Notch信号通路对IgA肾病大鼠外周血Th17细胞数量及功能的影响

doi:10.3877/cma.j.issn.2095-3216.2021.05.004

目的

分析Notch信号通路对IgA肾病大鼠肾间质纤维化过程中外周血Th17细胞数量及功能变化的影响。

方法

将大鼠分为空白组及模型组，模型建立后的大鼠随机分为3组：二甲基亚砜(DMSO)组(Notch信号通路激活剂干预)、TGF-β组和DAPT组(Notch信号通路抑制剂干预)，其外周血单个核细胞(PBMC)使用淋巴细胞分离液分离，再分别采用DMSO(0.1%)、TGF-β抑制剂(2.5 μmol/L)和DAPT(2.5 μmol/L)干预。留取大鼠肾脏组织，免疫荧光法观察大鼠肾脏IgA沉积情况，RT-PCR法检测肾脏及PBMC的Notch1、Hes1、Hes5、TGF-β及Smad3基因表达情况。采用流式细胞术检测血中Th17及Treg细胞；ELISA方法检测各组PBMC培养上清液中IL-23、IL-17、IL-10及TGF-β1水平。

结果

模型组大鼠Notch1、Hes1、Hes5、TGF-β及Smad3基因表达均明显高于空白组(P<0.05)。DAPT组及TGF-β组大鼠Notch1、Hes1、Hes5、TGF-β、Smad3基因和PBMC的磷酸化Smad3蛋白表达水平明显低于DMSO组(P<0.05)。此外，TGF-β组和DAPT组的Th17细胞、Treg细胞、Th17/Treg比值也低于DMSO组(P<0.05)。DAPT组及TGF-β组的培养上清中IL-17、IL-23、TGF-β、IL-10水平均低于DMSO组(P<0.05)。DAPT组IL-10水平显著高于TGF-β组(P<0.05)。

结论

在IgA肾病大鼠肾间质纤维化过程中，Notch信号通路异常激活。Notch信号通路可以影响外周血Th17细胞数量，对其功能也存在一定调控作用。

关键词: Notch信号通路, IgA肾病, 肾间质纤维化, Th17细胞, 功能, 大鼠

Objective

To analyze the effect of the Notch signaling pathway on the number and function of peripheral blood Th17 cells in the process of renal interstitial fibrosis of rats with IgA nephropathy.

Methods

The rats were divided into the blank group and the model group. When the model was established, the model group was randomly divided into 3 sub-groups: the dimethyl sulfoxide (DMSO) group (for Notch signaling pathway activating), TGF-β group, and DAPT group (for Notch signaling pathway inhibiting). After the peripheral blood mononuclear cells (PBMC) were separated with lymphocyte isolation solution, they were treated with DMSO (0.1%), TGF-β inhibitor (2.5 μmol/L), and DAPT (2.5 μmol/L), respectively. The kidney tissues of the rats were collected, and the IgA deposition in the rat kidneys was observed by immunofluorescence method. The expressions of Notch1, Hes1, Hes5, TGF-β, and Smad3 genes in both the kidneys and the PBMC was detected with RT-PCR method. Flow cytometry was used to detect Th17 and Treg cells in the blood, while ELISA method was used to detect the levels of IL-23, IL-17, IL-10, and TGF-β1 in the supernatant of PBMC culture of the groups.

Results

The expressions of Notch1, Hes1, Hes5, TGF-β, and Smad3 genes in the model groups were significantly higher than those in the blank group (P<0.05). The expressions of Notch1, Hes1, Hes5, TGF-β, and Smad3 genes as well as PBMC level of phosphorylated Smad3 protein in the DAPT group and TGF-β group were significantly lower than those in the DMSO group (P<0.05). Besides, the Th17 cells, Treg cells, and Th17/Treg ratios of TGF-β group and DAPT group were also lower than those of DMSO group (P<0.05). The levels of IL-17, IL-23, TGF-β, and IL-10 in the culture supernatant of the DAPT group and TGF-β group were all lower than those of the DMSO group (P<0.05). The IL-10 level of the DAPT group was significantly higher than that of the TGF-β group (P< 0.05).

Conclusion

In the process of renal interstitial fibrosis of the rats with IgA nephropathy, the Notch signaling pathway was abnormally activated. The Notch signaling pathway could not only affect the number of Th17 cells in the peripheral blood, but also had a certain regulatory effect on their function.

