Honeysuckle (Lonicera japonica Thunb.), a traditional Chinese herb, has widely been used to treat pathogen infection. However, the underlying-mechanism remains elusive.
Aims of the study
To reveal the host microRNA (miRNA) profile with the anti-viral activity after honeysuckle treatment.
Materials and methods
Here we reveal the differentially expressed miRNAs by Solexa® deep sequencing from the blood of human and mice after the aqueous extract treatment. Among these overexpressed innate miRNAs both in human and mice, let-7a is able to target the NS1 region (nt 3313-3330) of dengue virus (DENV) serotypes 1, 2 and 4 predicated by the target predication software.
We confirmed that let-7a could target DENV2 at the predicated NS1 sequence and suppress DENV2 replication demonstrated by luciferase-reporter activity, RT-PCR, real-time PCR, Western blotting and plaque assay. ICR-suckling mice consumed honeysuckle aqueous extract either before or after intracranial injection with DENV2 showed decreased levels of NS1 RNA and protein expression accompanied with alleviated disease symptoms, decreased virus load, and prolonged survival time. Similar results were observed when DENV2-infected mice were intracranially injected with let-7a.
We reveal that honeysuckle attenuates DENV replication and related pathogenesis in vivo through induction of let-7a expression. This study opens a new direction for prevention and treatment of DENV infection through induction of the innate miRNA let-7a by honeysuckle.
- Dengue virus replication
Dengue virus (DENV), a mosquito-borne virus, belongs to the Flaviviridae family and has four serotypes (DENV1 to DENV4). The DENV genome contains a 5′ untranslated region (UTR), 3 structural proteins (capsid, C; membrane, prM; and envelope glycoprotein, E), 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), and 3′ UTR (Deubel et al., 1990 and Henchal and Putnak, 1990). Among them, non-structure protein 1 (NS1) is highly conserved among DENVs and is a 48 kDa glycoprotein which functions as a cofactor for viral RNA replication, and is expressed either as the membrane or soluble form at the early phase of infection (Muller and Young, 2013 and Winkler et al., 1989). In DENV2-infected human endothelial cells, NS1 protein is abundantly present in membrane/organelle compared to the peri-nucleus and cytoskeleton at 12 h post-infection (Poungsawai et al., 2011). NS1 can be secreted from the infected cells and accumulated in the supernatant as well as on the cellular membrane (Avirutnan et al., 2007). In addition, NS1 is involved in the formation of immune complexes and complement activation during DENV pathogenesis (Avirutnan et al., 2011). DENV infection causes diseases from mild dengue fever to severe and fatal dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) (Deen et al., 2006, Gubler, 1998, Mackenzie et al., 2004 and Swaminathan and Khanna, 2009). Currently, there are no effective DENV medications.
Honeysuckle (Lonicera japonica Thunb.) is a perennial evergreen plant, which is widely cultivated in China, Japan, Korea, and India ( He et al., 2013). It is used in traditional medicine owing to its pharmacological properties including neuroprotection ( Kwon et al., 2012, Kwon et al., 2011 and Weon et al., 2011), anti-oxidation ( Ku et al., 2009 and Leung et al., 2006), anti-inflammation ( Chen et al., 2012, Kang et al., 2010, Lee et al., 2001 and Park et al., 2005; Park, K.I. et al., 2012; Ryu et al., 2010; Tae et al., 2003; Tzeng et al., 2014; Yoo et al., 2008; Ziyan et al., 2007), anti-carcinogenesis ( Lee et al., 2013, Leung et al., 2008, Leung et al., 2006, Leung et al., 2005 and Liao et al., 2013; Park, H.S. et al., 2012; Yoo et al., 2008; You et al., 2011), as well as anti-bacterial and anti-viral effects (Wang et al., 2013). The aqueous extract of honeysuckle can relieve fever and flu-like symptoms (Shang et al., 2011). The activity of symptom relief induced by honeysuckle can be demonstrated by purification of the ingredients and tested in vivo. Phytochemical studies have shown that the bud of honeysuckle flower contains active ingredients which include chlorogenic acid, isochlorogenic acid, linalool, luteolin, and shuangkangsu (a novel cyclic peroxide) ( Li et al., 2009 and Shang et al., 2011). Although honeysuckle extract has been shown to exhibit anti-COX-2, anti-inflammatory, anti-oxidative, anti-microbial and anti-viral activities in vitro and in vivo, it remains unclear whether honeysuckle could affect the infection and replication of DENVs through induction of the innate microRNA (miRNA) expression.
