To Explore The Elemene's Effects And Mechanisms On Airway Granulations And Fibroblasts

Purpose: To explore the effects of elemenes on human airway granulations and fibroblasts, and determine their underlying mechanism of action.Content:With higher incidence trends such as tuberculosis, endotracheal intubation,the incidence of airway stenosis alsol increased, some patients with interventional therapy guided by using bronchoscope, illness repeatedly, curative effect was poor. To explore new treatment approach had important clinical significance. How to adjust the balance of the body wounds and repair, extracts of Chinese herbal medicine because of its good curative effect and little poisonous to the cells was becoming more and more attention. Elemenes, a compound derived from Zingiberaceae Curcuma wenyujin rhizome, used in traditional Chinese medicine, had shown anti-cancer effects against a broad spectrum of tumors. In addition, there was also a research shows that ?-elemene could inhibit proliferation of human fibroblast and lens epithelial cells and even resisted effect of liver fibrosis. So to establish human airway primary fibroblasts model in vitro, elemenes intervention, local drug injection by bronchoscopy in airway guanulations of a patient with tracheobronchial stenosis. Observed the pharmacological effects, and from the molecules levels(caspase-3 protein ? caspase-3 m RNA ?Differentially expressed mi RNAs) to explore the potential mechanism. Once proven,it will be a new treatment of refractory airway stenosis, reduce the recurrence rate.Methods: 1. Human primary airway fibroblasts were cultured in vitro and purified. 2.The total purified cells were divided into two groups consisting of a negative control group(NS group) and an experimental group(elemenes emulsion group), and observed by light and electron microscopy. 3. The proliferation rate in each group of cells was measured with the MTS assay. The apoptosis rate in each group was determined by flow cytometry(FACS) analysis. 4. The cells were divided into three groups consisting of a negative control group(NS group), positive control group(DXM group), and an experimental group(elemenes emulsion group), and the caspase-3 protein content in each group was measured by western-blot. The levels of caspase-3 m RNA in the three groups of cells were measured by RT-PCR. The levels of mi-RNA expressed by airway fibroblasts in the negative control group(NS group) and experimental group(elemenes emulsion group) were determined in triplicate using a next generation high throughput sequencer, and the corresponding target genes were predicted. The differentially expressed mi-RNAs were identified by RT-PCR. 5. Patients with airway stenosis were treated with a bronchoscopy-guided local injection of elemenes, and observed for any curative effects. Before and after treatment, samples of granulation tissue were removed with a bronchoscope forceps, stained using HE and immunohistochemical methods, and observed under a light microscope.Results: 1. Human primary airway fibroblasts were successfully cultured and purified in vitro. 2. When examined under a light microscope, cells in the elemenes group had a conglobated appearance, and contained increased numbers of cytoplasmic granules when compared with cells in the control group. When examined by electron microscopy, cells in the elemenes group displayed deposits of cytoplasmic granules,signs of cellular necrosis, but no nuclei or organelles. The proliferation rate of cells in the NS group did not change as their concentration increased; however, cells in the elemenes group showed decreased rates of proliferation as their concentration increased(P<0.05). 3. When compared with cells in the NS group, cells in the elemenes group showed increased rates of apoptosis and necrosis as their concentrations gradually increased. Increased levels of caspase-3 expression were observed in the following order: NS group > DXM group > the elemenes emulsion group(P<0.05).Increased levels of caspase-3 m RNA expression were also observed in the following order: NS group > DXM group > the elemenes emulsion group. 4. High throughput sequencing methods identified decreased levels of hsa-mi R-146b-5p, hsa-mi R-34a-5p,hsa-mi R-574-5p, and hsa-mi R-185-5p in the elemenes emulsion group when compared with those levels in the NS group. After verification of their identities by Q-PCR,hsa-mi R-146b-5p were isolated(P<0.05) and their corresponding target genes were predicted. Which related target genes including ZNF253, FAM120 AOS, RHOBTB3,HSFY1, HSFY2, TRAF6, DIS3 L, SYT14, CLLU1, DCAF17, COX6B2, 5.Bronchoscopic examinations showed that the airway granulation tissue of all patients was partially absorbed following an elemenes injection. After treatment, granulation tissue edema decreased, and the fibroblasts transformed into mature fiber cells.Conclusion: Elemenes may induce apoptosis and necrosis in human primary airway fibroblasts and inhibit their proliferation. the potential mechanisms may be related to the increase of the expression of caspase-3 apoptosis protein and caspase-3 m RNA.2.Elemenes inhibited the proliferation of human airway primary fibroblast might be related to downregulation of hsa mi R- 146-b-5 p, which related target genes including ZNF253, FAM120 AOS, RHOBTB3, HSFY1, HSFY2, TRAF6, DIS3 L, SYT14,CLLU1, DCAF17, COX6B2, specific mechanism needed further research. 3. There were no obvious adverse reaction were found in a intractable airway stenosis patient who were treated by bronchoscopy guided local injection of elemene, it promoted the absorption of airway granulation, providing a new way in the treatment of refractory airway stenosis.