炎症是指在内源性或外源性损伤因子的刺激下具有血管系统的活体组织所做出的防御反应，炎症反应是剧烈免疫反应的结果。本实验室前期工作以钙调蛋白磷酸酶（CN）为靶酶，从JHH中筛选出效果较好的免疫抑制单体YDHH。前期研究证明YDHH在皮肤移植、胶原型关节炎等自身免疫性疾病的模型中均具有较好的疗效。由于炎症与免疫两者之间的关系密切，所以我们猜想YDHH在抗炎方面也具有很好的作用。本实验采用脂多糖(LPS)处理巨噬细胞，建立体外炎症模型，旨在探讨免疫抑制剂YDHH的抗炎免疫作用及其相关机制，这对于开发高效低毒的抗炎免疫药物具有重要意义。 MTT实验结果展示，YDHH在100μM~500μM浓度范围内对细胞活性基本没有影响。故实验浓度采用100μM、300μM和500μM这三个浓度梯度。 ELISA和Real-time PCR的结果表明LPS可显著上调细胞中NO、IL-6、TNF-α的分泌和它们mRNA的表达水平。YDHH可剂量依赖性抑制 NO、IL-6、TNF-α的分泌和它们mRNA的表达水平。YDHH对炎症过程中促炎和抗炎系统之间的平衡起重要的调节作用，它既可以抑制炎症细胞因子的表达上调，同时又促进抗炎因子IL-10的分泌，有效发挥抗炎作用。 MAPK和NF-κB信号通路是细胞内普遍存在的两种介导炎症反应的信号级联通路。YDHH能抑制p38、JNK和ERK1/2的磷酸化水平。YDHH能阻止LPS诱导下在胞质中的NF-κB核转位，抑制NF-κB的活性，抑制IκB的降解。因此我们认为YDHH的抗炎作用可能是通过抑制MAPK和NF-κB途径来实现的。 YDHH可以通过影响CN/NFAT信号通路发挥其免疫抑制作用；YDHH抑制CN的活性，进而抑制了其下游重要的核转录因子NFAT的活性及其入核，最终使IL-2等基因的转录、翻译受到抑制。说明YDHH是通过影响CN的活性发挥免疫和抗炎功能的。

Inflammatory is defined as the defense reaction of living organism with vascular system to endogenous or exogenous injury factors, which is the result of vigorous immune response. A molecular model with CN as the target enzyme for screening immunosuppressants had been established and finally a small molecular compound YDHH from JHH was founded.Our previous work have shown that YDHH have significant immunosuppressive effect in skin graft, collagen arthritis and other autoimmune diseases. Since there is a close relationship between inflammation and immune, so we guess YDHH also has a good anti-inflammatory effect.The objective of our research is to investigate the anti-inflammatory and immunosuppressive effect and the possible mechanisms of immunosuppressant YDHH, using the in vitro inflammatory model of RAW264.7 cell line stimulated by lipopolysaccharide.Which is important for the development of efficient and low toxicity of anti-inflammatory and immunosuppressive drugs. MTT indicated that with the change of YDHH concentration vary from 100μM~500μM,cell viability had unchanged.which provided reference dose of 100μM、300μM and 500μM concentration gradient for the following experiments. ELISA and Real-time PCR results showed that the secretion and the mRNA expression of NO、IL-6 and TNF-α were upregulated by LPS in RAW264.7 cells. Pretreated with YDHH significantly decresed them in a dose-dependent manner. YDHH play an important regulatory role in the balance between pro-inflammatory and anti-inflammatory systems,YDHH can achieve its anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokines and increasing the secretory levels of anti-inflammatory cytokines like IL-10. MAPK and NF-κB signaling pathway are two widespread signaling cascades that mediate inflammatory response in cells.YDHH can inhibit the phosphorylation expression levels of p38、JNK and ERK1/2.In addition, YDHH can inhibit the nucleus translocation and the activity of NF-κB, the degradation of IκB proteins. These findings suggested that the anti-inflammatory effects of YDHH are mediated by suppression of MAPK and NF-κB signal pathways. The results showed that YDHH can influence CN/NFAT signal pathway by inhibiting the activity of CN and its downstream nuclear transcription factor NFAT, which finally inhibited the expression and secretion of IL-2 gene. This showed YDHH exerts immunosuppressive and anti-inflammatory effect by inhibiting inhibiting the activity of CN.