# Term Identifier Porcine parvovirus infection activates mitochondria-mediated apoptotic signaling pathway by inducing ROS accumulation Sentence Abstract Sentence Background: Sentence Porcine parvovirus (PPV) infection primarily causes reproductive failure of pregnant swine and results in host cell death. Sentence Boars, as an important disseminator, shed PPV to sows via semen. Sentence PPV infects and numerously replicates in boar testicle, which results in damage of swine testicle in vivo. Sentence Reactive oxygen species (ROS), a mediator of cell apoptosis, play a crucial role in the mitochondria apoptotic pathway. Sentence However, whether PPV infection induces ST cells apoptosis and ROS accumulation is still unclear. Sentence Methods: Sentence To determine the effects of PPV infection on the apoptosis, we detected morphological changes, DNA ladder, activities of caspases, and expression of PARP in PPV-infected ST cells. Sentence Moreover, aiming to investigate the effect of PPV infection on the mitochondrial apoptotic pathway and ROS accumulation, we detected the Δψm, apoptosis-related genes, and ROS. Sentence To investigate the role of ROS in the process of PPV-induced apoptosis, the ST cells were infected with PPV and treated with the ROS antioxidants. Sentence The ROS level was measured using Reactive Oxygen Species Assay Kit and the Δψm, expression level of Bcl-2, translocation of Bax, and redistribution of mitochondria cytochrome c were tested. Sentence In this study, we demonstrated that PPV infection could induce apoptosis that was characterized by morphological changes, DNA fragmentation and activation of caspases. Sentence Moreover, PPV infection suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, decreased the mitochondrial transmembrane potential, and triggered the release of cytochrome c, which caused the subsequent activation of caspase-9 and caspase-3 and initiation of apoptosis. Sentence However, during the process of PPV-induced apoptosis, the protein levels of Fas and FasL were not affected. Sentence Further studies showed that PPV infection caused ROS accumulation. Sentence Inhibition of ROS could reduce mitochondrial transmembrane potential and could significantly block ST cells apoptosis via suppressing Bax translocation, cytochrome c and caspase-3 activation. Sentence Conclusions: Sentence All these results suggest that PPV-induced ROS accumulation mediates apoptosis in ST cells, which provided theoretical basis for the molecular pathogenesis of PPV infection. Sentence PPV is a member of parvoviridae, containing a negative single-stranded DNA genome [1, 2] . Sentence PPV infection may lead to reproductive failure, including infertility, embryonic death, stillbirth and fetal mummification in pregnant sows [3] . Sentence PPV infection mainly causes reproductive clinical syndrome, including infertility, abortion, stillbirth, neonatal death, and reduced neonatal vitality [4] . Sentence Boars play an important role in dissemination of PPV. Sentence It was reported that PPV infection damages both uterine [5] and testicles [6, 7] in vivo. Sentence Swine testicular is the reproductive organ and responsible for generating semen, which serve as one of the transmission routes of PPV. Sentence Therefore, we use ST cells to investigate the mechanism of PPV infection in this study. Sentence The infection of animal virus such as human parvovirus [8, 9] , canine parvovirus [10] may induce host cells apoptosis and is a major factor of cell death and tissue damage in viral diseases. Sentence Previous studies indicated that PPV infection caused apoptosis via p53 pathway in PK-15 cells [11] . Sentence Although, we previously demonstrated that PPV induced apoptosis PK-15 cells, whether PPV induces ST cells apoptosis and the role of ROS in apoptosis are unknown. Sentence Apoptosis, a programmed cell death (PCD), is an important pathological cellular response. Sentence In vertebrate cells, the apoptotic pathways mainly include the extrinsic and the intrinsic pathways [12] . Sentence The extrinsic apoptotic pathway is