Dengue virus (DENV) belongs to the Flaviviridae
family, and it comprises four antigenically distinct serotypes (serotypes 1–4). DENV is transmitted to humans by the Aedes
]. Specific antiviral treatment or a global licensed vaccine against DENV is currently unavailable. The manifestations of DENV infection include symptoms that range from mild dengue fever (DF) to the more severe forms that include dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [2
]. The pathogenesis of DENV infection has been extensively investigated; however, it is still not completely understood.
Liver injury is very frequently identified in severe dengue infection [3
], and it has been found to be correlated with the clinical pathology [4
]. An example of this relationship was the observed elevation of aminotransferases. Reactive hepatitis with hepatic failure was also reported in patients with DHF/DSS [6
]. The World Health Organization (WHO) has included liver injury as one of the major disease criteria for severe forms of DENV infection [7
]. Immunocompetent and immunocompromised mouse models, to study DENV pathogenesis and antiviral strategies, have been reviewed [8
]. In AG129 mice (deficient in the interferon-α/β and -γ receptors), DENV infection resulted in high viral loads in the organs, and led to systemic diseases including liver injury and vascular leakage [11
]. In another study, a higher dosage of DENV infection in the AG129 mice resulted in disease pathogenesis, and also resulted in a high mortality rate [12
]. However, in immunocompetent mice, the typical signs of liver injury were observed and correlated with elevated liver transaminases [13
]. Hepatocyte apoptosis has also been reported in severe dengue cases [17
], and was significantly observed in the immunocompetent mouse model of DENV infection [19
]. Using this animal model, the vital role of mitogen-activated protein kinase (MAPK) signaling in the modulation of DENV-induced apoptosis leading to liver injury was revealed [19
]. Our previous studies found that the inhibitors of MAPKs were unable to restrict DENV production, but they improved liver injury by improving host responses, including cellular apoptotic events [20
Crocetin is a natural compound that is obtained mainly from the crocus plant (Crocus sativus L.) and Gardenia jasminoides
. The anti-inflammatory and immunomodulatory properties of crocetin were comprehensively examined in various diseases characterizing liver injury and apoptosis [23
]. Crocetin was reported to protect against morphine (opioid analgesic)-induced liver toxicity in the mouse [24
]. In an experimental fulminant hepatitis in rats, crocetin treatment was reported to reduce apoptosis, inflammation and oxidative stress responses [25
]. Crocetin was also reported to improve post-hemorrhagic shock in patients [26
]. In both in vitro and in vivo models of various disease conditions, the pharmacokinetic properties of crocetin were reviewed; however, its effects on DENV infection have not been studied. Accordingly, the present study aimed to investigate the efficacy of crocetin for the treatment of DENV-induced liver injury in a mouse model, and to identify its possible mechanism(s) of action.
2. Materials and Methods
2.1. DENV Infection and Crocetin Treatment in Mice
The animal experiment protocol was approved by the Siriraj Animal Care and Use Committee (SI-ACUP 019/2555; dated 22 April 2014) under the ethical principles and guidelines of the National Research Council (NRC) of Thailand. Male 8-week-old BALB/c mice were purchased from the National Laboratory Animal Centre (NLAC), Mahidol University, Nakhon Pathom, Thailand. The mice were acclimated in pathogen-free conditions at the laboratory animal facility of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Intravenous infection with 4 × 105 focus-forming units (FFU) of DENV-2 (strain-16681) via the lateral tail vein was performed. Treatment with crocetin (MP Biomedicals, CA, USA) at a dose of 50 mg/kg was administered via the same route at 1 h before, and 1 h and 24 h after, DENV infection. Other groups of mice that were mock-infected or infected with DENV-2 were treated with 2% dimethyl sulfoxide (DMSO) and used as control groups. Blood samples were collected on the third and seventh days after DENV infection for the hematology analysis and serum preparation. The blood sample collection on day 7 was taken just before the sacrifice. All study mice were euthanized with an intraperitoneal injection of sodium pentobarbital. The liver tissue sample from the experimental mice was harvested, sectioned and immediately stored in RNA later solution (Invitrogen, CA, USA) as per the manufacturer’s protocol.
