糖肾方对糖尿病肾病蛋白聚糖介导肾脏脂质沉积的机制研究

Diabetic nephropathy (DN) is the leading cause of end stage renal disease. Lipotoxicity is one of major underlying mechanisms contributing to renal injury and the development of albuminuria in diabetic condition. Proteoglycans are the components of extracelluar matrix. They are covalently substituted with glycosaminoglycan (GAG) side chains which mediate lipidprotein retention by interacting with lipoproteins through ionic interaction. In the previous study, we found that Tangshen Formula (TSF) can significantly alleviate lipid accumulation in the kidney and delay DN progression. Meanwhile, proteoglyan expression and its GAG side synthesis in renal cortex were decreased markedly after TSF treatment. In the current proposal, we will investigate the effect of decorin and biglycan, the two proteoglycan, on the lipidprotein retention in renal cells by the plasmids with the site of GAG side conjuction mutated. The effect of TSF on the core protein of decorin and biglycan, GAGs chain synthetase molecular weight, disaccharide units, and low-density lipoprotein affinity are carried out in db/db mouse model and cells. In addition, the effect of TSF on regulating the activity of decorin/biglycan promoters is analyzed. The study will reveal the mechanism of TSF alleviates lipidprotein retention, which will provide scentific clues in clincal use in treating DN.

肾脏脂质沉积是糖尿病肾病(diabetic nephropathy,DN)发生发展的重要因素。蛋白聚糖（Proteoglycan, PG）是细胞外基质重要成份，与中医"津液"密切相关，DN 病理情况下PG 异常升高，导致"痰瘀互结"。PG中糖胺聚糖链与脂蛋白通过静电力结合是导致脂质在组织沉积的关键因素。课题组前期研究发现，中药糖肾方能够延缓DN进展，减少肾脏脂质沉积，同时显著下调肾皮质PG表达。本研究拟从两种关键PG（decorin和biglycan）入手，通过构建无糖胺聚糖链的突变质粒，明确两者中糖胺聚糖链在肾脏固有细胞脂质沉积中的作用；继而采用db/db小鼠验证糖肾方降低其表达，减少肾脏脂质沉积，延缓DN进展的科学假说；在细胞和启动子水平明确药物对decorin/biglycan核心蛋白表达、糖链合成、结构及其与低密度脂蛋白亲和力的调控机制，从而阐释糖肾方益气养阴、化浊泄痰的科学内涵。

肾脏脂质沉积是糖尿病肾病发生发展的重要因素。蛋白聚糖是细胞外基质重要成份，中药糖肾方能够延缓 DN 进展，减少肾脏脂质沉积，同时显著下调肾皮质蛋白聚糖表达。本研究从两种关键蛋白聚糖（decorin, DCN 和 biglycan, BGN）入手，1）采用诱发性2型糖尿病肾病大鼠、自发性2型糖尿病肾病db/db小鼠和诱发性1型糖尿病肾病三种糖尿病肾病动物模型证实了BGN和DCN参与了肾脏脂质沉积和炎症损伤，并发现中药糖肾方具有下调肾脏BGN和DCN表达的作用；2）构建BGN和DCN重组质粒，建立体外过表达系统，发现BGN和DCN促进肾脏小管细胞脂质沉积的作用，揭示了DCN具有下调自噬活性，提示抑制自噬可能是DCN促进细胞脂质沉积的重要机制；利用体外敲降系统和免疫共沉淀技术，发现BGN通过TLR4和NOX4结合加重肾脏细胞的炎症和氧化应激的作用机制；3）在诱发性1型糖尿病肾病大鼠模型中发现糖肾方具有减少肠源尿毒素的作用，课题组通过肠道菌群测序研究发现糖肾方能够抑制肠道内有害菌群，促进肠道菌群稳态。肠道菌群-免疫是肾脏病研究的新热点，BGN与免疫功能密切相关，该研究为后续研究BGN在肾脏损伤中的作用提供了新的思路和研究基础。本项目揭示了BGN/DCN途径在糖尿病肾病脂质沉积中的作用及糖肾方调控机制，推动了糖尿病肾病药物新靶标的发现及糖肾方治疗糖尿病肾病生物学内涵的阐释。