MicroRNA is a small RNA that is known to be involved in the regulation of cellular activity (Baulcombe, 2004, Chapman and Carrington, 2007 and Stefani and Slack, 2008). In addition, miRNAs are able to regulate viral replication by targeting various viral mRNAs or viral RNA genomes per se ( He et al., 2013 and He et al., 2009; Hsu et al., 2007; Scaria et al., 2006). MiRNAs can regulate the targeted mRNA by interacting with the 3′UTR, 5′UTR, and coding region (Breving and Esquela-Kerscher, 2010). The interaction between miRNA and its target is usually via the seed sequence of miRNAs, which is located at the nucleotide position 2–8 ( Bartel, 2009). The sub-cellular location for miRNAs to execute function is in the cytoplasm, nucleus, and mitochondria ( Breving and Esquela-Kerscher, 2010 and Kren et al., 2009). Moreover, miRNAs can be transported out of the cell through the exsosome to execute its regulation activity in a distant recipient cell (Valadi et al., 2007).
Let-7 miRNA was first reported in Caenorhabditis elegans in the regulation of development, and is highly conserved in different species including C. elegans, mice and human ( Reinhart et al., 2000). Let-7 is widely expressed in the brain, cortex, midbrain, lung, trachea, heart, stomach, small intestine, colon, muscle, white and brown adipose tissues, liver, spleen, kidney, thymus, and blood ( Lagos-Quintana et al., 2002, Pasquinelli et al., 2000, Sempere et al., 2004 and Sun et al., 2009). However, the level of let-7 expression is relatively low in bone marrow, which contains many immature cells (Pasquinelli et al., 2000). This is consistent with a characteristic of let-7a that can inhibit cell proliferation-related genes including Ras ( Akao et al., 2006, He et al., 2010 and Johnson et al., 2005), c-Myc ( He et al., 2010 and Sampson et al., 2007), HMGA2 ( Lee and Dutta, 2007, Motoyama et al., 2008 and Sun et al., 2009), NIRF (He, X. et al., 2009), E2F2, and CCND2 (Dong et al., 2010). In addition, let-7a is involved in the expression of inflammation-associated cytokine interleukin-6 (Meng et al., 2007). However, whether let-7a can interact with the RNA of DENV both in vitro and in vivo is not known.
In this study, we clarified how honeysuckle suppresses DENV2 activity both in vitro and in vivo. This is the first report to demonstrate that miRNA let-7a as an innate immune response can be induced by honeysuckle aqueous extract, and has both preventive and therapeutic potential against DENV2 infection in vivo.
2. Materials and methods
2.1. Cell culture and virus
Human hepatoma (Huh7; JCRB number: JCRB0403), baby hamster kidney (BHK-21; ATCC number: CCL-10), and Aedes albopictus cells (C6/36; ATCC number: CRL-1660) were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO, NY, USA) supplemented with 10% fetal bovine serum (FBS; Trace Biosciences, Sydney, Australia), penicillin (200 U/ml; Biowest, Nuaillé, France) and streptomycin (100 μg/ml; Bioweat). Huh7 and BHK-21 cells were grown at 37 °C with 5% CO2. C6/36 cells were grown at 28 °C with 5% CO2.
DENV2 (PL046) and DENV3 (strain 739079A) were grown in C6/36 cells with 2% FBS/DMEM. Virus in the culture supernatant was isolated by centrifugation and filtration through a 0.22 µm filter (Millipore, MA, USA) before storage at −80 °C. Virus titer was determined by plaque assay using BHK-21 cells.
DENV2 NS1 sequence (nt 3313-3333; based on DENV2 NGC strain) and its mutant, which are shown in Figs. 1 and 3A, were amplified from DENV2 infectious clone (Lee et al., 2005) and constructed into a pMIR-Report™ vector (Applied Biosystems, NY, USA) to form pMIR-DENV2-NS1-WT and pMIR-DENV2-NS1-Mut.
2.3. Luciferase assay
The reporter plasmid, pMIR-DENV2-NS1-WT or pMIR-DENV2-NS1-Mut, was co-transfected with pRL-TK (an internal control) and miRNA into Huh7 cells with Turbofect™ (Fermentas, PA, USA). The reporter and control plasmids were mixed at a ratio of 4:1 for transfection. After transfection and incubation, cells were lysed and the luciferase activity was measured using Dual-Luciferase Reporter assay kit (Promega, WI, USA) and determined by luminometer (Minilumate LB9506, Germany).
