固本通络方调控IgA肾病小鼠肠道细菌脂多糖介导的糖基化缺陷IgA产生的机制研究

The generation of galactose-deficient IgA1 (Gd-IgA1) is the trigger of IgA nephropathy (IgAN), which is also the key to the treatment of IgAN. Previous studies have found that "Gubentongluo Formula"(GBTL) can improve blood Gd-IgA1 and intestinal symptoms in IgAN patients, and can simultaneously regulate intestinal B cells and flora structure of IgAN mice. The abnormality of intestinal flora can lead to abnormal bacterial lipopolysaccharide in the intestine, affecting B cell activation and Gd-IgA1 production. Therefore, the efficacy of GBTL is related to the regulation of intestinal bacterial lipopolysaccharide. Therefore, we propose the hypothesis that GBTL regulates intestinal bacterial lipopolysaccharide, improves intestinal B cell activation, reduces Gd-IgA1 production and treats IgAN via the TLR4/MyD88/NF-κB pathway. This study intends to use ddY mice as IgAN model, lipopolysaccharide inhibitor eritoran as control, observe the effect of GBTL on the intestinal bacterial lipopolysaccharide-mediated Gd-IgA production to treat IgAN, and clear the GBTL regulation IgAN small Intestinal lipopolysaccharide-mediated intestinal B cell activation in the murine gut affects the mechanism of Gd-IgA production. This reveals the mechanism and scientific connotation of GBTL in the prevention and treatment of IgAN, and provides reliable evidence for the prevention and treatment of IgAN by "targeting the intestinal flora".

糖基化缺陷IgA1(Gd-IgA1)产生是IgA肾病（IgAN）的始动环节和治疗关键。前期研究发现固本通络方能改善IgAN患者血Gd-IgA1和肠道症状，且可同时调节IgAN小鼠的肠B细胞和菌群结构。因肠道菌群异常可导致肠道细菌脂多糖异常，影响B细胞激活以及Gd-IgA1产生，故固本通络方的疗效与调控肠道细菌脂多糖有关。因此，提出“固本通络方调控肠道细菌脂多糖，经由TLR4/MyD88/NF-κB途径，改善肠道B细胞激活，减少Gd-IgA1产生而治疗IgAN”的假说。本课题拟用ddY小鼠为IgAN模型，脂多糖抑制剂依立托仑为对照，观察固本通络方干预肠道细菌脂多糖介导的Gd-IgA产生以治疗IgAN的作用，明确该方调控IgAN小鼠肠道细菌脂多糖介导的肠B细胞激活以影响Gd-IgA产生机制。以此揭示固本通络方防治IgAN的作用机理及其科学内涵，并为“以肠道菌群为靶位”防治IgAN提供可靠证据。

糖基化缺陷IgA1(Gd-IgA1)产生是IgA肾病（IgAN）的始动环节和治疗关键。前期研究发现固本通络方能改善IgAN患者血Gd-IgA1和肠道症状，且可同时调节IgAN小鼠的肠B细胞和菌群结构。因肠道菌群异常可导致肠道细菌脂多糖异常，影响B细胞激活以及Gd-IgA1产生，故固本通络方的疗效与调控肠道细菌脂多糖有关。因此，提出“固本通络方调控肠道细菌脂多糖，经由TLR4/MyD88/NF-κB途径，改善肠道B细胞激活，减少Gd-IgA1产生而治疗IgAN”的假说。本课题拟用ddY小鼠为IgAN模型，脂多糖抑制剂依立托仑为对照，观察固本通络方干预肠道细菌脂多糖介导的Gd-IgA产生以治疗IgAN的作用。结果表明固本通络方可降低24小时尿蛋白定量水平及血清肌酐水平，减少肾小球系膜细胞、内皮细胞及基质增生，改善肾小球系膜区IgA沉积；降低小鼠血清和肠道粘膜组织LPS水平、Gd-IgA1和IgA水平；降低B细胞活化率及IgA前体B细胞占比比率，降低小鼠粪便培养中菌落蛋白表达水平，上调Cosmc蛋白和Cosmc 基因表达水平。LPS水平与B细胞活化率及IgA前体B细胞占比均呈正相关。后运用不同浓度的脂多糖(0、1、5、10和20 ng/mL) 及固本通络方处理固有层B淋巴细胞。检测结果提示LPS剂量依赖性地提高了IgA和半乳糖缺乏IgA1 的含量，提高了TLR4、Cosmc、MyD88和磷酸化(p)-NF-κB的水平，提高了产生IgA的B细胞的CD86+CD19+和比例。TLR4的敲除逆转了LPS的作用。提示TLR4介导LPS对固有层B淋巴细胞的作用。固本通络方可通过TLR4/MyD88/NF-κB通路，剂量依赖性地抑制LPS和TLR4过表达对固有层B淋巴细胞的影响。本研究结果也同国内外相关文献报道相吻合。为“以肠道菌群为靶位”防治IgAN提供可靠证据。