Xuebijing Injection Attenuates Endothelial Barrier Dysfunction In H₂S-induced Acute Lung Injury Of Rats By Upregulating Claudin-5 Expression

Objective:To study the mechanism of Xuebijing injection improving the function of pulmonary vascular barrier in acute lung injury of rats from the perspective of claudin-5 protein.Methods:Acute lung injury（ALI）model of animals and cells was induced by hydrogen sulfide（H₂S）/sodium hydrosulfide（Na HS）exposure.（1）In vivo studies:dividing Sprague-Dawley（SD）rats by random number method.（1）In H₂S exposure experiment,rats were divided into 0h,6h,12h,24h groups（n=6）according to the disposal time after 300ppm H₂S exposure for 3h and control group（no H₂S exposure）.The lung tissues were taken to measure wet/dry weight ratio（W/D）and detect expression of claudin-5 mRNA.（2）In Xuebijing intervention experiment（n=4）,there were control group,12h group（exposure to 300ppm H₂S for 3h,then stay 12h）,Xuebijing control group（4 m L/kg,twice a day for 3 days）,Xuebijing intervention group（H₂S exposure after pretreatment of Xuebijing injection）.The total protein content in plasma and bronchoalveolar lavage fluid（BALF）of rats were detected respectively,and the pulmonary permeability index（PPI）was calculated（PPI=protein content in BALF/protein content in plasma）.（2）In vitro studies:（1）In H₂S exposure experiment,human lung microvascular endothelial cells（HPMECs）were divided into1h,3h,6h,12h,24h groups based on co-incubation time with 500μmol/L Na HS and control group（no Na HS treatment）.The claudin-5 protein expression and permeability changes of HPMECs were measured at different co-incubation time,also the control group.（2）In Xuebijing intervention experiment,control group,12h group（co-incubated with 500μmol/L Na HS for 12h）,Xuebijing control group（2g/L Xuebijing injection for 24h）,Xuebijing intervention group（2g/L Xuebijing injection pre-treated for 24h,then co-incubated with 500μmol/L Na HS for 12h）.The HPMECs claudin-5protein expression and permeability were measured in 4 groups.Results:（1）In vivo studies:（1）In H₂S exposure experiment,compared with the control group,the lung W/D ratio of rats increased significantly at 6h and peaked at 12h after H₂S exposure（4.67±0.11vs.4.26±0.06,P<0.01）,and the expression of claudin-5mRNA in lung tissue was significantly decreased,6h group decreased to 89%and 12h group decreased to 85%in control group（P<0.01）.（2）In Xuebijing intervention experiment,the total protein content in BALF and PPI of the 12h group were significantly higher than those in the control group[total protein content（mg/L）:262.31±14.24vs.33.30±3.09,P<0.01;PPI:（11.72±0.57）×10^-3vs.（1.21±0.08）×10^-3,P<0.01];Based on the control group,the increase of those was significantly reduced in the Xuebijing intervention group compared with the 12h group[total protein content（mg/L）:153.25±7.32vs.262.31±14.24,P<0.01;PPI:（5.79±0.23）×10^-3vs.（11.72±0.57）×10^-3,P<0.01].（2）In vitro test:（1）In H₂S exposure experiment,compared with the control group,the permeability of monolayer HPMECs treated with Na HS gradually increased,reaching the highest level in the 12h group,almost twice that of the control group（1.95±0.13,P<0.01）,and claudin-5 protein expression decreased to the lowest level in 12h group（claudin-5/β-actin:0.42±0.03vs.1.03±0.05,P<0.01）.（2）In Xuebijing intervention experiment,based on the control group,the monolayer HPMECs permeability was significantly improved（fluorescence intensity of fluorescein sodium:1.46±0.10vs.1.89±0.11,P<0.01）and the decrease of claudin-5 protein expression was reduced（claudin-5/β-actin:0.68±0.04vs.0.38±0.03,P<0.01）in Xuebijing intervention group compared with that in 12h group.Conclusion:Xuebijing injection may upregulate claudin-5 expression to improve pulmonary vascular barrier function and alleviate ALI.