实验海洋生物学重点实验室知识库

    糖尿病肾病（DN）是主要的糖尿病微血管疾病，是最早出现的糖尿病并发症，也是慢性肾衰和终末期肾病需要透析的最主要病因。DN是糖尿病持续高血糖引起的代谢障碍并发症，其产生过程与多种病理过程相关。从海带中提取的低分子量褐藻多糖硫酸酯（LMWF）属于一类高度硫酸化杂多糖，由岩藻糖、硫酸基、半乳糖等组成。LMWF因结构独特具有多种生物活性，包括抗凝血、抗炎症、抗氧化、抗肿瘤等。LMWF作为天然褐藻多糖通过影响多种细胞因子抑制DN的发展速度，但LMWF对DN的治疗机制尚不清楚。本论文从体外细胞模型水平与体内动物模型水平入手，探讨LMWF改善DN的作用与机制。

    体外细胞实验结果证明LMWF能够显著改善晚期糖基化终末产物（AGEs）引发的人体肾小球系膜细胞（HRMCs）异常增殖与肥大，降低细胞培养基中内毒素含量与乳酸脱氢酶（LDH）活性，从而改善HRMCs损伤；通过定量蛋白质组学KEGG富集分析，发现细胞外基质-受体相互作用（ECM-receptor interaction）信号通路与LMWF关联性最大，其中纤维连接蛋白（FN）表达量变化受LMWF影响最显著；通过细胞免疫荧光和表面等离子体共振（SPR）检测发现LMWF与FN存在特异性结合，平衡解离常数K_D为453.7 μmol/L；结果证明带正电荷的鱼精蛋白硫酸盐（PS）能够促进LMWF与HRMCs结合，增强LMWF改善HRMCs损伤的治疗作用。

    在体内动物实验方面，通过STZ诱导成年雄性Wistar大鼠建立DN早期模型证实LMWF较微晶纤维素（MC）可更好地缓解DN大鼠多饮、多食、体态消瘦等症状，但其对血清生化指标影响不大，未产生明显改善DN大鼠高血糖的效果。在肾脏功能方面，LMWF能够明显改善DN大鼠多尿、尿蛋白排泄量过多、尿微量白蛋白与尿肌酐比值（ACR）过高等DN早期病理症状；能够抑制肾小球基底膜病变，阻止肾小球系膜及基底膜增厚，缓解肾小球结构病变，下调DN大鼠肾皮质层FN、肌营养不良聚糖蛋白（DG）、层粘连蛋白（LAMC1）、白细胞介素-6（IL-6）、细胞间黏附分子-1（ICAM1）等促炎症因子的水平，而LMWF与抗生素（Anti）联用时其改善DN的效果受到影响且大幅降低。LMWF能够改善DN大鼠肾脏功能损伤，降低肾脏组织因炎症引起的病变，且这种作用与肠道菌群相关。

    体内动物实验结果证实LMWF能够显著抑制DN大鼠肠道通透性增大、粪便量增多、结肠长度缩短等肠道功能损伤症状。通过对DN大鼠粪便16S rDNA测序并进行聚类统计分析，结果证明LMWF显著提高拟杆菌门（Bacteroidetes）丰度并降低厚壁菌门（Firmicutes）丰度，缓解DN大鼠肠道菌群紊乱，显著提高拟杆菌目S24-7（Bacteroidales S24-7 group）和瘤胃球菌科（Ruminococcaceae）等可产生短链脂肪酸（SCFAs）的菌种丰度，且LMWF对DN的治疗作用与肠道菌群存在关联性。

    综上所述，LMWF能够有效缓解DN细胞病变及肾脏损伤，其通过结合FN抑制细胞外基质-受体相互作用信号通路，缓解由AGEs引起的HRMCs异常增殖与肥大，LMWF能够调节肠道菌群结构并抑制肠道屏障损伤，下调肾脏FN和IL-6等ECM相关的促炎症因子水平，减轻肾脏病理改变程度。本研究结果为进一步探讨褐藻多糖等治疗DN的作用机理及开发大分子活性物质作为海洋药物提供参考。

