| Sepsis is one of the most common acute and critical illnesses in the clinic.Epidemiological results showed that the number of sepsis patients was 48.9 million in the globe in 2020 with a mortality of 25-30%,which suggested that sepsis was still a huge burden for the health of the human being.The intestine mechanical barrier is composed of a single layer of epithelial cells,which plays an essential role in absorbing nutrients and preventing bacteria and toxins from the intestinal lumen.The intestinal barrier function is disordered during sepsis,which allows the translocation of bacteria and toxins to circulation and leads to severe tissue damage,while effective treatment is absent.The lung tissue is damaged in the early stage of sepsis,which leads to organ hypoxia and dysfunction,and vascular leakage is an important pathophysiological process during lung injury,which results in tissue edema and organ dysfunction,while the treatment is very limited.Microvesicles(MV)are natural membranous vesicles carrying a large number of biologically active molecules such as proteins,lipids,and nucleic acids,and participate in the regulation of various pathophysiological processes.Mesenchymal stem cell-derived microvesicles(MMV)are proved to carry a large number of stem cell-related therapeutic substances,such as keratinocyte growth factor,m RNA,mi R-292,and mi R295,so MMVs are considered a promising treatment strategy for diseases as acute lung injury,glomerulonephritis,leukemia.Whether MMVs could improve the intestinal barrier and pulmonary vascular barrier function after sepsis is still unclear.Pulmonary vascular endothelial cells also could secrete a large amount of MVs(endothelial microvesicles,EMV)during sepsis due to the stimulation of various inflammatory factors.Our previous study indicated that EMVs led to pulmonary vascular leakage,which was related to the mi R-23 b in EMVs,while the mechanism was unclear.Besides,the production of MVs is regulated by many factors,whether the inhibition of the secretion of MVs could attenuate the EMV-induced injury is not clear.In the present study,we used cecal ligation and puncture(CLP)-induced sepsis rats and lipopolysaccharide(LPS)-stimulated intestinal epithelial cell(IEC-6)and vascular endothelial cells(VEC).Firstly,we studied the role and mechanism of MMVs in the protection of intestinal barrier function during sepsis,and the role of MMVs-carried mfn2,PGC-1α,and functional mitochondria in improving mitochondrial dynamic balance and cell function.Secondly,we studied the role of MMVs in the protection of pulmonary vascular leakage during sepsis,and the mechanism of MMVs-carried PKA and Wnt5 a in improving tight junctions and adhesion junctions.Finally,we studied the inhibitory effect of amitriptyline on EMV production during sepsis and its protective effect on pulmonary vascular leakage.Research contents:Part 1: The role and mechanism of MMVs in the protection of intestinal barrier function by restoring mitochondrial dynamic balance1.The protective effect of MMVs on intestinal barrier function(1)The changes of intestinal barrier function after sepsisThe rat sepsis model and the IEC-6 injury model were used to study the changes of intestinal barrier function of rats at different times after sepsis,and the changes of proliferation and barrier functions of IEC-6,so as to clarify the characteristic changes of intestinal barrier function after sepsis.(2)The protective effect of MMVs on the intestinal barrier function after sepsisMMVs were infused into sepsis rats and incubated with damaged IEC-6 to study the effect of MMVs on the intestinal barrier function of rats,and the effect on IEC-6 proliferation and barrier functions,so as to clarify the effect of MMVs on intestinal barrier function.2.MMVs restored the proliferation and barrier functions of IEC-6 by improving mitochondrial function(1)The protective effect of MMVs on mitochondria in IEC-6MMVs were incubated with damaged IEC-6 to study the effects of MMVs on mitochondrial morphology and quantity,mitochondrial oxidative phosphorylation,so as to clarify the effect of MMVs on the mitochondrial function.