Mechanism Of Diclofenac Sodium In Relaxing Mouse Tracheal Smooth Muscle And The Function Of PTPMT1 Gene In Lung Cancer Cells

In recent years,people's living environment has been threatened by poor air quality,air pollution,smog,tobacco smoke caused by smoking,automobile exhaust and dust mites,and the incidence of respiratory diseases,which are in close contact with the external environment,is also rising.The incidence and mortality of lung diseases?such as asthma,chronic non-obstruction,lung cancer,etc.?are increasing every year.Therefore,it is of great significance to further study the pathogenesis of lung diseases and explore new drugs and new programs for the treatment of lung diseases.This paper consists of two parts,all of which are about lung diseases?asthma,lung cancer?.The first part is to study the mechanism of relaxation of tracheal smooth muscle by a small molecule compound in mice,which is useful to develop bronchodilators for the treatment of obstructive respiratory diseases such as asthma and chronic obstructive pulmonary disease.Diclofenac sodium?DCF?is widely used in the treatment of analgesia and arthritis in the clinic.It has been reported that diclofenac sodium has the effect of treating epilepsy and relieving phlegm.So we wonder whether diclofenac sodium can relax the tracheal ring of mice?In our previous experiments,we have already found that diclofenac sodium can inhibit the contraction of the tracheal ring in mice caused by high K⁺/ACh.In order to explore the mechanism of diclofenac sodium of relaxing the tracheal ring,we further studied the ion channel that regulates smooth muscle contraction.At the tissue level,we explore the experimental results of which diclofenac sodium diastolic tracheal smooth muscle may be associated with ion channels,followed by patch clamp techniques at the cellular level to detect changes in ion channel currents at the tissue level.The experimental results show that DCF induced relaxation of the tracheal ring by inhibiting VDLCCs channel-mediated extracellular Ca²⁺influx,and inhibiting the opening of NSCCs channel.And the release of calcium,regulating the Na⁺/Ca²⁺exchange mechanism to exchange intracellular calcium ions to reduce intracellular Ca²⁺concentration,which leads to relaxation of the tracheal ring.At the same time,DCF also helps to activate the K⁺channel and the large-conductance Ca²⁺-activated K⁺channel?BK channel?to relax the smooth muscle cell to achieve the effect of relaxing the tracheal ring.Diclofenac sodium regulates the change of intracellular concentration of Ca²⁺by affecting a variety of ion channels,so as to achieve the relaxation effect of tracheal smooth muscle.The second part is to study the effect of the decrease in the expression of PTPMT1on lung cancer cell.Primary bronchogenic carcinoma is the most common condition in malignant tumors,and its morbidity and mortality are increasing year by year.In primary bronchogenic lung cancer,non-small cell lung cancer accounts for approximately 80%of all lung cancer.Non-small cell lung cancer is the most common type of disease in lung cancer,and the incidence is increasing year by year.On a global scale,the disease has been ranked among the leading causes of death worldwide.Due to the lack of early screening and treatment,many patients find themselves in the middle or advanced stages of the disease,so the disease is difficult to treat and the survival rate is low.Therefore,in-depth study of the nosogenesis and block the pathogenesis of lung cancer play important roles in the diagnosis of the program.PTPMT1 is a phosphatase specifically localized on the mitochondrial inner membrane.This enzyme plays an important role in the function of mitochondria,the deletion of PTPMT1 impairs the differentiation of stem cells or the death of early embryo.In order to further explore the function of this gene in lung cancer cells,this gene was knocked out from A549 cells by using the CRISPR/Cas9 system.In this way,we obtained two PTPMT1 stably knockdown A549 cell lines?sg2-2,sg6-1?.The gene editing of the knockdown cell line was examined at three different levels:DNA,RNA and protein,and the results showed that the efficiency of knock down is about 70%-80%.To investigate the effect of PTPMT1 knockdown on the proliferation of A549 cells,we recorded the proliferation of WT cells and KD cells in 5 days by MTT assay,collected datas and ploted a curve to compare the proliferation between WT cells and KD cells.The results showed that the proliferation of two KD cells was inhibited by compared to WT cells.Moreover,the cell growth of the sg6-1 strain was more pronounced in which the gene PTPMT1was knocked down more.In order to further study the reasons,we performed flow cytometry analysis with PI staining under the condition of non-synchronization.And we found that there is no big difference in cell cycle between the WT and KD cells.We then examined the expression levels of cell cycle-related proteins at the RNA level,and the results showed that the negative regulator of cell cycle,P27,was significantly up-regulated in the two KD cells.Our research can provide a theoretical basis of lung cancer for early lung cancer diagnosis and gene-targeted therapy.