【作者】 贾新华；

【导师】 张伟；

【作者基本信息】 山东中医药大学 ， 中西医结合临床， 2020， 博士

【摘要】 目的:肺纤维化(pulmonary fibrosis,PF)是一种慢性弥漫性肺间质性疾病,其特征是基底膜和间质组织中细胞外基质蛋白的大量沉积,以及损伤的上皮细胞和肌成纤维细胞的增殖。肺纤维化患者临床表现为进行性呼吸困难,限制性通气功能障碍以及低氧血症等,最终会导致患者出现难以治愈的呼吸困难,患者大多死于呼吸衰竭。Notch信号通路是一种高度保守的信号传导通路,对细胞的增殖、分化、凋亡等过程起到调控作用,同时也是多种组织和器官早期发育必需的细胞间调节信号。近年来,越来越多的研究发现Notch信号通路与肺纤维化密切相关。本研究是在国家自然科学基金“化痰类中药多途径调节肺间质纤维化抗氧化机制的研究(编号:81273704)”的实验结果基础上,通过使用TGF-β1对MRC-5细胞进行造模,给予不同贝母组分(贝母甲素、贝母乙素、贝母辛和西贝母碱)进行干预,观察不同贝母组分对造模后MRC-5细胞氧化损伤的保护作用;同时使用DAPT阻断干预Notch信号通路,观察贝母组分保护MRC-5细胞以抗氧化损伤的具体作用机制。方法:1.MRC-5氧化损伤模型的构建及贝母组分最大无毒浓度的选择。通过5ng/ml的TGF-β1对MRC-5细胞的不同作用时间,MTT法检测细胞增殖抑制率,Real-time PCR、Western Blotting法检测不同作用时间的TGF-β1对MRC-5细胞内collagenⅠ、collagenⅢ的表达,ELISA法检测细胞内8-OHdG蛋白浓度的表达,选取TGF-β1造模的最佳作用时间。贝母组分对MRC-5的最佳无毒浓度,选取IC10为最佳无毒浓度,以备后续实验进行。2.观察贝母组分对TGF-β1诱导的MRC-5细胞氧化损伤模型的保护作用。分为空白组、模型组(TGF-β1组)、贝母甲素组(贝母甲素+TGF-β1)、贝母乙素组(贝母乙素+TGF-β1)、贝母辛组(贝母辛+TGF-β1)和西贝母碱组(西贝母碱+TGF-β1),共6组。通过RT-PCR法检测并比较各组细胞中collagenⅠ、collagenⅢmRNA的表达;Western Blotting法检测并比较各组细胞中collagenⅠ、collagenⅢ、α-SMA蛋白的表达;免疫荧光观察各组细胞中α-SMA的阳性肌成纤维细胞的数量及荧光表达强度。并筛选对MRC-5细胞保护能力较好、抗纤维化能力较强的贝母组分,进行后续具体抗纤维化及抗氧化损伤机制的探寻。3.贝母乙素通过调控TGF-β1/Notch1信号通路介导的氧化损伤改善肺纤维化的机制。分为空白组、模型组(TGF-β1组)、贝母乙素组(贝母乙素+TGF-β1)、TGF-β1+DAPT组和贝母乙素组+DAPT,共5组。通过Western Blotting、RT-PCR法检测各组collagenⅠ、Ⅲ,Notch1-4、Jag1-2,Hes1和DLL1、3、4的蛋白及mRNA的表达并进行组内比较;ELISA法检测各组8-OHdG的表达水平;生化检测并比较各组SOD、GSH的表达;慢病毒转染Notch1目的基因至MRC-5细胞,检测贝母乙素用药后collagenⅠ、collagenⅢ、α-SMA和Notch1的蛋白表达。结果:1.MRC-5氧化损伤模型的构建及贝母组分最大无毒浓度的选择。1.1选定以5ng/ml TGF-β1预刺激MRC-5 48小时进行氧化损伤造模的构建。1.2 TGF-β1不仅可以诱导MRC-5细胞产生氧化损伤,同时也使其产生纤维化损害。1.3选定以贝母甲素3.125μg/ml、贝母乙素6.25μg/ml、贝母辛6.25μg/ml及西贝母碱6.25μg/ml作为该实验药物的最大无毒浓度,以备后续实验进行。2.观察贝母组分对TGF-β1诱导的MRC-5细胞氧化损伤模型的保护作用。2.1贝母甲素、贝母乙素、贝母辛和西贝母碱均可对TGF-β1所诱导的MRC-5细胞起到不同程度的保护作用,其中贝母乙素、贝母辛相对于其他的贝母组分而言,降低collagenⅠ、collagenⅢ、α-SMA纤维化因子的mRNA和蛋白表达量更明显,说明这两种贝母组分对TGF-β1所诱导的MRC-5细胞抗纤维化作用更强。2.2贝母乙素组相较贝母辛组的α-SMA阳性肌成纤维细胞数量及荧光表达改善更为明显,说明贝母乙素对MRC-5细胞的保护作用更强,抗纤维化作用更明显,故选择贝母乙素作为后续实验药物。3.贝母乙素通过调控TGF-β1/Notch1介导的氧化损伤改善纤维化的机制研究3.1贝母乙素组+DAPT与TGF-β1+DAPT组之间Notch1、Jag1、Hes1的mRNA和蛋白表达无明显差异性,说明贝母乙素可能通过抑制Notch1、Jag1、Hes1的表达,降低信号通路下游因子的释放,起到保护细胞,减轻纤维化损害的作用;而在Notch2-4、Jag2、DLL1、DLL3及DLL4中无表达或表达无意义。3.2贝母乙素可显著改善GSH和SOD的表达,减少8-OHdG的表达具有抗氧化作用;且DAPT+TGF-β1组可见GSH、SOD表达的上调,与贝母乙素组相比无明显差异性,说明贝母乙素可能通过阻断Notch信号通路起到抗氧化作用;同时贝母乙素组与DAPT+TGF-β1组相比,其collagenⅠ、collagenⅢ的蛋白和mRNA表达无明显差异,且较TGF-β1组明显减少,说明贝母乙素可能通过Notch信号通路抗氧化以减轻纤维化损害。3.3转染Notch1病毒的MRC-5细胞内collagenⅠ、collagenⅢ、α-SMA、Notch1的蛋白表达明显上升,贝母乙素组MRC-5细胞内collagenⅠ、collagenⅢ、α-SMA、Notch1的蛋白表达明显降低,说明贝母乙素可以抑制Notch信号通路,降低通路下游产物表达,进而可以起到保护细胞,减缓FB向MFB的转化过程,对纤维化具有治疗作用。结论:1.TGF-β1可诱导MRC-5细胞产生氧化损伤,导致其纤维化产生。2.给予最大无毒浓度的贝母组分均可使TGF-β1诱导的MRC-5细胞内降低纤维化因子的表达,说明安全浓度下的贝母组分可对氧化损伤的MRC-5细胞起到保护作用,减少纤维化损害。3.贝母乙素可通过上调SOD、GSH的表达而发挥抗氧化、减少纤维化因子的产生的作用,其机制可能与Notch1/Jag1/Hes1信号通路有关。4.贝母乙素可以通过抑制Notch信号通路,降低通路下游产物表达,进而起到保护细胞,减少氧化损伤,减缓FB向MFB的转化过程,对纤维化具有治疗作用。更多还原

【Abstract】 Objective:Pulmonary fibrosis is a chronic diffuse interstitial lung disease,which is characterized by large deposits of extracellular matrix proteins in the basement membrane and interstitial tissue,as well as damage to the epithelial cells and myofibroblasts proliferation.The clinical manifestations of patients with pulmonary fibrosis are progressive dyspnea,restrictive ventilation dysfunction,and hypoxemia,etc.,which will eventually lead to dyspnea that is difficult to cure,and most patients die of respiratory failure.Notch signaling pathway is a highly conserved signaling pathway that regulates cell proliferation,differentiation,and apoptosis,and