2.4. RT-PCR and real-time PCR
DENV NS1 RNA and negative-stranded NS1 RNA were reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). PCR was performed using YEA DNA polymerase (Yeastern Biotech, Taipei, Taiwan) and TaKaRa Gradient PCR machine. To reversely transcribe miRNAs into cDNA, a NCode™ VILO™ miRNA cDNA Synthesis Kit (Invitrogen, CA, USA) was used. Real-time PCR was conducted using the Step One Real-Time PCR System (Applied Biosystems).
2.4.1. Primers used in this study are listed as follows
The reverse transcription primer for negative-stranded DENV2 NS1 was Taq-3.2 (Wati et al., 2007), and the cDNA of DENV2 NS1 negative-stranded RNA was amplified by PCR using the primers Taq and Taq-3.1 (Wati et al., 2007).
2.4.2. Primer Taq-3.2 sequence
2.4.3. Primer Taq sequence
2.4.4. Primer Taq- 3.1 sequence
To detect all 4 serotypes of DENV, the universal PCR primers AD3 and AD4 were used (Henchal et al., 1991).
2.4.5. AD3 primer sequence
2.4.6. AD4 primer sequence
To detect endogenous beta-actin mRNA, beta-actin-F and beta-actin-R primers were used. The sequence of beta-actin-F was:
2.4.7. beta-actin-F primer sequence
2.4.8. beta-actin-R primer sequence
The miRNA levels of let-7a, mouse snoRNA55, and human U54 were measured by real-time PCR.
2.4.9. The hsa-let-7a PCR primer sequence was
2.4.10. The snoRNA55(Mus) PCR primer sequence was
2.4.11. The U54(homo) PCR primer sequence was
Primers DENV2-NS1-F and DENV2-NS1-R were used to amplify the DENV2 NS1 RNA by PCR.
2.4.12. The sequence of DENV2-NS1-F
2.4.13. The sequence of DENV2-NS1-R
The mRNA level of endogenous beta-actin and the level of infected DENV2 NS1 RNA were measured by real-time PCR using the primers of beta-actin-F, beta-actin-R, DENV2-NS1-F, and DENV2-NS1-R.
2.5. Western blot analysis
Protein samples were prepared by lysing the cells in RIPA buffer and centrifugation at 13,600 rpm for 15 min at 4 °C. RIPA buffer contained PMSF, EGTA, aprotinin, leupeptin, Na3VO4 (Sigma, MO, USA), and EDTA (Merck, Darmstadt, Germany). Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins in the gel were transferred onto Immobilon-P Membrane (PVSF) (IPVH00010; Millipore). Blocking solution (TBST) with 5% milk was applied onto the PVSF membrane for 1 h. After incubation with the specific primary antibody at 4 °C overnight, the secondary antibody was added. The PVSF membrane was stained with the secondary antibody for 1 h at room temperature (RT). Finally, the data were analyzed using the Biospectrum Imaging system (UVP, CA, USA). The expression level of the protein was quantified using Image-J software. Anti-NS1 primary antibody (dilution rate 1:540) was a gift from Dr. Huan-Yao Lei (National Cheng Kung University, Taiwan). Anti-beta-actin antibody (dilution rate 1:5000) was purchased from Sigma.
2.6. Immunofluorescence assay (IFA)
Cells were seeded on the slide and incubated with virus for various times. Then, the cells were washed with PBS and fixed in 3.7% formaldehyde/PBS solution for 30 min at RT. After washing with PBS, the slide was incubated with 0.1% Triton X-100 (Merck) for 30 min, and washed with PBS before incubating with 2% BSA (Sigma) for 1 h at RT. The slide was then stained with anti-DENV2-NS1 primary antibody (dilution rate 1:100; Abcam, Cambridge, UK) at 4 °C overnight. The slide was washed with PBS and incubated with the goat anti-mouse IgG-Alexa 488 secondary antibody (dilution rate 1:200; Invitrogen) for 1 h at 37 °C. After washing with PBS, the slide was stained with Hochest dye (dilution rate 1:200; Sigma) for 30 min at RT for nuclei staining. Finally, the slide was examined under a confocal microscope (FV-1000; Olympus, Tokyo, Japan). The images were merged using Photoshop software.