induced by stimulation of death receptors that belongs to the tumor necrosis factor receptor (TNFR) family, such as Fas. Sentence The induction of intrinsic apoptotic pathway is associated with mitochondria upon activation that is highly regulated by ROS [13] . Sentence ROS, known as toxic products of cellular metabolism, are mainly produced by the mitochondria in most vertebrate cells and act as signaling molecules. Sentence Moreover, growing evidence suggests ROS are involved in regulation of many pathological processes such as cellular apoptosis [14, 15] . Sentence However excessive ROS is toxic to cells, especially in the process of apoptosis. Sentence It is indicated that ROS accumulation was able to enhance apoptosis by collapsing the mitochondrial potential, inducing mitochondrial oxidation channel, and releasing cytochrome C from mitochondria to the cytosol [16] . Sentence ROS accumulation is also linked to virus-induced apoptosis. Sentence For instance, parvovirus H-1 infection induces apoptosis via mediating ROS accumulation [17, 18] . Sentence We have reported that PPV infection could be able to trigger cell apoptosis through mitochondria-mediated pathway [19] . Sentence However, whether ROS are associated with PPV-induced apoptosis is still unclear. Sentence In this study, we asked whether PPV infection could induce apoptosis via ROS accumulation in ST cells. Sentence To address this issue, we detected whether PPV infection causes apoptosis in ST cells. Sentence In addition, the ROS induced by PPV infection was assessed compared with mock infection. Sentence The results showed that PPV infection could triggers ST cell apoptosis via PPV-induced ROS accumulation. Sentence To determine the role of PPV infection in apoptosis of ST cells, we observed the morphological change of PPVinfected ST cells. Sentence PPV-infected cells started to show typical apoptotic features including chromatin condensation (Fig. 1a , indicated by yellow arrows) after 24 hpi and nuclear fragmentation compared with mock-infected cells (Fig. 1a , indicated by white arrows) after 36 hpi. Sentence Next, we detected the pattern of chromosomal DNA fragmentation and found that DNA ladders appeared at 48 hpi in PPV infected cells at 1.0 MOI (Fig. 1b) . Sentence Then the apoptotic rate was assessed using flow cytometry. Sentence As shown in Fig. 1c , the apoptotic rate of PPV-infected cells was increased at 24 hpi in comparison with the mockinfected cells and appeared time-dependent. Sentence To investigate the mechanism of apoptosis induced by PPV infection, we further examined the activities of several vital caspases, including caspase-3, -8 and -9. Sentence The results indicated that the caspase-9 and -3 were activated after PPV infection at 1.0 MOI as early as 12 hpi (Fig. 1d ). Sentence However the activity of caspase-8 was not affected by PPV infection. Sentence In addition, poly-ADP-ribose polymerase (PARP), an indicator of caspase-3 activation, was cleaved in PPV-infected ST cells at 12 hpi in a timedependent manner (Fig. 1e ). Sentence In addition, the Fas and FasL were not affected (Fig. 1f ) . Sentence Taken together, these results indicated that PPV infection induced apoptosis in ST cells through activating caspase-9 and caspase-3. Sentence Δψm plays an important role in the process of cell apoptosis. Sentence Once the Δψm is collapsed, mitochondria-mediated apoptotic signaling pathway will be activated. Sentence To determine the effect of PPV infection on Δψm, we detected the Δψm of PPV-infected and mock-infected ST cells. Sentence As shown in Fig. 2a , the percentage of Δψm-depolarized cells increased 6 % at 6 hpi and the level of depolarization was time-dependent. Sentence During mitochondria-mediated apoptosis, Δψm was regulated by Bcl-2 family members, such as pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. Sentence To explore the effects of PPV infection on Bcl-2 and Bax, western blot analysis was performed. Sentence The western blot results showed that PPV infection increased Bax expression and suppressed Bcl-2 expression (Fig. 2b) . Sentence To further detect whether the collapse of Δψm is associated with the release of cytochrome c and the translocation of Bax, the cytoplasmic and mitochondrial proteins were analyzed by western blot and respectively normalized to β-actin and Cox-4. Sentence The results showed that PPV-infection promoted the translocation of Bax from the cytosol to the mitochondria and enhanced the release of mitochondrial cytochrome c from mitochondria to cytosol at 12 hpi ( Fig. 2c and d). Sentence All these results indicated that PPV infection activated the mitochondrial apoptotic pathway in ST cells. Sentence ROS play an important role in the process of apoptosis [16, 17] and activate the mitochondria-mediated apoptotic pathway [19] . Sentence To investigate the role of ROS in the process of PPV-induced apoptosis, we detected ROS level in PPV-infected cells using DCFH-DA followed by observation under fluorescence microscopy. Sentence The results indicated that the fluorescence reached to the highest level at 24 hpi, and then decreased gradually along with the time course (Fig. 3a) . Sentence To further demonstrate the ROS level, quantitative analysis of ROS in PPV-infected cells was performed using flow cytometry. Sentence As shown in Fig. 3b and c, ROS was significantly accumulated at 24 hpi in comparison with mock-infected cells and subsequently decreased at 36 hpi continuously with the extension of time. Sentence All the data indicated that PPV infection could trigger ROS accumulation in ST cells. Sentence ROS accumulation may initiate cell apoptosis via activating the intrinsic mitochondrial pathway. Sentence Moreover, excessive ROS also leads to the damage of mitochondrial DNA and the permeabilization of mitochondrial outer membrane [20] . Sentence To further explore the effect of ROS on Δψm,we used ROS antioxidants PDTC (100 μM) (Merck, Germany) and NAC (100 μM) (Sigma-Aldrich, US) to suppress ROS production in ST cells infected with PPV at 1 MOI and 24 hpi and measured the ROS level and Δψm. Sentence Both PDTC and NAC, which did not show cytotoxicity at concentrations used (data not shown), significantly prevented the production of ROS (Fig. 4a) and reduced Δψm in PPV-infected cells (Fig. 4b) . Sentence These results indicated that PPV infection decreased mitochondrial membrane potential through inducting ROS accumulation. Sentence To further investigate the role of ROS accumulation in apoptosis, we analyzed the expression of Bcl-2, translocation of Bax, and the redistribution of mitochondria cytochrome c in ST cells infected with PPV at 1 MOI, 24 hpi and treated with PDTC and NAC. Sentence As shown in Fig. 4c , the decrease of Bcl-2, the translocation of Bax from cytoplasm to mitochondria, and the release of mitochondrial cytochrome c to cytoplasm in PPV infected ST cells were blocked by PDTC and NAC treatment in comparison with these in PPV-infected cells alone. Sentence In addition, caspase-3 activity and apoptotic rate were remarkably decreased in the ST cells infected with PPV at 1 MOI and 24 hpi in the presence of PDTC or NAC (Fig. 4d and e) . Sentence The data indicated that the accumulation of ROS induced by PPV infection contributed to the apoptosis at 1 MOI and 24 hpi. Sentence Apoptosis plays a predominant role in contributing to the pathogenic process of virus infection [21] and presents specific morphological and molecular changes, including chromatin margination, DNA fragmentation [22, 23] . Sentence Previous studies showed that PPV infection induced PK-15 apoptosis via mitochondria-mediated pathway [19] . Sentence Evidence shows that oxidative stress is an important regulatory factor of virus-induced apoptosis and induced by various triggers, including viral infection [24] . Sentence In this study, we found that PPV infection induced apoptosis of ST cells via activating mitochondrialmediated pathway and PPV-induced ROS accumulation was involved in regulating the process of apoptosis. Sentence Apoptosis is primarily classified into two pathways regulating cell death involving the continuous activation