Regulation And Molecular Mechanism Of Mitochondria Quality Of Murine Lung Mesenchymal Stem Cells Under Oxidative Stress By Wenfei Huaxian Decoction: A Preliminary Study

1 ObjectiveBased on the academic thought of“treating pulmonary diseases with warming therapeutic methods”by Hong Guangxiang,the Master of Chinese Medicine,this article thoroughly discusses the theoretical context and clinical experience of Wenfei Huaxian decoction.It is the representative prescription in“treating pulmonary interstitial fibrosis with warming therapeutic methods”.We combined the discovery of mitochondrial morphological changes of murine lung mesenchymal stem cells?MLMSCs?with the theory of“warming Yang and strengthening the body resistance”,then applied relevant experimental technologies to explore the regulation and molecular mechanism of Wenfei Huaxian decoction on the mitochondria quality of MLMSC under oxidative stress.2 Methods2.1 Theoretical ExplorationWe collected and sorted out ancient classic literatures on pulmonary interstitial fibrosis,and summarized the theoretical knowledge and clinical experience of representative physicians.Further,we described the exploration of our team with respect to this disease,explained the definition,principle,and key points of“treating pulmonary interstitial fibrosis with warming therapeutic methods in an entire period”,demonstrated the medicinal and curative effects of Wenfei Huaxian decoction,and with the macroscopic theory of Traditional Chinese Medicine,integrated the microscopic changes in mitochondria.2.2 Experimental Study2.2.1 Extraction,identification and modeling of MLMSCsWe extracted the cells from the lung tissues of C57BL/6 mice,purified by passage culture,and observed MLMSCs morphology under a microscope,drew cell proliferation curves,and identified their characteristic phenotypes by flow cytometry.We treated the cells with H₂O₂?2000,1500,1000,800,600,400,200,and 100?M?for 6h and then analyzed cell viability using the CCK8 method to establish the model.2.2.2 Grouping and observation of the effects of Wenfei Huaxian decoction on MLMSCs under oxidative stressIn order to determine the scientific concentrations,we used the CCK8method to detect the effects of Wenfei Huaxian decoction?2000,1000,500,250,125,62.5,31.25,and 15.625?g/m L?on the viability of healthy MLMSCs and others oxidized by H₂O₂,and established the NAC 5m M group as the positive control.We then detected and compared the activity of SOD,intracellular ROS level,and apoptosis rate in the cells of each group.2.2.3 Exploration of the regulation of Wenfei Huaxian decoction on the mitochondria quality of MLMSCs induced by H₂O₂We detected the mitochondrial membrane potential of MLMSCs by flow cytometry,measured the intracellular ATP levels using a microplate spectrophotometer,observed the mitochondrial ultrastructure of MLMSCs by transmission electron microscopy,gauged the m RNA levels of mitochondrial dynamic genes?FIS1,MFN1,MFN2,DRP1,OPA1,and PGC-1??by polymerase chain reaction,and estimated the expression of mitochondrial autophagy proteins?LC-3?,Beclin-1,and p62?by western blot.2.2.4 Preliminary study on the molecular mechanism of the mitochondria quality of MLMSCs regulated by Wenfei Huaxian decoctionWe assessed the m RNA levels of PINK1 and Parkin in MLMSCs by polymerase chain reaction,and estimated their protein expression in the mitochondria by western blot.3 Results3.1 Theoretical ExplorationThis article summarizes the evolution of cognition,representative treatments,and related research progress in pulmonary interstitial fibrosis in Traditional Chinese Medicine.Then,we introduced our view“treating pulmonary interstitial fibrosis with warming therapeutic methods in an entire period”.Based on the elaboration and discussion of our view and the representative prescription of Wenfei Huaxian decoction,we combined previous research,then integrated the mitochondrial changes into the macroscopic theory.Finally,we established a complete idea