黄芩苷对肺纤维化小鼠组织中纤维化因子TGF-β1及mmp2,timp2表达的影响

Effect of baicalin on expression of fibrogenic factors TGF-β1,mmp2 and timp2 in tissue of mice with pulmonary fibrosis

考察通过Biacore试验从芩百清肺浓缩丸垂钓的黄芩苷对博来霉素诱导的肺纤维化小鼠纤维化因子TGF-β1及mmp2,timp2表达的影响。采用Biacore技术检测芩百清肺浓缩丸与TGF-β1是否存在特异性结合,通过Biacore垂钓法,富集、再生和回收与TGF-β1结合的亲和活性成分,然后运用超高效液相色谱-质谱联用(UPLC-Q-TOF-MS)技术对回收到的样品分析鉴定单体成分是否为黄芩苷,再次运用Biacore对鉴定后的单体成分黄芩苷进行亲和力动力学分析加以验证,并采用体内药效学加以验证。将健康雌雄各半BALB/c小鼠30只任意分为3组,黄芩苷组、模型组、空白组;空白组予以气管内注射氯化钠注射液(0.08 mL·kg^-1),黄芩苷组、模型组复制肺纤维化模型(气管内注射4 mg·kg^-1博来霉素)。将上述4组小鼠饲养14 d后,运用免疫组化法确定因子在细胞中的位置及TGF-β1与mmp2,timp2蛋白的变化,RT-PCR法检测TGF-β1及mmp2,timp2 mRNA的表达。Biacore亲和力分析结果显示,芩百清肺浓缩丸与TGF-β1蛋白的结合响应峰值达到1524.0 RU,存在特异性结合。Biacore垂钓法钓取芩百清肺浓缩丸中与TGF-β1亲和的活性成分,运用UPLC-Q-TOF-MS,并对照数据库,显示化合物为黄芩苷,SPR亲和力动力学分析确定了黄芩苷与TGF-β1的亲和常数(KD)为1.62006μmol·L^-1,表明黄芩苷与TGF-β1存在稳定结合,验证了垂钓结果。免疫组化结果显示,与模型组相比,黄芩苷组纤维素沉积有显著减轻;从RT-PCR分析看出,与模型组相比,黄芩苷组能降低TGF-β1及mmp2,timp2 mRNA的表达。黄芩苷与靶蛋白TGF-β1结合能够抑制mmp2,timp2的表达,推延肺纤维化进程。

To investigate the effect of baicalin extracted from Qinbai Qingfei Concentrated Pills on the expressions of TGF-β1,mmp2 and timp2 in mice with pulmonary fibrosis induced by bleomycin.The Biacore technique was used to detect the specific binding between Qinbai Qingfei Concentrated Pills and TGF-β1,and the affinity components were enriched,regenerated and recovered by Biacore fishing.Then ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS)were used to determine whether the monomer was baicalin.Biacore was used to verify the affinity kinetics of baicalin,which was validated by pharmacodynamics in vivo.Totally 30 BALB/C mice were randomly divided into three groups:baicalin group,blank group and model group.The blank group was given sodium chloride injection(0.08 mL·kg^-1),while the model group and the baicalin group were injected with 4 mg·kg^-1 bleomycin.The localization of TGF-β1,mmp2 and timp2 protein in the cells and the mRNA expressions of TGF-β1,mmp2 and timp2 were detected by RT-PCR 14 days later.The results of Biacore affinity analysis showed that the peak of binding response between Qinbai Qingfei Concentrated Pills and TGF-β1 protein reached 1524.0 RU,with specific binding.The affinity constant KD of baicalin and TGF-β1 was 1.62006μmol·L^-1,which was determined by SPR kinetic analysis,suggesting a stable binding between baicalin and TGF-β1,which verified the results of angulation.The results of immunohistochemistry showed that the deposition of cellulose in baicalin group was significantly less than that in model group,the mRNA expressions of TGF-β1,mmp2 and timp2 were decreased in baicalin solution compared with the model group.Baicalin combined with TGF-β1 could inhibit the expressions of mmp2 and timp2 and delay the progress of pulmonary fibrosis.