ABSTRACT
Objective: To investigate the cell death-inducing effect of saikosaponin-d (SSD) on human hepatoma Hep3B cells and its potential mechanism. Methods: Hepatoma Hep3B cells were divided into five groups: blank control group, DMSO (vehicle) group, and three SSD treatment groups treated with various doses of SSD (5, 10, and 15 μmol/L). MTT assay was used to evaluate cell viability. Flow cytometer was employed to quantitatively detect the percentage of dead cells after Annexin V-FITC/PI double staining. Apoptosis was detected morphologically after Hoechst 33258 staining. The activity of caspase-3 apoptotic protease was determined by spectrophotometry. Cell morphologic changes were observed with an inverted microscope. Western blotting and Real-time PCR were employed to evaluate the expression levels of C/EBP homology (CHOP) protein and mRNA, respectively. Results: MTT assay showed that SSD inhibited the viability of human hepatoma Hep3B cells in concentration- and time-dependent manners. Sinomenine hydrochloride induced the death of Hep3B cells in a concentration-dependent manner indicated by flow cytometry. After staining with Hoechst 33258, the nuclei of SSD-treated cells showed nucleosomal agglutination, nucleosomal shrinkage and fragmentation under the fluorescence microscope, which are the characteristics of apoptotic cells. SSD significantly activated the key apoptotic executor caspase-3. The occurrence of paraptosis, characterized by extensive cytoplasmic vacuoles, was observed in SSD-treated cells under an inverted microscope. The pretreatment of a pancaspase inhibitor Z-VAD-FMK completely inhibited caspase-3 activity triggered by SSD, but only partially suppressed cell death and could not reduce the cytoplasmic vacuolation in SSD-treated cells. The protein and mRNA expressions of CHOP, a stress-inducible molecule, were upregulated by SSD, which could not be inhibited by Z-VAD-FMK. Conclusion: SSD can simultaneously induce caspase-dependent apoptosis and caspase-independent paraptosis in human hepatoma Hep3B cells. The upregulated expression of CHOP may be the mechanism involved in SSD-induced paraptosis.
ABSTRACT
Objective To investigate the expression of C/EBP homology protein (CHOP) in peripheral brain tissue of patients with severe traumatic brain injury (TBI) and its correlation with the injury severity.Methods The study included peripheral brain tissues of 41 TBI patients (TBI group).Another 16 autopsy specimens succumbed to other diseases (except for TBI or other central nervous system diseases) were selected as controls.The control group and TBI group were subdivided into immaturity group (≤18 years),adult group (18-59 years) and elderly group (＞59 years).According to Glasgow Coma Scale (GCS) on admission,TBI group was classified as severe TBI group (GCS of 6-8) and particularly-severe TBI group (GCS of 3-5).CHOP expression in peripheral tissues after TBI was compared in between different age,gender and GCS.Nerve cell apoptosis was detected by TUNEL technique and correlation between CHOP level and apoptotic number was analyzed.Results There were no age and gender differences regarding CHOP expression in control group (P ＜ 0.05).Compared with control group,expression of CHOP presented notable up-regulation in TBI group (P ＜ 0.05).Expression of CHOP presented no gender difference in TBI group (P ＞ 0.05),but its expression was lower in the aged than in adult or immaturity (P ＜ 0.05) as well as notably higher in particularly-severe TBI group than in severe TBI group (P ＜ 0.05).Nerve cell apoptosis in TBI group was far greater in number than that in control group (P ＜0.05).A positive correlation was observed between CHOP level and apoptotic index (r =0.72,P ＜ 0.05).Conclusion Expression level of CHOP after TBI is closely related to the injury severity and nerve cell apoptosis,but the apoptosis pathway induced by CHOP may not be a major factor in secondary brain injury after TBI in the aged patients.