熟大黄调控NF-κB和TGF-β甲基化-平衡酒精性胰腺炎性损伤-抗炎修复的机制

The targets and effective substance foundation were the challenge of Chinese materia medica for the management of alcoholic pancreatic injury (API). We found that alcohol exposure induced pancreatic inflammatory injury with hypo-methylation of nuclear factor -kappa B (NF-κB) and hyper-methylation of transforming growth factor -beta （TGF-β） within pancreas. NF-κB and TGF-β，a couple of oppositional inflammatory regulators, involved the process of pancreatic inflammatory injury and anti-inflammatory repair through antagonistic regulating the expression of downstream TNF alpha and IL-6. Combined with the progress of cooked rhubarb promoting pancreatic repair through TGF-β and the idea of adjusting of Yin and Yang with the aim of balance, we hypothesized: the cooked rhubarb exerts balanceable regulation on alcoholic inflammatory injury - anti-inflammatory repair process of pancreas through the regulating the methylation alteration of NF-κB and TGF-β. We planned: first, to explore the relationship between the methylation alteration, mRNA transcription, protein expression of NF-κB,TGF-β, TNF-α and IL-6, the level of serum TNF-α, IL-6 and the inflammatory injury - anti-inflammatory repair process of pancreas in rats after chronic alcohol administration with different times; Second, to study the effect of cooked rhubarb on the methylation of NF-κB and TGF - β, the expression of TNF-α and IL - 6, and the pancreatic pathological changes of alcohol treated rats, exploring the mechanism of cooked rhubarb to balance the inflammatory injury - anti-inflammatory repair process of API; Third, to clarify the mechanism of cooked rhubarb on regulating DNA methylation of NF-κB and TGF-β in pancreas acini to balance the inflammatory injury - anti-inflammatory repair process after cooked rhubarb pre-treating caerulin-stimulated acinar cells from chronic alcohol-facilitating rats; At last, to screen the effective components of cooked rhubarb on regulating DNA methylation of NF-κB and TGF-β in pancreatic acini to balance the inflammatory injury - anti-inflammatory repair process after pre-treating caerulin-stimulated acinar cells from alcohol-facilitating rats with components from cooked rhubarb, such as emodin, rhein, chrysophanol, et al. Totally, we aimed to elucidate the balanceable adjustment mechanism of cooked rhubarb for alcoholic inflammatory injury - anti-inflammatory repair process with Yin and Yang in balance through regulating the methylation alteration of NF-κB and TGF-β with pancreas, screen the therapeutic targets and pharmacodynamic components of cooked rhubarb for API, which is valuable and a guiding significance for the translational research of Chinese materia medica.

靶点和物质基础是中药防治酒精性胰腺损伤（API）研究的难题。我们发现酒精诱导胰腺NF-κB低甲基化和TGF-β高甲基化，两者拮抗调控下游炎症因子而致炎性损伤，结合熟大黄促胰腺TGF-β表达的进展和"调整阴阳，以平为期"理念提出假说：熟大黄调控胰腺NF-κB和TGF-β甲基化以平衡API的炎性损伤-抗炎修复过程。拟：一分析酒精暴露大鼠胰腺NF-κB与TGF-β甲基化、mRNA表达与炎性损伤的关系；二观察熟大黄对酒精暴露大鼠胰腺NF-κB与TGF-β甲基化及转录、炎症因子和病理的影响，探索熟大黄平衡炎性损伤-抗炎修复机理；三熟大黄处理雨蛙肽损伤的酒精易化胰腺腺泡，验证熟大黄调控甲基化以平衡炎性损伤-抗炎修复的机理和靶点；四大黄成分处理雨蛙肽损伤的酒精易化胰腺腺泡，筛选调控DNA甲基化的药效成分。最终阐明熟大黄平衡API损伤-修复的阴平阳秘机制，对靶点和药效成分筛选有转化意义。

对于胰腺疾病而言，酒精与遗传是致命的结合。前期研究发现酒精暴露可能诱导胰腺组织炎症因子甲基化改变而引起胰腺炎症损伤，结合熟大黄调控TGF-β表达以促进再生修复的研究进展和“调整阴阳、以平为期”的理念，我们假设：熟大黄调控NF-κB和TGF-β甲基化以平衡酒精性胰腺损伤的促炎损伤-抗炎修复过程。通过摸索建立了液体酒精饲料诱导酒精性胰腺损伤大鼠模型，进而摸索最佳酒精暴露时间、甲基化改变明显的炎症因子和熟大黄诱导甲基化的最佳剂量等，建立酒精暴露+熟大黄干预的急性胰腺炎模型。提取DNA、RNA，以焦磷酸测序法和实时荧光定量PCR检测炎症因子（TNF、NF-κB、TGF-β3、IL-1α和IL-10）DNA甲基化百分比及RNA浓度，SABC法检测胰腺组织炎症因子蛋白的表达，胰腺组织病理评分。结果发现，1）酒精暴露后不同时间点TNF、NF-κB、TGF-β3、IL-1α和IL-10等炎症因子DNA甲基化水平，证实酒精暴露能诱导IL-1α、IL-10以及TNF基因甲基化而形成炎性甲基化记忆，而对预实验中初步发现NF-κB与TGF-β甲基化无明显影响。2） 通过观察3、6、12g/kg.BW.D不同剂量熟大黄灌胃酒精暴露后大鼠胰腺炎性因子DNA甲基化变化情况，证实3g/kg.BW.D熟大黄能调控IL-1α、IL-10的甲基化变化。3） 熟大黄可能通过上调IL-1αDNA甲基化，下调IL-10 DNA甲基化从而降低IL-1α表达，增加IL-10表达以抑制炎症反应，防治酒精性胰腺炎性损伤。4）熟大黄可能通过上调IL-1αDNA甲基化，下调IL-10 DNA甲基化从而降低IL-1α表达，增加IL-10表达以抑制炎症反应，减轻酒精暴露后急性胰腺炎的炎症损伤，从而证实熟大黄调控炎性记忆、减轻酒精暴露所致炎性损伤的机理。研究结果为临床督促患者及时戒酒、预防酒精性胰腺炎的复发提供实验依据。