Study On Protective Mechanism Of Wenfei Huaxian Decoction Pretreatment On Oxidative Damage Of Mouse Lung Mesenchymal Stem Cells Induced By H₂O₂ Based On The Theory Of Warming Method

1 ObjectiveGuided by the academic thought of Guangxiang Hong,a master of Chinese medicine,on treating lung not far from warming,this paper explores the etiology and pathogenesis of IPF in traditional Chinese medicine,summarizes the specific application of warming therapy in IPF treatment,and concludes the evidence-based medical evidence of warming therapy in IPF treatment.Through experimental study,an oxidative stress model of mouse lung mesenchymal stem cells induced by hydrogen peroxide was established to detect protein molecules in the related pathways after pretreatment with Wenfei Huaxian Decoction.To reveal the molecular mechanism of antioxidation of Wenfei Huaxian Decoction.2 Methods2.1 Theoretical ExplorationCollect the classical literature of traditional Chinese medicine about IPF,explain the understanding of IPF by famous Chinese medicine experts in past dynasties;explain the specific application of warming method in IPF treatment and the clinical efficacy of Wenfei Huaxian Decoction combined with the thought of Chinese medicine master treating lung not far from warming;collect modern clinical research data on warming method for IPF treatment,and conduct systematic evaluation of warming method for IPF treatment.2.2 Experimental study2.2.1 Cell ModelingThree healthy C57BL/6 mice were taken and the mouse lung mesenchymal stem cells were obtained after cervical dislocation.The phenotype and cell cycle of the10th generation mouse lung mesenchymal stem cells were identified by flow cytometry,and adipogenic,osteogenic and chondrogenic differentiation were induced.Mouse lung mesenchymal stem cells were treated with H₂O₂（100,200,400,600,800,1000μM）for 6 hours.Cell viability was measured by MTT.2.2.2 Cell Grouping and AdministrationMouse lung mesenchymal stem cells were planted on 96-well or 6-well plates with 8×10⁴/ml.After 24 hours of cell adherence,the cells were divided into control group,model group,low,medium and high dose pretreatment groups of Wenfei Huaxian Decoction.（1）Control group:24 hours after adding culture medium,the culture medium was abandoned and fresh culture medium was added for 6 hours.（2）Model group:24hours after adding culture medium,the culture medium was abandoned and treated for 6 hours with the culture medium containing hydrogen peroxide（800μM）.（3）The low,medium and high dose groups of Wenfei Huaxian Decoction:24 hours after adding the culture solution containing Wenfei Huaxian Decoction（100,200,400μg/ml）,the culture medium was abandoned and treated for 6 hours with the culture medium containing hydrogen peroxide（800μM）.2.2.3 Detection Indicators2.2.3.1 Cell morphology and adipogenic oil red staining,osteogenic alizarin red staining and chondrogenic alcian blue staining were observed under microscope.Cell surface antigens Sca-1,CD54,CD45,CD44,CD11b and cell cycle were detected by flow cytometry.2.2.3.2 Cell viability was detected by MTT assay,and LDH,SOD and MDA levels were detected by related kits.2.2.3.3 Apoptosis was detected by Hoechst apoptotic staining and flow cytometry;expression of apoptotic related proteins Bax,Bcl-2 and Cleaved caspase-3 were detected by Western blot;expression of BaxmRNA,Bcl-2mRNA and Cytochrome CmRNA were detected by RT-PCR.2.2.3.4 Flow cytometry was used to detect ROS and mitochondrial membrane potential;Western blot was used to detect the expression of antioxidant pathway proteins Akt,p-Akt,c-Nrf2,n-Nrf2 and HO-1;RT-q PCR was used to detect the expression of Nrf2mRNA and HO-1mRNA.3 Results3.1 Theoretical ExplorationTCM etiology and pathogenesis of IPF are closely related to viscera deficiency,phlegm and blood stasis,obstruction of lung collaterals.Weakness of Qi and yang,cold congestion,phlegm and blood stasis,exogenous cold and dampness occupy an important position,which is also the basis for IPF to use warm therapy.Wenfei Huaxian Decoction is an empirical prescription for IPF.It integrates warming yang,dispersing cold and removing blood stasis,and is the wisdom crystallization of the academic thought of treating lung not far from warming.Through the systematic analysis of clinical research on the treatment of IPF by warming method,it is found that compared with western medicine alone,warming method can effectively improve the total effective rate,significantly improve the main symptoms of TCM,such as cough and wheezing,improve lung function,improve the quality of life,and avoid the toxic and side effects of long-term oral hormones.It is worth discussing and promoting the treatment strategy of IPF by using warming therapy reasonably according to syndrome.3.2 experimental study3.2.1 Isolation,purification,identification and differentiation of mouse lung mesenchymal stem cells（mLMSCs）Under the microscope,mouse lung mesenchymal stem cells exhibited spindle-like,fusiform,similar to fibroblasts.The cell growth curve exhibited a typical S shape,ie,stationary phase,multiplication phase,and plateau phase.After adipogenic differentiation,the cells appeared red under the microscope.After osteogenic differentiation,the calcified nodules bound to the dye and appeared orange-red under the microscope.After cartilage-induced differentiation,the chondrocytes bound to Alcian Blue and appeared blue under the microscope.The cell cycle showed that most of the cells were in quiescent phase,and a small number of cells were in the proliferative phase;flow cytometry showed that the phenotype of the cells was Sca-1+/CD54+/CD44+/CD45-/CD11b-.3.2.2 Preliminary evaluation of protective effect of Wenfei Huaxian Decoction on oxidative damage of mLMSCs induced by hydrogen peroxideDifferent concentrations of Wenfei Huaxian Decoction（50,100,200,400,800μg/ml）were used to treat mouse lung mesenchymal stem cells for 24hours.Excepted 800μg/ml group（P<0.05）,there was no significant difference in cell survival rate between the other groups and the control group（P>0.05）.Different concentrations of hydrogen peroxide（200,400,600,800,1000μM）were used to treat mouse lung mesenchymal stem cells for 6 hours.Compared with the control group,excepted 200μM group（P>0.05）,the cell survival rate of other groups decreased in a dose-dependent manner.