Study On P-selectin Release From Platelets Activated By Vibrio Vulnificus And Its Related Pathogenesis

Vibrio vulnificus（V.vulnificus）is a kind of moderately halophilic gram-negative bacteria commonly found in the sea,which is distributed in sea water,sea mud,oysters,shrimp,fish and other seafood in estuaries and coastal waters.As an opportunistic pathogen,it can infect fish or humans,mainly through ingestion of undercooked contaminated seafood or wound contact.About 50%of patients with foodborne infections eventually develop sepsis,with a mortality rate of>50%.V.vulnificus is an opportunistic pathogen with high mortality.Almost all cases occur in people with basic diseases（such as liver diseases,diabetes and malignant tumors）.Previous studies have shown that individuals with chronic liver diseases（such as cirrhosis）are 80 times more likely to suffer from primary sepsis after infection with V.vulnificus than healthy individuals.At present,the research on the pathogenesis of V.vulnificus is mostly limited to the identification of virulence factors,and there is a lack of research on animal models of V.vulnificus susceptible populations（such as liver disease background）.The known virulence factors and pathogenesis are still difficult to explain the mechanism of sepsis caused by V.vulnificus acute infection.It is believed that the results of this study will provide key clues for the treatment of V.vulnificus sepsis and the research and development of drugs and have important scientific significance.The main research content and results of this paper are as follows:（1）First of all,a highly efficient scheme for the preparation of natural VVH protein of V.vulnificus culture was established based on the"ammonium sulfate precipitation desalination column anion chromatography hydrophobic interaction",which provides a reference for the preparation of hemolysin of other Vibrios,such as Vibrio cholerae and Vibrio mimicus.Evaluating the hemolytic activity of VVH and the effects of cholesterol and estradiol on hemolytic activity,it was found that cholesterol and estradiol at physiological concentrations did not inhibit the hemolytic activity of VVH.This also shows that the specific role of VVH in the pathogenic process of V.vulnificus in the body deserves further exploration.（2）Explore the calcium dependent mechanism of VVH activating platelet to release P-selectin.Blocking evaluation using calcium channel inhibitors found that both extracellular and intracellular calcium channel inhibitors of platelets could not inhibit the release of P-selectin from platelets activated by VVH,while the intracellular calmodulin（Ca M）inhibitor trifluoperazine could completely inhibit the activation of platelets by VVH.This indicates that the mechanism of VVH inducing platelet activation may be mainly related to the oligomerization of VVH on the surface of some platelets to form"pores",which activate some platelets through the extracellular calcium influx Ca M-MLCK pathway,and then activate the release of other platelets through a cascade amplification reactionɑ-particulate matter,ultimately leading to the increase of platelet surface P-selectin.（3）The role of V.vulnificus in activating platelets to release P-selectin in the pathogenesis was evaluated by ALD mouse model and P-selectin-deficient mice.It is found that P-selectin may be related to the intensification of host inflammatory reaction in the early stage of V.vulnificus infection in alcoholic liver disease mice.Compared with WT mice,Selp^-/-mice have increased plasma inflammatory reaction,but weakened liver lesions,indicating that P-selectin-deficient may play an anti-inflammatory role in the model of V.vulnificus infection in alcoholic liver disease mice,However,VVH may play an early proinflammatory role in the inflammatory response caused by V.vulnificus infection in ALD patients.Therefore,the knockout of P-selectin and vvh A in Selp^-/-mice can largely alleviate the early sepsis response caused by Vibrio vulnificus infection.In summary,the natural VVH of V.vulnificus culture with high activity was successfully prepared and found that the purified VVH mainly activates the platelet release of P-selectin through the external calcium influx Ca M-MLCK pathway.In vivo infection model found that the activation of platelet release of P-selectin by V.vulnificus through VVH may be related to the intensification of inflammatory response after infection of the body to resist bacteria.The research results can provide a theoretical basis for the treatment plan and the development of drugs for V.vulnificus sepsis.