Abstract:

Objective To discuss the effect of exosomes derived from bone marrow mesenchymal stem cells（BMSCs） （BMSC-exos） on asthma in the mice， and to clarify the mechanism. Methods The BMSCs were extracted， and the expressions of its markers were detected. The BMSCs were transfected with microRNA （miR）-26a mimic （mimic-miR-26a）， miR-NC and miR-26a inhibitor （inh-miR-26a）， respectively， and the corresponding exosomes （mimic-miR-26a exos， miR-NC exos and inh-miR-26a exos） were extracted. Mouse asthma model was established by administering ovalbumin （OVA） and aluminum hydroxide to the mice. The 50 successfully modeled mice were randomly divided into model group， BMSC-exos group， miR-NC exos group， mimic-miR-26a exos group and inh-miR-26a exos group， with 10 mice in each group， and 10 non-modeled mice were regarded as control group. Each group was administered daily with 10 μg of BMSC-exos， miR-NC exos， mimic-miR-26a exos， inh-miR-26a exos and an equal volume of phosphate buffered saline （PBS）， respectively. HE staining was used to observe the pathomorphological manifestations of lung tissue of the mice in various groups； small animal lung function test instrument was used to detect the airway resistance of mice in various groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of miR-26a in the lung tissue of mice in various groups； dual luciferase reporter gene assay was used to verify the targeting relationship between miR-26a and Toll-like receptor 4 （TLR4）； Western blotting method was used to detect the expression level of TLR4 protein in the lung tissue of mice in various groups. Results The extracted BMSCs highly expressed their markers cluster of differentiation（CD）44 and CD105， but did not express CD45 and CD34， indicating that BMSCs were successfully extracted. The particle size of BMSC-exos was about 100 nm， with a bilayer membrane and a “cup-shaped” morphology， which conformed to the structural characteristics of exosomes. BMSC-exos expressed tumor susceptibility gene 101 （TSG101）， CD9 and CD81， which conformed to the biological characteristics of exosomes. HE staining results showed that the lung tissue structure of mice in control group was normal， the alveolar septum was not significantly widened， and no obvious inflammatory cell infiltration was observed； the lung tissue structure of mice in model group was disordered， the alveolar structure was obviously damaged， the alveolar septum was significantly widened， there was a large amount of inflammatory cell infiltration， goblet cells were hyperplastic， and lung injury was obvious， indicating successful modeling； in BMSC-exos group， the lung tissue structure disorder of mice was significantly alleviated， the inflammatory cell infiltration was reduced， and the alveolar structure damage was reduced； after treatment with miR-26a BMSC-exos， the lung tissue structure of mice in control group was normal； in model group， a large amount of inflammatory cell infiltration was observed in the lung tissue， the alveolar structure was obviously damaged， goblet cells were obviously hyperplastic， the alveolar septum was significantly widened， and lung injury was obvious； compared with model group， the degree of lung tissue lesions in miR-NC exos group was alleviated， and the degree of lung tissue lesions in mimic-miR-26a exos group was further alleviated. The small animal lung function test results showed that under the action of 6.25， 12.50， 25.00 and 50.00 g·L^-1 methacholine （Mch）， compared with control group， the airway resistance of mice in model group was significantly increased （P<0.05）； compared with model group， the airway resistance of mice in BMSC-exos group was significantly decreased （P<0.05）. Under the action of 6.25， 12.50， 25.00 and 50.00 g·L^-1 Mch， compared with model group， the airway resistance of mice in miR-NC exos group was significantly decreased （P<0.05）； compared with miR-NC exos group， the airway resistance of mice in mimic-miR-26a exos group was significantly decreased （P<0.05）， and the airway resistance of mice in inh-miR-26a exos group was significantly increased （P<0.05）. The real-time fluorescence quantitative PCR （RT-qPCR） method results showed that compared with miR-NC exos， the expression level of miR-26a in mimic-miR-26a exos was significantly increased （P<0.05）， and the expression level of miR-26a in inh-miR-26a exos was significantly decreased （P<0.05）； compared with control group， the expression level of miR-26a in the lung tissue of mice in model group was significantly decreased （P<0.05）； compared with model group， the expression level of miR-26a in the lung tissue of mice in BMSC-exos group was significantly increased （P<0.05）； compared with miR-NC exos group， the expression level of miR-26a in the lung tissue of mice in mimic-miR-26a exos group was significantly increased （P<0.05）， and the expression level of miR-26a in the lung tissue of mice in inh-miR-26a exos group was significantly decreased （P<0.05）. The dual luciferase reporter gene assay results showed that compared with the cells transfected with TLR4 wild type （WT） and miR-NC group， the relative luciferase activity of cells in group transfected with TLR4 WT and miR-26a was significantly decreased （P<0.05）， indicating that TLR4 was the target gene of miR-26a. The Western blotting method results showed that compared with control group， the expression level of TLR4 protein in the lung tissue of mice in model group was significantly increased （P<0.05）； compared with model group， the expression level of TLR4 protein in the lung tissue of mice in BMSC-exos group was significantly decreased （P<0.05）； compared with miR-NC exos group， the expression level of TLR4 protein in the lung tissue of mice in mimic-miR-26a exos group was significantly decreased （P<0.05）， and the expression level of TLR4 protein in the lung tissue of mice in inh-miR-26a exos group was significantly increased （P<0.05）. Conclusion BMSC-exos can reduce airway resistance and alleviate the degree of lung tissue lesions in the asthmatic model mice， its mechanism may be related to BMSC-exos promoting the delivery of miR-26a to the lung tissue and inhibiting the expression of TLR4.

Key words: Bone marrow mesenchymal stem cells, Exosomes, Asthma, Airway resistance, MicroRNA-26a, Toll-like receptor 4