	0.59±0.14
UUO	5.32±1.46^a	3.93±1.06^a	2.32±0.59^a	1.68±0.57^a
UUO＋IMD	6.75±1.61^b	4.97±1.08^b	3.86±0.83^b	2.75±0.69^b
UUO＋IMDshRNA	4.11±1.15^b	3.06±0.95^b	2.02±0.62^b	0.73±0.21^b
UUO＋空质粒	5.31±1.36	3.97±1.11	2.31±0.62	1.67±0.64

图2 UUO术后不同时间各组大鼠肾组织HO-1蛋白表达的比较(免疫组化×200倍)

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Intermedin attenuates renal interstitial fibrosis through inhibiting oxidative stress in rats with unilateral ureteral obstruction

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Intermedin attenuates renal interstitial fibrosis through inhibiting oxidative stress in rats with unilateral ureteral obstruction