| Objective：To explore the mechanisms of neutrophil migration dysfunction in severe sepsis and the preventiveeffects of PA-MSHA through establishing models of sepsis and severe sepsis, then investigating the effectsof PA-MSHA on neutrophil migration in vitro; the live time; the contents of the cytokine that include tumornecrosis factor-α (TNF-α), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10) and induciblenitric oxide synthase (iNOS);the histomorphology of organs; the serumal biochemical indicator whichinvolve alanine transarninase (ALT), aspartate aminotransferase (AST), Creatinine(Creat), Blood UreaNitrogen(BUN), Creatine kinase(CK), Creatine kinase MB(CK-MB) in rats with sepsis and severe sepsis.Methods:1.72healthy male SD rats were divided in three groups randomly: the sham operation group (C group), thesepsis group (S group), the severe sepsis group (SS group). For induction of sepsis and severe sepsis,1.6and4mL/kg body weight human stool suspension, diluted in saline to3ml, were injected i.p. into theabdomen.Intraperitoneal injection of equal normal saline were given to the C group. After modlesestablished, General condition and48h-survival rate of the rats were observed. After modles established7hours, the Clinical Status were valued,invasive mean arterial pressure (MAP),marterialblood gas index,aterial lactic acid (LA), the serumal biochemical indicator (ALT, AST, Creat, BUN, CK, CK-MB),theserum cytokines (IL-6,IL-10) were tested; blood were cultured. At last, rats were executed with soudiumpentobarbital, heart, liver, lung, kidney and intestine were observed with microscope.2.216healthy male SD rats were divided in eight groups randomly: the sham operation group(C group),7hours after sepsis modle completed group (S7group),15hours after sepsis modle completed group (S15group),7hours after severe sepsis modle completed group (SS7group), PA-MSHA group (PA group),PA-MSHA+S7h group (PA-S7group), PA-MSHA+S15h group (PA-S15group), PA-MSHA+SS7hgroup (PA-SS7group).0.3ml PA-MSHA were injected subcutaneously in every intervention group for7days. General condition and72h-survival rate of the rats were observed;Peripheral neutrophils wereacquired by discontinuous percoll gradient centrifugation and the number of neutrophil migration in vitrowere measured by transwell chambers; TNF-α, IL-6, IL-8, IL-10and iNOS in serum by the way ofenzyme linked immunosorbent assay (ELISA); biochemical indicator (ALT, AST, Creat, BUN, CK,CK-MB) were tested; heart, liver, lung, kidney and intestine were observed with microscope afte ratswere executed with soudium pentobarbital.Results：1.(1) general condition: mainly evaluating from eating, breathing, spontaneous activity and the alertness ofexogenous stimuli, the SS group were worse than S group;(2) the Clinical Status were: the clinical statusscore of C group, S group and SS group were separatly focus on1score,2-3score and3-4score,separately in the stage of normal, slight-midrange stage and midrange-severe stage;(3)48h-survival rate: C group100%; comparison of survival curve:log-rank test: C group＞S group: x2=16.94, P＜0.01;C group＞SS group: x2=16.94, P＜0.01; S group＞SS group: x2=6.12, P＜0.05; the median live timeof SS group was shorter than S group (17.25vs9.15, P＜0.01);(4) MAP: S group and SS group werelower than C group: group vs C group:107.25±13.57vs44.86±12.21, P＜0.05; SS group vs C group:89.75±13.30vs144.86±12.21, P＜0.05);(5) arterialblood gas index and LAC: compared with Cgroup, the PCO2, HCO3-, BE of S group and SS group were increased (P＜0.05); PO2and SaO2%of Sgroup were decreased (P＜0.05); the PO2, SaO2%of SS group were lower than S group (P＜0.05);(6)biochemical indicator: The BUN of S group and SS group, the ALT, AST, Creat and CK of SS groupwere all higher than C group (P＜0.05); the ALT, UN of SS group were higher than S group (P＜0.05);(7) the serum cytokines: compared with C group, the IL-6and IL-10of S group were increased (P＜0.05), and the IL-6and IL-10of SS group were significant increased (P＜0.01); the IL-6and IL-10of SSgroup were higher than S group;(8) blood culture: C group was negative,S group and SS group wereboth of positive (8/8), Escherichia coli;(9) pathological change: the heart,liver,lung, kidney and intestineof S group and SS group had pathological changes, and SS group was wores than S group.2.