Mechanism Of Mitochondrial-mediated Apoptosis Induced By WB Strain Of Giardia Duodenalis In Caco-2 Cells

Giardia is a parasite that mainly inhabits the small intestine of the host.It can infect a variety of mammals including humans and cause giardiasis.The clinical symptoms of the disease are generally abdominal pain,diarrhea,indigestion,and weight loss.Everyo ne can become a host for Giardia parasites,among which children,immunodeficiency people,and frail elderly people are more likely to be infected.For children,in addition to gastrointestinal symptoms,they also cause growth retardation and mental retardation problems.At present,there are no effective drugs and methods for the prevention and treatment of giardiasis,so the understanding of the pathogenic mechanism of Giardia plays an important role in the prevention and treatment of giardiasis.Previous studies have found that oxidative stress response is related to the occurrence of many diseases,and it also has a certain effect on intestinal damage,which may activate intestinal epithelial cell apoptosis.This study aims to explore the mechanism of Giardia-induced apoptosis of intestinal epithelial cells and the role of reactive oxygen species(ROS)in this process.In this study,Giardia trophozoites and Caco-2 cells were co-cultured to establish a Giardia infection cell model.After Giardia trophozoites acted on Caco-2 cells for different time durations,the cytotoxicity was detected by lactate dehydrogenase(LDH)kit.Flow cytometry was used to detect the effect of Giardia trophozoites on cell apoptosis and cell cycle,and the changes of cell morphology were observed with ordinary optical microscope and transmission electron microscope.Flow cytometry and fluorescence microscopy were used to detect changes in intracellular ROS levels after Giardia stimulation,and glutathione reductase kit was used to determine the content of glutathione(GSH).Then the molecules Bax and Bcl-2 were detected by immunofluorescence,q PCR,and Western Blot.JC-1 staining was used to detect intracellular mitochondrial membrane potential(MMP)after Giardia stimulation,and to detect cytochrome C(Cyt-C),caspase-3,9,and poly ADP ribose polymerase(PARP)content changes.ROS inhibitor NAC was used to detect its effect on cell activity and mitochondrial apoptosis pathway.Through in vivo experiments,NAC intervention was carried out on mice to observe their weight changes and diarrhea occurrence.Intestinal tissue sections were prepared and stained using HE assay to observe the changes of intestinal tissue after Giardia infection.The experimental results showed that,after stimulation of the Caco-2 cells with trophozoites at a ratio of 1:10,the LDH content was significantly increased in the culture medium(P < 0.01),indicating that trophozoite stimulation can cause Caco-2 cell damage.The following results showed that Giardia trophozoites can induce cell apoptosis and cause cell damage.At the same time,stimulation of Giardia trophozoites can hinder Caco-2 cell cycle(G1 phase)and promote the up-regulation of G0/G1 phase cell ratio and the down-regulation of S phase and G2/M phase cell ratio.It was found by electron microscopy that the trophozoites of Giardia can cause mitochondrial damage.Excessive ROS accumulation can lead to cell damage and induce cell apoptosis after Giardia trophozoite stimulation,as reflected by that the intracellular ROS content was increased in a time-dependent manner(P < 0.01),while the intracellular GSH level was decreased significantly(P < 0.01).The results indicated that trophozoite stimulation can affect the balance of redox reactions and cause oxidative stress reactions in cells.The increased permeability of mitochondrial membrane can cause the release of Cyt-C into cytoplasm,activate caspase cascade reaction,and induce cell apoptosis.Our results showed that the trophozoite stimulation promote the release of Cyt-C from mitochondria into cytoplasm and the activation of caspase-3,9,and PARP.We also used NAC to verify the effect of ROS on mitochondrial apoptotic pathway,showing that the activity of Caco-2 cells was significantly increased(P < 0.01),the level of Cyt-C released from mitochondria to cytoplasm and the level of activated caspase-3 were significantly reduced.These indicated that the endogenous apoptosis of Caco-2 cells induced by Giardia trophozoites was significantly dependent on ROS.In animal experiments,the effect of ROS on apoptosis induced by Giardia was verified by NAC intervention in mice.The results showed that the weight of mice infected with Giardia was reduced with diarrhea symptoms,and the intestinal segment was swollen after dissection.It was shown from HE staining that the villi of small intestine became shorter,atrophic,or even broken,the arrangement of intestinal epithelial cells was irregular,and the integrity of intestinal epithelial cell layer w as destroyed.The diarrhea of mice pretreated with NAC inhibitor was mild and there was no significant intestinal damage,which confirmed the role of ROS in inducing endogenous apoptosis.This study demonstrates that Giardia trophozoites stimulate Caco-2 cells to produce ROS to result in endogenous apoptosis.By regulating the balance of Bax and Bcl-2 levels in the cells,the mitochondrial outer membrane permeability is increased,the mitochondrial membrane potential is activated,the release of Cyt-C is promoted,caspase-3/9 are activated,and PARP is lysed,which eventually leads to apoptosis.Our findings should be helpful in understanding the pathogenic mechanism of Giardia and providing new directions for the prevention and treatment of giardiasis.