【作者】 张心月；

【导师】 张伟；

【作者基本信息】 山东中医药大学 ， 中西医结合临床， 2022， 博士

【摘要】 [目的]肺纤维化(Pulmonary fibrosis,PF)是间质性肺疾病的共同结局,其发病原因复杂,病理机制尚不清楚,患者临床表现为进行性肺组织顺应性减退,弥散功能降低,通气/血流比例失调,导致患者出现难治性呼吸困难,最终呼吸衰竭而死亡,严重危害人类生命健康。不同学者的研究发现,中医药对肺纤维化的早期干预及晚期康复,均能起到一定的作用,能够有效地提高患者肺功能、体耐力等。中医药治疗从病因病机入手,痰、瘀、虚贯穿在肺纤维化病程进展始终。导师张伟教授创新性提出了“气运失常”、“血运失常”、“津液代谢失常”、“脏腑功能失调”、“本虚标实”的五大病机,认为五大病机贯穿肺纤维化发病及病程演变始终。以五大病机理论为指导,前期我们在国家自然科学基金、省自然科学基金、泰山学者专项经费等资助下开展了益气类中药、化痰类中药以及活血化瘀、益气温阳等药物的多途径抗肺纤维机制的实验研究,证实海藻、蜈蚣、黄芪等代表性的中药具有明显的抗纤维化作用,可能与炎症机制、血管新生机制,免疫机制,上皮间充质转化机制,抗氧化机制,细胞外基质重塑等有关,具体作用机制需要进一步进行验证。本研究基于前期研究基础(海藻、蜈蚣、黄芪具有明显抗纤维化作用),结合肺纤维化“虚”“痰”“瘀”的病机,从细胞水平,深入探讨祛痰、化瘀、补虚类中药抗纤维化的具体作用机理,以期为肺纤维化的治疗以及新药的研发提供理论依据。[方法]1.川贝、海藻对博来霉素致肺纤维化大鼠的抗氧化作用机制研究选取大鼠90只,随机分为9组:空白对照组;模型组;吡非尼酮组;高、中、低浓度川贝组;高、中、低浓度海藻组。予以相应药剂灌胃处理28天后,断椎处理大鼠,取血、肺部组织分别保存于液氮和4%多聚甲醛固定液中,用于后续切片HE染色,观察病理变化,并检测造模情况。采用ELISA法检测各组大鼠肺组织中谷胱甘肽(Glutathione,r-glutamyl cysteingl+glycine,GSH)、超氧化物歧化酶(Superoxide dismutase,SOD)的表达水平,并筛选出表达效果最佳的海藻,通过海藻多糖干预人胚肺成纤维细胞(MRC-5)的氧化应激过程,来发掘海藻多糖的抗氧化机制,通过沉默相应的基因片段以及质粒过表达技术干预Nrf2通路,印证其作用靶点。2.桃仁、蜈蚣对博来霉素致肺纤维化大鼠血管新生作用机制研究选取大鼠90只,分为9组:空白对照组;模型组;吡非尼酮组;高、中、低浓度桃仁组;高、中、低浓度蜈蚣组。予以相应药剂灌胃处理28天后,断椎处理大鼠,取血、肺部组织分别保存于液氮和4%多聚甲醛固定液中,用于后续切片HE染色,观察病理变化,并检测造模情况。采用ELISA法检测各组大鼠肺组织中血管内皮生长因子(Vascular endothelial growth factor,VEGF)的变化,筛选出干预效果最佳的蜈蚣,对其作用机制展开研究,通过间充质干细胞(BMSc)观察蜈蚣毒素在血管新生过程中的作用机制,采用Jagged1蛋白干预Notch1通路,观察蜈蚣毒素对配体DLL4及VEGF受体Col-Ⅱ的调节作用,RT-PCR法检测Notch通路中DLL4、Col-Ⅱ mRNA 的表达。3.党参、黄芪对博来霉素致肺纤维化大鼠炎症反应作用机制研究选取大鼠90只,随机分为9组:空白对照组;模型组;吡非尼酮组;高、中、低浓度党参组;高、中、低浓度黄芪组。予以相应药剂灌胃处理28天后,断椎处理大鼠,取血、肺部组织分别保存于液氮和4%多聚甲醛固定液中,用于后续切片HE染色,观察病理变化,并检查造模情况。采用ELISA法检测各组大鼠肺组织中白细胞介素-8(Interleukin-8,IL-8)表达水平,并筛选出其中作用效果最佳的黄芪,对其抗炎机制展开研究。观察黄芪多糖(Astragalus Polysacharin,APS)对巨噬细胞(RAW264.7)内抗炎因子(Interleukin-10,IL-10)的干预作用,并采用RT-PCR方法检测IL-10、YM-1抗炎性巨噬细胞因子的表达。通过质粒过表达等技术印证APS对巨噬细胞的抗炎机制,发现其作用相关靶点。[结果]1.川贝、海藻对博来霉素致肺纤维化大鼠的抗氧化作用机制研究川贝高浓度、中浓度时能够显著提高GSH的含量(P<0.01),且与吡非尼酮作用相当,各组间比较无明显差异,中药海藻各组与吡非尼酮组均具有显著差异(P<0.01),且效果优于吡非尼酮组。与模型组比较,吡非尼酮组;高、低浓度海藻组SOD表达作用明显提高(P<0.01),中浓度海藻组、川贝组大鼠组织SOD表达作用有所提高(P<0.05,具有统计学意义);海藻各组组间对比无统计学意义。在本实验化痰组的两味中药当中,海藻的抗氧化作用优于其他各组,其GSH、SOD活性相对于其他中药各组表现最强,能够有效地提高GSH、SOD的抗氧化活性,后对其作用机制展开研究发现。同空白对照组相比,细胞内SOD、GSH的表达水平在siRNA沉默后有所下调,Nrf2沉默后的下调水平最为明显。海藻多糖的干预能够显著提高细胞内SOD、GSH的表达水平,但是siRNA沉默后的升高水平有限。细胞内HO-1的表达量在质粒过表达后有所升高,海藻多糖的干预能够进一步提高HO-1的表达。Keap1的表达水平与HO-1相反。2.桃仁、蜈蚣对博来霉素致肺纤维化大鼠血管新生作用机制研究实验各组的VEGF水平与模型组对比,均有明显的下降(P<0.01)。中药组中低浓度的桃仁、蜈蚣组;中浓度蜈蚣组下降水平最低,且组间差异无统计学意义(P>0.05)。高、中浓度桃仁组与高浓度蜈蚣组,对VEGF的表达有一定的抑制作用,但效果不如吡非尼酮组,其具有统计学意义(P<0.05),蜈蚣干预组对VEGF的抑制作用整体优于桃仁组,遂对蜈蚣的主要成分蜈蚣毒素的作用机制展开后续研究。蜈蚣毒素的实验组同对照组相比,DLL4、COl-Ⅱ的表达水平明显减少(P<0.01);加入VEGF催化蛋白Jagged1的实验组,能够显著的促进DLL4和COl-Ⅱ的表达水平升高(P<0.001),而在此基础上加入蜈蚣毒素,可再次下调基因DLL4、COl-Ⅱ的表达水平。3.党参、黄芪对博来霉素致肺纤维化大鼠炎症反应作用机制研究通过组间分析发现,中药各组与吡非尼酮的组间比较无明显差异,均能有效地对IL-8炎性细胞因子产生抑制作用。但黄芪中药组的方差最小,离散程度更低,表达稳定性更好。在Palmitate的诱导因素下,巨噬细胞内出现促炎反应,IL-10作为抗炎表达因子相对于对照组显著提高,具有统计学意义(P<0.001),而加入黄芪多糖(APS)的Palmitate组,IL-10含量降低,同对照组相比(P<0.01)具有显著统计学意义。在AMPK缺失的条件下,APS对IL-10、YM-1基因与蛋白的表达促进作用均显著降低。在pcDNA-Zeo组中APS可以有效地提高IL-10、YM-1的表达效力,发挥显著的抗炎作用。[结论]1.海藻多糖能够显著的提高GSH、SOD的蛋白含量,并作用于Nrf2/ARE信号通路中Nrf 2的转录激活过程,起到抗氧化的作用。能够有效地提高HO-1的抗氧化作用,下调Keap1的氧化反应。2.蜈蚣毒素可以有效降低VEGF在血清中的含量,并且作用于Notch1信号通路当中,通过干预Jagged1的表达,来抑制DLL4和COl-Ⅱ的表达水平,抑制无效血管的新生。说明蜈蚣毒素对于Jagged1诱导的BMSCs细胞血管新生具有抑制调节的作用。3.在炎症反应因子IL-8的水平上,黄芪多糖与吡非尼酮具有相似的作用效果,且表达稳定性优于其他中药各组。黄芪多糖能够有效地提高巨噬细胞的抗炎反应,促进IL-10活性的表达作用,并进一步证实黄芪多糖的抗炎机制依赖AMPK活性。更多还原