(2)The influence of inhibition of the mitochondrial oxidative phosphorylation on the effect of MMVsThe oxidative phosphorylation inhibitor 2,4-dinitrophenol was used to incubate with IEC-6 along with MMVs to study the influence of inhibiting mitochondrial oxidative phosphorylation on the effect of MMVs,so as to clarify the role of mitochondrial oxidative phosphorylation in the protective effect of MMVs.3.The mechanism of MMVs in improving the mitochondrial dynamic balance(1)MMVs delivered mfn2 to promote mitochondrial fusion in IEC-6Mfn2 overexpressing and sh RNA adenoviruses were used to transfect MSCs,and modified MMVs were harvested to incubate with damaged IEC-6,to study the role of MMVs-carried mfn2 in the protective effect of mitochondrial fusion and function.(2)MMVs delivered PGC-1α to promote mitochondrial biogenesis in IEC-6PGC-1α overexpressing and sh RNA adenoviruses were used to transfect MSCs,and modified MMVs were harvested to incubate with damaged IEC-6,to study the role of MMVs-carried PGC-1α in the protective effect of mitochondrial biogenesis and function.(3)MMVs delivered functional mitochondria to improve the mitochondrial function in IEC-6Transmission electron microscopy and flow cytometry were used to observe the quantity and function of mitochondria in MMVs;the mitochondrial red fluorescent plasmid was used to study the fusion of the mitochondria delivered by MMVs and the mitochondria in IEC-6,so as to clarify the effect of MMV-delivered mitochondria on the function of mitochondria in IEC-6.Part 2: The role and mechanism of MMVs in the protection of pulmonary vascular barrier function during sepsis1.The protective effect of MMVs on pulmonary vascular barrier functionMMVs were infused into sepsis rats and incubated with damaged VECs,to study the protective effect of MMVs on pulmonary vascular leakage and lung injury.2.Proteomics analysis of MMVsThe protein mass spectrometer was used to investigate the protein profile in MMVs,and KEGG and GO analyses were used to study the vascular leakage-related proteins.3.The mechanism of MMVs in protecting pulmonary vascular barrier function(1)MMVs delivered PKA to protect pulmonary vascular barrier functionPKA activator,inhibitor,and PKA-si RNA were used to study the effect of the MMV-carried PKA on VASP and ZO-1 in VEC,so as to clarify the mechanism of MMV-carried PKA in protecting tight junctions.(2)MMVs delivered Wnt5 a to protect pulmonary vascular barrier functionWnt5a activators,inhibitors,and Wnt5a-si RNA were used to study the effect of the MMV-carried Wnt5 a on cdc42 and VE-cadherin in VEC,so as to clarify the mechanism of MMV-carried Wnt5 a in protecting adherent junctions.Part 3: The role of EMVs in pulmonary vascular barrier function after sepsis1.The role of EMV-carried mi R-23 b in pulmonary vascular leakageMi R-23 b overexpressing and interfering plasmids were used to study the effect of EMVs on pulmonary vascular leakage and lung injury,so as to clarify the mechanism of EMVs in regulating pulmonary vascular leakage.2.The effect of inhibition of EMV production on pulmonary vascular barrier functionAmitriptyline was used to treat VECs to study the change in EMV production;the amitriptyline-treated EMVs were infused into rats and VECs to clarify the effect of the inhibition of EMV production on pulmonary vascular barrier function.Research results:Part 1: The role and mechanism of MMVs in the protection of intestinal barrier function by restoring mitochondrial dynamic balance1.The protective effect of MMVs on intestinal barrier functionThe intestinal barrier function of rats was severely damaged after sepsis in a time-dependent manner.The infusion of MMVs significantly restored the intestinal barrier function and restored the proliferation and barrier functions of IEC-6.2.MMVs restored the proliferation and barrier functions of IEC-6 by improving mitochondrial functionMMVs improved the mitochondrial dynamic balance in IEC-6,thereby improving mitochondrial membrane potential and mitochondrial oxidative phosphorylation.The inhibition of mitochondrial oxidative phosphorylation inhibited the protective effect of MMVs on IEC-6,suggesting that mitochondrial