2.7. Plaque assay
BHK-21 cells were cultured on the 12-well plate overnight at 37 ℃ before infection with dengue viruses. Cells and virus were incubated for 2 h with shaking at RT. The incubation solution was discarded and 1 ml of 0.8% carboxy methyl cellulose (Sigma) with 2% FBS/DMEM was added. Cells were maintained for 4 days at 37 °C. The cells were washed with PBS twice and stained with 2% crystal violet solution (Sigma) for 1 h at RT followed by washing with water. The plaques on the plate were then counted (Lee et al., 2008).
2.8. Animal model
To test the effect of honeysuckle and let-7a on DENV infection and replication in vivo, C57/B6 and ICR suckling mouse models were used ( Lee et al., 2005). Despite these two models of DENV infection could not show the clinical symptoms mimic to infection in human, they are good animal models to evaluate DENV virulence and replication. The C57/B6 mice were maintained at the Model Animal Research Center (MARC) of Nanjing University and the ICR suckling mice were maintained at the Animal Facility of National Cheng Kung University. Mice were bought from Nanjing University (Nanjing, China) and National Laboratory Animal Center (Taipei Taiwan). Mice were housed at 22 °C with a 12-hr light-dark cycle in an on-site animal facility.
Animal welfare and experimental studies complied with the guidelines of the Care and Use of Laboratory Animals (National Research Council, 1996) and the Model Animal Care and Use Program (MACUP) supervised and this study was specifically approved by the Institutional Animal Care and Use Committee (IACUC) of the Model Animal Research Center (MARC) of Nanjing University and the IACUC of National Cheng Kung University (the approval number: No. 101291).
Intracranial injection of DENV2 was administered to the ICR suckling mice, and 2% FBS/DMEM was used for the mock infection.
To study the effect of let-7a on DENV2-infected mice, let-7a was i.c. injected after the mice had been i.c. inoculated with DENV2. The negative control was 15 μM negative control miRNA in DMEM.
DENV2-infected mice that received honeysuckle (40 μl) or let-7a treatment were examined for the expression levels of viral NS1 RNA and protein as well as viral titer in the brains by real-time PCR, Western blotting, and plaque assay. The disease symptoms and survival rate of the treated mice were also determined. Anti-NS1 and anti-beta-actin antibodies were used to examine the DENV2 NS1 protein level in infected mice brains. Beta-actin was the internal control for both RNA and protein investigation. Clinical score was defined as follows: 0: healthy; 1: weight loss; 2: reduced mobility; 3: difficulty moving with forelimb or hind limb weakness; 4: paralysis and very ill; 5: death.
2.9. Honeysuckle aqueous extract preparation and blood miRNA profile in human participants and mice
Human subject assessment was conducted according to the Declaration of Helsinki, and this study was specifically approved by the Institutional Review Board (IRB) of National Cheng Kung University Hospital (IRB approval number: IBR-99-127). Written informed consent was obtained from each of the participants. For each volunteer in this study, a dosage of honeysuckle aqueous extract was prepared as immersed 10 g of honeysuckle dry flower bud in 500 ml double distill-water and cook for 20 min before drinking. Volunteer ingested 2 dosages a day (one in the morning and one in the evening) for continue 4 days as a complete treatment course. One day after honeysuckle treatment, blood samples were taken and immediately mixed with TRIzolTM (Invitrogen). The total RNAs in the blood samples was analyzed by Solexa® deep sequencing.
Dried honeysuckle flower was purchased from Shandong province, China (Linyi Jin Tai Yao Ye Co. LTD, Shandong, China; voucher specimen number: 2011). The honeysuckle (L. japonica Thunb.) was identified by Dr. Guanghai Liu, a taxonomist of Linyi Honeysuckle Institute, Shandong, China. One dose of honeysuckle aqueous extract was prepared by boiling 10 g of honeysuckle flower in 500 ml double distill-water for 20 min before consumption. The fingerprint of compounds in honeysuckle aqueous extract was determined by UPLC-Q/TOF MS (Supplementary data 2 and Supplementary Table 2), and was used to ensure the consistency of honeysuckle usage in this study. Volunteers ingested 2 dosages a day (one in the morning and one in the evening) for 4 consecutive days. One day after honeysuckle treatment, blood samples were taken and immediately mixed with TRIzol™ (Invitrogen). The total RNA in the blood samples was analyzed by Solexa® deep sequencing.
To study honeysuckle-induced miRNA expression profile in human blood, a total of 17 health volunteers were recruited. In the treatment group, 6 men (aged between 23 and 29 years old) and 2 women (aged 23–25 years) ingested honeysuckle aqueous extract. Three men (age 23–30) and 6 women (age 23–27) drank water and their blood samples were used as the control group.
