竹节参皂苷IVa通过抗炎和抗氧化减轻脂多糖诱导的大鼠急性肺损伤

Saponin from Panax japonicus IVa alleviates lipopolysaccharide-induced acute lung injury in rats via its anti-inflammatory and antioxidative effects

目的:探讨竹节参皂苷IVa(saponin from Panax japonicus IVa,SPJ IVa)对大鼠急性肺损伤的影响,并对其可能的保护机制进行初步的探索。方法:将60只SD大鼠随机均分为4组,每组15只:对照组、模型组、低剂量SPJ IVa组和高剂量SPJ IVa组。以脂多糖(LPS,2 mg/kg)气管内滴注法建立大鼠急性肺损伤模型,低、高剂量SPJ IVa组大鼠在造模后30 min分别腹腔注射15和45 mg/kg SPJ IVa。造模后24 h,收集血清、支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和肺组织。HE染色法评估肺组织病理形态学变化;称重法测定肺组织湿重/干重值;ELISA法检测血清和BALF中白细胞介素1β(interleukin-1β,IL-1β)、IL-6和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)水平;试剂盒法检测丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽(glutathione,GSH)的水平;免疫组织化学标记cleaved caspase-3法和TUNEL染色法评估肺组织中细胞凋亡情况;Western blot法测定肺组织中核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)、血红素加氧酶1(heme oxygenase-1,HO-1)、核因子κB(nuclear factor-κB,NF-κB)p65和Toll样受体4(Toll-like receptor 4,TLR4)表达。结果:与对照组比较,模型组肺组织发生明显损伤病变,肺损伤积分(0.21±0.22 vs 2.98±0.46)和湿/干重比值(3.09±0.41 vs 6.36±0.61)显著上升(均P<0.01);与模型组比较,低、高剂量SPJ IVa组肺损伤积分(1.80±0.31和1.05±0.25 vs 2.98±0.46)和湿/干重比值(5.25±0.44和3.89±0.35 vs 6.36±0.61)显著降低(均P<0.01)。LPS可致血清和BALF中促炎因子(IL-1β、IL-6、TNF-α)和氧化指标MDA水平升高(P<0.01),抗氧化指标SOD、GSH降低(P<0.01),低、高剂量SPJ IVa治疗均可降低LPS导致的上述促炎因子和氧化指标MDA(P<0.01),并升高SOD和GSH水平(P<0.05或P<0.01)。模型组肺组织中可见显著细胞凋亡,低、高剂量SPJ IVa治疗可抑制LPS导致的肺组织中TUNEL阳性细胞和cleaved caspase-3表达(P<0.01)。模型组肺组织中Nrf2、HO-1、NF-κB p65和TLR4表达高于对照组(P<0.01);低、高剂量SPJ IVa治疗后,Nrf2和HO-1表达进一步上调(P<0.01),而NF-κB p65和TLR4表达降低(P<0.01)。结论:SPJ IVa可抑制LPS所致ALI大鼠肺损伤,此作用可能与其抑制TLR4/NF-κB和Nrf2/HO-1介导的炎症反应和氧化应激有关。

AIM:To investigate the effects of saponin from Panax japonicus IVa(SPJ IVa)on acute lung injury in rats and to explore its possible protective mechanism.METHODS:Sixty SD rats were randomly divided into four groups,15 rats in each group:the control group,model group,low-dose SPJ IVa group,and high-dose SPJ IVa group.A rat model of ALI was established via intratracheal instillation of lipopolysaccharide(LPS,2 mg/kg).Rats in the low-and high-dose SPJ IVa groups were intraperitoneally injected with 15 and 45 mg/kg SPJ IVa,respectively,30 min after modeling.Serum,bronchoalveolar lavage fluid(BALF),and lungs were collected 24 h after modeling.Pathomorphological changes in lung tissues were assessed using HE staining.The wet weight/dry weight ratio of lung tissues was measured using the weighing method,whereas ELISA was used to measure the levels of interleukin-1β(IL-1β),IL-6,and tumor necrosis factor-α(TNF-α)in the serum and BALF.The levels of malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)were assessed using the kit method.Cell apoptosis in lung tissues was evaluated by immunohistochemical staining of cleaved caspase-3 and TUNEL.Western blot was used to measure the expression of nuclear factor E2-related factor 2(Nrf2),heme oxygenase-1(HO-1),nuclear factor-κB(NF-κB)p65,and Toll-like receptor 4(TLR4)in lung tissues.RESULTS:Compared with control group,the lung tissues of the model group were significantly damaged,and the lung injury scores(0.21±0.22 vs 2.98±0.46)and lung wet/dry weight ratios(3.09±0.41 vs 6.36±0.61)were significantly increased(P<0.01).Compared with model group,the lung injury scores(1.80±0.31 and 1.05±0.25 vs 2.98±0.46)and lung wet/dry weight ratios(5.25±0.44 and 3.89±0.35 vs 6.36±0.61)in low-and high-dose SPJ IVa groups were significantly reduced(P<0.01).The administration of LPS resulted in elevated levels of pro-inflammatory cytokines(IL-1β,IL-6,and TNF-α)as well as the oxidative marker MDA in both serum and BALF(P<0.01).Additionally,it led to a decrease in antioxidant markers SOD and GSH(P<0.01).However,treatment with both low and high doses of SPJ IVa effectively attenuated the LPS-induced production of pro-inflammatory factors and oxidative markers MDA(P<0.01),while also increasing SOD and GSH levels(P<0.05 or P<0.01).In the model group,evident apoptosis was observed in lung tissues,whereas treatment with low and high doses of SPJ IVa significantly suppressed TUNEL-positive cells and the expression of cleaved caspase-3(P<0.01).The expression levels of Nrf2,HO-1,NF-κB p65,and TLR4 in lung tissues were significantly higher in the model group than in the control group(P<0.01);in turn,after treatment with low and high doses of SPJ IVa,Nrf2 and HO-1 were further upregulated(P<0.01),whereas NF-κB p65 and TLR4 were downregulated(P<0.01).CONCLUSION:The inhibitory effect of SPJ IVa on LPS-induced ALI in rats may be attributed to its ability to suppress the TLR4/NF-κB-and Nrf2/HO-1-mediated inflammatory response and oxidative stress.