| With the advent of aging society,the number of patients with idiopathic pulmonary fibrosis(IPF)is increasing.It has been confirmed that oxidative stress-induced cell oxidative/antioxidant imbalance is the key to promote the formation and progress of pulmonary fibrosis,and oxidative stress-induced mitochondrial dysfunction and changes in energy metabolism(oxidative phosphorylation and aerobic glycolysis)are closely related to the occurrence of IPF.At present,there is no cure for IPF.Therefore,it is urgent to reveal its pathogenesis and develop more safe and effective drugs or treatment strategies.Traditional Chinese medicine believes that the disease belongs to the category of "lung flaccidity",the basic pathogenesis is Yang deficiency and cold coagulation,phlegm stagnation and blood stasis,and lung Yang deficiency is the key of IPF pathogenesis.Warming Yang and dispersing cold,clearing phlegm and removing blood stasis are the basic methods to treat IPF.According to Professor Hong Guangxiang's academic thought of "treating lung not far away from temperature",the team proposed the idea of "treating pulmonary interstitial fibrosis with whole course warming method",and created Wenfei Huaxian Decoction on the basis of Yanghe Decoction.After nearly 20 years of clinical practice and observation,the clinical application of Wenfei Huaxian Decoction in the treatment of IPF has achieved good results.In order to further clarify the mechanism of Wenfei Huaxian Decoction,the team conducted a lot of basic research.In the theoretical study of traditional Chinese medicine,we believe that lung mesenchymal stem cells(LMSCs)are the Yang in the lung,and mitochondria are the center of energy metabolism,which is called "Yang in Yang".Aerobic glycolysis can not only release the rapid production of ATP to provide energy for cells,but also transform nutrients into precursors of mitochondrial biosynthesis.Therefore,we think that aerobic glycolysis is also a part of lung Yang in function.Wenfei Huaxian Decoction can treat IPF by warming lung Yang(mitochondria and aerobic glycolysis).On the basis of basic experimental research,the team found that Wenfei Huaxian Decoction has antioxidant effect.Traditional Chinese medicine attaches great importance to holistic view and syndrome differentiation and treatment.Compound Chinese medicine contains a variety of material basis,which has multi-target and multi-channel network effect.Therefore,in order to further understand the target of Wenfei Huaxian Decoction,this study used hydrogen peroxide(H2O2)to intervene LMSCs to establish oxidative stress model to simulate the changes of lung microenvironment,in order to reveal the mechanism of Wenfei Huaxian Decoction in the treatment of IPF.Objective Taking LMSCs of mice in vitro oxidative stress model as the research object and AMPK/SIRT3 pathway as the breakthrough point,this paper discusses the protective mechanism of Wenfei Huaxian Decoction on LMSCs in vitro oxidative stress model from two aspects of mitochondrial function and energy metabolism,so as to provide effective theoretical support for clinical treatment of IPF.Methods1.The LMSCs of C57BL/6 mice were isolated and extracted by gradient density centrifugation.The flow cytometry was used to identify the surface markers of cells and to conduct the induction and differentiation of osteoblasts,adipogenesis and chondrogenesis.The model of oxidative stress injury was established by H2O2 intervention method,and the concentration of Wenfei Huaxian Decoction was screened by MTT method;The effect of Wenfei Huaxian Decoction on the proliferation of LMSCs was detected by EDU;The effects of Wenfei Huaxian Decoction on the levels of superoxide dismutase(SOD),malondialdehyde(MDA)in LMSCs induced by H2O2 were detected by enzyme standard instrument. 2.The oxidative stress model was established after 24 hours of LMSCs intervention with different concentrations of Wenfei Huaxian Decoction.The levels of reactive oxygen species(ROS)were measured by flow cytometry;The effects of Wenfei Huaxian Decoction on the mitochondrial membrane potential,mitochondrial quantity,mitochondrial transmembrittility pore(m PTP)induced by H2O2 were observed by fluorescence microscope;Seahorse XFe24 was used to detect the protective effect of Wenfei Huaxian Decoction on the oxygen consumption rate(OCR)of LMSCs;RT-PCR was used to detect the m RNA levels of PGC-1 ? and SOD2;Western blot was used to detect the expression levels of AMPK,SIRT3,PGC-1 ? and SOD2.After 24 hours of silence,SIRT was given Wenfei Huaxian Decoction(400 ?g/ml)for 24 hours,then H2O2 was used to make the model,and the ROS level of each group was detected by flow cytometry;Seahorse XFe24 detects the change of OCR;Western blot was used to detect the expression level of AMPK/SIRT3 and its pathway protein.After 24 hours of LMSCs intervention,the model was made by H2O2,and ROS levels in each group were detected by flow cytometry;Seahorse XFe24 was used to detect whether OCR of each group changed;Western blot was used to detect the expression of AMPK/SIRT3 pathway protein.3.The different concentrations of Wenfei Huaxian Decoction were used to treat LMSCs for 6 hours after 24 hours of treatment.The effects of Wenfei Huaxian Decoction on the aerobic glycolysis of LMSCs were evaluated.The glucose content and lactate level were measured by enzyme standard instrument;Seahorse XFe24 