To study the honeysuckle-induced miRNA expression profile in mice, eight hundred male mice (C57/B6 strain; aged 10 weeks and 64 weeks; four hundred mice/age group; 200 mice/treatment group) were treated with honeysuckle aqueous extract or double distilled water. To mimic the human medicine treatment and prevent side effects from overdose, the body weight of each mouse was measured and the amount of honeysuckle was calculated based on the dosage for a 50 kg man. Thus, for a 25 g C57/B6 mouse (age either 10 weeks or 64 weeks), the dosage of honeysuckle aqueous extract was 0.005 g of honeysuckle in 0.2 ml double distilled water. For each 4 g ICR suckling mouse, the dosage of honeysuckle aqueous extract was 0.0008 g of honeysuckle in 40 μl double distilled water. Honeysuckle aqueous extract was prepared as follows: 3 g of dried honeysuckle flower were boiled in 150 ml double distilled water for 20 min until there was 50 ml water left. This extract solution was stored at −80 °C. The mice were fed with the honeysuckle aqueous extract (40 μl or 0.2 ml according to their body weight) twice a day for 4 consecutive days and the blood samples were taken at the indicated times for RNA extraction. Total RNA was extracted using TRIzol™ reagent. The total RNA including miRNA were analyzed by deep sequencing. The level of let-7a miRNA in the blood of the ICR mice was evaluated by real-time PCR.
Solexa® deep sequencing and data analysis were performed following the manufacturer's protocol (Beijing Genomics Institute, Shenzhen, China; http://www.genomics.cn/en/index). Briefly, small RNAs were annealed with 5′ and 3′ adaptors before RT-PCR and sequencing analysis. The sequencing results were then compared with miRNAs in the miRNA database to identify candidate miRNAs. The sequencing result revealed the copy number of the matched miRNAs in the examined blood samples. The expression level of each matched miRNA among the total miRNAs was normalized and represented as the expression of transcriptions per million counts, and was then statistically analyzed by standard deviation (std). The normalized data were marked as “std number”. The normalized miRNA expression level after log2 conversion (treatment/control) was shown as the fold change of each identified miRNA. The significance of the fold change of miRNA expression levels was determined by the p-values. A p-value of <0.05 was defined as significant.
The miRBase website (http://www.mirbase.org) was used to search for human homologues of the endogenous matched miRNAs of the mice (Kozomara and Griffiths-Jones, 2011). The dengue virus type 2 PL046 sequence (DENV2 PL046; NCBI accession number AJ968413; http://www.ncbi.nlm.nih.gov) was queried on the ViTa viral target prediction website (http://vita.mbc.nctu.edu.tw) to predict the miRNA targets of virus genome sequences (Hsu et al., 2007). The clustering family and seed sequence group family of the miRNAs were analyzed using the smirnaDB website (http://www.mirz.unibas.ch/cloningprofiles).
2.10. UPLC-Q/TOF MS and UPLC analysis
UPLC-Q/TOF MS experiments were performed on a Dionex Ultimate 3000 HPLC system (Thermo, Dionex, Germany) coupled with impact HD Q/TOF MS (Bruker Daltonics, German). The aqueous extract of L. japonica was separated by Kinetex C18 column (2.1×150 mm, i.d., 2.6 mm particle size, Phenomenex, Torrance, USA) with gradient eluting system at a flow rate of 0.5 ml/min with (A) 0.1% formic acid in 100% acetonitril and (B) 0.1% formic acid in Mili-Q water as elute of 2.5–5.0% A at 0–30 min, 5.0–25.0% A at 30–60 min, 25.0–100.0% A at 60–70 min, 100.0–75.0% A at 70–75 min, and 75.0–50.0% A at 75–80 min. The MS source was operated in negative ion mode. Nitrogen was used as a nebulizing (0.3 bar) and drying gas (4.0 L/min, 200 °C). Helium was used as the collision gas.
2.11. Statistical analysis
Data are presented as mean±standard error for the indicated number of separate experiments. Statistical differences were analyzed by two-way analysis with Mann-Whitney U test. The statistical difference of Kaplan-Meier survival curves of mice was analyzed with Log Rank test. All statistics were calculated using SigmaState version 3.5 (Systat Software, San Jose, CA, USA).
