Research On The Mechanisms Of Giardia Duodenalis-Induced Apoptosis And Anti-Apoptosis In Intestinal Epethelial Cells

Giardia duodenalis is a flagellated protozoan.Giardia is transmitted through the ingestion of the infective cyst stage shed in human or animal faeces and might be present in faecally contaminated water,food,or fomites.Giardia is a non-invasive protozoan parasite infecting the upper small intestine causing acute,watery diarrhea or giardiasis in 280 million people annually.Studies have shown that Giardia trophozoites or their excretory-secretory proteins（ESPs）can induce apoptosis of intestinal epithelial cells（IECs）,and the apoptosis of IECs is the main reason for the symptoms of giardiasis.The molecular mechanisms by which Giardia induces apoptosis of IECs and the cellular defense against the parasite remain unclear.This study established an in vitro model of Giardia and IECs,further revealed the mechanism of IEC apoptosis caused by Giardia infection and the anti-apoptotic mechanism of host cells,and provided new insights into the interaction mechanisms between Giardia and IECs and the pathogenesis of giardiasis,as well as provided important implications for the development of new treatment strategies for giardiasis.When IECs（Caco-2 cells and HT-29 cells）were stimulated by Giardia,cellular RNA and protein were extracted for quantitative PCR（q PCR）and Western blot experiments.The results showed that cyclooxygenase-2（COX-2）transcription and protein levels were significantly upregulated in IECs during Giardia infection.In the CCK-8 assays,the COX-2 inhibitor NS398reduced the viability of Caco-2 and HT-29 cells exposed to Giardia.It is clear that cells must keep the levels of ROS and NO in check for survival,growth,and apoptosis.Here,I investigated if COX-2 could regulate IEC ROS/NO production during in vitro infection of Caco-2 and HT-29cells by Giardia.Giardia exposure increased the cellular ROS levels,and COX-2 inhibition promoted this process.In contrast,the release of NO from IECs decreased gradually following Giardia exposure,and COX-2 inhibition augmented the reduction of NO release.The data indicated that COX-2 could regulate the levels of ROS and NO during Giardia-IEC interactions.Acridine orange（AO）/ethidium bromide（EB）double staining was performed to assess the potential regulatory role of COX-2 in Giardia-induced IEC apoptosis.Upon Giardia exposure,more apoptotic cells stained orange were seen in NS398-pretreated groups.In contrast,apoptosis induced by Giardia exposure was blunted by preadministration of rebamipide to overexpress COX-2 level in IECs.Therefore,COX-2 potentially operated as an anti-apoptotic regulator during Giardia infection.I further examined the effects of COX-2 on the expression levels of apoptosis-related proteins.As expected,exposure of IECs to Giardia induced the cleavage of caspase-3（CASP3）and PARP,and preincubation of cells with NS398 before exposure enhanced the cleavage of CASP3 and PARP.We also found that NS398 pretreatment could promote the downregulation of the inhibitors of apoptosis proteins c IAP-2 and survivin induced by Giardia exposure.By contrast,COX-2 overexpression by its agonist rebamipide had remarkable inhibitory effect on IEC apoptosis induced by Giardia exposure.Taken together,Giardia exposure upregulated COX-2 expression in IECs,and it played a role in modulating cell survival and anti-apoptotic process.MAPK（p38/ERK）and AKT signaling pathways affect multiple cellular processes,including cell proliferation and apoptosis.The changes in the phosphorylation levels of MAPK/AKT-related proteins were first evaluated.There was a significant increase in the phosphorylation of p38,ERK1/2,and AKT within hours after Giardia exposure.p38 inhibitor SB202190,ERK1/2 inhibitor SCH772984,and AKT inhibitor MK-2206 2HCl were able to block the upregulation of COX-2 induced by Giardia exposure,revealing an