连翘叶酶-醇提取物抗药物性肝损伤作用研究

Protective mechanism of Forsythia suspense leaves extract against drug-induced liver injury

【目的】研究连翘叶酶-醇提取物(FLEAE)抗药物性肝损伤(DILI)的作用及其机制。【方法】将48只昆明小鼠随机分为6组,每组8只,分别为正常对照组(NC)、FLEAE对照组(FC)、DILI模型组(DILI)及FLEAE高、中、低剂量治疗组(HFC、MFC、LFC),其中NC组和FC组小鼠腹腔注射生理盐水,其余各组小鼠腹腔注射300 mg/kg APAP注射液,每天2次,连续注射4 d,第5天开始NC组和DILI组小鼠灌胃生理盐水,FC、HFC、MFC和LFC组小鼠分别灌胃FLEAE 600,600,400和200 mg/kg,每天2次,连续灌胃3 d。最后1次灌胃12 h后,检测各组小鼠谷丙转氨酶(ALT)、谷草转氨酶(AST)水平及氧化应激指标谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、过氧化氢(H_(2)O_(2))和丙二醛(MDA)水平,肝组织切片苏木精伊红(HE)染色观察肝损伤情况。用网络药理学方法构建FLEAE调控DILI的靶点集,GO和KEGG分析靶点的基因功能及通路,筛选FLEAE抗DILI的核心靶点。qRT-PCR和Western Blot检测核心靶点PIK3CA及其下游凋亡抑制分子B细胞淋巴瘤-2(Bcl-2)基因和蛋白的表达水平。【结果】FLEAE可显著降低DILI组小鼠血液中的ALT和AST水平,改善药物性肝损伤。FLEAE可显著降低DILI小鼠的GSH和SOD水平,提高H_(2)O_(2)和MDA水平。网络药理学分析表明,FLEAE抗DILI靶点与凋亡过程的负调控、氧化应激等有关,主要富集于PI3K/Akt信号通路,可能通过PIK3CA发挥作用;qRT-PCR和Western Blot结果显示,FLEAE可能通过提高PIK3CA基因和蛋白的表达发挥抗DILI及抑制细胞凋亡的作用。【结论】FLEAE通过上调DILI中PIK3CA的表达抑制细胞凋亡和改善氧化应激,发挥抗DILI的作用。

【Objective】This study investigated the effect and mechanism of Forsythia suspense leaves enzyme-alcohol extract(FLEAE)on drug-induced liver injury(DILI).【Method】Forty-eight Kunming mice were randomly divided into 6 groups with 8 mice in each group.The groups included normal control group(NC),FLEAE control group(FC),DILI model group(DILI),high,medium and low dose FLEAE treatment groups(HFC,MFC and LFC).Mice in NC group and FC group were intraperitoneally injected with normal saline and mice in other groups were intraperitoneally injected with 300 mg/kg APAP injection twice a day for 4 days.On the fifth day,mice in NC group and DILI group were gavaged with normal saline and mice in FC,HFC,MFC and LFC groups were gavaged with FKEAE at levels of 600,600,400 and 200 mg/kg twice a day for 3 days.Twelve hours after the last gavage,alanine transaminase(ALT)and aspartate aminotransferase(AST)levels were determined and liver tissue sections were stained with hematoxylin and eosin(HE)to observe liver injury.The levels of glutathione(GSH),superoxide dismutase(SOD),hydrogen peroxide(H_(2)O_(2))and malondialdehyde(MDA)were measured.Network pharmacology was used to construct the target set of FLEAE regulating DILI.GO and KEGG were used to analyze the gene function and signal pathway of the target and the core targets of FLEAE against DILI were screened.Quantificational real-time polymerase chain reaction(qRT-PCR)and Western Blot were used to detect the expression levels of core target PIK3CA and its downstream apoptosis-inhibiting molecule B-cell lymphoma-2(Bcl-2).【Result】FLEAE significantly reduced blood ALT and AST levels and improved liver injury in DILI group.FLEAE significantly increased GSH and SOD levels and decreased H_(2)O_(2) and MDA levels in DILI group.Network pharmacological analysis showed that FLEAE anti-DILI targets were related to the negative regulation of apoptosis and oxidative stress and were mainly enriched in PI3K/Akt signaling pathway,indicating that it may play a role through PIK3CA.The qRT-PCR and Western Blot results showed that FLEAE may improve the expression of PIK3CA gene and protein to anti-DILI and inhibit cell apoptosis.【Conclusion】FLEAE can inhibit apoptosis and improve oxidative stress by up-regulating PIK3CA expression and anti-DILI.