is also an intercellular regulatory signal necessary for the early development of a variety of tissues and organs.In recent years,more and more studies have found that Notch signaling pathway is closely related to pulmonary fibrosis.This research is based on the experimental results of the National Natural Science Foundation of China "Study on Phlegm-Traditional Chinese Medicines to Regulate the Antioxidant Mechanism of Pulmonary Interstitial Fibrosis(No.81273704)",and used TGF-β1 to construct MRC-5 cells.Model,given different Fritillaria components(Peimine,Peiminine,Peimisine and Imperaline)to observe the protective effect of different Fritillaria components on oxidative damage of MRC-5 cells after modeling.At the same time,DAPT was used to block the Notch signal pathway and observe the specific mechanism of action of Fritillaria component in protecting MRC-5cells against oxidative damage.Method:1.Construction of MRC-5 oxidative damage model and selection of maximum nontoxic concentration of Fritillaria component.According to the different action time of 5ng / ml TGF-β1 on MRC-5 cells,MTT method was used to detect the cell proliferation inhibition rate,and Real-time PCR and Western Blotting method were used to detect the effects of TGF-β1 on MRC-5 cells.The expression of the concentration of 8-OHdG protein in the cells was detected by ELISA,and the optimal time for TGF-β1 modeling was selected.The optimal non-toxic concentration of Fritillaria component to MRC-5,IC10 was selected as the optimal nontoxic concentration for subsequent experiments.2.To observe the protective effect of Fritillaria component on TGF-β1-induced MRC-5 cell oxidative damage model.Divided into blank group,model group(TGF-β1 group),Peimine group(Peimine+TGF-β1),Peiminine group(Peiminine + TGF-β1),Peimisine group(Peimisine + TGF-β1)and Imperaline group(Imperaline + TGF-β1),a total of 6 groups.RT-PCR was used to detect and compare the expression of collagenⅠand collagenⅢ mRNA in each group of cells;Western Blotting was used to detect and compare the expression of collagen Ⅰ,collagen Ⅲ,and α-SMA proteins in each group of cells;Immunofluorescence was used to observe the number of α-SMA positive myofibroblasts and the intensity of fluorescence expression in each group of cells.3.Peimine improves fibrosis by regulating TGF-β1 / Notch-mediated oxidative damageIt was divided into blank group,model group(TGF-β1 group),Peimine group(Peimine+ TGF-β1),TGF-β1 + DAPT group and Peimine group+ DAPT,a total of 5groups.Western Blotting and RT-PCR were used to detect the protein and mRNA expressions of collagenⅠ,collagen Ⅲ,Notch1-4,Jag1-2,Hes1 and DLL1,3,4 in each group and compare within groups;ELISA method was used to detect 8-OHdG in each group Expression level;Biochemically detect and compare the expressions of SOD and GSH in each group;Lentivirus transfected the Notch1 target gene into MRC-5 cells,and detect the protein expression of collagenⅠ,collagen Ⅲ,α-SMA and Notch1 of Peimine after treatment.Result:1.Construction of MRC-5 oxidative damage model and selection of maximum nontoxic concentration of Fritillaria component.1.1 Selected 5 ng / ml TGF-β1 pre-stimulated MRC-5 for 48 hours for oxidative damage modeling.1.2 TGF-β1 can not only induce oxidative damage to MRC-5 cells,but also cause fibrotic damage.1.3 Peimine 3.125 μg/ml,Peiminine 6.25 μg / ml,Peimisine 6.25 μg / ml,and Imperaline 6.25 μg / ml were selected