髓源性抑制细胞通过抑制炎症反应减轻小鼠肾脏缺血再灌注损伤

doi:10.3877/cma.j.issn.1674-3903.2023.01.006

目的

探讨髓源性抑制细胞(MDSC)对小鼠肾脏缺血再灌注损伤(IRI)的作用及其可能机制。

方法

15只雄性C57BL/6小鼠分为假手术组、IRI组、IRI＋MDSC组，每组5只。所有小鼠采取背部双侧切口，切除左肾。假手术组小鼠暴露右肾50 min后缝合，IRI组夹闭右侧肾蒂50 min后开放灌注，IRI＋MDSC组于开放灌注1 h内经尾静脉输注使用C57BL/6小鼠骨髓细胞诱导的MDSC。恢复灌注后24h，留取小鼠血液及肾脏和脾脏组织标本，观察肾组织病理变化，检测血尿素氮和肌酐水平。ELISA检测血清中细胞因子TNF-α、IFN-γ、IL-6和IL-10水平。流式细胞术检测脾脏中T细胞活化。符合正态分布的计量数据以均数±标准差(±s)表示，采用单因素方差分析比较。

结果

与IRI组相比，IRI＋MDSC组小鼠肾功能明显改善，血尿素氮和血清肌酐水平均低于IRI组([(29.6±3.1)和(34.1±1.4)mmol/L，(105±13)和(176±20)μmol/L，P均<0.05]。IRI＋MDSC组肾小管上皮细胞坏死程度、范围及炎性细胞浸润程度较IRI组减轻，Paller评分优于IRI组[(55.0±3.0)和(40.6±6.7)分，P<0.05)]。IRI＋MDSC组血清中促炎性细胞因子水平均低于IRI组(P均<0.05)，TNF-α分别为(452±55)和(605±73)pg/mL，IFN-γ分别为(405±101)和(576±76)pg/mL，IL-6分别为(459±37)和(863±20)pg/mL；而具有抗炎作用的细胞因子IL-10水平则高于IRI组，分别为(1519±244)和(926±66)pg/mL。同时，IRI＋MDSC组小鼠脾脏中活化的CD4^＋T细胞和CD8^＋T细胞水平均低于IRI组，分别为(15.6±1.3)%和(20.2±1.4)%、(31.8±1.8)%和(40.4±2.0)%，差异均有统计学意义(P均<0.05)。

结论

体外诱导MDSC可通过抑制IRI后机体的炎症反应，减轻肾损伤。

关键词: 髓源性抑制细胞, 缺血再灌注损伤, 肾脏, 炎症反应, 小鼠

Objective

To investigate the effect and mechanism of myeloid-derived suppressor cells (MDSCs) on renal ischemia-reperfusion injury (IRI) in mice.

Methods

A total of 15 male C57BL/6 mice were randomly divided into Sham group (n=5), IRI group (n=5), IRI combined with MDSCs transfusion group (IRI+ MDSC group, n=5). Twenty-four hours after perfusion, blood and kidney tissue samples of mice were collected, and the pathological changes of kidney tissue were observed by hematoxylin-eosin (HE) staining, and the contents of blood urea nitrogen (BUN) and serum creatinine (Cr) were detected. Serum levels of cytokines TNF-α, IFN-γ, IL-6 and IL-10 were detected by ELISA. T-cell activation in spleen was detected by flow cytometry. One-way ANOVA was used to compare the differences between multiple groups.

Results

Compared with the IRI group, the IRI+ MDSC group showed significantly improved renal function [Cr: (105±13) and (176±20) μmol/L, BUN: (34.1±1.4) and (29.6±3.1) mmol/L, P<0.05]. The necrosis degree and range of renal tubular epithelial cells and inflammatory cell infiltration degree were reduced in the IRI+ MDSC group. Paller scores were better in the IRI+ MDSC group than those in the IRI group [(55.0±3.0) vs. (40.6±6.7), P<0.05]. Serum levels of pro-inflammatory cytokines [TNF-α: (452±55) vs. (605±73) pg/mL, IFN-γ: (405±101) vs. (576±76) pg/mL, and IL-6: (459±37) vs. (863±20) pg/mL] in the IRI+ MDSC group were lower than those in IRI group (all P<0.05), respectively. However, the level of anti-inflammatory cytokine IL-10 was higher in the IRI+ MDSC group [(1519±244) pg/mL]. At the same time, the levels of activated CD4⁺ T cells and CD8⁺ T cells in spleen of the IRI+ MDSC group were lower than those of the IRI group, which were (15.6±1.3)% and (20.2±1.4)%, (31.8±1.8)% and (40.4±2.0)%, respectively, and the differences were statistically significant (all P<0.05).

Conclusion

MDSCs induced in vitro can alleviate renal injury by inhibiting the inflammatory response after IRI.

Key words: Myeloid-derived suppressor cell, Ischemia-reperfusion injury, Renal, Inflammatory response, Mice

图1 GM-CSF联合PGE2体外诱导的MDSC表面标志物及其抑制功能实验注：GM-CSF.粒细胞-巨噬细胞集落刺激因子；PGE2.前列腺素E2; MDSC.髓源性抑制细胞；a和b.流式细胞术检测MDSC表面标志物及比例；c．流式细胞术检测MDSC对CD8^＋ T细胞增殖的抑制功能；^*.P<0.05

表1 三组小鼠肾功能、细胞因子和T细胞活化水平比较(±s)

组别	只数	肾功能		细胞因子(pg/mL)				T细胞活化(%)
组别	只数	BUN(mmol/L)	Cr(μmol/L)	TNF-α	IFN-γ	IL-6	IL-10	CD4^＋CD44^＋CD62L^－	CD8^＋CD44^＋CD62L^－
假手术组	5	15.8±1.8	27±6	86±23	104±18	96±29	114±8	13.0±2.2	21.6±2.2
IRI组	5	34.1±1.4^a	176±20^a	605±73^a	576±76^a	863±20^a	926±66^a	20.2±1.4^a	40.4±2.0^a
IRI＋MDSC组	5	29.6±3.1^a，b	105±13^a，b	452±55^a，b	405±101^a，b	459±37^a，b	1 519±244^a，b	15.6±1.3^a，b	31.8±1.8^a，b

表1 三组小鼠肾功能、细胞因子和T细胞活化水平比较(±s)

组别	只数	肾功能		细胞因子(pg/mL)				T细胞活化(%)
组别	只数	BUN(mmol/L)	Cr(μmol/L)	TNF-α	IFN-γ	IL-6	IL-10	CD4^＋CD44^＋CD62L^－	CD8^＋CD44^＋CD62L^－
假手术组	5	15.8±1.8	27±6	86±23	104±18	96±29	114±8	13.0±2.2	21.6±2.2
IRI组	5	34.1±1.4^a	176±20^a	605±73^a	576±76^a	863±20^a	926±66^a	20.2±1.4^a	40.4±2.0^a
IRI＋MDSC组	5	29.6±3.1^a，b	105±13^a，b	452±55^a，b	405±101^a，b	459±37^a，b	1 519±244^a，b	15.6±1.3^a，b	31.8±1.8^a，b

图2 三组小鼠恢复灌注后24 h肾组织病理检查结果(HE ×400)注：IRI.缺血再灌注损伤；MDSC.髓源性抑制细胞；a.假手术组；b. IRI组；c. IRI＋MDSC组

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