Berberine Alleviates Endothelial Glycocalyx Degradation And Promotes Glycocalyx Restoration In LPS-induced ARDS

Objective:Acute respiratory distress syndrome(ARDS)is a clinically common disease that life-threatening respiratory disease until now.In the pathogenesis of acute respiratory distress syndrome(ARDS),an increase in vascular endothelial permeability may trigger pulmonary edema and ultimately lead to respiratory failure.Endothelial glycocalyx damage is an important factor that causes an increase in vascular endothelial permeability.Although many new treatment strategies have been developed over the years,but the morbidity and mortality of patients with ARDS is still high that threatens human health.Therefore,it is very necessary and urgent to continue to develop effective therapeutic drugs.Berberine(BBR)is an isoquinoline alkaloid extracted from Coptis chinensis,which has been reported to have multiple pharmacological activities,such as antibacterial,anti-inflammatory and antioxidant.In addition,berberine has a better effect on hyperlipidemia and hyperglycemia This experiment in C57BL/6 mice and HUVECs(human umbilical vein endothelial cells)as the basic research,from the antioxidant,inhibition of HP A?MMP9 enzyme degradation and anti-inflammatory to explore the effect of BBR on the endothelial glycocalyx in LPS-induced ARDS and its possible related mechanisms,which provides a new theoretical basis for clinical prevention and treatment of acute respiratory distress syndrome.Methods:1.BBR pretreatment:? In vivo study:We selected male C57BL/6 mice aged about 8-10 weeks.Mice were randomly separated into five groups:the control group,LPS group,and BBR+LPS(50,100,and 200 mg/kg)groups.The BBR+LPS groups received BBR administered orally for 7 days,and the control and LPS groups were given an equal volume of sterile saline instead.Then,mice in the LPS group and BBR+LPS(50,100,and 200 mg/kg)groups were intraperitoneally injected with LPS(20 mg/kg)to induce ARDS.Mice in the control group received an equal volume of normal saline.After infusion of LPS for 6 h,mice in each groups were killed,the lung tissues,blood and bronchoalveolar lavage fluid were collected for later use.HE staining technique and lung injury scores were used to observe the pathological morphological changes,evaluate the severity of lung injury.Evans blue dye(EBD)was used to detect pulmonary vascular permeability.The activity of inflammatory factors(TNF-??IL-1??IL-6)in bronchoalveolar lavage fluid and ROS,MDA contents in lung tissues were determined by ELISA.Western blot technique was used to determine the level and activation of key regulatory proteins at the NF-?B signaling pathway(NF-?B/P65,p-I?B?/I?B?).IF was used to detect the expression of HS,SDC-1,HPA,and MMP9 in lung tissue.? In vitro study:the HUVECs were separated into five groups:the control group,the LPS group,and BBR+LPS(1.25,2.5,and 5 ?M)groups.In the LPS group,HUVECs were treated with LPS(1?g/mL)for 6 h.In the LPS+ BBR groups,HUVECs were pretreated with BBR(1.25,2.5,and 5 ?M for 1 h prior to LPS(1 ?g/mL)stimulation for 6 h.After 6 h,the content of MDA was detected by ELISA.The production of ROS in cells was detected by DCFH-DA and the expression of mtROS was detected by MitsoxTM.The level of HS,SDC-1,HPA,and MMP9 in cells were detected by immunofluorescence.2.BBR post-treatment:? In vivo study:Mice were randomly separated into six groups:the control group,LPS group,self-repairing group,and BBR-repairing(50,100,and 200 mg/kg)groups.Mice in the LPS group,self-repairing group,and BBR-repairing(50,100,and 200 mg/kg)groups were intraperitoneally injected with LPS(20 mg/kg)to induce ARDS.The control was given an equal volume of sterile saline instead.After 6 h,the mice in the LPS group were killed,and those in the BBR-repairing groups received BBR(50,100,and 200 mg/kg)administered orally for 3 days.The mice in the control and self-repairing groups received an equal volume of sterile saline.Three days later,the mice in each group were killed and lung tissue was collected.Immunofluorescence was used to detect the expression of endothelial glycocalyx in lung tissue.? In vitro study:HUVECs were separated into six groups:the control group,LPS group,self-repairing group,and BBR-repairing(1.25,2.5,and 5 ?M groups.HUVECs in the LPS group,self-repairing group,and BBR-repairing(1.25,2.5,and 5 ?M groups were stimulated with LPS(1 ?g/mL)for 6 h.Afte 6 h,the HUVECs in the self-repairing group were replaced with complete medium,whereas the HUVECs in the BBR-repairing groups were replaced with complete medium containing BBR(1.25,2.5,and 5 ?M)for an additional 12 h incubation.The levels of HS,SDC-1 in cells were detected by immunofluorescence.Results:HE and Evans blue results showed that berberine(BBR)can significantly improve lung injury and significantly reduce lung injury score,attenuated the increase in pulmonary vascular permeability.In addition,the results of in vitro and in vivo experiments further showed that berberine significantly reduced the production of ROS in LPS-induced ARDS mice,reduced the expression of intracellular ROS and mtROS in LPS-stimulated ARDS,reduced oxidative stress and the release of inflammatory factors(TNF-?,IL-1?,IL-6)and inhibited the activation of NF-?B signaling pathway.Immunofluorescence results showed that BBR reduced the expression of HPA and MMP9 in LPS-induced ARDS,thereby alleviates endothelial glycocalyx degradation and protecting the integrity of endothelial glycocalyx.The results of BBR post-treatment showed that berberine could promotes glycocalyx restoration in LPS-induced ARDS.Conclusion:This study carried out a series of studies on berberine from the aspects of anti-inflammatory,antioxidant,inhibition of HPA,MMP9 expression.The results showed that BBR pretreatment alleviated the degradation of endothelial glycocalyx,thereby reducing the increase of vascular permeability and protecting the integrity of endothelial glycocalyx.In addition,BBR post-treatment can also promote the repair of endothelial glycocalyx.In general,BBR has a good therapeutic effect on LPS-induced ARDS injury.