of caspases. Sentence "Of these, the extrinsic pathway participates in the ligation of particular ""death"" receptors (e.g., Fas) by recruitment of adaptor molecules to form the deathinducing signaling complex (DISC) and activate upstream caspases via caspase-8 [25] ." Sentence The mitochondrion-mediated (intrinsic) pathway is characterized by changed mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytoplasm. Sentence "In the cytoplasm, cytochrome c then is involved in the formation of a multiprotein complex known as the ""apoptosome"" and activates the caspase cascade through caspase-9 [26] ." Sentence In this study, we determined caspase-3 and caspase-9 were activated by PPV infection, but caspase-8 was not activated. Sentence This suggests that PPV infection induced the apoptosis of ST cells via mitochondrion-mediated pathway. Sentence Previous studies indicated that Bcl-2 family proteins as major regulators, including Bcl-2 and Bax, regulated the integrity of mitochondrial membranes, and the destruction of the mitochondrial membrane potential leaded to [27, 28] . Sentence Apoptosis can be triggered by the decreased mitochondrial permeability, which causes the release of apoptotic initiating factors, and the activation of caspase such as caspase-3. Sentence Some parvoviruses infection may lead to the increase of membrane permeability, subsequently inducing cell apoptosis. Sentence For example, bocavirus minute virus of canines (MVC) results in the time-dependent disruption of mitochondrial outer membrane potential (MOMP) [29] . Sentence Here we showed that mitochondrial membrane potential was significantly reduced in manner of time-dependent, which is consistent with the finding that MVC infection induced the disruption of MOMP. Sentence The oxidative damage also leads to the release of cellular toxic proteins such as cytochrome c from the mitochondria to the cytosol, subsequently triggering apoptosis [14] . Sentence To explore whether ROS was involved in the apoptosis induced by PPV infection, we investigated the direct effect of PPV infection on the oxidative profile. Sentence Time course analysis of PPV infected ST cells showed that ROS accumulation increased significantly, suggesting that PPV infection induced ROS accumulation. Sentence In this study, two ROS inhibitors, PDTC and NAC, significantly attenuated PPV-induced Bax translocation, the release of cytochrome c, caspase-3 activity, and apoptotic rate. Sentence The data indicate that ROS accumulation induced by PPV infection increases mitochondrial outer membrane. Sentence Our finding is consistent with that the canine parvovirus (CPV) and rat parvovirus H-1 (H-1PV) nonstructureal NS1 protein induced apoptosis via accumulation of intracellular reactive oxygen species (ROS) [18, 30] . Sentence Our data provide a link between ROS and apoptosis induced by PPV infection. Sentence The results demonstrated that PPV infection induced ST cells apoptosis via activating ROS accumulation and mitochondria-mediated apoptotic signaling. Sentence Our findings may provide insights into the interaction between PPV and host, and uncover the pathogenesis of PPV infection. Sentence Further studies on the mechanisms of PPV-induced apoptosis should give us new insights on the interplays between PPV and its host. Sentence ST cells were purchased from American type culture collection (ATCC, CRL-1746) and cultured in DMEM (Gibco, US) culture medium supplemented with 10 % heat-inactivated fetal bovine serum (Gibco, US) at 37°C in a 5 % CO 2 humidified atmosphere. Sentence The PPV YL strain was used as previously described [19] . Sentence Virus titers were determined by 50 % tissue culture infective doses (TCID 50 ) according to Reed and Muench method [31] . Sentence Mouse monoclonal antibodies against Bcl-2, Bax, Cytochrome c, Cox-4, PARP, Fas, FasL, and β-actin were purchased from Santa Cruz Biotechnology. Sentence Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Promega Corporation. Sentence Caspases activities assay of caspase-3, -8, and -9 Sentence ST cells were infected