association between p38/ERK/AKT signaling and COX-2.TLR4 inhibition by its inhibitor TAK-242 significantly inhibited both the p38 phosphorylation and COX-2 expression in IECs exposed to Giardia,while there were no observed changes for ERK and AKT.This research then assessed the potential influence of TLR4/MAPK/AKT signaling on ROS/NO generation and the downstream apoptotic cascades.Inhibition of those signaling proteins led to a significant increase in ROS production and a significant decrease in NO production in IECs exposed to Giardia,except for the effect of TLR4inhibition on ROS production.As reflected in AO/EB staining analysis,after Giardia exposure,more obvious apoptosis-inducing effects were observed when TLR4,p38,ERK1/2,and AKT were inhibited.In addition,inhibition of TLR4,p38,ERK1/2,and AKT significantly increased the levels of CAPS3 and PARP cleavage and repressed the expressions of c IAP-2 and survivin in IECs after exposure to Giardia.In the context of Giardia-IEC interactions,considering the formerly established close links between TLR4/MAPK/AKT signaling and COX-2 and between COX-2 and ROS/NO/apoptosis,it can be inferred that COX-2-mediated ROS/NO regulation and anti-IEC apoptosis was possibly controlled by p38/ERK/AKT signaling or TLR4-dependent p38 signaling.Immunofluorescence and Western blot analyses showed that nuclear NF-κB p65 was significantly increased after exposure of IECs to Giardia.We also observed that COX-2 up-expression in IECs caused by Giardia exposure could be blocked by NF-κB p65 inhibition with its inhibitor JSH-23.After Giardia exposure,NF-κB p65 inhibition could result in a significant increase/decline in ROS/NO production in IECs and also enhance IEC apoptosis as reflected by both AO/EB staining and Western blot analyses.This research assayed if NF-κB p65 nuclear translocation was associated with TLR4-dependent p38 signaling.As expected,NF-κB p65 nuclear translocation in IECs caused by exposure to Giardia could be attenuated by both TLR4 and p38 inhibition,and this was confirmed in the following immunofluorescence analysis.Combined with former findings,this research could clearly figure out that COX-2-mediated anti-IEC apoptosis during Giardia infection is possibly dependent on TLR4-p38 signaling-controlled NF-κB nuclear translocation.The present study was conducted to determine the potential anti-apoptotic function of COX-2 in IECs exposed to Giardia trophozoites;investigate the probable signaling mechanisms regulating COX-2 expression,ROS/NO production,and IEC apoptosis;and try to reveal the interrelation.Based on a model of interaction between Giardia,excretory-secretory proteins（ESPs）or purified protein and IECs（Caco-2 and LS174 cells）,the study found that Giardia stimulation increased the up-regulated expression of the endoplasmic reticulum（ER）stress marker protein GRP78 at both transcription and protein levels,and activated the PERK receptor,resulting in phosphorylation of e IF2α,upregulation of ATF-4 expression,and activation of PERK/e IF2α/ATF-4/CHOP pathway.According to the q PCR results,Giardia stimulation increased the transcription levels of several chaperone proteins in IECs.The fluorescence microscope and fluorescence microplate found that Giardia stimulation increased the ROS level of LS174 cells,and the Western blot results showed that treating cells with ROS inhibitor NAC alleviated the increase of GRP78 and CHOP levels and p38 phosphorylation level caused by Giardia stimulation,and inhibited the activation of PERK/e IF2α/ATF-4 pathway.When the cells were pretreated with p38 inhibitor SB202190,the activation of GRP78/PERK/e IF2α/ATF-4 pathway was also mitigated.When Fluo-4 probe was used to detect the intracellular calcium ion(Ca²⁺)level,the fluorescence microscope and fluorescence microplate found that Giardia stimulation caused the increase of intracellular Ca²⁺level.The cells were pretreated