外源性硫化氢抑制TLR2和TLR4表达并减轻大鼠肾脏缺血再灌注损伤

doi:10.3877/cma.j.issn.2095-3216.2022.02.001

目的

观察外源性硫化氢(H₂S)供体硫氢化钠(NaHS)能否抑制Toll样受体2(TLR2)和Toll样受体4(TLR4)表达、减轻大鼠肾脏缺血再灌注损伤(IRI)。

方法

24只6~8周龄雄性SD大鼠随机分为3组：假手术(Sham)组、肾脏缺血再灌注(I/R)组、NaHS+I/R组。采用右肾切除联合左肾动脉夹闭45 min后再灌注24 h的方法诱导肾IRI。夹闭左肾动脉前，NaHS+I/R组给予NaHS(300 nmol/min)连续输注10 min，Sham组和I/R组则给予等体积生理盐水。分别留取各组腹主动脉血及肾组织标本。Western印迹法检测肾组织TLR2、TLR4蛋白的表达；免疫组织化学法检测肾组织白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的表达；比色法检测血尿素氮(BUN)、血肌酐(Scr)。HE染色观察肾脏组织学改变；TUNEL法检测肾组织细胞凋亡。

结果

与Sham组比较，I/R组的TLR2、TLR4、IL-6、TNF-α表达均增加(P<0.05)，BUN、Scr亦明显升高(P<0.05)，肾小管上皮损伤评分较高(P<0.05)，肾组织凋亡细胞增加(P<0.05)。与I/R组比较，NaHS+I/R组的TLR2、TLR4、IL-6、TNF-α表达均减少(P<0.05)，BUN、Scr亦明显下降(P<0.05)，肾小管上皮损伤评分较低(P<0.05)，肾组织凋亡细胞减少(P<0.05)。

结论

外源性H₂S可以抑制TLR2、TLR4途径，减少炎症因子释放及细胞凋亡，减轻大鼠肾脏IRI。

关键词: 硫化氢, 肾脏, 缺血再灌注损伤, Toll样受体

Objective

To observe whether exogenous hydrogen sulfide (H₂S) donor sodium hydrosulfide (NaHS) can alleviate renal ischemia-reperfusion injury (IRI) in rats by inhibiting the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4).

Methods

Twenty-four male SD rats aged 6-8 weeks were randomly divided into 3 groups: sham group (renal sham operation), I/R group (renal ischemia-reperfusion), and NaHS+ I/R group. Renal IRI was induced by right nephrectomy combined with left renal artery clipping for 45 minutes followed by 24 hours of reperfusion. Before the left renal artery clipping, the NaHS+ I/R group was given a continuous infusion of NaHS (300 nmol/min) for 10 minutes, while the sham group and I/R group were given an equal volume of normal saline. Abdominal aortic blood and kidney tissue samples were collected from each group. The expression of TLR2 and TLR4 protein in renal tissue was detected by western blotting. The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in renal tissue was detected by immunohistochemistry. Blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by colorimetry. HE staining was used to observe renal histological changes, and TUNEL method to detect renal tissue apoptosis.

Results

Compared with the sham group, the I/R group showed more expression of TLR2, TLR4, IL-6 and TNF-α (P<0.05), higher levels of BUN and Scr (P<0.05), higher scores of the renal tubular injury (P<0.05), and more apoptotic cells in the renal tissue (P<0.05). Compared with the I/R group, the NaHS+ I/R group showed less expression of TLR2, TLR4, IL-6 and TNF-α (P<0.05), lower levels of BUN and Scr (P<0.05), lower scores of the renal tubular injury (P<0.05), and less apoptotic cells in the renal tissue (P<0.05).

Conclusion

Exogenous H₂S inhibited the expression of TLR2 and TLR4, reduced the release of inflammatory factors and apoptosis, and alleviated the renal IRI in rats.

Key words: Hydrogen sulfide, Kidney, Ischemia-reperfusion injury, Toll-like receptor

图1 各组大鼠肾组织Toll样受体2和Toll样受体4蛋白检测结果及半定量分析注：A：TLR2和TLR4蛋白Western印迹检测结果；B：各组大鼠肾组织TLR2蛋白表达分析；C：各组大鼠肾组织TLR4蛋白表达分析；TLR2：Toll样受体2；TLR4：Toll样受体4；与Sham组比较，^aP<0.01；与I/R组比较，^bP<0.01

图2 各组大鼠肾组织IL-6表达注：A：Sham组(免疫组化×200)；B：I/R组(免疫组化×200)；C：NaHS+I/R组(免疫组化×200)；D：各组大鼠肾组织IL-6相对表达水平分析；IL-6：白细胞介素6；与Sham组比较，^aP<0.05；与I/R组比较，^bP<0.05

图3 各组大鼠肾组织TNF-α表达注：A：Sham组(免疫组化×200)；B：I/R组(免疫组化×200)；C：NaHS+I/R组(免疫组化×200)；D：各组大鼠肾组织TNF-α相对表达水平分析；TNF-α：肿瘤坏死因子-α；与Sham组比较，^aP<0.05；与I/R组比较，^bP<0.05

图4 各组大鼠血尿素氮和血肌酐水平注：A：各组大鼠BUN水平分析；B：各组大鼠Scr水平分析；BUN：血尿素氮；Scr：血肌酐；与Sham组比较，^aP<0.05；与I/R组比较，^bP<0.05

表1 各组大鼠BUN、Scr水平比较(n＝6，±s)

项目	Sham组	I/R组	NaHS+I/R组
BUN(mmol/L)	4.49±0.89	18.10±0.77^a	7.64±1.46^b
Scr(μmol/L)	41.72±4.79	140.73±12.73^a	70.80±3.77^b

表1 各组大鼠BUN、Scr水平比较(n＝6，±s)

项目	Sham组	I/R组	NaHS+I/R组
BUN(mmol/L)	4.49±0.89	18.10±0.77^a	7.64±1.46^b
Scr(μmol/L)	41.72±4.79	140.73±12.73^a	70.80±3.77^b

图5 各组大鼠肾组织病理学改变注：A：Sham组(HE×400)；B：I/R组(HE×400)；C：NaHS+I/R组(HE×400)；D：各组大鼠肾组织肾小管损伤评分分析；与Sham组比较，^aP<0.05；与I/R组比较，^bP<0.05

图6 各组大鼠肾组织细胞凋亡比较注：A：Sham组(TUNEL×200)；B：I/R组(TUNEL×200)；C：NaHS+I/R组(TUNEL×200)；D：各组大鼠肾组织凋亡细胞阳性率分析；照片箭头指示凋亡细胞；与Sham组比较，^aP<0.05；与I/R组比较，^bP<0.05

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Exogenous hydrogen sulfide inhibited TLR2 and TLR4 expression and alleviated the renal ischemia-reperfusion injury in rats

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Exogenous hydrogen sulfide inhibited TLR2 and TLR4 expression and alleviated the renal ischemia-reperfusion injury in rats