[Exploring inhibitory effect and mechanism of Jianpi Huayu Jiedu Decoction inhibits gastric cancer HGC-27 cells by regulating ROS/PARP1/GSDME-mediated pyroptosis].

This study aims to investigate the mechanism by which Jianpi Huayu Jiedu Decoction(JHJD) inhibits gastric cancer HGC-27 cells via regulation of the reactive oxygen species(ROS)/poly(ADP-ribose) polymerase 1(PARP1)/gasdermin E(GSDME) pathway. HGC-27 cell xenografts were established in Balb/c-nu mice. After tumor formation, mice were allocated into a low-dose(7.5 g·kg~(-1) per day) JHJD group, a high-dose(15 g·kg~(-1) per day) JHJD group, and a model group. The mice in the JHJD groups were administrated with corresponding doses of JHJD and those in the model group with an equal volume of normal saline by gavage. The body mass, subcutaneous xenograft volume, and tissue mass of the mice were measured, and the tumor inhibition rate was calculated. The pathological changes of the xenograft tissue in each group were observed under a microscope after hematoxylin-eosin(HE) staining. The ROS level in the tissue was measured by a ROS kit, and the mRNA levels of PARP1 and GSDME in the xenograft were determined by RT-qPCR. HGC-27 cells were assigned into 5%, 10%, and 15% blank serum groups and 5%, 10%, and 15% JHJD-containing serum groups. Cell viability was detected by the CCK-8 assay, and intracellular ROS levels were quantified by the ROS kit. The morphological changes of cells were observed under a microscope. The protein levels of PARP1 and GSDME were determined by Western blot. The lactate dehydrogenase(LDH) release rate and adenosine triphosphate(ATP) levels in the cell supernatant were determined by LDH and ATP kits, and high mobility group box-1 protein(HMGB1) levels in the cell supernatant were determined by ELISA. In vivo experiments revealed that the low-and high-dose JHJD groups had lower volume and mass of subcutaneous xenografts than the model group(P<0.05), with the tumor inhibition rates of 20.43% and 30.75%, respectively. HE staining showed that cells in the model group exhibited pleomorphism, clustered arrangement, and nuclear division. Cells in the low-dose JHJD group showed reduced nuclear volume, deeply stained nuclei, lack of mitotic figures, and numerous vacuoles. Cells in the high-dose JHJD group showed nuclear pyknosis, nuclear fragmentation, and numerous cell debris. The mRNA levels of PARP1 and GSDME and the levels of ROS in the tissue in both the low-dose and high-dose JHJD groups raised compared with those in the model group(P<0.05). In vitro experiments showed that serum containing JHJD at all concentrations inhibited the proliferation of HGC-27 cells at the time points of 24, 48, and 72 h(P<0.05), and the inhibitory effect increased in a concentration-dependent manner. Morphological features of pyroptosis, including swelling, rupture, and release of vesicular contents at the cell edges, were induced by JHJD-containing serum to varying degrees, being the most pronounced in the 15% JHJD-containing serum group. The ROS levels and the protein levels of PARP1 and GSDME in all JHJD-containing serum groups were higher than those in the 5% blank serum group(P<0.05), and the differences between JHJD-containing serum groups were significant(P<0.05). Compared with the 5% blank serum group, JHJD-containing serum increased the LDH release rate and the ATP and HMGB1 levels in the supernatant(P<0.05) in a concentration-dependent manner, and the differences between groups were significant(P<0.05). In conclusion, JHJD may inhibit the proliferation of gastric cancer HGC-27 cells by inducing pyroptosis through upregulating the ROS/PARP1/GSDME pathway.