The Role Of Artesunate Influence On The Process Of TGF-β1Induced Alveolar Epithelial Cells Ⅱ Typetranslate To Mesenchymal

Objective: Idiopathic pulmonary fibrosis is progressive disease which mesenchymal cell proliferation and extracellular matrix for abnormal sedimentary，lung tissue is mainly composed of alveolar epithelial cells，alveolar epithelial cells containⅠtype andⅡtype．The alveolar epithelial cellsⅡtypehave stem cell properties，cell factor TGF-β1can induce epithelial cells turning into mesenchymal cells (EMT)，thus aggravating pulmonary fibrosis， and its possible mechanism for activation of the substrate Smad，P38MAPK．Artesunate is antimalarials for traditional，previous studies have confirmed that it have anti fibrosis effection．This study is after TGF-β1induced epithelial cells-mesenchymal cells transition (EMT)，give differentdoses of artesunate play role in TGF-β1mesenchymal cells induced by different period，detection Smad3、Smad7、P38MAPK、ACTA2、VIM mRNA and protein expression, to study the artesunate influence the process ofEMT through the TGF-β1/Smad and TGF-β1/P38MAPK path and study the most significant dose and time．Methods: experiment divided to two parts．part one：Culture rat alveolarepithelial cells typeⅡ (RLE-6TN) in vitro；Preliminary experimental detection TGF-β1induced concentration；To establish experimental-model of Rat’s alveolar epithelial type II (AT2) cells（RLE-6TN）in vitro．to detect artesunate IC50by MTT assay and select the concentration of drug Interven tion based on artesunate IC50．Choose artesunate’s intervention concentration according to artesunate’s IC50；Cells divide into6groups(dosage effect)，blank group；TGF-β1(3ng/ml) group；TGF-β1(3ng/ml) combine with artesunate (1ug/ml) group；TGF-β1(3ng/ml) combine with artesunate (2ug/ml)group； TGF-β1(3ng/ml) combine with artesunate(4ug/ml) group；TGF-β1(3ng/ml) combine with artesunate (8ug/ml) group，Culture24hours，to observed cells form under inverted microscope；Collect cells，extracting totalRNA，detect the the mRNA expression of Smad7、Smad3、ACTA2、Vim assessed by RT-PCR；Extracting total protein，detect the protein expression of P-Smad3、Smad7、ACTA2、Vim assessed by Western bloting．Cells divide to four groups(time effect)，blank group (1%FBS)；TGF-β1group (3ng/ml)；TGF-β1(3ng/ml) combine with artesunate (1ug/ml) group；Artesunate group (1ug/ml), Culture12、24、48hours，observed cells formunder inverted microscope；Collect cells，extracting total RNA，to detectthe mRNA expression of Smad7、Smad3、ACTA2、Vim assessed by RT-PCR；to extract total protein after culture24hour，to detect the proteinexpression of P-Smad3、Smad7、ACTA2、Vim assessed by Western bloting。Part two: Cell divide to6groups，like part one，Colle cells，extractionmRNA，to detect the mRNA expression of P38MAPK、ACTA2、Vim assessed by RT-PCR；Extracting total protein， to detect the protein expression of P38MAPK、ACTA2、Vim assessed by Western bloting.Results: part one:Artesunate’s IC50is8.86ug/ml when it play role in TGF-β1induced RLE-6TN cells24hours and artesunate’s IC50is5.04ug/ml when it play48hours. Detecte RLE-6TN cell’s morphology，Compared with the normal group，TGF-β1group paving stone shape epitheliumfor m into a spindle，and its cell gap slightly bigger，the connection becomeloose between the cells，after artesunate play role in TGF-β1induced RLE-6TN cells24hours, Compared with the TGF-β1group，cells of the spindle structure reduce，some recovery paving stone shape epithelium form,but the connection between the cells is loose, TGF-β1combine with artesunate1ug/ml group showed the most obvious；Compared with the blank control group，The TGF-β1group’s mRNA levels of Smad7、Smad3、ACTA2、Vim mRNA and protein are higher (P<0.05)；And Compared with TGF-β1group，TGF-β1combine with artesunate group Smad7mRNA andprotein are higher (P<0.05)；Smad3、ACTA2、Vim mRNA are lower (P<0.05), P-Smad3、ACTA2、Vim protein is lower (P<0.05).This trend is obvious when artesunate is1ug/ml and when it affect24hours.Part three：Compared with the blank control group，The TGF-β1group’smRNA levels of p38MAPK、ACTA2、Vim mRNA and protein are higher(P<0.05)；And Compared with TGF-β1group，TGF-β1combine with artesunate group P38MAPK、ACTA2、Vim mRNA and protein are lower (P<0.05)．Conclusion:1．TGF-β1can induce epithelial cells turn into mesenchymalcells．2．Artesunate can inhibition TGF-β1inducted epithelial cells-mesenchymal cells tanslation in a certain extent，The possible mechanism is up-regulation Smad7mRNA and protein expression and down-regulationSmad3mRNA expression，or down-regulation p38MAPK mRNA and protein expression．Inhibition of Smad3protein phosphorylation.3．Artesunate plays the role of anti fibrosis. The possible mechanism is inhibite EMT．...