ER stress及PUMA对5-FU诱导的肝脏细胞损伤与凋亡的影响

Effects of ER Stress and PUMA on 5-FU-induced Liver Cell Injury and Apoptosis

【目的】探讨内质网应激(ERS)及p53上调凋亡调控因子(PUMA)在5-氟尿嘧啶(5-FU)诱导的肝脏细胞的损伤与凋亡中发挥的作用。【方法】给予10只PUMA基因敲除小鼠(PUMA-KO)和20只野生型小鼠(PUMA-WT)腹腔注射等剂量的5-FU,建立小鼠肝脏损伤模型,其中10只WT小鼠同时给予腹腔注射ER stress抑制剂4-苯基丁酸(4-PBA),对照组10只KO小鼠及20只WT小鼠给予等量生理盐水,造模完成后收集小鼠的血清及肝脏组织,评估肝脏病理损伤程度,检测血清中ALT及AST表达水平以及肝脏组织中PUMA及GRP78表达水平,观察这些指标在不同处理组小鼠的变化。【结果】与WT对照组相比,5-FU组小鼠血清ALT及AST水平明显升高,H&E染色可见点状局灶性坏死,伴出血和炎症,TUNEL染色可见凋亡细胞明显增多(Z=3.78,P <0.001),PUMA及GRP78表达明显升高,提示PUMA及ER stress均参与了5-FU诱导肝脏细胞损伤和凋亡。同等剂量的5-FU刺激下,4-PBA组小鼠肝脏组织GRP78及PUMA表达均下调,同时TUNEL结果显示肝脏细胞凋亡减轻(χ~2=32.99,Z=3.78,P <0.001),进一步证实PUMA及ER stress均参与了肝脏细胞损伤和凋亡过程;随后发现在等剂量的5-FU诱导时,Cleaved caspase-3免疫组化染色显示PUMA基因敲除小鼠肝脏组织凋亡信号较WT小鼠明显减少(χ~2=33.99,Z=3.78,P <0.001),但两组小鼠GRP78的表达差异不具有统计学意义。以上结果说明,抑制ER stress会降低PUMA的表达并减轻肝脏细胞损伤与凋亡,敲除PUMA并不影响ER stress的激活,仅仅会减轻肝脏细胞损伤与凋亡。【结论】5-FU通过激活内质网应激上调PUMA的表达,进而促进了肝脏细胞的损伤以及凋亡。

【Objective】To investigate the role of ER stress and PUMA in 5-FU-induced liver cells injury and apoptosis.【Methods】We established 5-FU-induced liver injury models by intraperitoneally injecting the isodose 5-FU to 10 PUMA knockout mice(PUMA-KO)and 20 PUMA Wild type mice(PUMA-WT). Meanwhile,10 WT mice were intraperitoneally injected with 4-Phenylbutyric acid,the ER stress inhibitor. In the control group,10 KO mice and 20 WT mice were given the same amount of normal saline.After the modeling,serum and liver tissues of the mice were collected to assess the degree of liver pathological injury,measure the expression levels of ALT and AST in serum,and detect the expression levels of PUMA and GRP78 in liver tissues. The changes of these indicators in different treatment groups were observed and compared.【Results】Compared with the WT control group,serum ALT and AST levels were significantly increased in the 5-FU group,HE staining showed punctate focal necrosis,accompanied by hemorrhage and inflammation. TUNEL staining showed apoptotic cells were markedly added(Z = 3.78,P<0.001),and expressions of PUMA and GRP78 were obviously augmented,suggesting that both PUMA and ER stress were involved in 5-FU-induced liver cells injury and apoptosis. Then,in the 4-PBA group,we found that the expression levels of GRP78 and PUMA were down-regulated,and apoptosis of liver cells was reduced under the same dose of 5-FU(χ^2= 32.99,Z = 3.78,P<0.001),further confirming that both PUMA and ER stress were involved in this process. Subsequently,it was found that,when induced by the same dose of 5-FU,cleaved caspase-3 staining showed that the liver apoptosis signal of the PUMA knockout mice was lower than the WT mice(χ^2= 33.99,Z = 3.78,P<0.001),but the difference in the expression of GRP78 between the two groups was not statistically significant. In summary,the expression of PUMA was reduced and the apoptosis of liver cells was attenuated after the inhibition of ER stress;PUMA knockdown could not influence the activation of ER stress but alleviated apoptosis of liver cells.【Conclusions】PUMA mediates ER stress-up-regulated liver cells apoptosis in 5-FU-induced Chemotherapeutic liver injury.