基于TGF-β1/Smad信号通路研究丹参调控Ly6C(high)巨噬细胞与自然杀伤细胞关系的抗肝纤维化作用机制

Ly6C(high) macrophages were comfirmed to inhibit functions of natural killer (NK) cells via up-regulating TGF-β1/Smad signal pathway, which was the key link in the formation of liver fibrosis. We found Salvia Miltiorrhiza (SM) had anti-fibrotic effects on regulating the activity of NK cells and inhibiting the expression of pro-inflammatory macrophages in the current research of National Science Foundation of China for Youth. Hence we put forward the hypothesis that the important pharmacological mechanism of SM against liver fibrosis is involved with releasing the negative control of pro-inflammatory Ly6C(high) macrophage on NK cells and restoring the immune killing function of NK cells. This project will be developed as follows: Firstly, with the positive control of receptor of TGF-β1(TβRI), CCl4-induced liver fibrosis model and Bone marrow reconstruction model will be used to estimate the effect of treatment with SM. Secondly, primary hepatic Ly6C(high) macrophaged will be isolated and pre-incubated with SM before co-cultured with NK cells (with gene silencing or over-expressing of TβRⅠ). Theses research are intend to prove the anti-fibrotic mechanism of SM on down-regulating TGF-β1/Smad signal pathway to release the negative control of pro-inflammatory Ly6C(high) macrophage on NK cells. This project will reveal the immunologic mechanism of SM against the liver fibrosis and strengthen the foundation to promote the pharmacological research of traditional Chinese medicine of promoting blood circulation and removing stasis.

研究证实Ly6C(high)巨噬细胞上调TGF-β1/Smad信号通路抑制NK细胞功能是促肝纤维化形成的重要环节。在研青年基金项目研究发现丹参的抗肝纤维化作用与调节NK细胞活性和抑制促炎型巨噬细胞表达有关，本项目提出“丹参调控TGF-β1/Smad信号通路，解除促炎型Ly6C(high)巨噬细胞对NK细胞负性调控以恢复NK细胞的免疫杀伤功能是丹参抗肝纤维化的重要药理机制”之科学假说。拟开展：体内以TGF-βⅠ型受体(TβRⅠ)抑制剂为对照，复制CCl4染毒模型和骨髓重建模型，丹参给药治疗；体外分离原代肝脏Ly6C(high)巨噬细胞，与丹参预孵育后再与NK细胞(TβRⅠ沉默/过表达)共培养，验证丹参下调TGF-β1/Smad信号通路，解除促炎型巨噬细胞对NK细胞功能负性调控的抗肝纤维化作用机制。该研究将阐明丹参抗肝纤维化的免疫药理机制，为推进活血化瘀中药的药理研究夯实基础。

本项目旨在研究丹参TGF-β1/Smad信号通路研究抑制Ly6C(high)巨噬细胞对自然杀伤细胞负性调控而抗肝纤维化作用机制。首先复制CCl4肝纤维化模型和巨噬细胞清除的CCl4肝纤维化小鼠模型；再分离小鼠骨髓细胞并诱导至Ly6C(high)Mo-mac，予丹参预孵育后再与和正常小鼠原代NK细胞共培养，收集NK细胞再与HSC共培养；最后构建TβRⅠ基因沉默或过表达的NK92细胞，与促炎型巨噬细胞共培养后，再与LX-2细胞共培养。通过上述研究发现，丹参可抑制CCl4诱导小鼠肝纤维化，减少肝纤维化小鼠肝脏Mo-Mac及Ly6C(high)Mo-Mac细胞数量，增加肝脏NK细胞数量和活性，减少NK细胞表达p-Smad2/3。巨噬细胞清除后，丹参可减轻纤维化小鼠死亡率，对肝脏TGF-β1表达无明显影响，对NK细胞数量无明显影响，抗肝纤维化作用无明显改善。丹参在体外可减轻Ly6C(high)Mo-Mac表达促炎症因子水平，减少CCR2和CX3CR1表达；经丹参孵育后的Ly6C(high)Mo-Mac再与NK细胞共培养，可增加NK细胞存活率、IFN-γ分泌水平，增加T-bet和Emoes转录因子水平，减弱HSC表达α-SMA的能力。NK细胞TβRⅠ基因沉默后，经丹参孵育后的Ly6C(high)Mo-Mac再与NK细胞共培养，NK细胞对HSC表达α-SMA无明显抑制作用，T-bet和Emoes转录因子水平无明显变化。NK细胞TβRⅠ基因过表达时，经丹参孵育后的Ly6C(high)Mo-Mac再与NK细胞共培养，NK细胞可明显一直HSC表达α-SMA，NK细胞T-bet和Emoes转录因子水平明显增加。在完成研究目标的基础上，进一步明确了隐丹参酮、二氢丹参酮I、丹酚酸A是丹参在体外抑制促炎型巨噬细胞活化的重要活性成分，可抑制NLRP3炎症小体活化。三者摩尔质比为8μM：16μM：12μM和16μM：12μM：4μM时对促炎型巨噬细胞分泌IL-1β的抑制作用最为明显，可通过促进细胞自噬，抑制NLRP3炎症小体活化而发挥作用。同时在体外H2O2诱导NK细胞氧化损伤模型，发现NK细胞以细胞晚期凋亡为主，其病理机制与激活JNK1信号通路诱导细胞凋亡，进而引发细胞程序性死亡相关。综上所述，本项目研究结果可为促进活血化瘀中药丹参抗肝纤维化的的药理机制研究拓宽新视野。