was used to detect the extracellular acidizing rate(ECAR);RT-PCR was used to detect the m RNA levels of HK2 and PKM2;The expression levels of HK2 and PKM2 were detected by Western blot.Whether Wenfei Huaxian Decoction can still play a protective effect after silent SIRT3 expression,that is,the condition of ECAR in each group and the expression level of HK2 and PKM2 protein in each group.After 24 hours of LMSCs intervention,the model was made by combination of AMPK activator(CC)and AICAR.Seahorse XFe24 was used to detect ECAR in each group,and the expression level of aerobic glycolysis enzyme was detected by Western blot.Results1.Flow cytometry showed that the immunophenotype of LMSCs was positive for Sca-1,CD44,CD90,CD54 and CD105,and CD11 b,CD45 and CD31 were negative.Further,the differentiation induction experiments of LMSCs were carried out,which confirmed that LMSCs can be differentiated into osteoblasts,chondrocytes and adipocytes in vitro,which is in line with the characteristics of MSCs.The results of EDU showed that LMSCs could be proliferated in vitro under the intervention of temperature lung chemical fiber Decoction with 25,100 and 400? g/ml concentration.In order to further verify the protective effect of Wenfei Huaxian Decoction on oxidative stress injury,we tested the content of SOD and MDA in cells.The results showed that the SOD activity of LMSCcs decreased after oxidative injury,the MDA content increased,25,100,400 ?g/ml warm lung chemical fiber decoction could enhance SOD activity(P<0.05),and down regulate the expression level of MDA(P< 0.05).2.Wenfei Huaxian Decoction can reduce the intracellular ROS level of LMSCs induced by H2O2(P<0.05).The fluorescence microscope showed that Wenfei Huaxian Decoction could close the LMSCs m PTP hole,enhance the mitochondrial membrane potential and increase the number of mitochondria.The level of basic OCR and ATP in mitochondria was enhanced by Wenfei Huaxian Decoction(P<0.05).Wenfei Huaxian Decoction(25,100,400 ?g/ml)could improve the reverse transcription expression of PGC-1? and SOD2(P<0.01).Wenfei Huaxian Decoction(100,400 ?g/ml could significantly improve the expression of SIRT3,PGC-1? and SOD2(P< 0.05),and inhibit P-AMPK/AMPK phosphorylation level(P<0.05).After silent SIRT3 expression,LMSCs showed further damage,which showed ROS increased(P<0.05);OCR was further injured but there was no significant statistical significance(P>0.05);The levels of SIRT3,PGC-1 ? and SOD2 were further decreased(P<0.05);But P-AMPK/AMPK had no statistical significance(P>0.05).Compared with NC + WFHX group,the mitochondrial basic OCR level was improved after SIRT3 si RNA + Wenfei Huaxian Decoction dry,but there was no statistical significance(P>0.05);The ROS level was decreased(P<0.05).The low knockdown of SIRT3 further reduced the protein expression of SIRT3,PGC-1? and SOD2 in SIRT3 si RNA + Wenfei Huaxian Decoction group(P<0.05),and P-AMPK/AMPK was up regulated,but there was no statistical significance(P>0.05).Compared with the model group,the expression of ROS was decreased after the intervention of Wenfei Huaxian Decoction(400 ?g/ml)and AMPK inhibitor(CC),which enhanced the basic OCR and ATP levels of mitochondria(P<0.05),promoted the expression of SIRT3,PGC-1 ? and SOD2 protein(P<0.05),and decreased the phosphorylation level of AMPK(P<0.05).After the intervention of AICAR,AMPK activator,the oxidative stress damage of cells was further aggravated.Compared with the model group,AICAR + Wenfei Huaxian Decoction(400 ?g/ml)could clear ROS(P<0.05),up regulate the basic OCR and ATP(P<0.05),down regulate the level of P-AMPK/AMPK(P<0.05),and promote the expression of PGC-1? protein(P<0.01),and SIRT3 and SOD2 had no statistical significance(P>0.05).3.The glucose intake and lactate content decreased after LMSCs oxidative stress injury(P<0.01),the function of glycolysis ECAR(P<0.01),the reverse transcription level and protein expression of HK2 and PKM2 decreased(P<0.05),and the indexes of warming lung chemical fiber Decoction(100,400 ?g/ml)could be improved(P<0.05).After silence of SIRT3 expression,the expression of glycolysis ECAR,HK2 and PKM2 decreased further(P<0.05).Compared with NC + WFHX group,Wenfei Huaxian Decoction(400 ?g/ml)+SIRT3 si RNA could improve the function of glycolysis ECAR(P<0.05),and promote the protein expression of HK2 and PKM2(P<0.05).Compared with the model group,CC combined with Wenfei Huaxian Decoction(400 ?g/ml)enhanced the level of glycolysis ECAR(P<0.01),and promoted the protein expression level of HK2 and PKM2(P<0.05);AICAR further weakened the function of aerobic glycolysis.AICAR combined with Wenfei Huaxian Decoction(400 ?g/ml)enhanced the level of glycolysis ECAR(P<0.01),but did not promote HK2 and PKM2(P>0.05).Conclusion(1)Wenfei Huaxian Decoction can promote Yang Qi(mitochondrion and aerobic glycolysis)by warming the Yang in the lung(LMSCs),so as to achieve the purpose of "strengthening the body and eliminating pathogenic factors".(2)Wenfei Huaxian Decoction can protect LMSCs from H2O2 induced injury.(3)Wenfei Huaxian Decoction can alleviate mitochondrial dysfunction and metabolic imbalance of LMSCs induced by H2O2 through AMPK/SIRT3 pathway.(4)Wenfei Huaxian Decoction can target AMPK/SIRT3 signaling pathway,enhance the aerobic glycolysis function of LMSCs,and then reduce the damage of oxidative stress. |
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