as the maximum non-toxic concentrations of the experimental drug.Subsequent experiments were performed.2.To observe the protective effect of Fritillaria component on TGF-β1-induced MRC-5 cell oxidative damage model.2.1 Peimine,Peiminine,Peimisine and Imperaline all can protect MRC-5 cells induced by TGF-β1 to varying degrees.Compared to other Fritillaria components Peiminine and Peimisine decrease the mRNA and protein expression of collagenⅠ,collagen Ⅲ,and α-SMA fibrosis factor,which indicates that these two components of Fritillaria are resistant to TGF-β1 induced MRC-5 cells Fibrosis is stronger.2.2 The number of α-SMA positive myofibroblasts and the fluorescence expression of the Peimine group were more obvious than those of the Fritillin group,indicating that Peimine has a stronger protective effect on MRC-5 cells and an anti-fibrotic effect.More obviously,Peimine was selected as the subsequent experimental drug.3.Peimine improves fibrosis by regulating TGF-β1 / Notch-mediated oxidative damage3.1 There was no significant difference in the mRNA and protein expressions of Notch1,Jag1,Hes1 between the Peimine+DAPT and TGF-β1+DAPT groups,indicating that Peimine may reduce the signal by inhibiting the expression of Notch1,Jag1,Hes1.The release of downstream factors of the pathway plays a role in protecting cells and reducing fibrotic damage;however,it is not expressed or meaningless in Notch2-4,Jag2,DLL1,DLL3 and DLL4.3.2 Peimine can significantly improve the expression of GSH and SOD,and reduce the expression of 8-OHdG has anti-oxidant effect;and the DAPT+TGF-β1 group shows an up-regulation of GSH and SOD,which is not significant compared with Peimine group.The difference indicates that Peimine may play an antioxidant role by blocking the Notch signaling pathway.At the same time,there is no significant difference in the expression of collagenⅠand collagen Ⅲ in the Peimine group compared with DAPT +TGF-β1 group.And compared with the TGF-β1 group,it was significantly reduced,indicating that Peimine may be oxidized through the Notch signaling pathway to reduce fibrotic damage.3.3 The protein expressions of collagenⅠ,collagenⅢ,α-SMA,and Notch1 in MRC-5 cells transfected with Notch1 virus were significantly increased,and the protein expressions of collagenⅠ,collagen Ⅲ,α-SMA,and Notch1 in MRC-5 cells of the Peimine group were significantly reduced.This shows that Peimine can inhibit the Notch signaling pathway and reduce the expression of downstream products of the pathway,which in turn can protect cells,slow down the conversion of FB to MFB,and have a therapeutic effect on fibrosis.Conclusion:1.TGF-β1 can induce oxidative damage in MRC-5 cells,leading to fibrosis.2.The maximum non-toxic concentration of Fritillaria component can reduce the expression of fibrosis factors in MRC-5 cells induced by TGF-β1,indicating that Fritillaria component at a safe concentration can damage oxidatively damaged MRC-5cells.It plays a protective role and reduces fibrotic damage.3.Peimine can play an antioxidant and reduce the production of fibrotic factors by up-regulating the expression of SOD and GSH,and its mechanism may be related to the Notch1/Jag1/Hes1 signaling pathway.4.Peimine can inhibit the Notch signal pathway and reduce the expression of downstream products,thereby protecting cells,reducing oxidative damage,slowing the conversion process of FB to MFB,and has a therapeutic effect on fibrosis.更多还原