丁酸钠通过组蛋白丁酰化修饰改善糖尿病肾脏病炎症和纤维化

Sodium butyrate ameliorates inflammation and fibrosis in diabetic kidney disease through histone lysine butyrylation

目的探讨丁酸钠(NaB)是否通过组蛋白丁酰化修饰改善糖尿病肾脏病(DKD)炎症和纤维化。方法无特定病原体(SPF)级C57BL/6雄性小鼠24只,8周龄、16~17 g。采用随机数字表法将其分为正常对照组(NC组)、高脂饮食联合链脲佐菌素(50 mg/kg)复制的DKD模型组(DKD组)、NaB溶液(每48小时40 mg/kg)腹腔注射干预组(DKD+NaB组),每组8只。体外培养肾系膜细胞,分为正常葡萄糖(NG)组(5.56 mmol/L葡萄糖)、高糖(HG)组(30 mmol/L葡萄糖)、HG+NaB组(30 mmol/L葡萄糖和1 mmol/L NaB)、HG+NaB+乙酰转移酶活性转录共激活因子(P300)抑制剂(A485)组(30 mmol/L葡萄糖、1 mmol/L NaB和10μmol/L A485)。采用酶联免疫吸附测定(ELISA)法检测各组小鼠血清炎症因子[单核细胞趋化蛋白-1(MCP-1)、白细胞介素-6(IL-6)]水平,及尿微量白蛋白和尿肌酐,并计算尿微量白蛋白/肌酐比值(UACR),采用Western blotting法检测各组小鼠肾脏组织和肾系膜细胞IL-6、MCP-1和转化生长因子-β(TGF-β)、泛丁酰化、H3K9丁酰化的蛋白表达水平,采用qRT-PCR检测各组小鼠肾脏组织和肾系膜细胞TGF-β、纤连蛋白(Fn)、P300的mRNA表达水平。采用HE、Masson染色观察小鼠肾脏组织硬化及纤维化程度。采用单因素方差分析进行组间比较。结果体内实验结果表明,与DKD组比较,DKD+NaB组小鼠UACR、血清IL-6、MCP-1水平均下降,肾脏组织的TGF-β和Fn的蛋白、mRNA表达均下降,泛丁酰化修饰、H3K9丁酰化修饰以及P300的蛋白、mRNA表达均明显上调(均P<0.05);HE、Masson染色显示,小鼠肾脏组织硬化及纤维化程度改善。体外实验结果表明,NaB呈浓度依赖性上调肾系膜细胞泛丁酰化和H3K9丁酰化修饰水平;与HG组比较,HG+NaB组肾系膜细胞泛丁酰化和H3K9丁酰化修饰均明显上调,IL-6和TGF-β的蛋白表达均下调;与HG+NaB组相比,HG+NaB+A485组的肾系膜细胞泛丁酰化和H3K9丁酰化修饰均下调,MCP-1、IL-6的蛋白、TGF-β的蛋白及mRNA、Fn的mRNA表达均上调(均P<0.05)。结论NaB可能通过组蛋白丁酰化修饰途径,抑制肾脏炎症及纤维化基因表达,改善DKD肾脏损伤。

Objective To investigate whether sodium butyrate(NaB)ameliorates inflammation and fibrosis in diabetic kidney disease(DKD)through histone lysine butyrylation(Kbu).Methods Twenty-four eight-week-old C57BL/6 male mice(16-17 g)without specific pathogen free(SPF)were randomly divided into normal control group(NC group),high-fat diet combined with streptozotocin(50 mg/kg)intraperitoneal injection of DKD model group(DKD group),intraperitoneal injection of NaB solution(40 mg/kg every 48 hours)intervention group(DKD+NaB group)using the random number table method,with 8 mice in each group.Normal renal mesangial cells were cultured in vitro,moreover,they were divided into normal glucose(NG)group(5.56 mmol/L glucose),high glucose(HG)group(30 mmol/L glucose),HG+NaB group(30 mmol/L glucose,and 1 mmol/L NaB),HG+NaB+acetyltransferase active transcriptional coactivator(P300)inhibitors(A485)group(30 mmol/L glucose,1 mmol/L NaB,and 10μmol/L A485).The levels of serum inflammatory factors[monocyte chemotactic protein-1(MCP-1),interleukin-6(IL-6)],urinary albumin and urinary creatinine in each group of mice were detected by enzyme-linked immunosorbent assay(ELISA),and the urinary albumin-to-creatinine ratio(UACR)was calculated.The expression levels of IL-6,MCP-1 and transforming growth factor-β(TGF-β),PanKbu and H3K9bu proteins in mouse renal tissue and renal mesangial cells in each group were detected by Western blotting.Quantitative real-time PCR(qRT-PCR)was used to detect the mRNA expression levels of TGF-β,fibronectin(Fn),and P300 in mouse renal tissue and renal mesangial cells in each group.HE and Masson staining were used to observe the degree of hardening and fibrosis of mouse renal tissue.One-way analysis of variance was used for comparison between groups.Results Invivo experiments showed that compared with the DKD group,the levels of UACR,serum IL-6 and MCP-1 in the DKD+NaB group decreased,the expression of TGF-β,and Fn proteins and mRNA in renal tissues decreased,and the expressions of PanKbu,H3K9bu modification,and P300 protein and mRNA in renal tissues were significantly up-regulated(all P<0.05).HE and Masson stains showed that the degree of hardening and fibrosis of renal tissues improved.In vitro experiments showed that NaB upregulated PanKbu and H3K9bu modification levels in renal mesangial cells in a concentration-dependent manner.Compared with the HG group,the PanKbu and H3K9bu modification in the HG+NaB group were significantly increased,and the expression of IL-6 and TGF-βprotein in renal mesangial cells was down-regulated.Compared with HG+NaB group,the PanKbu and H3K9bu modification in renal mesangial cells were down-regulated in HG+NaB+A485 group,and the expression of MCP-1,IL-6 protein,TGF-βprotein,mRNA,and Fn mRNA was up-regulated(all P<0.05).Conclusion NaB may inhibit renal inflammation and fibrosis gene expression,and improve DKD kidney injury through Kbu pathway.