Study On The Mechanism Of Feishuai Mixture Serum On The Expression Of IL-8,ROS And Cell Proliferation Induced By Cigarette Smoke Extractin In 16HBE Cells And ASMC Cells

Research Purpose16HBE cells and Airway Smooth Muscle Cells(ASMC)were used to study the therapeutic effect of Feishuai Mixture in terms of anti-inflammatory,antioxidant,and cell proliferation inhibition.Revealing the mechanism of Feishuai Mixture in treating chronic obstructive pulmonary disease.To provide theoretical and scientific evidence for new targets for treating COPD in Chinese medicine.Research MethodsThe subject experiment is divided into two parts:In the first part,16 HBE cells were stimulated with CSE to construct a COPD model,and the traditional Chinese medicine Feishuai Mixture serum and blank serum were used to intervene in the culture treatment.CCK-8 was used to determine the effect of different concentrations of CSE on cell viability of 16 HBE cells;ELISA was used to determine the expression of IL-8 protein in 16 HBE cells;RT-PCR was used to determine the expression of IL-8 m RNA in 16 HBE cells;Fluorescein Gene Reported system was used to detect IL-8 transcriptional activity;the level of migration-related proteins in 16 HBE cells was determined by Western blotting.To investigate the effect of Feishuai on the expression of IL-8 m RNA and protein in 16 HBE cells induced by CSE.In the second part,a COPD model was established by stimulating ASMC cells with CSE,and the serum of Feishuai Mixture,NAC and Mito Q10 were used to intervene the culture treatment.Determination of mitochondria in ASMC cells by MTG-DAPI staining method;and the cell cycle was detected by flow cytometry to study the effect of Feishuai Mixture on mitochondrial damage and cell cycle of ASMC cells induced by CSE.Results1.CCK-8 test results:In the 1% CSE group,2.5% CSE group,5% CSE group,7.5% CSE group,and 10% CSE group,as the CSE concentration increased,the cell viability did not decrease significantly(P>0.05);after that,cell viability in the subsequent 15% CSE group and 20% CSE group decreased significantly(P<0.05).2.RT-PCR and Elisa test results:the expression level of IL8 m RNA and protein in 16 HBE cells was low without any stimulation;Compared with the blank control group,After 12 h,24 h,36 h,48 h,60 h and 72 h of stimulation with 10% CSE,the expression of IL-8 m RNA and protein in 16 HBE cells increased significantly,and the difference was statistically significant(P<0.05).After intervention with Chinese medicine-containing serum,compared with the CSE cell group,the changes in IL-8 m RNA and protein content of the blank serum control group weren’t statistically significant difference(P>0.05).Compared to the blank serum control group,the IL-8 m RNA and protein content decreased significantly,the difference was statistically significant(P<0.05);After 12 h,24 h,36 h,48 h,60 h and 72 h of stimulation with 10% CSE,the expression of IL-8 m RNA and protein in 16 HBE cells increased significantly,and the difference was statistically significant3.Fluorescein reporter gene system test results:Compared with the CSE cell group,the IL-8 transcription activity of the blank serum control group was not statistically significant(P>0.05);compared with the blank serum control group,the IL-8 transcriptional activity decreased significantly,and the difference was statistically significant(P<0.05).4.Western-Blot test results:The traditional Chinese medicine Feishuai Mixture can inhibit the expression of N-cadherin and Vimentin in 16 HBE cells stimulated by 10% CSE,and at the same time enhance the expression of E-cadherin.This effect on the expression of migration-related proteins in 16 HBE cells varies with concentration increase.5.MTG-DAPI staining results:Compared with the blank model group,the fluorescence intensity of the 2.5% CSE group was significantly enhanced;while the fluorescence intensity of the cells in the NAC control group and the drug-containing serum group decreased compared with the 2.5% CSE group,but it was still lower than the blank model group.6.Flow cytometry test results: Compared with the blank model group,the ratio of G1/G0 in the 2.5% CSE group decreased and the proportion of S phase increased;compared with the 2.5% CSE group,the NAC group,Mito Q10 group,and Chinese medicine were low,medium,and The proportion of G1/G0 cells in the high-dose group increased significantly,and the difference was statistically significant(P<0.05).Conclusions1.Cigarette smoke extract can inhibit 16 HBE cell activity in a concentration-dependent manner,up-regulate the m RNA and protein level of IL-8 in 16 HBE cells,and induce EMT in 16 HBE cells.2.The traditional Chinese medicine Feishuai Mixture can reverse the regulation effect of CSE on the transcriptional activity of IL-8m RNA in 16 HBE cells.3.Cigarette smoke extract can inhibit the activity of ASMC cells in a concentration-dependent manner,induce mitochondrial damage by inducing the production of ROS in ASMC cells,which can promote cell division and proliferation.4.The traditional Chinese medicine Feishuai Mixture can effectively reduce the accumulation of ROS in ASMC cells,inhibit mitochondrial damage,and inhibit cell proliferation.Its effectiveness is not inferior to the antioxidants NAC.5.The mechanism of traditional Chinese medicine Feishuai mixture in the treatment of COPD involves the regulation of inflammatory response,oxidative stress and cell proliferation.