沙苑子总黄酮通过抑制内质网应激和JNK通路过度活化减轻百草枯中毒大鼠肺损伤

Total flavonoids from astragalus complanatus attenuates lung injury following paraquat poisoning in rats through inhibiting excessive endoplasmic reticulum stress and c-Jun N-terminal kinase pathway

目的 探讨沙苑子总黄酮（FAC）是否可通过抑制内质网应激和c-Jun氨基末端激酶（JNK）通路过度活化来减轻大鼠百草枯诱导的肺损伤.方法 48只SD大鼠按随机数字表法分为对照组、模型组、二甲基亚砜（DMSO）对照组和FAC低、中、高剂量组6组,每组8只.模型组采用PQ 80 mg/kg灌胃诱导肺损伤;对照组则给予等量生理盐水灌胃;DMSO组于PQ灌胃前2h腹腔注射10％ DMSO 20 mL/kg;FAC低、中、高剂量组于PQ灌胃后腹腔注射40、80、160 mg· kg-1· d-1的FAC溶液.制模后72 h处死大鼠取肺,检测肺湿/干质量（W/D）比值、总肺水含量（TLW）;光镜下观察肺组织病理学改变,并进行肺组织损伤评估;逆转录-聚合酶链反应（RT-PCR）检测肺组织JNK和葡萄糖调节蛋白78（GRP78）的mRNA表达;蛋白质免疫印迹试验（Western Blot）检测肺组织JNK、磷酸化JNK（p-JNK）和GRP78的蛋白表达.结果 与对照组比较,模型组和DMSO组肺W/D比值、TLW、肺泡损伤定量评估指数（IQA）均明显升高,JNK、GRP78的mRNA表达和JNK、p-JNK、GRP78的蛋白表达均明显升高.与模型组比较,FAC各剂量组肺W/D比值、TLW、IQA以及JNK mRNA表达和p-JNK蛋白表达均明显下降,其中以FAC高剂量组降低最为显著[肺W/D比值：3.0±0.3比5.5±0.5,TLW：2.2±0.3比4.7±0.4,IQA：（15.4±3.0）％比（40.0±5.7）％,JNK mRNA：0.21 ±0.08比0.82±0.27,p-JNK蛋白：0.31±0.09比0.78±0.25,均P＜0.01];FAC低、中、高剂量组GRP78 mRNA和JNK、GRP78蛋白均为高表达,与模型组比较差异均无统计学意义（GRP78 mRNA：0.54±0.18比0.74±0.20,JNK蛋白：0.76±0.27比0.80±0.28,GRP78蛋白：0.51 ±0.18比0.69±0.21,均P＞0.05）.结论 百草枯中毒后肺组织发生了过度的内质网应激损伤;FAC可能通过抑制内质网应激及JNK信号通路过度活化起到保护肺组织的作用.

Objective To investigate the effect of total flavonoids from astragalus complanatus （FAC） on attenuating lung injury resulted from paraquat （PQ） poisoning by inhibiting excessive endoplasmic reticulum stress （ERS） and c-Jun N-terminal kinase （JNK） pathway in rat.Methods Forty-eight Sprague-Dawley （SD） rats were randomly divided into six groups （n=8 in each group）,including control group,model group,dimethyl sulfoxide （DMSO） vehicle control group,and FAC in low,medium,and high dosage groups.The model was reproduced by giving PQ 80 mg/kg orally to induce lung injury.The rats in control group were treated with saline by gavage.The rats in DMSO group were given 10％ DMSO 20 mL/kg by gavage 2 hours before intraperitoneal injection of PQ,and those in FAC low,medium and high dosage groups received 40,80,160 mg·kg-1· d-1 of FAC solution intraperitoneally after the PQ administration.The rats were sacrificed 72 hours after giving PQ,and the left lung tissue was harvested 72 hours after the reproduction of experimental model.The ratio of wet/dry weight （W/D） and total lung water content （TLW） were determined.The pathohistological changes of the left lung was observed under light microscope,and scored with alveolar damage index of quantitative assessment （IQA）.The mRNA expressions of JNK and glucose regulated protein 78 （GRP78） were determined by reverse transcription-polymerase chain reaction （RT-PCR）,and the protein expression of JNK,phosphorylation-JNK （p-JNK）,and GRP78 were determined by Western Blot.Results Compared with control group,the W/D ratio,TLW and IQA were increased significantly in model group and DMSO group,and the mRNA expressions of JNK and GRP78 and the protein expressions of JNK,p-JNK and GRP78 were markedly increased.Compared with the model group,the W/D ratio,TLW and IQA,and the expressions of JNK mRNA and p-JNK protein were significantly decreased in the FAC groups,especially in FAC high dosage group [W/D ratio：3.0 ± 0.3 vs.5.5 ± 0.5,TLW：2.2 ± 0.3 vs.4.7 ± 0.4,IQA：（15.4 ± 3.0）％ vs.（40.0 ± 5.7）％,JNK mRNA：0.21 ± 0.08 vs.0.82 ±0.27,p-JNK protein：0.31 ±0.09 vs.0.78 ±0.25,all P＜0.O1].The mRNA expression of GRP78 and the protein expressions of JNK and GRP78 were highly expressed in FAC low,medium and high dosage groups,and there was no significant difference compared with those in model group （GRP78 mRNA：0.54 ± 0.18 vs.0.74 ± 0.20,JNK protein：0.76 ± 0.27 vs.0.80 ± 0.28,GRP78 protein：0.51 ± 0.18 vs.0.69 ± 0.21,all P＞0.05）.Conclusions PQ induces excessive ERS in the lung tissue resulting in lung injury.FAC has a protective effect on lung against PQ injury,and it may be related with inhibition JNK pathway in ERS.