2.2. Hematology and Liver Transaminases
Blood samples were collected on day 7 into an EDTA-containing vacutainer tube (BD Vacutainer® Blood Collection Tubes, NJ, USA). The hematology analysis was performed using a CELL-DYNTM 3700 automated hematology analyzer (Abbott, Chicago, IL, USA). For the preparation of serum, blood samples were allowed to clot and then centrifuged at 2000× g for 10 min. The liver transaminases were estimated using a Roche/Hitachi Model 902 chemistry analyzer (Roche Diagnostics, Rotkreuz, Switzerland).
2.3. Histopathology Analysis
Liver tissues that were harvested from study mice were fixed in 10 formalin in PBS. The liver tissues were thoroughly washed and paraffin-embedded. The paraffin-embedded blocks were sectioned and mounted on a glass slide for hematoxylin and eosin (H&E) staining. Results were obtained from 6 mice/group and the data was presented as a representative from each group.
2.4. DENV-NS1 Viral RNA Quantification from the Liver
DENV-NS1 viral RNA was quantified from the liver tissue using a previously reported protocol [22
]. Briefly, the total RNA from the liver tissue homogenates was prepared using a ZR Viral RNA Kit (Zymo Research, USA) and Invitrap Spin Universal RNA Mini Kit (Stratec Molecular, Birkenfeld, Germany), respectively. An in vitro transcription-derived DENV-NS1 RNA with known copies was 10-fold serially diluted (standard) and the samples prepared from the liver homogenates simultaneously underwent qRT-PCR in a Roche LightCycler 480 Instrument (Roche Applied Science, Penzberg, Germany). The Ct values of the serially diluted standard were used to plot a standard curve and were compared with the test samples. The Ct values were analyzed for determining the DENV NS1 copies in the test samples. Data were obtained from 8 mice/group. Results were represented as virus copies/µg of total RNA obtained.
2.5. Focus-Forming Unit (FFU) Assay in Liver Homogenates
FFU assay was performed to count the viral titers in the livers of the study mice, as previously described [22
]. Briefly, liver tissue samples were homogenized in Roswell Park Memorial Institute (RPMI) Medium and centrifuged at 14,000 revolutions per minute (RPM) for 12 min to obtain a clear supernatant. The obtained supernatant was filter-sterilized and used to determine the viral titer by FFU assay. Data were obtained from 6 mice/group and presented as FFU/mg of the liver tissue sample.
2.6. Superoxide Dismutase and Catalase Activity
The enzyme activity of superoxide dismutase (SOD) and catalase (CAT) was evaluated using commercial kits purchased from Abcam (Cambridge, UK) (ab65354 and ab83464, respectively). Liver homogenates were prepared from at least 6 mice/group, in a lysis buffer, and centrifuged at 14,000× g for 5 min at 4 °C to obtain a supernatant. The experiments were performed with the obtained supernatant according to the manufacturer’s instructions. The absorbance of the SOD and CAT reaction mixtures was read at 450 nm and 570 nm, respectively, using a Synergy™ microplate reader (Bio-tek Instruments, Inc., Winooski, VT, USA).
2.7. Gene Expression Profiler (RT-PCR Array)
RNA samples from the livers of study mice were prepared using an Invitrap Spin Universal RNA Mini Kit (Stratec Molecular, Birkenfeld, Germany). Equal concentrations of RNA samples were reverse-transcribed to cDNA using the SuperScript® III First-Strand Synthesis System (Invitrogen, CA, USA). The obtained cDNA was mixed with SYBR Green RT2 qPCR Mastermix (QIAGEN, Hilden, Germany) and aliquoted into an RT2 Profiler™ PCR Array (Qiagen, Hilden, Germany) containing 84 selected apoptosis-related genes. PCR amplification was performed in a Roche LightCycler 480 Instrument (Roche Applied Science, Rotkreuz, Switzerland), and Ct values were analyzed using the ‘Qiagen Data Analysis Center’ web platform (2–Ct analysis).
