hanges of H22 cells have been observed by inverted microscope. B The viability of H22 cells was measured by MTT assay just after MPEE remedy for 24 and 48 h. D The viability of BEL-7404, HepG2 and NCTC1469 cells just after MPEE remedy for 24 h. G The viability of splenocytes from C57BL/6 mice immediately after MPEE remedy for 24 h. Information were analyzed by ANOVA. p 0.05; p 0.01; p 0.001 when compared with untreated groupZhou et al. Chin Med(2021) 16:Web page 7 ofFig. 2 Nuclear morphology and cell cycle distribution of H22 cells upon MPEE remedy. H22 cells have been treated with unique CYP11 Inhibitor manufacturer concentrations of MPEE for 24 h. A Following EP Inhibitor Biological Activity staining with Hoechst 33258, nuclear morphology of H22 cells was observed by inverted fluorescence microscopy. The arrows indicated the chromosomal condensation. B Cell cycle phase distribution was analyzed by flow cytometry following PI staining. D Heatmap of clustered cell cycle linked genes as evaluated by transcriptome analysis. E The mRNA levels of Cdk2, Cyclin D1, Gadd45, Cdk1, Mcm2, Mcm4, Cyclin B1 and Cdc25b had been analyzed by qRT-PCR. F The protein levels of Cyclin B1, Cyclin D1 and Cdk2 were detected by Western blot. Information were analyzed by ANOVA. p 0.01; p 0.001 compared to untreated groupZhou et al. Chin Med(2021) 16:Web page 8 ofFig. 3 The apoptosis of H22, BEL-7404 and HepG2 cells induced by MPEE remedy. Distinctive concentrations of MPEE have been applied to treat H22, BEL-7404 and HepG2 cells for 24 h. A The apoptosis and necrosis of H22 cells were analyzed by flow cytometry following Annexin V/PI staining. D The apoptosis and necrosis of BEL-7404 and HepG2 cells had been shown. Information were analyzed by ANOVA. p 0.05; p 0.01 p 0.001 in comparison with untreated groupmitochondria-dependent pathway and found that the levels of cleaved caspase-9 and -3 had been considerably increased by MPEE remedy compared together with the untreated handle. At the same time, MPEE promoted the cleavage of caspase-8 (Fig. 4E; Further file 1: Fig. S1). Sequentially, the upregulated level of cleaved DNA repair enzyme of poly (ADP-ribose) polymerase (PARP) was observed. The results recommended that caspase cascade was involved within the apoptosis induced by MPEE. To investigate the part of caspase in the induction of apoptosis, H22 cells were pretreated with Z-VAD-FMK(FMK, a broad-spectrum caspase inhibitor) and AcDEVD-CHO (CHO, a caspase 3 inhibitor), and after that treated with MPEE. Soon after 24 h, the apoptosis of H22 cells was analyzed by flow cytometry. The pretreatment of FMK and CHO significantly decreased the apoptosis of H22 cells induced by MPEE (Fig. 5A ), suggesting that mitochondria-dependent pathway partially mediated MPEE-induced apoptosis.Zhou et al. Chin Med(2021) 16:Web page 9 ofFig. 4 The effects of MPEE on m and caspase cascade in H22 cells. H22 cells had been treated with various concentrations of MPEE for 24 h. A, B Cells had been stained with JC-1 along with the fluorescence alterations have been analyzed by flow cytometry. C The protein levels of Bax, Bcl-2 and cytochrome c were detected by Western blot. D The mRNA levels of Bax and Bcl-2 were analyzed by qRT-PCR. E The levels of cleaved-caspases and -PARP were detected by Western blot. Data were analyzed by ANOVA. p 0.001 when compared with untreated groupMPEE induced reactive oxygen species (ROS) production and endoplasmic reticulum (ER) stressIt has been reported that ROS production was involved inside the induction of mitochondrial dysfunction and ER strain [26]. We identified that MPEE significantly induced ROS production working with both flow cytometry a