【Abstract】 Objective:Pulmonary fibrosis(PF)is the common outcome of interstitial lung disease.Its etiology is complex and the pathological mechanism is not clear.The clinical manifestations of patients are progressive decreased lung tissue compliance,decreased diffusion function and imbalance of ventilation / blood flow ratio,resulting in refractory dyspnea and death due to respiratory failure,which seriously endangers human life and health.Scholars have found that the early intervention of traditional Chinese medicine can effectively improve the lung function and endurance of patients with advanced fibrosis.TCM treatment starts from the etiology and pathogenesis,and phlegm,blood stasis and deficiency run through the course of pulmonary fibrosis.Professor Zhang Wei,the tutor,creatively put forward the five pathogenesis of "abnormal qi circulation","abnormal blood circulation","abnormal body fluid metabolism","dysfunction of Zang Fu organs" and "deficiency and excess",and believed that the five pathogenesis ran through the pathogenesis and course evolution of pulmonary fibrosis.Guided by the five pathogenesis theory,in the early stage,with the support of the National Natural Science Foundation of China,the Provincial Natural Science Foundation and the special fund for Taishan Scholars,we carried out the experimental research on the multi-channel anti lung fiber mechanism of Qi supplementing traditional Chinese medicine,phlegm resolving traditional Chinese medicine,blood activating and stasis removing drugs,Qi supplementing and Yang warming drugs,and confirmed that seaweed,centipede Representative traditional Chinese medicines such as Astragalus have obvious anti fibrosis effect,which may be related to inflammatory mechanism,angiogenesis mechanism,immune mechanism,epithelial mesenchymal transformation mechanism,antioxidant mechanism,extracellular matrix remodeling and so on.The specific mechanism needs to be further verified.Based on the previous research(seaweed,centipede and Astragalus membranaceus have obvious anti fibrosis effect),combined with the pathogenesis of "deficiency","phlegm" and "blood stasis" of pulmonary fibrosis,this study deeply discusses the specific anti fibrosis mechanism of traditional Chinese medicine for resolving phlegm,promoting blood circulation and Supplementing Qi at the cellular level,in order to provide a theoretical basis for the treatment of pulmonary fibrosis and the research and development of new drugs.Method:1.Study on antioxidant mechanism of scallop and seaweed on bleomycin induced pulmonary fibrosis in ratsNinety rats were randomly divided into 9 groups: blank control group;Model group;Pirfenidone group;High,medium and low concentration Chuanbei group;High,medium and low concentration seaweed groups.After intragastric administration of corresponding drugs for 28 days,the rats were treated with vertebral transection.The blood and lung tissues were saved in liquid nitrogen and 4% paraformaldehyde fixed solution respectively for subsequent slice HE staining,pathological changes were observed,and the modeling was detected.The expression levels of glutathione(r-glutamyl)and superoxide dismutase(SOD)in lung tissue of rats in each group were detected by ELISA,and the seaweed with the best expression effect was selected.Seaweed polysaccharide was used to intervene the oxidative stress process of human embryonic lung fibroblast(MRC-5),To explore the antioxidant mechanism of seaweed polysaccharide,intervene Nrf2 pathway by silencing corresponding gene fragments and plasmid overexpression technology,and confirm its target.2.Effect of peach kernel and centipede on angiogenesis in rats with bleomycin induced pulmonary fibrosisNinety rats were divided into 9 groups: blank control group;Model group;Pirfenidone group;High,medium and low concentration peach kernel group;High,medium and low concentration centipede group.After intragastric administration of corresponding drugs for28 days,the rats were treated with vertebral transection.The blood and lung tissues were saved in liquid nitrogen and 