果糖通过尿酸和活性氧簇诱导人肾小管上皮细胞单核细胞趋化蛋白-1的表达

Fructose induces HK-2 cells to express monocyte chemoattractant protein-1 through uric acid and reactive oxygen species

目的·探讨果糖诱导肾小管上皮细胞表达单核细胞趋化蛋白-1(monocyte chemoattratant protein-1,MCP-1)的机制。方法·将HK-2细胞分为对照组,果糖孵育(1、5和10 mmol/L)组,果糖、己酮糖激酶抑制剂(KHK-IN)共孵育(果糖5 mmol/L,KHKIN分别为12、100和1 000 nmol/L)组,尿酸孵育(5、15和50 mg/d L)组,果糖、别嘌醇共孵育(果糖5 mmol/L,别嘌醇分别为0.01、0.1和0.5 mmol/L)组,尿酸、别嘌醇共孵育(尿酸50 mg/d L,别嘌醇分别为0.01、0.1和0.5 mmol/L)组,H2O2孵育(0.1和0.3 mmol/L)组,果糖、N-乙酰半胱氨酸(NAC)共孵育(果糖5 mmol/L,NAC分别为5、10和50 mmol/L)组,尿酸、NAC共孵育(尿酸50 mg/d L,NAC分别为5、10和50 mmol/L)组。采用实时荧光定量PCR和蛋白免疫印迹实验检测MCP-1 m RNA和蛋白的表达;利用活性氧(ROS)荧光探针观察果糖和尿酸对HK-2细胞ROS产生的影响。结果·果糖剂量及时间依赖性诱导HK-2细胞MCP-1 m RNA转录和蛋白表达,此过程可被KHK-IN阻断。外源性尿酸诱导HK-2细胞产生MCP-1,别嘌醇抑制果糖引起的MCP-1表达,但不能阻断外源性尿酸的作用。果糖和尿酸均诱导HK-2细胞产生ROS,别嘌醇抑制了果糖而非外源性尿酸诱导的ROS产生。H2O2诱导HK-2细胞生成MCP-1,NAC则抑制这种作用。结论·果糖在己酮糖激酶催化下,通过引起细胞内尿酸升高和产生ROS,导致人肾小管上皮细胞产生MCP-1。

Objective · To investigate the mechanism of fructose-induced monocyte chemoattratant protein-1（MCP-1） production in HK-2 cells. Methods · The HK-2 cells were divided into fructose incubated（1, 5 and 10 mmol/L） group, fructose and ketohexo-kinase inhibitor（KHK-IN） coincubation（fructose 5 mmol/L, KHK-IN was 12, 100 and 1 000 nmol/L, respectively） group, uric acid incubation（5, 15 and 50 mg/d L） group, fructose and allopurinol co-incubation（fructose 5 mmol/L, allopurinol were 0.01, 0.1 and 0.5 mmol/L） group, uric acid and allopurinol co-incubation（uric acid 50 mg/d L, allopurinol respectively 0.01, 0.1 and 0.5 mmol/L） group, H2 O2 incubation（0.1 and 0.3 mmol/L） group, fructose and N-acetylcysteine（NAC） coincubation（fructose 5 mmol/L, NAC respectively 5, 10 and 50 mmol/L） group, and uric acid and NAC co-incubation（uric acid 50 mg/d L, NAC was 5, 10 and 50 mmol/L, respectively） group. The quantitative PCR method and Western blotting method were used to observe the expression of MCP-1 m RNA and protein. The effects of fructose and uric acid on the production of ROS in HK-2 cells were observed by using a fluorescent probe. Results · Fructose doseand time-dependently induced MCP-1 gene transcription and protein production in HK-2 cells, which could be blocked by the ketohexo-kinase blockers. Exogenous uric acid induced MCP-1 production in HK-2 cells. Allopurinol inhibited fructose, but not exogenous uric acid-induced MCP-1 expression. Both fructose and uric acid induced ROS generation. Incubation with H2 O2 promoted MCP-1 production in HK-2 cells. NAC completely inhibited MCP-1 production induced by fructose and H2 O2. Conclusion · Catalyzed by the ketohexo-kinase, fructose resultes the production of MCP-1 through uric acid and reactive oxygen species.