驴乳清蛋白通过抑制MAPK和NF-κB信号通路缓解对乙酰氨基酚诱导的小鼠急性肝损伤

Donkey Whey Protein Alleviates Acetaminophen-induced Acute Liver Injury in Mice Through Inhibiting Mitogen-Activated Protein Kinase and Nuclear Factor-κB Signaling Pathway

为了探讨驴乳清蛋白(DWP)补充对对乙酰氨基酚(APAP)诱导小鼠急性肝损伤(ALI)的保护作用及潜在的分子机制,本试验将6周龄雄性C57BL/6J小鼠随机分为6个组(n=6)。正常对照组(Control组)和APAP诱导肝损伤组(APAP组)小鼠每天灌服0.3 mL生理盐水,DWP对照组(DWP组)小鼠每天灌服400 mg/(kg•bw)DWP,DWP低、中、高剂量组(L、M、H组)小鼠每天分别灌服100、200和400 mg/(kg•bw)DWP,连续7 d。末次给药24 h后,除Control组和DWP组外,其余各组小鼠经腹腔注射300 mg/(kg•bw)APAP,16 h后收集各组小鼠血清和肝脏组织样品。苏木素-伊红(H.E.)染色观察小鼠肝脏组织学变化,试剂盒检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平,酶联免疫吸附试验(ELISA)检测小鼠肝脏细胞因子含量,免疫印迹(Western blot)方法检测核转录因子-κB(NF-κB)p65和有丝分裂原活化蛋白激酶(MAPK)相关信号分子,包括细胞外调节激酶(ERK)、Jun N-端激酶(JNK)、p38的磷酸化水平。结果显示,与APAP组相比,H组小鼠血清ALT、AST水平和肝脏病理组织学评分极显著降低(P<0.01),肝脏组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)含量显著降低(P<0.05),NF-κB p65和p38蛋白的磷酸化水平显著降低(P<0.05),ERK和JNK蛋白的磷酸化水平极其显著降低(P<0.001)。结果表明,DWP可能通过抑制MAPK和NF-κB信号通路,进而缓解APAP诱导的小鼠急性肝损伤和炎症反应。

To investigate the protective effect of donkey whey protein(DWP)supplementation on acetaminophen(APAP)-induced acute liver injury(ALI)in mice and its potential molecular mechanisms,this study randomly divided 6-week-old male C57BL/6J mice into six groups(n=6).The Control group and the APAP group received daily oral administration of 0.3 mL saline;the DWP group received daily oral administration of 400 mg/(kg•bw)DWP;the DWP low-,medium-,and high-dose groups(L,M,H groups)received daily oral administration of 100,200,and 400 mg/(kg•bw)DWP,respectively,for 7 consecutive days.After 24 hours of the final administration,except for the Control group and DWP group,the remaining groups of mice were intraperitoneally injected with 300 mg/(kg•bw)APAP.After 16 hours,serum and liver tissue samples were collected from each group of mice.Hepatic histological changes were observed using hematoxylin-eosin(H.E.)staining,serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured using assay kits,hepatic cytokine levels were determined using enzyme-linked immunosorbent assay(ELISA),and the phosphorylation levels of nuclear factor-κB(NF-κB)p65 and mitogen-activated protein kinase(MAPK),including extracellular regulated protein kinases(ERK),c-Jun N-terminal kinase(JNK),and p38,were analyzed using Western blot.The results showed that compared to the APAP group,the H group exhibited extremely significantly reduced serum ALT and AST levels,as well as liver pathological scores(P<0.01),significantly decreased levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)in liver tissue(P<0.05),as well as the phosphorylation levels of NF-κB p65 and p38 proteins(P<0.05),and extremely significantly reduced the phosphorylation levels of ERK and JNK proteins(P<0.001).These results indicate that DWP may alleviate APAP-induced ALI and inflammatory response by inhibiting the MAPK and NF-κB signaling pathways.