CAJ | 학술논문

目的探讨体外高糖培养的大鼠肾小球系膜细胞(RMCs)中抗衰老蛋白 Klotho 及中性粒细胞明胶酶相关脂质运载蛋白(NGAL)两者表达水平的变化及其相关作用,同时观察 Toll 样受体-4(TLR4)/核因子-kB(NF-kB) p65通路在此过程中的作用。方法设计并合成针对 NGAL 基因的3个特异性 siRNA序列转入 RMCs,筛选抑制效率最佳的 siRNA 用于后续实验；以吡咯烷二硫基甲酸盐(PDTC)或 Klotho 重组蛋白干预体外高糖培养的 RMCs；采用实时定量 PCR 检测 Klotho、TLR4、NGAL mRNA 的表达,Western 印迹检测 Klotho、TLR4、NF-kB p65、NGAL、纤连蛋白、结缔组织生长因子(CTGF)的表达,ELISA 检测单核细胞趋化因子-1(MCP-1)、趋化因子配体5(CXCL5)的分泌。结果高糖抑制 Klotho 表达(P<0.05)并激活 TLR4/NF-kB p65通路促进 NGAL、纤连蛋白、CTGF、MCP-1、CXCL5表达,基因沉默 NGAL 表达后纤连蛋白、CTGF、MCP-1、CXCL5表达明显下降(P<0.01),PDTC 干预后 NGAL 蛋白表达明显下降(P<0.01),Klotho 重组蛋白干预可抑制 TLR4/ NF-kB p65通路活性,下调 NGAL、纤连蛋白、CTGF、MCP-1、CXCL5的表达(P<0.01)。结论在 RMCs 中,Klotho 可通过抑制高糖刺激的 TLR4/ NF-kB p65通路活性下调 NGAL 的表达,从而抑制纤连蛋白、CTGF、MCP-1、CXCL5的表达,可能对糖尿病肾脏组织起保护作用。
목적탐토체외고당배양적대서신소구계막세포(RMCs)중항쇠로단백 Klotho 급중성립세포명효매상관지질운재단백(NGAL)량자표체수평적변화급기상관작용,동시관찰 Toll 양수체-4(TLR4)/핵인자-kB(NF-kB) p65통로재차과정중적작용。방법설계병합성침대 NGAL 기인적3개특이성 siRNA서렬전입 RMCs,사선억제효솔최가적 siRNA 용우후속실험；이필각완이류기갑산염(PDTC)혹 Klotho 중조단백간예체외고당배양적 RMCs；채용실시정량 PCR 검측 Klotho、TLR4、NGAL mRNA 적표체,Western 인적검측 Klotho、TLR4、NF-kB p65、NGAL、섬련단백、결체조직생장인자(CTGF)적표체,ELISA 검측단핵세포추화인자-1(MCP-1)、추화인자배체5(CXCL5)적분비。결과고당억제 Klotho 표체(P<0.05)병격활 TLR4/NF-kB p65통로촉진 NGAL、섬련단백、CTGF、MCP-1、CXCL5표체,기인침묵 NGAL 표체후섬련단백、CTGF、MCP-1、CXCL5표체명현하강(P<0.01),PDTC 간예후 NGAL 단백표체명현하강(P<0.01),Klotho 중조단백간예가억제 TLR4/ NF-kB p65통로활성,하조 NGAL、섬련단백、CTGF、MCP-1、CXCL5적표체(P<0.01)。결론재 RMCs 중,Klotho 가통과억제고당자격적 TLR4/ NF-kB p65통로활성하조 NGAL 적표체,종이억제섬련단백、CTGF、MCP-1、CXCL5적표체,가능대당뇨병신장조직기보호작용。
Objective To explore the changes in expression of Klotho, an aging-suppression protein, and neutrophil gelatinase associated lipocalin ( NGAL) and their relationship with rat mesangial cells ( RMCs) cultured with high glucose in vitro, and to explore the role played by Toll-like receptor-4 (TLR4) / nuclear factor-kB(NF-kB) p65 pathways in this process. Methods Three NGAL-siRNA sequences were designed and synthesized. The effective sequence in subsequent experiments was chosen. RMCs were preincubated with pyrrolidinedithiocarbamate (PDTC)or exogenously added Klotho prior to high glucose treatment. Realtime PCR was used to analyze Klotho, TLR4, NGAL mRNA expressions. Western blot was used to observe Klotho, TLR4,NF-kB p65, NGAL,fibronectin (FN), and connective tissue growth factor ( CTGF) protein expression. ELISA assay was used to detect monocyte chemoattractant protein-1 ( MCP-1) and CXCL5 secretions. Results High glucose suppressed Klotho expression significantly(P<0. 05) and activated TLR4 / NF-kB p65 pathway. Meanwhile,the levels of NGAL,FN,CTGF, MCP-1, and CXCL5 were highly expressed ( P < 0. 01). NGAL gene silencing obviously down-regulated the increased expressions of FN, CTGF, MCP-1, and CXCL5 ( P < 0. 01). After PDTC treatment the overexpression of NGAL protein was markedly lowered(P<0. 01). In addition, Klotho treatment significantly inhibited the activity of TLR4 /NF-kB p65 pathways and down-regulated the expressions of NGAL, FN, CTGF, MCP-1 and CXCL5 stimulated by high glucose(P<0. 01). Conclusion Klotho inhibits the activity of TLR4 / NF-kB p65 pathways and thus inhibits NGAL expression in RMCs cultured with high glucose in vitro. And then it suppresses the expressions of FN, CTGF, MCP-1, and CXCL5. This provides a new basis to illustrate the protection mechanism of the anti-aging protein Klotho in diabetic nephropathy, and may provide new ideas and therapeutic targets for prevention and treatment.