4% paraformaldehyde fixed solution respectively for subsequent slice HE staining,pathological changes were observed,and the modeling was detected.The changes of vascular endothelial growth factor(VEGF)in the lung tissue of rats in each group were detected by ELISA,the centipede with the best intervention effect was selected,and its mechanism was studied.The mechanism of centipede toxin in the process of angiogenesis was observed by mesenchymal stem cells(BMSC),and Jagged1 protein was used to intervene the Notch 1 pathway,To observe the regulatory effect of centipede toxin on ligand Dll4 and VEGF receptor col II.The m RNA expressions of Dll4 and col II in Notch pathway were detected by RT-PCR.3.Study on the mechanism of Codonopsis pilosula and Astragalus membranaceus on inflammatory response of bleomycin induced pulmonary fibrosis ratsNinety rats were randomly divided into 9 groups: blank control group;Model group;Pirfenidone group;High,medium and low concentration dangshen group;High,medium and low concentration Astragalus group.After intragastric administration of corresponding drugs for 28 days,the rats were treated with vertebral fracture.The blood and lung tissues were taken and stored in liquid nitrogen and 4% paraformaldehyde fixed solution respectively for subsequent slice HE staining,pathological changes were observed,and the modeling was checked.The expression level of interleukin-8(IL-8)in lung tissue of rats in each group was detected by ELISA,and Astragalus membranaceus with the best effect was selected to study its anti-inflammatory mechanism.To observe the intervention effect of Astragalus Polysaccharide(APS)on anti-inflammatory factor IL-10 in macrophages(RAW264.7),and to detect the expression of IL-10 and YM-1 anti-inflammatory macrophage factor by RT-PCR.The anti-inflammatory mechanism of APS on macrophages was confirmed by plasmid overexpression and other techniques,and its related targets were found.Results:1.Study on the antioxidant mechanism of scallop and seaweed on bleomycin induced pulmonary fibrosis ratsAt high and medium concentrations,scallop could significantly increase the content of GSH(P < 0.01),which was equivalent to that of pirfenidone,and there was no significant difference between the groups.There was significant difference between the traditional Chinese medicine seaweed group and the pirfenidone group(P < 0.01),and the effect was better than that of the pirfenidone group.Compared with the model group,pirfenidone group;The expression of SOD in high and low concentration seaweed group was significantly increased(P < 0.01),and the expression of SOD in medium concentration seaweed group and Chuanbei group was significantly increased(P < 0.05);There was no statistical significance in the comparison between seaweed groups.Among the two traditional Chinese medicines in the phlegm resolving group in this experiment,the antioxidant effect of seaweed is better than that of other groups,and its GSH and SOD activities are the strongest compared with other traditional Chinese medicine groups,which can effectively improve the antioxidant activities of GSH and SOD.Later,it is found that its mechanism of action is studied.Compared with the blank control group,the expression levels of SOD and GSH in cells were down regulated after si RNA silencing,and the down regulation level after Nrf2 silencing was the most obvious.The intervention of seaweed polysaccharide can significantly increase the expression levels of SOD and GSH in cells,but the increase level after si RNA silencing is limited.After intervention,the expression of HO-1 in seaweed cells was further increased.The expression level of Keap1 was