2.8. RT-PCR Analysis
RNA was prepared from the livers of study mice and quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent concentrations of RNA samples were prepared and converted to cDNA. The obtained cDNA was mixed with LightCycler®
480 SYBR Green Mastermix (Invitrogen, CA, USA) and the primer set for the individual gene of interest. The primer blast program was used for customized oligo designs that were used in the current study and the designs are shown in Table 1
. RT-PCR reactions were performed in a Roche LightCycler 480 Instrument (Roche Applied Science, Rotkreuz, Switzerland), and the obtained Ct values were normalized to the Ct value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—the housekeeping gene control. The results were further analyzed by the 2–Ct
method and the results of that analysis represent data from at least 6 mice/group.
2.9. Cytosolic and Nuclear Fractionation of Proteins from Liver Tissues
Liver tissue samples were homogenized and fractionated to cytosolic and nuclear fractions using a Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific, Waltham, MA, USA). Samples from 6 mice/group were used in the experiments. Briefly, the stored frozen tissues were thawed and homogenized in protease inhibitor-containing buffers. The cytoplasmic and nuclear fractions were separately collected by centrifugation according to the manufacturer’s instructions. The concentration of the cytoplasmic and nuclear protein fractions obtained were estimated and stored at −70 °C.
2.10. Western Blot Analysis
Protein samples that were prepared from the liver tissue homogenates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto a nitrocellulose membrane, and blocked with 5% bovine serum albumin (BSA) or 5% skim milk for an hour to prevent non-specific binding. The nitrocellulose membrane was incubated overnight with mouse-anti-glutathione peroxidase 1/2 (Gpx 1/2), or rabbit anti-total JNK1/2, or mouse anti-phosphorylated JNK1/2, or mouse anti-total p38 or mouse anti-phosphorylated p38, or rabbit anti-cleaved caspase-3 or mouse anti-heamoxygenase-1 (HO-1), or mouse anti-cycloxygenase-2 (COX-2), all of which were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The membrane was further incubated in the dark at room temperature with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Dako Denmark A/S, Glostrup, Denmark). Immune complexes were detected by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as the housekeeping gene control to normalize the experiments.
To characterize the nuclear translocation of NF-kB, the proteins obtained from the nuclear and cytoplasmic fractions were separately subjected to Western blot analysis using rabbit anti-NF-kB p65 or mouse anti-NF-kB p50 primary antibodies. The corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, CA, USA) was used to detect the immune complexes. GAPDH and Lamin B1 were used as the housekeeping gene controls for cytosolic and nuclear fractions, respectively.
The immunoblots obtained from 6 mice/group underwent densitometry analysis using the ImageJ program (United States National Institutes of Health, Bethesda, MD, USA), and the image intensity was reported.
2.11. Statistical Analysis
The results obtained were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered to be statistically significant.
Liver injury is most commonly observed in the severe forms of dengue infection [27
], and hematology parameters were used to predict the severity of disease in dengue patients [29
]. DENV-associated clinical indications, including leucopenia and thrombocytopenia, were explained by the previous finding that liver injury was linked with elevated transaminases [30
]. In immunocompetent mice (Male BALB/c mice of age 8 weeks), DENV-associated clinical symptoms were observed [32
]. These clinical responses were consistently observed in our experimental model in mice, and crocetin treatment at a dosage of 50 mg/kg of mice improved leucopenia, thrombocytopenia and liver transaminases in DENV-infected mice. Previously, the immunomodulatory and anti-inflammatory properties of crocetin with different dosage regimens were reviewed [34
]; the dosage of 50 mg/kg was widely used in mice with different disease conditions [36
]. The histopathological observations supported the improvements in liver injury when DENV-infected mice were treated with crocetin. In the present study, we injected DENV into mice via the intravenous (IV) tail vein, and treatment with crocetin was also given via the same route, which responded in improving the DENV-induced thrombocytopenia and leucopenia.
