ABSTRACT
Objective To discuss the protective effect of bone marrow mesenchymal stem cell(BMSC)on lung injury induced by vibrio vulnificus sepsis and its mechanism. Methods BMSCs were isolated by whole bone marrow adherent culture from mouse. Male ICR mice were randomly divided into normal saline control group(NS group),normal saline+BMSC control group(NSB group),vibrio vulnificus sepsis group(VV group),vibrio vulnificus sepsis + BMSC group(VVB group)according to random number table,with 40 mice in each group. Sepsis mouse model was reproduced by injecting vibrio vulnificus(1×107 cfu/mL)5 mL/kg through the left side peritoneal cavity, and caudal intravenous injection of BMSC(4×105 cfu/mL)5 mL/kg for intervention after model reproduction. Ten mice in each group were sacrificed at 6,12,24 or 48 hours after injecting vibiro vulnificus,and their lung tissues were harvested. The lung wet/dry(W/D)ratio was calculated. The expression of nuclear factor-κBp65(NF-κBp65)in nucleus was measured by Western Blot. The levels of tumor necrosis factor-α(TNF-α)and interleukins(IL-1β, IL-6)in lung tissue were detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes in lung tissue were observed after hematoxylin-eosin(HE)staining and uranyl acetate-lead citrate staining. Results After vibrio vulnificus injection,lung W/D ratio,the expression of NF-κBp65 in nucleus,and the levels of TNF-α, IL-1β,IL-6 in the lung tissues were significantly increased in VV group compared with those in NS group at all the time points,and peaked at 12 hours. Compared with the VV group,the VVB group had significantly decreased levels of lung W/D ratio,NF-κBp65 expression,and the levels of TNF-α,IL-1β,IL-6,with significant differences at all the time points〔VV group vs. NS group at 12 hours:lung W/D ratio 7.22±0.03 vs. 5.21±0.02,NF-κBp65 expression (glay scale)1.86±0.74 vs. 0.75±0.07,TNF-α(ng/L)433.24±3.23 vs. 106.57±1.21,IL-1β(ng/L)35.64±0.15 vs. 10.64±0.48,IL-6(ng/L)58.84±0.55 vs. 17.69±1.35,all P<0.05;VVB group vs. VV group at 12 hours:lung W/D ratio 6.49±0.06 vs. 7.22±0.03,NF-κBp65 expression(A value)1.16±0.08 vs. 1.86±0.74,TNF-α(ng/L)357.22±3.25 vs. 433.24±3.23,IL-1β(ng/L)27.77±0.59 vs. 35.64±0.15,IL-6(ng/L)38.68±1.29 vs. 58.84±0.55,all P<0.05〕. There were no significant differences in above indexes between NS group and NSB group. In the NS and NSB groups pathological changes were not obvious under light microscopy,in the VV group lung tissue hyperemia and edema was significant,the edema fluid,red blood cells and inflammatory cells also could be seen, and in the VVB group lung damage that mentioned above could be alleviated. In the NS and NSB groups epithelial cell structure of type Ⅰ and type Ⅱ was completed,and the changes were not obvious under the transmission electron microscopy. In the VV group the alveolar walls were damaged significantly,with type Ⅰ epithelial cell cytoplasm swelling,bubbling and rupture,with type Ⅱ epithelial cells visible cytoplasm decrease,cavitation,addiction to osmium lamellar corpuscle emptying,lysosome hyperplasia,microvilli reduction,and in the VVB group the above damage was alleviated. Conclusion Vibrio vulnificus sepsis can cause acute lung damage and edema,and BMSC can down regulate inflammatory cytokines,reduce lung injury caused by vibrio vulnificus sepsis.
ABSTRACT
BACKGROUND: Pro-inflammatory cytokines, such as tumor necrosis factor(TNF)-alpha and interleukin (IL)-1, have been incriminated in the pathogenesis of septic shock in animals and humans. IL-10 reduces the release of pro-inflammatory cytokines, nitric oxide and reactive oxygen species. IL-10 prevents endotoxin-induced toxicity. OBJECTIVE: The aim of this study was to investigate the relation between IL-10 and TNF-alpha in toxemic mice according to concentrations of V. vulnificus cytolysin. METHODS: First, after administration of V.vulnificus cytolysin(2, 4, 6, 8, 10 hemolytic units; HU) and physiologic saline through a mouse tail vein, we obtained blood samples from the heart at 60 minutes which was a peak time of IL-10 and TNF-alpha release. We measured serum concentration of circulating TNF-alpha and IL-10 using commercially available enzyme-linked immunosorbent assay methods. Second, after administration of 1,000U recombinant mouse IL-10 through a mouse tail vein 30 min before infusion of the lethal dose(8HU) of V. vulnificus cytolysin. We obtained blood samples from the heart at 60 minutes and measured serum concentration of circulating TNF-alpha level. RESULTS: Both IL-10 and TNF-alpha levels were significantly correlated with V. vulnificus cytolysin concentration(P=0.002). TNF-alpha levels were 76.9+/-9.5 pg/ml in 2HU, 315.8+/-39.8 pg/ml in 4HU, 426.1+/-27.9 pg/ml in 6HU, 931.3+/-22.3 pg/ml in 8HU, 1825.2+/-18.8 pg/ml in 10HU and 23.6+/-5.1 pg/ml in physiologic saline. IL-10 levels were 80.2+/-21.5 pg/ml in 2HU, 244.4+/-35.4 pg/ml in 4HU, 382.2+/-22.6 pg/ml in 6HU, 740.1+/-33.0 pg/ml in 8HU, 997.3+/-16.8 pg/ml in 10HU and 35.8+/-15.0 pg/ml in physiologic saline. After administration of 1,000U recombinant mouse IL-10, comparing to control group(931.3+/-22.3 pg/ml), TNF-alpha level was reduced to 307.2+/-23.9 pg/ml(P=0.003). CONCLUSION: IL-10 has an inhibitory effect on the production of TNF-alpha although it is released together with TNF-alpha in toxemic mice. IL-10 blood levels are directly related to the severity of toxemia. We conclude that IL-10 is a prognostic factor for the development of sepsis and might be used for the treatment of sepsis.