(1) general condition: mainly evaluating from eating, breathing, spontaneous activity and the alertness ofexogenous stimuli, the S7group, S15group and SS7group were separatly worse than PA-S group,PA-S15group and PA-SS7group; SS7group was worse than S7group;(2)72h-survival condition: thesurvival time (median): PA-S group was obviously longer than S group (28.65h vs17.75h, P＜0.05; SS7group was lower than S7group (9.15h vs17.75h, P＜0.05); PA-SS7was longer than SS7group (14.1h vs9.15h, P＜0.05); survival rate: C group and PA group s72h survival rate were both100%; comparison ofsurvival curve:log-rank test:C group＞S7group: x2=16.96, P＜0.01; C group＞SS7group; x2=16.96, P＜0.01; S7group＞SS7group: x2=11.55, P＜0.01; PA-S7group＞S7group: x2=8.477, P＜0.01; PA-SS7group＞SS7group: x2=5.798, P＜0.05;(3) the number of neutrophil migration invitro: comparedwith C group, except PA group,the other groups were significantly decreased (P＜0.01) invitro: C group:355.90±21.51; S7group:121.40±37.71; S15group:21.98±6.06; SS7group:58.7±4.77; S15group,PA-S15group, SS7group, PA-SS7group were significant decreased than S7group (P＜0.01); SS7groupwas higher than S15group (P＜0.05); ompaired with PA-group, A-S7group, PA-S15group, PA-SS7group were obviously decreased (P＜0.01), PA-S15group, PA-SS7group were less than PA-S7group (P＜0.01); PA-SS7group was more than PA-S15group(P＜0.05); PA-S7group was significantlyincreased than S7(P＜0.01)..(1) the serum cytokines concentration①T NF-α concentration: compared with C group, exceptPA group, the other groups were significantly increased (P＜0.01); compared with PA group, S7group,PA-S7group were increased (P<0.05); SS7group was higher than S7group;②IL-6concentration:compared with C group,except PA group was no difference, S7group and PA-S7group were increased(P<0.0), SS7group and PA-SS7group were significantly increased (P<0.01); compared with PA group,S7group, PA-S7group were increased (P<0.05), SS7group and PA-SS7group were significantly increased (P<0.01); SS7group was higher than PA-S7group and PA-SS7group (P<0.05), and wasobviously hingher than S7group (P<0.01);③I L-8concentration:compared with C group, except PAgroup was no difference, S7group were increased (P<0.05), PA-S7group, SS7group and PA-SS7groupwere significantly increased (P<0.01); SS7group was higher than S7group;④I L-10concentration:compared with C group, PA group, S7group, PA-S7group were increased (P<0.05), SS7group and PA-SS7group were significantly increased (P<0.01); compared with PA group, PA-SS7groupwas increased (P<0.05), SS7group was significantly increased (P<0.01); PA-SS7group was obviouslylower than SS7group;⑤i NOSconcentration: S7group and SS7group were higher than C group, PAgroup and PA-S7group (P<0.05); PA-SS7group was lower than SS7group (P<0.05);(2) the serumalbiochemical indicator:①ALT: compared with Cgroup, S7group and PA-SS7group were higher(P<0.05), SS7group was significantly increased (P<0.01); compared with PA group, S7group andPA-SS7group were increased (P<0.05), SS7group was significantly increased (P<0.01); SS7group werehigher than S7group and PA-SS7group, and was obviously higher than PA-S7group (P<0.01);②AST:SS7group was higher than the other groups (P<0.05); PA-SS7group was higher than C group (P<0.05);③C reat and UBN: SS7group was higher than the other groups (P<0.05);④C K:SS7group was higherthan S7groups and PA-SS7group (P<0.05), and was obviously higher than C group and PA-S7group(P<0.01);⑤CK-MB: SS7group were obviously higher than C group and PA group (P<0.05), and washigher than S7groups, PA-S7and PA-SS7group (P<0.01).(3) pathological changes of organs are asfollow: from modest to serious: PA-S7group, S7group, PA-SS7group, SS7group.Conclusions：1. The neutrophil migration in vitro could be inhibited in the early stage of sepsis, and the inhibition gotworse with the progress of the disease, neutrophil migration in vitro was dysfunction in severe sepsis.2. PA-MSHA preconditioning could enhance the function of neutrophil migration in vitro in sepsis rats bydown regulating the level of iNOS in serum, then reduce injuries,slow down the progress of the disease,prolong the survival time.3.0.3ml PA-MSHA preconditioning for7days could not enhance the function of neutrophil migration invitro in severe sepsis, but it could reduce injuries and prolong the survival time in part by downregulating the level of cytokines in serum. |
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