高糖腹膜透析液激活神经酰胺表达并通过Src途径诱导腹膜透析模型小鼠腹膜损伤

High glucose-peritoneal dialysis solution activates ceramide expression and induces peritoneal injury via Src pathway in peritoneal dialysis model mice

目的:探讨高糖腹膜透析液(peritoneal dialysis solution,PDS)通过激活神经酰胺(ceramide,CER)诱导腹膜透析模型小鼠腹膜损伤的作用机制。方法:采用30只5周龄体重约22 g的雄性C57BL;6小鼠建立腹膜透析模型,并分成假手术组(1.5 ml灭菌注射用水,n=7)、高糖PDS组(1.5 ml 4.25%PDS,n=8)、高糖PDS+酸性鞘磷脂酶(acid sphingomyelinase,ASMase)抑制剂地昔帕明(desipramine,DES)组(1.5 ml灭菌注射用水+10 mg;kg DES,n=8)、高糖PDS+Src激酶抑制剂PP2组(1.5 ml灭菌注射用水+1 mg;kg PP2,n=7),每日腹腔注射1次,28 d后处死小鼠留取腹膜组织。HE、Masson染色观察小鼠腹膜组织的病理学变化,免疫组化检测腹膜组织中TLR4、巨噬细胞阳性染色细胞,免疫荧光检测ASMase、CER蛋白表达,高压液相色谱、高压液相色谱串联质谱法检测ASMase活性和CER水平,实时荧光定量PCR检测胞质型(c)-Src、磷酸化(p)-Src、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)mRNA表达,Western印迹检测c-Src、p-Src蛋白表达,酶联免疫吸附测定法检测血清C反应蛋白、IL-6、TNF-α表达水平。结果:(1)高糖PDS可导致腹膜透析模型小鼠腹膜增生、胶原沉积、纤维化,提示造模成功。与高糖PDS组比较,DES及PP2干预后小鼠腹膜增生、胶原沉积、纤维化均明显改善(均P<0.05)。(2)与假手术组比较,高糖PDS组小鼠腹膜组织中ASMase活化、CER水平均明显较高,DES可以明显抑制高糖PDS引起的ASMase活性、CER表达增加(均P<0.05),PP2对ASMase活性、CER水平均无明显影响(均P>0.05)。(3)与假手术组比较,高糖PDS组小鼠腹膜组织TLR4、巨噬细胞阳性染色细胞均较多,IL-6、TNF-αmRNA表达以及血清C反应蛋白、IL-6、TNF-α水平均较高(均P<0.05)。DES、PP2可以明显抑制高糖PDS引起的TLR4、巨噬细胞增多以及相关炎性因子表达增高(均P<0.05)。(4)与假手术组比较,高糖PDS组小鼠腹膜组织中c-Src、p-Src mRNA和蛋白表达均明显较高,PP2可以明显抑制高糖PDS引起的p-Src mRNA和蛋白表达增加(均P<0.05),对c-Src mRNA和蛋白表达均无明显影响,DES对c-Src、p-Src mRNA和蛋白表达均无明显影响(均P>0.05)。结论:高糖PDS可能通过活化ASMase刺激CER表达增加,磷酸化Src激酶,启动TLR4信号转导,诱导腹膜透析小鼠腹膜组织的炎症损伤。

Objective To explore the mechanism of peritoneal dialysis solution(PDS)-induced peritoneal microinflammation through activation of ceramide(CER)in peritoneal dialysis model mice.Methods Thirty 5-week-old male C57BL;6 mice weighing about 22 g were used to set up peritoneal dialysis models,and then were randomly divided into 4 groups:sham operation group(1.5 ml sterilized water,n=7),high glucose-PDS group(1.5 ml 4.25%PDS,n=8),high glucose-PDS+acid sphingomyelinase(ASMase)inhibitor desipramine(DES)group(1.5 ml sterilized water+10 mg;kg DES,n=8),high glucose-PDS+Src kinase inhibitor PP2 group(1.5 ml sterilized water+1 mg;kg PP2,n=7),with intraperitoneal injection once a day.After 28 days,the mice were sacrificed to retain peritoneal tissues.HE staining and Masson staining were used to observe the histological changes of peritoneum.Immunohistochemistry was used to detect the Toll-like receptor 4(TLR4)and macrophages.High performance liquid chromatography,liquid chromatography;mass spectrometry and immunofluorescence were used to detect the expression of ASMase and CER.Real-time quantitative PCR was used to detect the mRNA levels of c⁃Src,p⁃Src,interleukin-6(IL⁃6),and tumor necrosis factor-α(TNF⁃α).Western blotting was used to detect the protein levels of c⁃Src,and p⁃Src.Enzyme-linked immunosorbent assay was used to detect the serum C reactive protein(CRP),IL⁃6 and TNF⁃α.Results(1)High glucose-PDS led to peritoneal hyperplasia,collagen deposition and fibrosis in the peritoneal dialysis mice,indicating successful modeling.Compared with high glucose-PDS group,peritoneal hyperplasia,collagen deposition and fibrosis of mice treated with DES and PP2 were significantly improved(all P<0.05).(2)Compared with sham operation group,ASMase activation and CER level of peritoneal tissues were significantly higher in high glucose-PDS group,and DES could significantly inhibit activated ASMase and increased CER expression caused by high glucose-PDS(both P<0.05).PP2 had no significant effect on ASMase activation and CER level(both P>0.05).(3)Compared with sham operation group,there were more TLR4 and macrophage positive staining cells in peritoneal tissues in high glucose-PDS group,and the mRNA expression levels of IL⁃6 and TNF⁃αin peritoneal tissues and serum CRP,IL⁃6 and TNF⁃αwere higher(all P<0.05).DES and PP2 could significantly inhibit the increased TLR4,macrophages and related inflammatory factors induced by high glucose-PDS(all P<0.05).(4)Compared with sham operation group,c⁃Src and p⁃Src mRNA and protein expression levels of peritoneal tissues in high glucose-PDS group were significantly higher(all P<0.05).PP2 significantly inhibited the increased p⁃Src mRNA and protein levels caused by high glucose-PDS(both P<0.05),but had no significant effect on the mRNA and protein expression levels of c⁃Src(both P>0.05).DES had no significant effect on the mRNA and protein expression levels of c⁃Src and p⁃Src(all P>0.05).Conclusions High glucose-PDS may enhance the expression of CER through stimulating the activity of ASMase,phosphorylate Src,activate TLR4 and induce inflammatory damage of peritoneum in peritoneal dialysis model mice.