Preliminary Profiling Of Transcriptome Of Human Umbilical Vein Endothelial Cell Treated By Posthemorrhagic Shock Mesenteric Lymph

Hemorrhagic shock is common in clinic.The cell injury caused by hemorrhagic shock is an important cause of acute lung injury,intestinal barrier dysfunction and multiple organ dysfunction syndrome.It is also a basic mechanism for developing refractory shock.The mechanism of distal organ damage and dysfunction caused by hemorrhagic shock is concerned.It is now believed that enteric toxic substances return through the intestinal lymphatic pathway to the whole body,which is a major factor of aseptic multiple organ damage caused by hemorrhagic shock.After hemorrhagic shock,the intestinal lymph flow into the subclavian vein and into the blood circulation system through the thoracic duct directly.In the process,the blood vessel is the first part exposed to post hemorrhagic shock mesenteric lymph(PHSML).Cell damage and dysfunction caused by PHSML are the main causes of increased vascular permeability,decreased vascular reactivity and organ damage caused by hemorrhagic shock.Many studies have shown that the treatment measures to reduce the return of PHSML as a target are of positive significance in the prevention and treatment of severe shock.Vascular endothelial cell injury is the basis of high permeability of blood vessels.PHSML reflux is involved in the pathological process of increased vascular permeability induced by hemorrhagic shock,but the detailed molecular mechanism is not very clear.Therefore,this study uses the transcriptome sequencing technique to observe the effect of PHSML on human umbilical vein endothelial cells(HUVECs)gene transcriptome.By clustering analysis(GO)and pathway enrichment analysis(KEGG)of differentially expressed genes,the main signal molecules and signaling pathways of PHSML damage HUVECs were identified.We hope to deepen the understanding of the molecular mechanism of PHSML damaged endothelial cells,so as to provide a new way of thinking on the prevention and treatment for PHSML of severe hemorrhagic shock.A model used healthy male rats of hemorrhagic shock was established by routine methods: the femoral artery bleed,and the blood pressure was maintained at 40 mm Hg for 90 min,and the fluid resuscitation.After the end of the fluid resuscitation,the abdominal operation was performed and the intestinal lymph was drained by routine method,which was PHSML.Another group of healthy male rats,after general anesthesia,was used routinely to drain intestinal lymph,which was normal intestinal lymph(normal mesenteric lymph,NML).Subsequently,HUVECs were incubated with the physiological saline(Control),NML and PHSML(final concentration 10%).After 3 h,HUVECs was collected,m RNA was extracted,and a new generation sequencing technology was used to sequence and analyze the three groups of cells.The sequencing results showed that there was almost no difference in the HUVECs transcriptome between the Control group and the NML group.Compared with NML,there are multiple differentially expressed genes of HUVECs after the treatment by PHSML.GO and KEGG were used to further analyze the differentially expressed genes between PHSML and NML.It is found that these differentially expressed genes are mainly related to biological processes such as apoptosis and stress.They are mainly enriched in the nucleotide oligomerization domain-like receptor(NLR)signaling pathway,nuclear factor ?B(NF-?B)signaling pathway and tumor necrosis factor(NOD)signaling pathway.These three signaling pathways and related differentially expressed genes are related to the inflammatory response.In order to verify the differentially expressed genes found in the sequencing results of the transcriptome,we detected the m RNA level of 7 differentially expressed genes(BIRC3,NFKBIA,CXCL1,ICAM-1,CCL2,TNFAIP3,JUNB)by real-time quantitative PCR(RT-PCR).The results showed that the RT-PCR results of these differentially expressed genes were consistent with the results of RNA-Seq.In view of the role of chemokine CCL2 in cell injury,we observed the effect of CCL2 inhibitor on PHSML damage HUVECs.The results showed that CCL2 inhibitor could significantly improve HUVECs cell proliferation after PHSML treatment.Based on the above studies,it is preliminarily revealed that the mechanism of PHSML damaging endothelial cells is related to inflammatory reaction mediated by NLR signaling pathway,NF-?B signaling pathway and TNF signaling pathway.Inhibition of CCL2 is beneficial to the reduction of endothelial cell damage caused by PHSML.The results of the study deepen the understanding of the molecular mechanism of vascular endothelial cells damaged by shock intestinal lymph,and provide a new perspective for the prevention and treatment of severe hemorrhagic shock.