Key words: Notch signaling pathway, IgA nephropathy, Renal interstitial fibrosis, Th17 cells, Function, Rat

表1 引物表

基因	序列
Notch1	上游	5'-TCGTGCTCCTGTTCTTTGTG-3’
	下游	5'-TTCTCTCCCGCTTCTTCTTGC-3’
Hes1	上游	5'-GTGGGTCCTAACGCAGTGTC-3’
	下游	5'-GTCAGAAGAGAGAGGTGGGCTA-3’
Hes5	上游	5'-ATGTCTCAGTCCCAAGGAGAA-3’
	下游	5'-CTCCAGCAGCAGTTCTCT-3’
TGF-β	上游	5'-ATTCCTGGCGTTACCTTGG-3’
	下游	5'-AGCCCTGTATTCCGTTCTCT-3’
Smad3	上游	5'-GGTAAAGGATTGCCACCAAA-3’
	下游	5'-GAACAGCCAGGAAAGGGACT-3’
β-actin	上游	5'-CACCGCGAGTACAACCTTC-3’
	下游	5'-CCCATACCCACCATCACACC-3’

表1 引物表

基因	序列
Notch1	上游	5'-TCGTGCTCCTGTTCTTTGTG-3’
	下游	5'-TTCTCTCCCGCTTCTTCTTGC-3’
Hes1	上游	5'-GTGGGTCCTAACGCAGTGTC-3’
	下游	5'-GTCAGAAGAGAGAGGTGGGCTA-3’
Hes5	上游	5'-ATGTCTCAGTCCCAAGGAGAA-3’
	下游	5'-CTCCAGCAGCAGTTCTCT-3’
TGF-β	上游	5'-ATTCCTGGCGTTACCTTGG-3’
	下游	5'-AGCCCTGTATTCCGTTCTCT-3’
Smad3	上游	5'-GGTAAAGGATTGCCACCAAA-3’
	下游	5'-GAACAGCCAGGAAAGGGACT-3’
β-actin	上游	5'-CACCGCGAGTACAACCTTC-3’
	下游	5'-CCCATACCCACCATCACACC-3’

图1 大鼠肾脏组织IgA沉积免疫荧光染色情况和Masson染色结果(×400)

图2 大鼠肾脏组织和PBMC中Notch信号通路相关因子表达水平

图3 Smad3和磷酸化Smad3蛋白的Western印迹检测结果

表2 信号通路干预后各基因表达水平检测结果

指标	DMSO组	DAPT组	TGF-β组
Notch1/β-actin	0.98±0.12	0.32±0.07	0.39±0.05
Hes1/β-actin	1.08±0.09	0.13±0.02	0.17±0.02
Hes5/β-actin	1.43±0.11	0.33±0.04	0.12±0.03
TGF-β/β-actin	1.10±0.08	0.12±0.01	0.08±0.01
Smad3/β-actin	1.04±0.10	0.14±0.02	0.37±0.03

表2 信号通路干预后各基因表达水平检测结果

指标	DMSO组	DAPT组	TGF-β组
Notch1/β-actin	0.98±0.12	0.32±0.07	0.39±0.05
Hes1/β-actin	1.08±0.09	0.13±0.02	0.17±0.02
Hes5/β-actin	1.43±0.11	0.33±0.04	0.12±0.03
TGF-β/β-actin	1.10±0.08	0.12±0.01	0.08±0.01
Smad3/β-actin	1.04±0.10	0.14±0.02	0.37±0.03

图4 Th17及Treg水平检测结果

表3 上清中各指标检测结果(pg/mL)

组别	IL-17	IL-23	TGF-β	IL-10
DMSO组	88.32±1.92	52.38±1.23	194.38±3.32	83.19±1.15
DAPT组	77.02±2.58	42.19±1.41	133.43±4.12	61.26±1.05
TGF-β组	75.21±1.34	43.09±0.97	131.29±3.11	54.58±1.03

表3 上清中各指标检测结果(pg/mL)

组别	IL-17	IL-23	TGF-β	IL-10
DMSO组	88.32±1.92	52.38±1.23	194.38±3.32	83.19±1.15
DAPT组	77.02±2.58	42.19±1.41	133.43±4.12	61.26±1.05
TGF-β组	75.21±1.34	43.09±0.97	131.29±3.11	54.58±1.03

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Effect of Notch signaling pathway on the number and function of Th17 cells in peripheral blood of rats with IgA nephropathy

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Effect of Notch signaling pathway on the number and function of Th17 cells in peripheral blood of rats with IgA nephropathy