葛根素抑制自噬改善脂多糖诱导的小鼠肝脏损伤作用研究

Puerarin Alleviated Liver Injury in LPS-Induced Mice by Suppression of Autophagy

目的观察葛根素对脂多糖(Lipopolysaccharide,LPS)诱导的小鼠肝脏损伤表型的改善作用,并探讨相关分子机制。方法选取18只C57BL/6J小鼠,随机分为3组:0.9%:NaCl(腹腔注射,20 mg·kg^(-1))处理为对照组(6只)、LPS(腹腔注射,20 mg·kg^(-1))为脓毒血症肝损伤模型组(6只)、及模型基础上予以葛根素(腹腔注射,160 mg·kg^(-1))的干预组(6只)。24 h后,检测小鼠血清中的丙氨酸氨基转移酶(Alanine aminotransferase,ALT)和谷草转氨酶(Aspartate aminotransferase,AST)水平,HE染色、免疫组化、免疫荧光检测实时定量PCR(Quantitative real-time polymerase chain reaction,qRT-PCR)、蛋白质印迹法(Western blot)检测肝脏组织相关基因表达,并用人正常肝细胞株LO2验证。结果与正常对照组比,模型组小鼠的血清ALT和AST水平显著增高,肝脏组织中凋亡相关基因P53和分解半胱氨酸天冬氨酸蛋白酶3(Cleaved Caspase3,cl.Cas3)表达增多,促炎因子白细胞介素-6(Interleukin-6,IL-6)、γ干扰素(Interferon-γ,IFN-γ)、肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)水平增加,抗炎因子白细胞介素-10(Interleukin-10,IL-10)水平降低。p62表达减少,Beclin1、LC3B表达增多。给予模型小鼠葛根素干预后,可有效逆转上述指标变化。体外实验重现上述结果。结论在LPS诱导的肝损模型中,葛根素可通过抑制自噬相关蛋白的过度表达,减少肝细胞死亡,改善小鼠的肝脏损伤病理表型,其机制可能是通过下调Toll样受体4(Toll-like receptor4,TLR4)实现的。提示葛根素早期干预可作为一种治疗肝脏损伤的新方法。

Objective To study the effects of puerarin on liver injury of lipopolysaccharide(LPS)induced mice and the possible molecular mechanism.Methods 18 C57BL/6J mice were randomly divided into three groups including the control group(n=6),the liver injury model group(n=6),and puerarin intervention group(n=6).The animals were treated by PBS intraperitoneally(200 ul,ip),LPS(20 mg·kg^(-1),ip)and/or puerarin treatment(160 mg·kg^(-1),ip)respectively and were sacrificed 24 h later.Serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST),and histopathological examination(HE)were used to evaluate liver injury.In addition,immunohistochemistry(IHC),immunofluorescence(IF),quantitative real-time polymerase chain reaction(qRT-PCR)and western blot were used to detect expressions of mRNA and proteins.Finally,LO2 cell line was used to confirm the above molecular expressions.Results Compared with the control mice,the serum ALT and AST levels of LPS-induced mice were significantly increased,as well as the pro-inflammatory factors(IL-6,IFN-γ,TNF-α)in the liver tissue,while the anti-inflammatory factor(IL-10)was significantly decreased.HE detecting showed that the expression of apoptosis-related p53 and cleaved Caspase3(cl.Cas3)in the liver were significantly increased.In addition,the expressions of autophagy-related proteins Beclin1 and LC3 were increased while the expressions of p62 were decreased in liver tissues of model animals.Puerarin intervention effectively reversed the above factors.Conclusion In the LPS-induced liver injury model,puerarin inhibits the overexpression of autophagy-related proteins,reduces liver cell death,and improves the pathological phenotype of liver damage in mice.The mechanism may be achieved by down-regulating the expression of TLR4.It is suggested that puerarin treatment can be used as a new way to treat liver injury.