with intracellular Ca²⁺chelating agent and ER Ca²⁺pump inhibitor,and then Western blot was used to detect the ER stress.The results showed that ER Ca²⁺pump inhibitor alleviated the activation of GRP78/PERK/e IF2α/ATF-4/CHOP pathway caused by Giardia stimulation.The q PCR results showed that the pretreatment of cells with the TLR4 inhibitor TAK-242 mitigated the up-regulation of chaperone expression for Giardia stimulation.Western blot analysis found that the pretreatment with TLR4 inhibitor also alleviated the activation of GRP78/PERK/e IF2α/ATF-4 pathway and increase of ROS level induced by Giardia stimulation.CCK-8 and AO/EB double staining results showed that the inhibition of TLR4advanced the decrease of cell viability induced by Giardia infection and accelerated the apoptosis.Griess Reagent test showed that the inhibition of CHOP transcription by si RNA technology alleviated the reduction of NO level in cells caused by Giardia stimulation,of TLR4/ROS/p38furthered the reduction of NO release,and of ER Ca²⁺pump alleviated the reduction of NO level in cells caused by Giardia stimulation.The reduction of NO level was mitigated through supplementing arginine.LPS was used to induce the expression of i NOS in cells in advance,and then the influence of TLR4/ROS/p38/Ca2+/CHOP on the transcription level of i NOS was explored by relevant inhibitors.The q PCR results showed that inhibition of ER Ca²⁺pump or CHOP alleviated the reduction of i NOS transcription level caused by Giardia stimulation,and of TLR4/ROS/p38 further decreased the i NOS transcription level.The CCK-8 results showed that inhibition of ROS/CHOP or ER Ca2+pump mitigated the decrease of cell viability caused by Giardia stimulation,and of TLR4/p38 further reduced the cell viability caused by Giardia stimulation.Western blot results showed that inhibition of ROS/CHOP or ER Ca²⁺pump alleviated the decrease of c IAP-2 and surviving expression levels and endogenous apoptosis resulting from Giardia stimulation,and of TLR4/P38 exacerbated the above phenomena.The test results showed that the stimulation of intestinal epithelial cells by Giardia-secreted proteins also induced ER stress and activated the GRP78/PERK/e IF2α/ATF-4/CHOP pathway,while inhibition of TLR4/ROS/p38 or ER Ca2+pump could alleviate the ER stress.Giardia-secreted proteins reduced the activity of IECs and induced apoptosis.In this study,10 Giardia-secreted proteins（PNPO,PPIB,2 kinds of tenascin and 6 kinds cathepsin B）were expressed and purified in vitro.After screening by q PCR and Western blot,the study found that recombinant protein PPIB could activate the GRP78/PERK/e IF2α/ATF-4/CHOP pathway in IECs,and inhibition of TLR4/ROS/p38 could alleviate the ER stress caused by PPIB stimulation.However,apoptosis induced by PPIB stimulation was not found within 12 h.Overall,this research first found that COX-2 involved the process of attenuating Giardia-induced IEC apoptosis.The anti-apoptotic process mediated by COX-2 was confirmed to be regulated by MAPK/AKT signaling or by TLR4-dependent p38-NF-k B signaling.The Giardia-induced ROS increase and NO decrease have been identified as IEC apoptosis stimulators.Here,the research provided new findings that the levels of ROS/NO could be influenced by MAPK/AKT/NF-k B signaling-controlled COX-2 regulation.On the other hand,sub-stimulation of Giardia induces the occurrence of ER stress in IECs,and ER stress induces apoptosis.Both TLR4/ROS/p38 and Ca²⁺in the ER can affect the GRP78/PERK/e IF2α/ATF-4 pathway in terms of the underlying mechanism of ER stress.The Giardia-secreted protein PPIB can also cause ER stress in IECs,and its potential impact needs to be further studied.This study not only provides new evidence for the pathogenic mechanism of giardiasis and the immune mechanism of the host against Giardia infection,but also provides new ideas for clinical prevention and treatment of giardiasis.