硫酸酯酶1对高糖诱导小鼠肾足细胞自噬损伤的影响

Influence of sulfatase 1 on high glucose induced podocyte autophagy injury

目的探讨小鼠肾足细胞在高糖状态下硫酸酯酶1(sulfatase 1, SULF1)表达情况及其在高糖诱导肾足细胞自噬损伤中的作用机制。方法小鼠肾足细胞根据干预方式分为对照组(采用5.5 mmol/L葡萄糖处理)、高渗组(采用5.5 mmol/L葡萄糖+19.5 mmol/L甘露醇处理)和高糖组(采用25 mmol/L葡萄糖处理),各组分别处理24 h。小鼠肾足细胞再分为空白对照组、阴性对照组和转染组,空白对照组细胞正常培养,转染组细胞转染Sulf1 siRNA,阴性对照组细胞转染negative siRNA,转染48 h。采用反转录PCR法检测各组细胞Sulf1 mRNA相对表达量,采用Western blot法检测各组细胞SULF1蛋白、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3, LC3)-Ⅱ、LC3-Ⅰ、蛋白激酶B(protein kinase B, Akt)、磷酸化Akt(phosphorylated Akt, p-Akt)、哺乳动物雷帕霉素蛋白(mammalian target of rapamycin, mTOR)和磷酸化mTOR(phosphorylated mTOR, p-mTOR)相对表达量,比较p-Akt/Akt p-mTOR/mTOR、LC3-Ⅱ/LC3-Ⅰ比值。结果高糖组细胞SULF1蛋白相对表达量和LC3-Ⅱ/LC3-Ⅰ(0.84±0.20、0.67±0.60)低于对照组(1.42±0.19、1.99±0.31)和高渗组(1.33±0.25、1.75±0.22)(P<0.05),p-Akt/Akt(0.41±0.03)、p-mTOR/mTOR(0.61±0.01)高于对照组(0.21±0.02、0.27±0.05)和高渗组(0.18±0.02、0.20±0.04)(P<0.05),对照组与高渗组比较差异均无统计学意义(P>0.05)。转染组细胞Sulf1mRNA和SULF1蛋白、LC3-Ⅱ/LC3-Ⅰ(0.627±0.074、0.73±0.07、0.55±0.07)低于空白对照组(1.000±0.013、1.63±0.06、1.17±0.10)和阴性对照组(0.924±0.141、1.65±0.14、1.20±0.06)(P<0.05),p-Akt/Akt(0.78±0.04)、p-mTOR/mTOR(0.58±0.01)高于空白对照组(0.30±0.04、0.44±0.03)和阴性对照组(0.34±0.03、0.44±0.04)(P<0.05),空白对照组与阴性对照组比较差异均无统计学意义(P>0.05)。结论高糖环境下,肾足细胞SULF1表达降低,对PI3K/Akt/mTOR信号通路的抑制作用减弱,导致通路活性增加,从而抑制自噬,致足细胞损伤;SULF1对高糖诱导的小鼠肾足细胞自噬具有保护作用。

Objective To investigate the expression of sulfatase 1(SULF1) in podocyte at high glucose level in mice and its mechanism in podocyte autophagy injury induced by high glucose. Methods The mice podocytes were divided into control group(treated with 5.5 mmol/L glucose), hypertonic group(treated with 5.5 mmol/L glucose + 19.5 mmol/L mannitol) and high-glucose group(treated with 25 mmol/L glucose).All three groups were treated for 24 h. The mice podocytes were redivided into blank control group, negative control group and transfection group. Blank control group was cultured normally. Transfection group was transfected with Sulf1 siRNA and negative control group was transfected with negative siRNA for 48 h. Reverse transcription PCR was used to detect the relative expressions of Sulf1 mRNA, and Western blot was used to detect the relative expressions of SULF1 protein, microtubule-associated protein 1 light chain 3(LC3)-Ⅱ, LC3-Ⅰ, protein kinase B(Akt), phosphorylated Akt(p-Akt), mammalian target of rapamycin (mTOR)and phosphorylated mTOR(p-mTOR)in three groups,The ratios of p-Akt/Akt,p-mTOR/mTOR and LC3-Ⅱ/LC3-Ⅰ were compared.Results The relative expression of SULF1 protein and LC3-Ⅱ/LC3-Ⅰratio were lower in high-glucose group(0.84±0.20,0.67±0.60)than those in control group(1.42±0.19,1.99±0.31)and hypertonic group(1.33±0.25,1.75±0.22)(P<0.05),the ratios of p-Akt/Akt and p-mTOR/mTOR were higher in high-glucose group(0.41±0.03,0.61±0.01)than those in control group(0.21±0.02,0.27±0.05)and hypertonic group(0.18±0.02,0.20±0.04)(P<0.05),and all the above indexes showed no significant differences between control group and hypertonic group(P>0.05).The relative expression of Sulf1 mRNA,SULF1 protein and LC3-Ⅱ/LC3-Ⅰ ratios were lower in transfection group(0.627±0.074,0.73±0.07,0.55±0.07)than those in blank control group(1.000±0.013,1.63±0.06,1.17±0.10)and negative control group(0.924±0.141,1.65±0.14,1.20±0.06)(P<0.05),the ratios of p-Akt/Akt and p-mTOR/mTOR were higher in transfection group(0.78±0.04,0.58±0.01)than those in blank control group(0.30±0.04,0.44±0.03)and negative control group(0.34±0.03,0.44±0.04)(P<0.05),and all the above indexes showed no significant differences between blank control group and negative control group(P>0.05).Conclusion High glucose decreases the expression of SULF1 in podocytes and weakens the inhibition of PI3 K/Akt/mTOR signaling pathway,which leads to the increase of pathway activity so as to inhibit autophagy and induce podocyte injury.SULF1 could protect the podocyte autophagy injury by high glucose in mice.