We evidenced viral replication in the liver of DENV-infected mice. Similarly, the viral copies in the serum samples on day 3 and day 7 were also seen in DENV-infected mice. In severe DENV-infected patients, a much higher viral load in the liver and serum samples was commonly observed [3
], which was consistently exhibited in our experimental model of DENV infection in mice. Moreover, our findings lead us to believe the liver is one of the major sites for DENV replication leading to liver injury. Previously, the hepatoprotective effects of crocetin on carbon tetrachloride (CCl4
)-mediated liver injury, via the modulating of the inflammatory responses, was reported [38
]. In an experimentally induced fulminant hepatic failure (FHF) model in rats, crocetin treatment suppressed the pro-inflammatory cytokines, oxidative stress and NF-κB activations [25
]. Based on these supportive studies, we aimed to primarily evaluate the effect of crocetin on DENV-induced liver damage. Our research group recently established the effectivity of N-acetyl cysteine (NAC) in reducing viral replication in DENV-infected HepG2 cells, as well as in an immunocompetent mouse model of DENV-induced liver injury [39
]. However, the JNK1/2 inhibitor, SP600125, was previously reported to reduce DENV replication in macrophages [40
], not in the liver of DENV-infected mice [20
]. Our results suggest that crocetin treatment was unable to restrict DENV production in the liver and serum of DENV-infected mice.
In the current study, we applied pre-, co- and post-treatments together, only aiming to identify whether crocetin treatment has any effect on DENV replication or host response, or both. In DENV-infected Huh7 cells, the effects of pre-, co- and post-treatment of sunitinib were explained [41
]. Our study in DENV-infected mice has this limitation, and more detailed studies are required to investigate the impact of crocetin treatment with pre-, co- or post-treatments, or their synergistic treatment options, and determine which treatment strategy works the best. That said, we identified that crocetin treatment did not have any direct impact on DENV production in the liver of DENV-infected mice, which suggests the improvements in the liver injury were possibly attained via modulating these host responses.
DENV replication was reported to initiate inflammatory and immunomodulatory responses in the host, causing liver damage [19
]. Evidence suggests that thrombocytopenia is associated with inflammatory responses [45
], and various oxidative stress-associated proteins were reported to contribute inflammatory responses and liver injury to DENV infection [46
]. A higher expression of cleaved caspase-3 was observed both in in vitro cultures [48
] and in DENV-infected mice exhibiting liver injury [22
]. This suggests the vital role of hepatic cell apoptosis in causing DENV-induced liver injury. Our findings are consistent with these findings, and interestingly, we found that DENV-infected mice treated with crocetin displayed a significant reduction in the expression of cleaved caspase-3. Our findings demonstrate the effectiveness of crocetin in restricting hepatic cell apoptosis in DENV-infected mice, and this finding is consistent with our observations of when MAPK inhibitors were used to treat DENV-infected mice [20
]. In an experimental model in rats, the administration of crocetin during resuscitation from hemorrhagic shock improved post-shock survival via the apoptotic pathways [49
]. Our results concluded that crocetin treatment did not directly influence DENV production in mice. However, and interestingly, crocetin treatment was able to improve liver injury by restricting hepatic cell apoptosis.