opposite to that of HO-1.2.Study on the mechanism of peach kernel and centipede on angiogenesis in rats with bleomycin induced pulmonary fibrosisCompared with the model group,the level of VEGF in the experimental group decreased significantly(P < 0.01).In the traditional Chinese medicine group,low concentration of peach kernel and centipede group;The decrease level of medium concentration centipede group was the lowest,and there was no significant difference between the groups(P > 0.05).High,medium concentration peach kernel group and high concentration centipede group had a certain inhibitory effect on the expression of VEGF,but the effect was not as good as that of pirfenidone group,which was statistically significant(P < 0.05).The inhibitory effect of centipede intervention group on VEGF was better than that of peach kernel group.Therefore,a follow-up study was carried out on the mechanism of centipede toxin,the main component of centipede.Compared with the control group,the expression levels of Dll4 and co1-ii in the experimental group of centipede toxin decreased significantly(P < 0.01);The experimental group with VEGF catalytic protein Jagged1 could significantly increase the expression levels of Dll4 and co1-II(P < 0.001).On this basis,the addition of centipede toxin could down regulate the expression levels of genes Dll4 and co1-ii again.3.Study on the mechanism of Codonopsis pilosula and Astragalus membranaceus on inflammatory response of bleomycin induced pulmonary fibrosis ratsThrough the analysis between groups,it was found that there was no significant difference between the traditional Chinese medicine group and the pirfenidone group,and they could effectively inhibit IL-8 inflammatory cytokines.However,the variance of Astragalus group was the smallest,the degree of dispersion was lower,and the expression stability was better.Under the inducing factors of palmitate,there was a pro-inflammatory reaction in macrophages.As an anti-inflammatory expression factor,IL-10 increased significantly compared with the control group(P < 0.001),while the content of IL-10 decreased in the palmitate group added with Astragalus Polysaccharide(APS),which was statistically significant compared with the control group(P < 0.01).Under the condition of AMPK deletion,the promoting effect of Astragalus Polysaccharide(APS)on the expression of IL-10 and YM-1 genes and proteins decreased significantly.In pc DNA Zeo group,APS can effectively improve the expression of IL-10 and YM-1 and play a significant antiinflammatory role.Conclusion:1.Seaweed polysaccharide can significantly increase the protein content of GSH and SOD,act on the transcriptional activation of Nrf2 in Nrf2 / ARE signaling pathway,and play an antioxidant role.It can effectively improve the antioxidant effect of HO-1 and down regulate the oxidation reaction of Keap1.2.Centipede toxin can effectively reduce the content of VEGF in serum and act on Notch 1signaling pathway.It can inhibit the expression level of Dll4 and co1-ii and inhibit the angiogenesis of ineffective blood vessels by interfering with the expression of Jagged1.It shows that centipede toxin can inhibit and regulate the angiogenesis of BMSCs cells induced by Jagged1.3.In the level of inflammatory response factor IL-8,astragalus polysaccharide and pirfenidone have similar effects,and the expression stability is better than other traditional Chinese medicine groups.Astragalus polysaccharide can effectively improve the antiinflammatory response of macrophages,promote the expression of IL-10 activity,and further confirm that the anti-inflammatory mechanism of Astragalus Polysaccharide depends on AMPK activity.更多还原