circuit from the perspective of“cell Yang”,mitochondria,lung mesenchymal stem cells,pulmonary interstitial fibrosis,Wenfei Huaxian decoction to“warming therapeutic methods in an entire period”which lays a solid theoretical foundation for subsequent experimental research.3.2 Experimental Study3.2.1 Identification of MLMSCs and establishment of the oxidative stress modelThe tenth generation MLMSCs was fusiform,arranged in a radial pattern,and tended to be uniform in morphology.After 24h of planting,the cells entered the logarithmic growth phase,which was maintained for at least 48h.The positive expression rate of CD11b was 3.01%,the CD45expression rate was 2.79%,while the positive expression rates of CD44,CD54,and Sca-1 were 97.8%,95.2%,and 98.3%?CD11b-,CD45-,CD44-,CD54+,and Sca-1+?.The oxidative stress model:H₂O₂ 600?M for 6h was selected.The cell survival rate decreased to 59.8%of the rate in control group?P<0.01?.3.2.2 Wenfei Huaxian decoction presented antioxidant effects on MLMSCs induced by H₂O₂The CCK8 test showed that the effects of Wenfei Huaxian decoction in the range of 500 to 15.625?g/m L was similar to that in the control group on healthy MLMSCs?P>0.05?.When the concentration was increased to 1000 and 2000?g/m L,the effects were different?P<0.01?.For MLMSCs induced by H₂O₂,when the concentration of Wenfei Huaxian decoction reached 62.5?g/m L,the effect was significantly different from that in the model group?P<0.05?.According to the above results,500 and62.5?g/m L of Wenfei Huaxian decoction were selected as the large and small dose groups.After modeling,the SOD level decreased?P<0.01?,intracellular ROS level increased?P<0.01?,and apoptosis rate increased?P<0.01?.Wenfei Huaxian decoction and the NAC groups can improve SOD levels?P<0.01?and reduce intracellular ROS and apoptosis rates?P<0.01?.The 500?g/m L Wenfei Huaxian decoction group was better than the NAC group?P<0.01?.3.2.3 Wenfei Huaxian decoction regulated mitochondria quality of MLMSCs induced by H₂O₂After modeling,the membrane potential and ATP level decreased significantly?P<0.01?,which were increased in Wenfei Huaxian decoction and NAC groups?P<0.05?,and the 500?g/m L Wenfei Huaxian decoction group was better?P<0.05?.Transmission electron microscopy revealed that the mitochondria in the model group were deformed long stripes,the ridges were loose and broken,and the matrix density was uneven.The mitochondria in the NAC group and 62.5?g/m L Wenfei Huaxian decoction group were still abnormal,but the ridges were relatively clear.In the 500?g/m L Wenfei Huaxian decoction group,the mitochondria were mostly oval,and the ridges and membrane were clear.In addition,autophagy bodies were found in the Wenfei Huaxian decoction groups.In the model group,the m RNA levels of FIS1,MFN1,MFN2,DRP1,OPA1,and PGC-1?were decreased?P<0.01?.The m RNA levels of FIS1,MFN1,MFN2,and PGC-1?in the 500?g/m L Wenfei Huaxian decoction group were much higher than those in the model?P<0.01?,and the m RNA level of DRP1 was increased?P<0.05?,while the m RNA level of OPA1 was not significantly different?P>0.05?.Compared with the model group,the protein expression of LC-3?and p62 in the 500?g/m L Wenfei Huaxian decoction group increased significantly?P<0.01?,and the expression of Beclin-1increased slightly?P<0.05?.3.2.4 Wenfei Huaxian decoction may regulate the mitochondria quality of MLMSCs via the PINK1/Parkin pathwayAfter modeling,the m RNA levels of PINK1 and Parkin decreased?P<0.01?;these increased in the Wenfei Huaxian decoction group?P<0.01?.Compared with the control group,the protein expression of PINK1increased while Parkin decreased in the model group?P<0.05?.The expression of these proteins in the Wenfei Huaxian decoction group were higher than those in the model group?P<0.05?.4 ConclusionWenfei Huaxian decoction can regulate the mitochondrial quality of MLMSCs and play an antioxidant protective role.Moreover,the mechanism may be related to the activation of the PINK1/Parkin signaling pathway.