The interplay between apoptosis and oxidative stress in the liver of severe DENV-infected patients was correlated with DENV-associated disease pathogenesis and disease severity [50
]. In DENV-infected dendritic cells, regulating the cellular oxidative stress responses was reported to modulate DENV-induced apoptosis [46
]. Apoptosis-associated gene profiling, in association with caspase 3 expression, was thoroughly studied in DENV-infected Huh7 cells [51
]. In DENV-infected patients exhibiting thrombocytopenia, the effects of oxidative stress responses were previously described [52
]. Dendritic cells infected with DENV exhibited higher productions of intracellular reactive oxygen species (ROS) and were reported to cause apoptosis [46
]. The major antioxidant enzymes, including SOD, CAT and Gpx, were previously reported to significantly influence the maintenance of redox status in DENV-infected patients [53
]. Imbalance in these antioxidant enzymes contributed to elevated pro-inflammatory cytokines, including TNF-α and interleukin-6 [56
]. Our findings are consistent in DENV-infected mice, and crocetin treatment effectively maintained the enzyme activities of these antioxidants. Interestingly, Crocetin was previously reported to improve cardiac stress via the modulation of antioxidant enzymes, including SOD, CAT and glutathione (GSH), and apoptosis was observed to be significantly decreased [57
In porcine epidemic diarrhea virus-infected cells, ROS production led to apoptosis via the induced phosphorylation of p38 MAPK and JNK [58
]. Extrinsic and intrinsic pathways of apoptosis via caspases were previously reported to contribute to p38 MAPK signaling when A549 cells were infected with Newcastle disease virus (NDV) [59
]. Our results are consistent with these findings. Specifically, the higher expression of cleaved caspase-3 is the result of a higher phosphorylation of p38 and JNK, which leads to liver injury. In our study, higher expressions of inflammatory cytokines, including TNF-α, TRAIL, IL-6 and IL-10, as well as the pro-apoptotic factors, including Fas and Fas-L, were found to contribute to the phosphorylation of p38 and JNK. Higher expressions of pro-inflammatory cytokines were previously reported to be influential in DENV infection and apoptosis [60
]. In DENV-infected monocytes, the expression of pro-inflammatory cytokines, including TNF-α and IL-6, was reported to be elevated and influential in the disease progression [61
]. TRAIL was also found to be highly expressed in different cell lines when they were infected with DENV [62
]. The serum levels of the anti-inflammatory cytokine, IL-10, were significantly higher in the severe DENV-infected patients, and were established as a prognostic marker for severe DENV infection [63
]. This observation was similar in different studies [64
], and similarly, we found a significantly higher expression of IL-10 in DENV-infected mice. The plasma levels of FasL, TRAIL and TNF-α were identified to be crucial pro-apoptotic factors in DENV-infected patients [66
]. The higher expression of FasL was also reported to be a marker for the early course of DENV infection [67
]. In human primary monocytes, DENV-induced apoptosis was initiated via the death receptor, Fas [68
], and interestingly, the interaction of Fas with its ligand (FasL) was reported to initiate TNF-induced apoptosis in vascular endothelial cells [48
]. These inflammatory cytokines and pro-apoptotic factors were reported to exhibit a higher phosphorylation of p38 and JNK [20
], and our results were in agreement with this study. Crocetin treatment in DENV-infected mice effectuated a reduction in the expressions of pro-inflammatory cytokines, apoptosis, and phosphorylation of p38 and JNK—all of which suggest improvement in liver injury.
NF-kB is one of the major modulators of both inflammatory and immune responses via the canonical or non-canonical signaling pathway. The canonical pathway gets activated through a p65/p50 heterodimer when pathogens or inflammation interrupts the cellular homeostasis [69
]. This may lead to the translocation of p65 and p50 into the nucleus [70
]. In liver injury, inflammatory cytokines, including TNF-α and IL-6, were reported to initiate apoptotic cell death via caspase activation and JNK phosphorylation, finally resulting in NF-κB translocation [71
]. In another study, JNK and p38 signaling were reported to be crucial for the translocation of cytoplasmic NF-kB towards the nucleus [72
]. Crocetin was previously identified to moderate the NF-κ p65 signals leading to reduced inflammatory responses in mice [73
]. In the current study, we observed the translocation of both NF-κB p65 and NF-κB p50 in the liver of DENV-infected mice. Interestingly, crocetin reduced this nuclear translocation, leading to improvement in liver injury. The vital role of NF-kB in DENV infection was previously explained by the nuclear translocation of NF-kB-induced cytokine production [74
], and NF-kB and JNK signaling were reported to be crucial for DENV-induced COX-2 expression, which was previously shown to facilitate DENV replication [75
]. Besides, the inhibition of this NF-kB translocation was reported to activate HO-1 [76
], which is required for maintaining antiviral immunity [77
]. In the present study, we found HO-1 to be induced when DENV-infected mice were treated with crocetin. Therefore, NF-kB may be one of the important proteins that is modulated by crocetin treatment.