with PPV at 1.0 MOI and incubated in a 5 % CO 2 humidified incubator at 37°C. Sentence The cells were harvested at 0, 6, 12, 24, 36, and 48 hpi, washed with ice-cold PBS for three times, and then lysed with 200 μl lysis buffer on ice for 1 h. Sentence The caspases activities were tested with colorimetric assay kit (Abcam, US) following the manufacture's manual. Sentence Briefly, the protein concentration was measured using BCA Protein Assay Reagent (Pierce, US). Sentence 150 μg total protein of each sample was incubated with each caspase substrate in a 96-well plate for 4 h at 37°C. Sentence The absorbance was measured at 400 nm with spectrophotometer (BioTek, US). Sentence The cells were harvested and stained with Tetrechlorotetraethyl benzimidazol carbocyanine iodide 1 (JC-1) for 15 min at room temperature in the dark. Sentence Then the Δψm was assessed using JC-1 Mitochondrial Potential Detection Kit (Biotium Inc, US) following the manufacture's protocol. Sentence The fluorescence signal was measured at 550 nm and 485 nm. Sentence ST cells were harvested and then lysed on ice for 1 h with radioimmunoprecipitation assay (RIPA) buffer with 1 mM phenylmethyl sulfonylfluoride (PMSF). Sentence Isolation of mitochondrial and cytosolic proteins was carried out using the Mitochondria/cytosol Fractionation Kit (Pierce, US) according to the manufacture's protocol. Sentence Protein concentration was detected using BCA Protein Assay Reagent (Pierce, US). Sentence Equivalent amount of protein was loaded and (See figure on previous page.) Fig. 4 The role of ROS in PPV-induced apoptosis. a The effects of ROS inhibitors PDTC and NAC on ROS production. Sentence PPV-infected cells were treated with PDTC or NAC and stained with DCFH-DA. Sentence The ROS production was measured by flow cytometry. b The effect of ROS on Δψm in PPV-infected ST cells. Sentence The Δψm of cells infected with PPV and treated with PDTC or NAC was detected by flow cytometry. c The effects of ROS scavengers on the Bcl-2, Bax translocation, cytochrome c redistribution. Sentence ROS inhibitors-treated cells were infected with PPV and analyzed using western blot assay. Sentence The mitochondrial and the cytosolic proteins were respectively normalized to Cox-4 and β-actin. d The effect of ROS scavengers on the activity of caspase-3 in PPV-infected ST cells. Sentence Caspase-3 activity was measured using the caspase activity kit. e The effect of ROS scavengers on the apoptotic rate of PPV-infected cells. Sentence The apoptotic rate was measured by flow cytometry. Sentence The data represent three independent experiments ± SD. **, p < 0.01 in comparison with PPV infection alone subjected to electrophoresis on 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Sentence Subsequently, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp, US). Sentence The membranes were blocked with 5 % non-fat dry milk for 1 h at room temperature, and incubated with primary antibody at the dilution of 1:500 in antibody dilution buffer (PBS, 2 % non-fat dry milk) over night at 4°C. Sentence The blots were subjected to secondary antibody at the dilution of 1:10000 in antibody dilution buffer (PBS, 2 % non-fat dry milk) at room temperature for 1 h and detected with ECL reagent (Pierce, US). Sentence The ROS were detected using Reactive Oxygen Species Assay Kit (Sigma-Aldrich, US). Sentence Briefly, ST cells were seeded into 24-well plate and infected with PPV at 1.0 MOI. Sentence Then the cells were stained with 10 μM 2' , 7'dichlorodihydrofluorescein (DCFH-DA) at 0, 12, 24, 36, and 48 hpi and observed under the fluorescence microscope (AMG EVOS Inc, Israel). Sentence Mock-infected and PPV-infected cells were harvested at indicated times and washed twice with DMEM without fetal bovine serum. Sentence Then the cells were incubated with 10 μM DCFH-DA at 37°C for 30 min in the dark and analyzed using flow cytometry (Beckman Coulter EPICS ALTRA, US). Sentence Statistical analyses were calculated using one way analysis of variance (ANOVA). Sentence Significance was attained at p < 0.05. Sentence