H2S硫巯基化修饰NLRP3炎性体延缓狼疮性肾炎进展的分子机制研究

狼疮性肾炎（LN）是常见且严重的炎症性免疫相关性肾病。申请人前期研究发现：H2S可抑制NLRP3炎性体表达及活化，延缓LN的进程，但其机制尚不明确。H2S可通过硫巯基化修饰发挥抗炎效应，而NLRP3炎性体具有发生该修饰的氨基酸序列基础。本项目提出“H2S硫巯基化修饰NLRP3炎性体延缓狼疮性肾炎发生发展”的假说。围绕该假说拟检测临床样本（LN患者血、尿和肾组织）中H2S及其合成酶和NLRP3炎性体的表达水平，并通过两种狼疮小鼠和H2S合成酶CSE基因敲除小鼠在体内观察外源性补充H2S和CSE基因敲除对狼疮小鼠肾组织中NLRP3炎性体活化及硫巯基化修饰作用的影响，采用质谱分析筛选硫巯基化修饰位点及其它可能的作用靶点，最后通过修饰位点突变转染等方法，从细胞分子水平验证H2S对NLRP3炎性体的硫巯基化修饰作用，探索H2S抑制NLRP3炎性体活化的作用机制，为LN防治提供新策略。

Lupus nephritis (LN) is a common and severe inflammatory immune-mediated nephropathy and is the most common cause of morbidity and mortality in patients with Systemic Lupus Erythematosus. Immune system dysfunction is the hallmark of the pathophysiology of LN, and both innate and adaptive components of the immune system have been implicated in LN progression. Broad-spectrum immunosuppressant therapy using steroids or cyclophosphamide are still the mainstay of treatment and are associated with significant adverse effects in patients. Eventually, there is an unmet need of novel therapeutic strategies that address specific immune and inflammatory processes in LN, a treatment that should be guided by pathophysiological investigation of the inflammatory changes which occur during disease progression. One specific area of interest in autoimmune and inflammatory diseases, including LN, is the role of the inflammasome in promoting progression of disease by triggering the activation of a wide range of inflammatory cytokines and upregulating autoantibody production. Prior work from our lab has demonstrated that H2S (Hydrogen sulfide) can inhibit the expression and activation of NLRP3 inflammasome and delay the progression of LN; yet, the detailed pathophysiological process that underlies the inhibitory effects of H2S is still unknown. H2S can exert anti-inflammatory effects through the sulfur-sulfhydrylation of protein residues, and the NLRP3 inflammasome has the amino acid sequence susceptible to sulfur-sulfhydrylation. Thus, this project aims to explore the mechanism of H2S-mediated delay in the progression of LN, and the central hypothesis is that H2S-mediates the sulfur-sulfhydrylation and subsequent inhibition of the NLRP3 inflammasome. We will first assess the expression levels of H2S, H2S synthase and NLRP3 inflammasome in clinical samples (blood, urine and kidney tissues) from patients with LN. Using two murine models of lupus, we will study the effect of exogenous H2S exposure and CSE (cystathionine-γ-lyase) gene knockout on NLRP3 inflammasome activation and sulfur-sulfhydrylation modification in the renal tissues of lupus mice. We will use mass spectrometry to screen for the different sulfur-sulfhydrylation modification sites on the NLRP3 gene and other possible therapeutic targets in the lupus kidney. Finally, we will verify the sulfur-sulfhydrylation sites of H2S on NLRP3 inflammasome in-vitro via transfection of plasmids carrying site-specific mutations of different potential target residues in the NLRP3. Collectively, successful completion of this proposal will provide a better insight on the pathophysiologic effect of hydrogen sulfide on the inflammatory response in lupus nephritis, and guide future therapeutic strategies targeting the inflammasome pathway in lupus nephritis.