| 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. |
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