白藜芦醇联合姜黄素腹腔预注射小鼠脓毒症诱发过程中的肺损伤情况观察

Lung injury during sepsis induction in mice pre-injected intraperitoneally with resveratrol and curcumin

目的观察白藜芦醇联合姜黄素腹腔预注射小鼠脓毒症诱发过程中的肺损伤情况,并探讨其作用机制。方法90只雄性C57BL/6小鼠随机分为1~5组,每组18只。1~3组小鼠分别腹腔注射100 mg/kg姜黄素、40 mg/kg白藜芦醇、100 mg/kg姜黄素+40 mg/kg白藜芦醇,然后用盲肠结扎穿刺法(CLP)诱发脓毒症,4组小鼠腹腔注射等量0.9%生理盐水后CLP建立脓毒症模型,5组仅腹腔注射等量0.9%生理盐水。记录各组小鼠存活情况。第5天处死各组小鼠取肺组织,计算肺组织湿/干重(W/D),观察肺组织损伤情况,采用ELISA法检测各组肺组织中促炎因子(TNF-α、IL-1β、IL-6)与抗炎因子(IL-10)的表达情况,采用WESTERN Blotting法检测各组各组肺组织中促凋亡蛋白(Bax、cleaved caspase-3、cleaved PARP)和抗凋亡蛋白(Bcl-2)的表达情况,采用双荧光素酶报告基因测算各组肺组织中NF-κB通路的激活情况。结果第5天时1、2、3、4、5组小鼠分别存活4、5、13、1、18只。(1)与5组比较,第5天时4组小鼠存活率低,肺组织W/D与损伤评分高(P均<0.05);与4组比较,第5天时3组小鼠存活率高,1、2、3组小鼠肺组织W/D与损伤评分低(P均<0.05);与1、2组比较,第5天时3组小鼠存活率高,肺组织W/D与损伤评分低(P均<0.05)。(2)与5组比较,4组小鼠肺组织中TNF-α、IL-1β、IL-6、IL-10浓度高,Bax、cleaved caspase-3、cleaved PARP蛋白相对表达量高,Bcl-2蛋白相对表达量低,NF-κB通路相对活性高(P均<0.05);与4组比较,1、2、3组小鼠肺组织中TNF-α、IL-1β、IL-6浓度低,IL-10浓度高,Bax、cleaved caspase-3、cleaved PARP蛋白相对表达量低,Bcl-2蛋白相对表达量高,NF-κB通路相对活性低(P均<0.05);与1、2组比较,3组小鼠肺组织中TNF-α、IL-1β、IL-6浓度低,IL-10浓度高,Bax、cleaved caspase-3、cleaved PARP蛋白相对表达量低,Bcl-2蛋白相对表达量高,NF-κB通路相对活性低(P均<0.05)。结论白藜芦醇联合姜黄素腹腔预注射可有效减轻小鼠脓毒症诱发过程中的肺损伤程度,其效果优于二者单独用药,作用机理与抑制炎症反应和细胞凋亡以及NF-κB通路激活有关。

Objective To observe lung injury during sepsis induction in mice pre-injected intraperitoneally with res⁃veratrol and curcumin,and to explore its mechanism of action.Methods Ninety male C57BL/6 mice were randomly divided into groups 1 to 5,with 18 mice in each group.The mice in the groups 1 to 3 were injected intraperitoneally with 100 mg/kg curcumin,40 mg/kg resveratrol,and 100 mg/kg curcumin+40 mg/kg resveratrol,respectively,and then the models of sepsis were induced by cecal ligation and puncture(CLP).The mice in the group 4 were injected intraperito⁃neally with an equal amount of 0.9%saline,and then we established the models of sepsis by CLP;the mice in the 5 group were only injected intraperitoneally with an equal amount of 0.9%saline.The survival of each group of mice was record⁃ed.The lung tissues of mice in each group were collected on day 5,the wet weight/dry weight(W/D)of lung tissues was calculated,and the damage of lung tissues was observed.The expression of pro-inflammatory factors(TNF-α,IL-1β,IL-6)and anti-inflammatory factor(IL-10)in the lung tissues of each group was measured by ELISA.The expression of pro-apoptotic protein(Bax,cleaved caspase-3,cleaved PARP)and anti-apoptotic protein(Bcl-2)in lung tissues of each group was measured by Western blotting.The activation of NF-κB pathway in the lung tissues of each group was measured by dual luciferase reporter gene.Results Four,5,13,1,and 18 mice survived in the groups 1 to 5 on day 5,respec⁃tively.①Compared with the group 5,the mice in the group 4 had a lower survival rate and a higher W/D and injury scores of lung tissues on day 5(all P<0.05);compared with the group 4,the mice in the group 3 had a higher survival rate,and the mice in the groups 1,2 and 3 had lower W/D and injury scores of lung tissues on day 5(all P<0.05);com⁃pared with the groups 1 and 2,the mice in the group 3 had a higher survival rate and lower W/D and injury scores of lung tissues on day 5(all P<0.05).②Compared with the group 5,the lung tissues of mice in the group 4 had higher concen⁃trations of TNF-α,IL-1β,IL-6 and IL-10,higher relative expression levels of Bax,cleaved caspase-3 and cleaved PARP protein,a lower relative expression level of Bcl-2 protein,and higher relative activity of NF-κB pathway(all P<0.05).Compared with the group 4,the lung tissues of mice in the groups 1,2 and 3 had lower concentrations of TNF-α,IL-1βand IL-6,a higher concentration of IL-10,lower relative expression levels of Bax,cleaved caspase-3 and cleaved PARP protein,a higher relative expression levels of Bcl-2 protein,and lower relative activity of NF-κB pathway(all P<0.05).Compared with the groups 1 and 2,the lung tissues of mice in the group 3 had lower concentrations of TNF-α,IL-1βand IL-6,a higher concentration of IL-10,lower relative expression levels of Bax,cleaved caspase-3 and cleaved PARP pro⁃tein,a higher relative expression level of Bcl-2 protein,and lower relative activity of NF-κB pathway(all P<0.05).Con⁃clusions Intraperitoneal pre-injection of resveratrol combined with curcumin effectively reduces the degree of lung inju⁃ry,during sepsis induction in mice and its effect is better than that of resveratrol and curcumin alone;the mechanism is re⁃lated to the inhibition of inflammatory response,apoptosis,and NF-κB pathway activation.