The Distribution Of TCM Symdrome As Well As Gene Expression Profiles In IPF And The Mechanism Of MALAT1 Promoting Fibrosis Via Rb-HDAC1-E2F1 Axis

Part 1: Literature research of IPF and evidence-based medicine research on the frequency distribution of TCM syndromesObjective: Idiopathic pulmonary fibrosis(IPF)is a progressive chronic fibroproliferative lung disease with a poor prognosis due to a lack of effective treatment.Traditional Chinese Medicine has been used in clinical treatment for many years,and it also shows certain advantages in the treatment of IPF.But the dialectical thinking of IPF is complex and varied,which causes the disease of dialectical classification and lvcorresponding treatment agent are diverse.Thus we performed this study to systematically discuss the current situation of TCM research on IPF and explore the distribution characteristics of TCM syndromes of IPF in China.Then we systematically analyze the RCT studies on the treatment of IPF with the certain TCM.Methods: We summarized and analyzed the dialectical classification,diagnosis and treatment methods of IPF through literature research.First we analyzed the distribution frequency of TCM syndromes of IPF in China by meta-analysis of single rate.Then we analyzed the clinical treatment effect with the TCM treatment of invigorating qi and activating blood by means of evidence-based medicine.Results:(1)We found that the major TCM syndromes of IPF in China are qi deficiency and blood stasis syndrome,qi deficiency in lung,spleen and kidney syndrome,and phlegm-turbidity obstructing lung syndrome by means of the meta-analysis.Through subgroup analysis,we found that the incidence of qi deficiency and blood stasis syndrome was higher in the middle latitudes.(2)The treatment of invigorating qi and activating blood can effectively improve the symptoms of IPF patients.Through further sorting out the Traditional Chinese Medicine included studies,we found that the TCM used to nourish qi deficiency are mainly astragalus,the TCM used to nourish lung and kidney are mainly honeysuckle and dendrobium,the TCM used to invigorate blood and remove blood stasisi are mainly chuanxiong and salvia miltiorrhiza.Part 2: Analysis and verification of lnc RNAs expression profile of IPFObjective: lnc RNA is a kind of multi-functional adapter molecules which can combine with proteins,DNA and other RNA,and play a variety of biological functions.(1)we integrated the transcriptome results through the way of database mining and obtained the key regulatory genes.(2)we verified the expression of these key regulatory genes between the IPF patients with qi deficiency and blood stasis syndrome and healthy subjects,respectively.Methods:(1)We integrated and analyzed the three datasets(GSE2052,GSE44723 and GSE24206)in GEO database.(2)IPF patients who met the diagnostic criteria for qi deficiency and blood stasis syndrome from January 2018 to June 2019 were collected,as well as the corresponding healthy physical examiners.We isolated the peripheral mononuclear cells of these subjects.Then we detected the expression of key regulatory genes by RT-PCR method.Results:(1)We obtained candidate genes,such as MALAT1,FENDRR,RNU1-1,E2F1 and so on through using bioinformatic analyses.(2)Compared with the healthy control group,MALAT1 and E2F1 in the IPF with qi deficiency and blood stasis syndrome were highly expressed,while YBX1 was low expressed,with a statistically significant difference,while FENDRR was not significantly different in the two groups.Part 3: Regulatory role of MALAT1 and E2F1 in phenotypic transformation of fibroblastObjective: To explore how MALAT1 and E2F1 play a regulatory role in the process of pulmonary fibersisMethods: We used TGF-β1 to induce the pulmonary fibrosis model in vitro.Then we evaluated the efficiency of pulmonary fibrosis model in morphology,m RNA level and protein level respectively.We detectd the expression of MALAT1 in cell models and performed the functional verification of MALAT1 in the process of pulmonary fibrosis by gain of function and loss of function methods.Then we observed the expression change of E2F1 according to the expression change of MALAT1.Finally,after knock down or overexpress MALAT1 accompanied with overexpress or knock down E2F1,we observed the transformation process of EMT and the expression change of Rb/HDAC1/E2F1Results: We found the expression of MALAT1 and E2F1 was high in both IPF patients with phlegm and blood stasis obstructing lung syndrome and pulmonary fibrosis cell model.Overexpressed MALAT1 can promot epithelial-mesenchymal transition(EMT)of fibroblast cell model using western blot.The EMT effect of fibrosis can be reversed when we silence/overexpress MALAT1 and co-transfect overexpress/silence E2F1 via Rb/HDAC1.Conclusion: MALAT1 and E2F1 are the differential expressed genes of phlegm and blood stasis obstructing lung syndrome of IPF.There is a high positive correlation between MALAT1 and E2F1.These two genes are the key regulation genes involved in lung fibrosis.We identified that MALAT1 can promot lung fibrosis via Rb-HDAC1-E2F1 axis using function and rescue experiments.