（P<0.05,P<0.01）.Different concentrations of Wenfei Huaxian Decoction（100,200,400μg/ml）were used to treat mouse lung mesenchymal stem cells for 24 hours,and then treated with H₂0₂（800μM）for 6 hours.The results showed that compared with the control group,the cell survival rate of the model group was significantly lower（P<0.01）.Compared with the model group,Wenfei Huaxian Decoction could improve the cell survival rate in low,medium and high dose groups（P<0.01）.Compared with the normal control group,LDH and MDA in the model group increased significantly,while SOD decreased significantly（P<0.01）.compared with the model group,LDH and MDA in the low,medium and high concentration groups decreased significantly,while SOD increased significantly（P<0.01）.3.2.3 Effect of Wenfei Huaxian Decoction on Apoptosis of mLMSCs Induced by H₂O₂.The results of Hoechst apoptosis staining showed that the nuclei of mLMSCs in the control group were blue and evenly distributed,and there was no condensed cell nucleus with apoptotic necrosis.The mLMSCs in the model group showed condensed nucleus with white color and even dense and densely stained nuclei.The pretreatment group of Wenfei Huaxian Decoction significantly reduced apoptotic cells and showed a positive correlation with the concentration of Wenfei Huaxian Decoction;Flow cytometry showed that the apoptotic rate of the model group was significantly higher than that of the normal control group（P<0.01）.Compared with the model group,the apoptotic rate of the low,medium and high concentration groups of Wenfei Huaxian Decoction were significantly lower（P<0.01）.Compared with the normal control group,the Bcl-2/Bax ratio in the model group decreased significantly（P<0.01）,Cleaved caspase-3 increased significantly（P<0.01）,the relative expression of BaxmRNA and Cytochrome CmRNA increased significantly,and the Bcl-2mRNA decreased significantly（P<0.01）.Compared with the model group,the Bcl-2/Bax ratio was significantly increased（P<0.01）,Cleaved caspase-3 was significantly decreased（P<0.01）,the relative expression of Bcl-2mRNA was significantly increased,and the Bax and Cytochrome CmRNA were significantly decreased（P<0.05,P<0.01）.Compared with the low dose group,the Bcl-2/Bax ratio in the middle and high dose groups of Wenfei Huaxian Decoction increased significantly（P<0.01）,Cleaved caspase-3 decreased significantly（P<0.01）,the relative expression of Bcl-2mRNA increased significantly（P<0.01）,and the Bax and Cytochrome CmRNA decreased significantly（P<0.01）.3.2.4 Study on Antioxidant Mechanism of Wenfei Huaxian DecoctionCompared with the normal control group,the ROS production in the model group increased significantly（P<0.01）,and the mitochondrial membrane potential decreased significantly（P<0.01）.Compared with the model group,the ROS production in the low,medium and high concentration groups of Wenfeihua Decoction decreased significantly（P<0.01）,and the mitochondrial membrane potential increased significantly（P<0.01）.Compared with the low concentration group,the ROS production in the medium and high concentration groups of Wenfeihua Decoction was significantly higher（P<0.01）.The mitochondrial membrane potential increased significantly（P<0.01）.After staining with JC-1,each group was observed under a fluorescence microscope.A large amount of red fluorescence and a small amount of green fluorescence were observed in the control group after combined visual field.A lot of green fluorescence can be seen in the model group.After pretreatment with low,medium and high concentrations of Wenfei Huaxian Decoction,the visible green fluorescence of each treatment group was gradually reduced,and the red fluorescence gradually increased.Compared with the normal control group,the p-Akt/t-Akt ratio and the expressions of c-Nrf2,n-Nrf2 and HO-1 in the model group had no significant difference（P>0.05）,while the relative expressions of Nrf-2 mRNA and HO-1 mRNA were significantly increased（P<0.01）.Compared with the model group,the ratio of p-Akt/t-Akt and the expression of c-Nrf2,n-Nrf2 and HO-1 in the low,medium and high concentration groups of Wenfei Huaxian Decoction were significantly increased（P<0.01）,and the relative expression of Nrf-2mRNA and HO-1mRNA were significantly increased（P<0.01）.Compared with the low dose group,the p-Akt/t-Akt ratio,c-Nrf2,n-Nrf2 and HO-1 expression in the middle and high dose group of Wenfei Huaxian Decoction were significantly increased（P<0.01）,and the relative expression of Nrf-2mRNA and HO-1mRNA were significantly increased（P<0.01）.4 ConclusionWenfei Huaxian Decoction based on warming method can significantly improve cell viability,reduce LDH and MDA production,enhance SOD activity,inhibit ROS production,repair mitochondrial membrane potential and reverse cell apoptosis,thereby improving oxidative damage induced by hydrogen peroxide in mouse lung mesenchymal stem cells.The mechanism may be related to the activation of Akt phosphorylation,the promotion of Nrf-2 nuclear translocation,the up-regulation of HO-1 protein expression and the positive regulation of Akt/Nrf-2/HO-1 signaling pathway.