| Background and purpose:Sepsis refers to life-threatening severe organ dysfunction caused by dysregulated immune response of the host to bacterial infection.This cndition progresses rapidly,and may lead to septic shock,mortality and morbidity,accounting for one of the major causes of death among critically ill patients.Currently,only symptomatic and supportive treatments are available for sepsis,and the efficacy is far from satisfactory.Pathogenesis of sepsis is a complex process involving multiple organs and multiple phases.At first,large amount of cytokines and chemokines and other inflammatory mediators are released in response to bacteria and endotoxins.The collective effect of these endogenous factors leads to uncontrolled inflammation,resulting in damage to and dysfunction of coagulation,immunity,endocrine and other systems and related organs.Previous treatments all targeted individual factors or a single aspect of sepsis,and achieved poor efficacy,suggesting that treatment of sepsis shall be a systematic combination of infection control,toxin neutralization,regulation of inflammation,and tissue repair.A number of animal experiments showed that treatment with MSCs can significantly improve survival of the animal model of sepsis by regulating inflammatory response,balancing the immune system,improving organ function,and reducing the bacterial load.While bactericidal/permeability-increasing protein(BPI)are a group of positively charged peptides that competes against LBP to bind to LPS on the outer membrane of Gram-negative bacteria,which leads to increased membrane permeability and lysis of bacterial cells.BPI can also bind to and neutralize soluble LPS.LL-37 has been proved effective against a broad spectrum of both Gram-positive and-negative bacteria.It can bind to both LPS and CD14,effectively preventing formation of LPS-CD14 complex and thus preventing LPS-induced inflammation.We have constructed fusion proteins of BPI and LL37(BPI-LL37 and LL37-BPI),and delivered these fusion proteins into human umbilical cord-derived MSCs to produce functionally enhanced MSCs(BPI-LL37-h UC-MSCs and LL37-BPI-hUC-MSCs,or in short,BL-h UC-MSCs and LB-hUC-MSCs).This study shall evaluate the efficacy of such enhanced MSCs by both in vitro assays and in vivo animal studies.Methods:Expression of CD29,CD34,CD45,CD90,and CD105 on the surface of the BL-hUC-MSCs and LB-hUC-MSCs were determined by flow cytometry.And the potential of these cells for osteogenic,chondrogenic,and adipogenic differentiation.Conditional medium of these two antibacterial MSCs were collected and used to treat LPS-induced mouse peritoneal macrophages,followed by measurement of inflammatory factors in the supernatant by ELISA.Then,the bacteriostat effect and endotoxin-neutrolization effect of these conditional media were evaluated by bacteriostatic test and limulus test.Mouse model of sepsis was constructed by cecal ligation and puncture,and the models were further divided into two groups: Group A that received antibiotics 6h after operation and Group B that received antibiotics 24 h after operation.Mice of both groups were divided into 4 subgroups: a sham group,a PBS group that received 0.2 ml PBS by tail-vein injection,a WT group that received equal volume of h UC-MSCs solution(7.5x105 cells/ml),and a BL group that received equal volume of BL-hUC-MSCs solution(7.5x105 cells/ml).General condition of these mice was monitored,and a 120 h survival curve was drawn.Serum of mice of Group B were collected 48 h after surgery,liver and kidney function of these mice were evaluated by biochemical tests,TNF-α,IL-1β,IL-6 and IL-10 levels were measured by ELISA,pathological changes of liver,kidney,and lung were evaluated by HE staining,and expression of cleaved-caspase-3 protein in liver,kidney,spleen,and lung were determined by immunohistochemical staining.Results:Part I:1.Among the BL-hUC-MSCs and LB-h UC-MSCs,rate of CD29+ cells,CD44+ cells,CD90+ cells,CD34-cells,and CD35-cells were all over 95%,rate of CD105+ cells decreased and CD105 expression was decreased on some cells;2.Both BL-h UC-MSCs and LB-hUC-MSCs can be induced for osteogenic,chondrogenic,and adipogenic differentiation;3.Both BL-h UC-MSCs and LB-hUC-MSCs decreased expression of inflammatory factors by macrophages;4.The conditional medium of both BL-hUC-MSCs and LB-hUC-MSCs inhibited proliferation of a variety of bacteria;5.The conditioned medium of both BL-hUC-MSCs and LB-hUC-MSCs were able to neutralize endotoxin.Part II:1.hUC-MSCs showed better efficacy in improving survival of model mice of Group A,while BL-hUC-MSCs showed better efficacy in improving survival of model mice of Group B;2.BL-h UC-MSCs transplantation reduced serum TNF-α,IL-1β,IL-6,and IL-10 levels;3.BL-h UC-MSCs transplantation reduced serum levels of alanine aminotransferase,aspartate aminotransferase,amylase,creatinine and urea;4.BL-h UC-MSCs transplantation alleviated damage to liver,kidney,and lung tissues;5.BL-hUC-MSCs transplantation reduced expression of cleaved caspase-3 in liver,kidney,and lung.Conclusion:1.The BL-h UC-MSCs showed stronger anti-bacterial,anti-inflammatory and endotoxin neutralizing effect than LB-hUC-MSCs.2.BL-hUC-MSCs transplantation significantly reduced inflammation of septic mice,protected organ function,repaired damaged tissues and organs,and ultimately improved survival of these mice.Reduced apoptosis as indicated by reduced expression of cleaved caspase-3 in the tissues and organs may play a significant role in the protective effect of BL-hUC-MSCs. |