dynamic balance and oxidative phosphorylation played an important role in the protection of IEC-6.Further study found that MMVs restored the concentration of aspartate by promoting the expression of GOT1,suggesting that aspartate participated in the protection of IEC-6.3.The mechanism of MMVs in improving the mitochondrial dynamic balance(1)MMVs carried the mitochondrial fusion protein mfn2 and delivered it to IEC-6,and improved mitochondrial dynamic balance by promoting mitochondrial fusion,and restoring mitochondrial membrane potential and oxidative phosphorylation,thereby improving the proliferation and barrier functions of IEC-6.(2)MMVs carried the mitochondrial biogenesis protein PGC-1α and delivered it to IEC-6,and improved mitochondrial dynamic balance by promoting mitochondrial biogenesis,and restoring mitochondrial membrane potential and oxidative phosphorylation,thereby improving the proliferation and barrier functions of IEC-6.(3)MMVs carried mitochondria with respiratory function and delivered them to IEC-6,which were able to fuse with the mitochondria in IEC-6,and directly improve the function of mitochondria in IEC-6.PGC-1α in MMVs could increase the quantity of mitochondria in MMVs and promoted the improvement of mitochondrial function in IEC-6.Part 2: The role and mechanism of MMVs and EMVs in the protection of pulmonary vascular leakage during sepsis1.The protective effect of MMVs on pulmonary vascular leakageMMVs improved pulmonary vascular leakage and lung injury in rats,and restored the barrier function of VECs;MMVs also improved the arterial blood gas levels,and increased the survival rate,and prolonged the survival time.2.Proteomics analysis of MMVsKEGG and GO analysis were used to investigate the protein profile of MMVs,and 34 kinds of vascular leakage-related proteins are highly expressed.Among them,PKA and Wnt5 a are closely related to vascular leakage.Further study confirmed that MMVs carried PKA and Wnt5 a,which might be related to the protection of pulmonary vascular leakage.3.The mechanism of MMVs in protecting pulmonary vascular leakage(1)MMVs delivered PKA to VECs,and recruited ZO-1 to the cell membrane by promoting the phosphorylation of VASP,thereby restoring the function of tight junction and improving pulmonary vascular leakage.(2)MMVs delivered Wnt5 a to VECs,and restored the expression of VE-cadherin on the cell membrane by promoting the expression of cdc42,thereby restoring the function of adherent junction and improving pulmonary vascular leakage.Part 3: The role of EMVs in pulmonary vascular barrier function after sepsis1.The role of EMV-carried mi R-23 b in pulmonary vascular leakageEMVs delivered mi R-23 b to VECs and inhibited the expression of ZO-1 by binding to the 3’UTR end of ZO-1 m RNA,thereby destroying the structures of tight junction and aggravating pulmonary vascular leakage.2.The effect of inhibition of EMV production on pulmonary vascular barrier functionAmitriptyline significantly inhibited the production of EMVs,thereby reducing EMV-induced pulmonary vascular leakage and lung injury.Conclusions:1.The intestinal barrier function is severely damaged after sepsis.MMVs can deliver mfn2,PGC-1α,and functional mitochondria to intestinal epithelial cells,and restore the mitochondrial dynamic balance and improve mitochondrial oxidative phosphorylation and metabolism,thereby improving the intestinal barrier function.2.MMVs can deliver PKA and Wnt5 a to vascular endothelial cells.On the one hand,MMV-delivered PKA promotes the phosphorylation of VASP and promotes the recruitment of ZO-1 to improve tight junctions and reduce pulmonary vascular leakage.On the other hand,MMV-delivered Wnt5 a promotes the expression of cdc42 and restores the expression of VE-cadherin to improve adherent junctions and reduce pulmonary vascular leakage.3.EMVs can deliver mi R-23 b to vascular endothelial cells,which leads to pulmonary vascular leakage and lung damage by inhibiting the expression of ZO-1.Amitriptyline can reduce the production of EMV,thereby reducing EMV-induced pulmonary vascular leakage and lung injury. |
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