  Diabetic nephropathy (DN), which occurred as the earliest diabetic complication, is one of diabetic microvascular diseases, and it is also the main reason for dialysis for chronic renal failure and end-stage renal disease. DN is a metabolic disorder complication caused by persistent hyperglycemia in diabetes mellitus, and it was closely related to various pathological processes. With the application of low molecular weight fucoidan (LMWF) extracted from Saccharina japonica, which consisted of fucose, sulfate, galactose, etc., it was verified that LMWF exhibited various biological activities, such as anticoagulation, anti-inflammation, anti-oxidation, anti-tumor, etc. Moreover, polysaccharides and polysaccharide complexes play an important role in the occurrence and development of DN. As one of natural brown seaweed polysaccharides, LMWF could intervene the key polysaccharide complexes and other cytokines in the process of DN, nevertheless the mechanism was not been illustrated yet. Here, we aimed to explore the mechanism of LMWF in improving DN.

  Through the establishment of cell model with advanced glycation endproducts (AGEs), we verified that LMWF could significantly improve abnormal HRMC proliferation and hypertrophy caused by AGEs. It was proved that the LMWF could reduce the endotoxin content and lactate dehydrogenase (LDH) activity in cell culture medium. Quantitative proteomics KEGG enrichment analysis indicated that LMWF was mostly related to extracellular matrix (ECM)-receptor interaction pathway. In the ECM-receptor interaction, fibronectin (FN) protein expression was significantly influenced by LMWF. Cell immunofluorescence and surface plasmon resonance (SPR) detection reflected the co-localization and the specific binding of LMWF and FN, with an equilibrium dissociation constant K_D of 453.7 μmol/L. Moreover, we found that positively charged protamine sulfate (PS) can promote LMWF binding to HRMCs and enhance the therapeutic effect of LMWF on HRMC injury.

  Through the establishment of early DN rat model induced by STZ, we verified that LMWF could obviously alleviate the over drink, hyperphagia and emaciation compared with microcrystalline cellulose (MC). LMWF exhibited less effects on serum biochemical indexes and did not significantly improve the hyperglycemia of DN rats. LMWF could obviously improve DN early symptoms including urorrhagia, excessive urinary protein excretion ratio, high ACR, etc. It inhibited glomerular basement membrane lesions, prevented the thickening of glomerular mesangial and basement membrane, and alleviated glomerular structure lesions. LMWF decreased the protein expression of FN, muscle protein malnutrition chitosan (DG), laminin (LAMC1), interleukin 6 (IL-6), intercellular adhesion molecule-1 (ICAM1), and other proinflammatory factors. Nevetheless, the effects was greatly reduced when it was combined with antibiotics (Anti). We deduced that LMWF could improve kidney dysfunction in DN rats and reduce kidney tissue lesions caused by inflammation, which was related to gut microbiota.

  LMWF could significantly inhibit the intestinal dysfunction of DN rats, including reducing intestinal permeability and fecal weight, and increasing the length of colon. 16S rDNA sequence analysis of DN rat faeces indicated that LMWF relieved the gut microbiota disorder, increased the abundance of Bacteroidetes and reduced the abundance of Firmicutes. Additionally, LMWF significantly increased the abundance of Bacteroidales S24-7 group, Ruminococcaceae, and other species which could yield short chain fatty acids (SCFAs). Therefore, LMWF could regulate the gut micobiota in DN rats.

  In summary, LMWF could effectively inhibited kidney damage, alleviating the abnormal HRMC proliferation and hypertrophy which caused by AGEs via binding FN and inhibiting ECM-receptor interaction in HRMCs. It regulated gut microbiota and improved intestinal dysfunction, and down-regulated the protein levels of ECM-related proinflammatory factors, such as FN and IL-6. LMWF alleviated renal pathological changes and inhibited the renal dysfunction. Our study provided references for further mechanism study on function of brown algal polysaccharides on DN, and potentially developed macromolecular bioactive substances as marine drugs in the future.