骆驼乳清蛋白对不同细菌脓毒症小鼠急性肺损伤的作用

Effects of camel whey protein on sepsis-associated acute lung injury caused by different bacteria in mice

  • 摘要:
    目的 探讨骆驼乳清蛋白(CWP)对不同细菌所致脓毒症小鼠急性肺损伤的作用。
    方法 雄性C57L/6小鼠适应性饲养1周后,取40只随机分为5组,每组8只,分别为假手术组(Sham组)、革兰阳/阴性脓毒症组(G+/Gsepsis组)、革兰阳/阴性脓毒症+CWP干预组(G+/Gsepsis+CWP组)。G+/Gsepsis+CWP组小鼠采用200 mg/kg剂量的CWP冻干粉溶液进行同步灌胃预处理,每天1次,连续干预7天;Sham组和G+/Gsepsis组小鼠给予等体积的生理盐水同步灌胃。于末次干预24 h后,除Sham组外,其余各组小鼠构建G+/G细菌感染所致脓毒症相关急性肺损伤动物模型。以各组小鼠造模时间为起始点,分别在第1天、3天和7天对小鼠进行取材和指标检测。造模后观察并记录各组小鼠的一般情况;HE染色观察小鼠肺组织病理形态学变化;肺组织湿/干重比(W/D)评估小鼠肺水肿程度;检测小鼠肺泡灌洗液(BALF)中炎症因子:肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IL-1β和IL-10水平;TUNEL染色观察小鼠肺组织细胞凋亡情况;Western Blotting法检测小鼠肺组织凋亡相关蛋白表达水平。
    结果 与Sham组相比,在造模后第1天、3天和7天,G+/Gsepsis组小鼠肺组织病理损伤评分增加,肺组织W/D增加,BALF中促炎因子TNF-α、IL-6、IL-1β水平升高、抗炎因子IL-10水平降低,肺组织细胞凋亡数目增加,Bcl-2蛋白表达降低,Cleaved-Caspase-3、Caspase-3蛋白表达升高(P<0.05);予CWP干预后,在第1天、3天和7天,G+/Gsepsis+CWP组较G+/Gsepsis组小鼠一般情况改善,肺组织病理形态改善,肺损伤评分降低,肺组织W/D降低,BALF中抗炎因子IL-10水平升高、促炎因子TNF-α、IL-6、IL-1β水平降低,肺组织细胞凋亡数目减少,Bcl-2蛋白表达升高,Cleaved-Caspase-3、Caspase-3蛋白表达降低(P<0.05),呈时间依赖性改变(P<0.001)。
    结论 CWP可减轻脓毒症小鼠肺组织病理损伤、减少肺组织水肿和调控机体炎症反应,其作用机制可能与调控肺组织细胞凋亡相关途径有关。

     

    Abstract:
    OBJECTIVE To investigate the effects of camel whey protein (CWP) on sepsis-associated acute lung injury caused by different bacteria in mice.
    METHODS  After C57L/6 mice were adaptively fed for one week, 40 mice were randomly divided into five groups, with eight mice in each group: sham operation group (Sham group), gram-positive/negative sepsis group (G+/Gsepsis group) and gram-positive/negative sepsis + CWP intervention group (G+/Gsepsis + CWP group). Mice in the G+/Gsepsis + CWP group were pretreated with a synchronous gavage of 200 mg/kg CWP freeze-dried powder solution once a day for seven consecutive days. Mice in the Sham group and G+/Gsepsis group were given an equal volume of saline for synchronous gavage. After 24 hours of the last intervention, except for the Sham group, the remaining groups were chosen to establish models of sepsis-related acute lung injury caused by G+/Gbacterial infection. Taking the modeling time of each group as the starting point, sample collection and indicator detection were performed on the 1st, 3rd and 7th day, respectively. After modeling, the general conditions were observed and recorded. The pathomorphological changes in lung tissue were observed by HE staining. The lung edema degree was evaluated by the wet/dry weight ratio (W/D) of lung tissue. The levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and IL-10 in the bronchoalveolar lavage fluid (BALF) were detected. The apoptosis of lung tissue cells was observed by TUNEL staining. The expression levels of apoptosis-related proteins in the lung tissue were detected by Western Blotting.
    RESULTS  Compared with the Sham group, on the 1st, 3rd and 7th day after modeling, the pathological injury scores and W/D of lung tissues in the G+/Gsepsis groups increased, the levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in BALF increased, the level of anti-inflammatory cytokine IL-10 decreased, the number of apoptotic cells in lung tissue increased, the expression of Bcl-2 protein decreased, and the expression of Cleaved-Caspase-3 and Caspase-3 proteins increased (P<0.05). On day 1, day 3 and day 7 after CWP intervention, the general conditions of mice in the G+/Gsepsis + CWP group improved compared to those in the G+/Gsepsis group, with improved pathomorphology of lung tissue, reduced lung injury scores, decreased lung tissue W/D, increased level of anti-inflammatory cytokine IL-10 in BALF, decreased levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and number of apoptotic cells in lung tissue, increased expression of Bcl-2 protein and decreased expression of Cleaved-Caspase-3 and Caspase-3 proteins (P<0.05), all showing time-dependent changes (P<0.001).
    CONCLUSION  CWP can alleviate pathological lung tissue damage, reduce lung tissue edema, and regulate the body's inflammatory response in mice with sepsis, and action mechanisms may be related to the regulation of pathways associated with apoptosis in lung tissue cells.

     

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