title: [Reproduction of a model of "two-hit" sepsis model with complication of pneumonia in rat].
abstract: OBJECTIVE
  To reproduce a clinically relevant "two-hit" model of sepsis complicated by pneumonia and to explore the correlation between "two-hit" and immune state.
  
  
  METHODS
  Eighty-one male Sprague-Dawley (SD) rats were divided into groups according to the random number table. Forty-five male rats were assigned respectively to sepsis-alone group, pneumonia 4 days and 7 days after sepsis groups, respectively. Survival rate of each group was observed. Another group of 36 male rats were divided into normal control group, sepsis-alone for 1, 4 and 7 days groups, and sepsis complicated by pneumonia for 4 days and 7 days after sepsis groups, each group consisted of 6 rats. Cecal ligation and puncture (CLP) was done in rats, and Streptococcus pneumoniae suspension (bacteria count 1 x 10(10) cfu/mL) was injected via the nose on the 4th day or 7th day after CLP. Rats were sacrificed at corresponding time points, and 1 day after challenge of Streptococcus pneumoniae on the 4 days or 7 days post CLP for the collection of blood and tissue samples to make bacterial count of the blood, splenocyte count, biochemical indices, cytokines concentration, pathological changes in spleen and apoptotic cells.
  
  
  RESULTS
  (1) Compared with the rats of sepsis-alone group, the rats in pneumonia 4 days after CLP group had poor survival rate (4 vs. 11, χ2 = 6.533, P = 0.011), while no difference was found between pneumonia 7 days after CLP group and sepsis-alone group (9 vs. 11, χ2 = 0.600, P = 0.439). (2) The blood bacterial count and all the biochemical indexes were sharply increased on 1 day post-CLP in the rats of sepsis-alone group, and then they gradually lowered. Compared with the rats of 1 day post-CLP, the proportion of splenocytes were decreased on the 4th day post-CLP [dendritic cells (DC): (0.69 ± 0.09)% vs. (0.87 ± 0.31)%, CD4+ T cells: (21.05 ± 2.89)% vs. (24.84 ± 4.59)%, CD8+ T cells: (10.62 ± 1.79)% vs. (13.40 ± 1.31 )%, all P < 0.05], but T-regulatory cell (Treg) count was higher on the 4th day after CLP compared with sepsis-alone rats [(3.14 ± 0.74 )% vs. (2.87 ± 1.08)%, P < 0.05]. The biochemical indices, including alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (SCr) were obviously lowered on 7 days post-CLP compared with 1 day after CLP [ALT (U/L): 35.33 ± 11.52 vs. 81.00 ± 38.40, AST (U/L): 70.33 ± 42.16 vs. 156.00 ± 28.11, BUN (mmol/L): 5.30 ± 2.27 vs. 9.13 ± 4.04, SCr (1μmol/L): 55.33 ± 10.67 vs. 96.67 ± 45.79, all P < 0.05]. The serum levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) peaked on the 1st day after CLP [TNF-α: (18.03 ± 2.88) ng/L, IL-6: ( 10.37 ± 4.20) ng/L, IL-1β: ( 102.44 ± 51.46) ng/L], and high mobility group box-1 (HMGB1) peaked on the 4th day after CLP [(1.76 ± 0.71) μg/L]. The levels of anti-inflammatory cytokines transforming growth factor-β1 (TGF-β1) and soluble tumor necrosis factor receptor-I (sTNFR-I) maintained at high levels [7 days post-CLP: TGF-β1 was (0.90 ± 0.56) ng/L, sTNFR-I was (1.56 ± 0.39) ng/L]. The spleen pathology became more marked with the time in the group of sepsis-alone, meanwhile the number of apoptotic spleencytes increased 4 days post-CLP as compared with that of the 1st day post-CLP (cells/HP: 52.99 ± 20.79 vs. 16.05 ± 3.28, P < 0.05). (3) Compared with the same period of sepsis-alone group, the rats with pneumonia 4 days post-CLP group showed a higher blood bacterial count (log cfu/mL: 1.78 ± 0.54 vs. 0.25 ± 0.18, P < 0.05), while no difference was found between 7-day of post-CLP pneumonia group and sepsis-alone group (log cfu/mL: 0.57 ± 0.46 vs. 0.13 ± 0.12, P > 0.05). The same trend of changes, with slight reduction in splenocytes and biochemical indices were found between the groups of sepsis followed by pneumonia and sepsis-alone, but no significant difference was found. The level of HMGB1 in the 4-day group of sepsis with complication of pneumonia was further decreased compared with sepsis-alone group (μg/L: 1.17 ± 0.74 vs. 1.76 ± 0.71, P < 0.05), and IL-1β in the 7-day group of sepsis complicated pneumonia was further higher than those of sepsis-alone group in the same period (ng/L: 105.73 ± 25.06 vs. 61.04 ± 31.29, P < 0.05), while there were no differences in levels of other cytokines between "two-hit" group and sepsis-alone group. Apoptosis of spleencytes in the 4-day group of sepsis complicated pneumonia was more marked than that of sepsis-alone group at the same period (cells/HP: 74.48 ± 22.47 vs. 52.99 ± 20.79, P < 0.05), while no difference was found between the 7-day groups of sepsis complicated pneumonia and the sepsis-alone group (cells/HP: 28.70 ± 4.13 vs. 30.43 ± 14.55, P > 0.05).
  
  
  CONCLUSIONS
  The mortality of this "two-hit" model with complication of pneumonia 4 days after CLP was significantly higher than that of single sepsis model. The ability of bacteria clearance was decreased, and immunocyte apoptosis was exacerbated. These findings may be with the result of the occurrence of immunoparalysis in the mid stage of sepsis. The "two-hit" model reproduced on 7 days after CLP might suggest reconstruction of host immune function, and maybe associated with the recovery of immune response.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Chen
authors.forename: Huixian
authors.initials: H
authors.lastname: Li
authors.forename: Shuai
authors.initials: S
authors.lastname: Ma
authors.forename: Bo
authors.initials: B
authors.lastname: Deng
authors.forename: Jianxin
authors.initials: J
authors.lastname: Lu
authors.forename: Feng
authors.initials: F
authors.lastname: Ding
mesh_terms: D000410:Alanine Transaminase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001806:Blood Urea Nitrogen; D003404:Creatinine / Q000097:blood; D004195:Disease Models, Animal; D024243:HMGB1 Protein / Q000097:blood; D053583:Interleukin-1beta / Q000097:blood; D015850:Interleukin-6 / Q000097:blood; D008297:Male; D011014:Pneumonia / Q000150:complications / Q000503:physiopathology*; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000097:blood; D018805:Sepsis / Q000150:complications / Q000503:physiopathology*; D013154:Spleen / Q000473:pathology; D053773:Transforming Growth Factor beta1 / Q000097:blood; D014409:Tumor Necrosis Factor-alpha / Q000097:blood
chemical_list: D024243:HMGB1 Protein; D053583:Interleukin-1beta; D015850:Interleukin-6; D047888:Receptors, Tumor Necrosis Factor, Type I; C508962:Tgfb1 protein, rat; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D003404:Creatinine; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase