Liang-Ge-San Ameliorates Lipopolysaccharide-induced Acute Lung Injury Through Up-regulating Exosomal MiR-21/PI3K/AKT Signaling Pathway

BackgroundAcute lung injury(ALI)is a type of acute respiratory system disease that seriously threatens the life of patients.Due to the lack of effective drug,the mortality rate of ALI has been high substantially.Therefore,it is crucial to find potential therapeutic drugs.Liang-Ge-San(LGS),a traditional Chinese medicine formula,can be used for the treatment of ALI in clinic.We have found that LGS could upregulate miR-21 to alleviate lung inflammation,but the underlying molecular mechanisms are still unclear.PurposeThis study aims to investigate the mechanism by which LGS inhibit lipopolysaccharide(LPS)-induced ALI,providing experimental basis for the clinical application of LGS and new perspective of the mechanisms of ALI.Methods and results1.MTT assay was used to determine that LGS had no toxicity on mouse macrophage cell line(RAW264.7)at concentrations of 50,100,and 200 μg/mL.ELISA was used to measure the effects of LGS on the expression of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β)in the LPS-induced RAW264.7 cells.The results showed that LGS inhibited the expression of inflammatory factors in a dose-dependent manner.qPCR results showed that LGS pretreatment upregulated the expression of miR-21 in RAW264.7 and its secreted exosomes(LGS-exosomes).Subsequently,a co-culture system was established between RAW264.7 and mouse type Ⅱ alveolar epithelial cell line(MLE-12).Cy3-labeled miR-21 mimic was used and flow cytometry results showed that MLE-12 could phagocytose the LGS-exosomes containing miR-21,which could be inhibited by the exosome secretion inhibitor GW4869.Western blotting was used to detect protein expression in MLE-12 cells in the co-culture system.The results showed that LGS and miR-21 mimic significantly upregulated the PI3K,p-PI3K(Tyr607),and p-AKT(Ser473)protein expression after LPS stimulation.Transfection of miR-21 inhibitor or treatment of GW4869 could inhibit the effects of LGS.Furthermore,LGS-exosomes were labeled with PKH67 and co-cultured with MLE-12 cells.Confocal microscopy showed that MLE-12 could phagocytose the LGS-exosomes.Western blotting was used to detect the protein expression in MLE-12 cells after co-culture with LGS-exosomes,and the results showed that LGS-exosomes upregulated the protein expression of PI3K,p-PI3K(Tyr607),and p-AKT(Ser473).2.LGS-exosomes inhibits LPS-induced ALI in vivo.40 mice were randomly divided into the CTL group,LPS group,CTL-exosomes group,LGS-exosomes group,and LGS-exosomes with miR-21 antagomir group.Mice were injected with miR-21 antagomir intravenously on days 1,2,and 4.On day 8,ALI model was induced by intratracheal instillation of LPS(5 mg/kg)and exosomes were administered simultaneously.Mice were sacrificed after 9 h,and bronchoalveolar lavage fluid(BALF)and lung tissue were collected.H&E staining showed that LGS-exosomes effectively alleviated pathological changes in lung,which may be related to the upregulation of miR-21.Meanwhile,LGS-exosomes also reduced lung edema,decreased the expression of IL-6,TNF-α,and total proteins in BALF.Western blotting results shown the LGS-exosomes upregulated the protein expression of PI3K,p-PI3K(Tyr607),and p-AKT(Ser473).However,administration of miR-21 antagomir could reverse the effect of LGS-exosomes.ConclusionThis study confirms that LGS could inhibit LPS-induced ALI.LGS could mediate the communication between macrophages and alveolar epithelial cells by upregulating the expression of miR-21 in macrophages exosomes and upregulate the activation of PI3K and p-AKT(Ser473)in alveolar epithelial cells,thereby inhibi18ting LPS-induced ALI.