Abstract:

Objective To study the protective effect of Pien-Tze-Huang on acetaminophen-induced liver injury，and to clarify the possible mechanism. Methods The human normal hepatocytes（L02 cells） were divided into control group， APAP group （10 mmol·L^-1 APAP），APAP+PZH group （10 mmol·L^-1 APAP and 0.4 mg·mL^-1 PZH）， and PZH group （0.4 mg·mL^-1 PZH）. The survival rates of the cells in various groups were determined by MTT method， the morphology was observed by inverted microscope， and the apoptotic rates were detected by flow cytometry. The activities of superoxide dismutase （SOD） and lactate dehydrogenase （LDH） and the levels of malondialdehyde （MDA） in the cell supernant in various groups were detected by the kits， the reactive oxygen species（ROS） levels and the mitochondrial membrane potential （MMP） in the hepatocytes in various groups were detected by fluorescence probe. Western blotting method was used to examine the expression levels of apoptosis-related proteins， phosphatidylinositol 3 kinase （PI3K）/protein kinase B（AKT） signaling pathway proteins， nuclear factor-κB（NF-κB） signaling pathway proteins and inflammatory factors in the cells in various groups. Results The results of MTT showed that compared with control group， the survival rate of the cells in APAP group was markedly decreased （P<0.05）； compared with APAP group， the survival rate of the cells in APAP+PZH group was significantly increased （P<0.05）. Compared with control group， the number of the L02 cells in APAP group showed a decreasing and loosely arranged trend； compared with APAP group， the number and arrangement of the L02 cells in APAP+PZH group were notably improved. The results of flow cytometry showed that compared with control group， the apoptotic rate of the L02 cells in APAP group was significantly increased（P<0.05）； compared with APAP group， the apoptotic rate of the cells in APAP+PZH group was significantly decreased（P<0.05）. Compared with control group，the activity of LDH and level of MDA in the cells in APAP group were significantly increased （P<0.05）， while the activity of SOD was significantly decreased （P<0.05）； compared with APAP group，the activity of LDH and level of MDA in the cells in APAP+PZH group were significantly decreased（P<0.05）， while the activity of SOD was significantly increased（P<0.05）. Compared with control group， the fluorescence intensity of ROS in the cells in APAP group was significantly increased；compared with APAP group，the fluorescence intensity of ROS in APAP+PZH group was significantly decreased. Compared with control group， the MMP of the L02 cells in APAP group was significantly decreased； compared with APAP group，the MMP of the L02 cells in APAP+PZH group was significantly increased. The results of Western blotting showed that compared with control group， the expression levels of caspase-9， B-cell lymphoma 2（Bcl-2） associated X protein（BAX）， phosphorylated PI3K （p-PI3K）， phosphorylated AKT （p-AKT）， phosphorylated NF-κB inhibitor alpha （p-IKBα）， p-P65， phosphorylated inhibitor of kappaB kinaseβ （p-IKKβ）， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α（TNF-α） proteins in the cells in APAP group were significantly increased （P<0.05）， while the level of Bcl-2 proteins in the cells in APAP group was significantly decreased （P<0.05）； compared with APAP group， the levels of caspase-3， BAX， p-PI3K， p-AKT， p-IKBα， p-P65， p-IKKβ， IL-1β， IL-6， and TNF-α proteins in the cells in APAP+PZH group were significantly decreased， while the level of Bcl-2 protein was significantly increased（P<0.05）. Conclusion PZH may reduce the oxidative stress and inflammatory response in the cells by regulating the PI3K/AKT and NF-κB signaling pathway， therefore attenuate the L02 cell injury induced by APAP.

Key words: Pien-Tze-Huang, Drug-induced liver injury, Oxidative stress, Phosphatidylinositol 3 kinase, Protein kinase B, Nuclear factor-κB