Neferine Protects Endothelial Glycocalyx Via Mitochondrial ROS In Lipopolysaccharide-induced Acute Respiratory Distress Syndrome

Objective:Acute respiratory distress syndrome(ARDS),a common emergency of respiratory system,is characterized by high mortality.The pathogenesis of ARDS is comlplexity.An increase in pulmonary vascular permeability is the basic pathological feature of ARDS.Damage to the endothelial glycocalyx is a critical factor in increased pulmonary vascular permeability.Neferine(Nef),a bisbenzylisoquinoline alkaloid isolated from green seed embryos of Nelumbo nucifera Gaertn,has extensive pharmacological activity,such as anti-inflamatory and anti-oxidant.Our previous studies have shown that the pretreatment of Nef could decreased the pulmonary vascular permeability and ameliorated inflammation cytokine release in LPS induced ARDS mice.But the therapeutic effects of Nef whether endothelial glycocalyx is exist in the process or not are not yet been studied and valued.Therefore the key of this study is to explore the correlation between Nef and endothelial glycocalyx in C57BL/6 mice and human umbilical vein endothelial cells(HUVECs).Methods:1.In vivo study:Experimental mice were randomly allocated into control,LPS,Nef + LPS,and Nef groups.Mice in the Nef + LPS and Nef groups were gavaged with a single dose of Nef solution(20 mg/kg/day)for seven days,and mice in the control and LPS groups received equal volume of normal saline instead of Nef in the same manner.Subsequently,the Nef + LPS and LPS groups were intraperitoneally injected with LPS(20mg/kg)to induce ARDS.The control and Nef groups received an equal volume of normal saline without LPS.At 6 h after LPS administration,mice lung tissue,bronchoalveolar lavage fluid,and serum samples were collected for further analysis.Staining lung tissue with hematoxylin and eosin(H&E)was used to evaluate pathological changes.Lung W/D weight ratio was used to assess pulmonary edema.Evans blue(EB)dye extravasation technique was used to determine lung-capillary permeability.Enzyme-linked immunosorbent assay(ELISA)was used to determine lung tissue homogenate malondialdehyde(MDA)and superoxide dismutase(SOD)activity and the activity of IL-1β,IL-6,TNF-α,and IL-10 in bronchoalveolar lavage fluid(BALF).Western blot technique was used to detect NF-kB signaling pathways in the key regulatory proteins(NF-κB/P65,Ik B a/p-Ik B a)activation state.Fluorescent probe was used to detect the production of ROS and Mitochondrial ROS(mtROS).The immunofluorescence technique was used to detect the level of HS and SDC-1 in lung tissue and HUVECs.2.In vitro study:HUVECs was used to carry out our experiment.Control cells were not exposed to LPS.LPS group:HUVECs were treated with LPS(1 μg/mL)for 6 h.Nef + LPS group:HUVECs were pretreated with 10 uM Nef for 1 h followed by LPS(1 μg/mL)stimulation for 6 h.Nef group:HUVECs were treated with 10 μM Nef for 7 h.Fluorescent probe was used to detect the production of ROS and Mitochondrial ROS(mtROS)in HUVECs.The level of lung endothelial HS and SDC-1 was measured by immunofluorescence.The activity of MDA and SOD in HUVECs and the level of IL-1β IL-6,TNF-,and IL-10 in BALF were all measured by ELISA.Results:In this study,we showed that Nef reduced the change of histopathological,reduce the level of W/D ratio and lung-capillary permeability,down-regulated the production of cytokines(IL-1β,IL-6,TNF-α and IL-10)and inhibited the activation of the NF-kB signaling pathway in mice with lipopolysaccharide(LPS)-induced ARDS.Further analysis indicated that Nef provided protection against endothelial glycocalyx degradation in LPS-induced ARDS mice(in vivo)and in LPS-stimulated human umbilical vein endothelial cells(in vitro).The glycocalyx-protective effect of Nef may be initiated by suppressing the production of mitochondrial ROS(mtROS)and decreasing oxidative damage.Conclusion:Neferine protects endothelial glycocalyx via mitochondrial ROS in lipopolysaccharide-induced acute respiratory distress syndrome.