3.1. Honeysuckle up-regulated miRNAs could target DENV NS1 sequence
MiRNAs play important roles in suppressing pathogens in human and animals. Whether honeysuckle suppresses microbial infection through the regulation of miRNAs has not been explored. To identify miRNAs which participate in honeysuckle-mediated suppression of DENV2 replication, the miRNA expression profiles of a honeysuckle-treated group and an untreated control group were compared by Solexa® deep sequencing analysis. Here, we determined the profile of differentially expressed miRNA in the blood of mice (400 young and 400 old mice) and human volunteers (17 young people) after ingestion of either honeysuckle aqueous extract or double distilled water for four days followed by next generation sequencing analysis. In the human Solexa® deep sequencing data, a total of 1921 miRNAs were screened and 80 of them were found to be up-regulated 1.2 fold in the honeysuckle-treated groups. Among them, twenty-two miRNAs were predicted to target the RNA genomes of DENV2 (Supplementary Table 1), and three miRNAs (let-7a, let-7b, let-7i) were predicted to target the RNA genomes of DENV1, DENV2, and DENV4 strains at the conserved NS1 region from nucleotide position 3313–3333 (Fig. 1; Table 1). In summary, above predicated miRNAs may play suppressive roles in dengue virus replication both in mice as well as in human. Among the up-regulated miRNAs, let-7a showed the highest expression level compared to other up-regulated miRNAs (Supplementary Table 1, 48%).
miRNAs predicated to target DENV2 genome aTargeted region in DENV2 genome and corresponding protein in bracket b10 week-old mice c64 week-old mice dHuman eHuman precursor cluster let-7a 3313-3333 (NS1) fX X X hsa-mir-98 let-7b 3313-3333 (NS1), X X X hsa-mir-98 10639-10660 (3′UTR) let-7i 3314-3333 (NS1), X X X hsa-let-7i 10327-10349 (3′UTR) miR-148b 8208-8229 (NS5) X X X hsa-mir-148b miR-221 4651-4673 (NS3), X X X hsa-mir-221 8973-9005 (NS5)
- DENV genome sequence was from DENV2 PL046 strain (NCBI accession number #AJ968413).
- Ten week-old C57B6 mice represent young age mice.
- Sixty four week-old mice represent old age mice.
- Human volunteers were between 23 and 30 year-old.
- The cluster family was listed based on human miRNA profile.
- X: Endogenous miRNAs induced after honeysuckle treatment
3.2. Honeysuckle treatment induced let-7a expression both in vivo and in vitro
Because let-7a expression was increased in the blood of mice and human after honeysuckle ingestion twice a day for four consecutive days, we then clarified whether honeysuckle could induce let-7a expression. Honeysuckle induction of endogenous let-7a expression in mice was investigated according to the schematic procedure shown in Fig. 2A. Our data showed significant up-regulation of let-7a in the blood of the ICR suckling mice at day 2 (11.25 fold) and day 4 (2 fold) after ingestion of honeysuckle aqueous extract. This induction was diminished at day 6 post-treatment (Fig. 2B). Similarly, our cell line data showed a significant increase of let-7a expression in hepatoma Huh7 cells after treatment with honeysuckle water extraction for 36 h and 48 h detected by real-time PCR (Supplementary data 1). In summary, honeysuckle can induce endogenous let-7a expression both in vitro and in vivo.
3.3. Let-7a specifically targets DENV2 NS1 sequence, suppresses NS1 expression and viral replication
To confirm that let-7a can specifically target DENV2 NS1 sequence and suppresses its expression, the suppression of exogenous let-7a on DENV2 NS1 expression in human hepatoma Huh7 cells was evaluated. Either wild type DENV2 NS1 (3313–3333) or NS1 sequence with mutations at the targeting region were cloned into the 3′UTR region of the reporter plasmid pMIR-Report™, which together with let-7a (miR let-7a) or non-specific miRNA (N.C.) were co-transfected into Huh7 cells by transient transfection (Fig. 3A). Our data reveal that the luciferase activity of the wild type pMIR-DENV2-NS1-WT was significantly suppressed by the exogenous let-7a as compared to the non-specific N.C. miRNA, and the NS1 mutant pMIR-DENV2-NS1-Mut showed no change by either let-7a or N.C. (Fig. 3A). Moreover, let-7a suppressed the luciferase activity of the wild type pMIR-DENV2-NS1-WT when let-7a dosage was increased (Fig. 3B). These results indicate that let-7a specifically targets the DENV2 NS1 sequence at the 3313-3333 region.
Because DENV3 genome RNA could not be targeted by let-7a (predicated by ViTa software), we used it to confirm the specific targeting of let-7a on DENV2 but not DENV3 genome sequence. Huh7 cells were transiently transfected with let-7a followed by DENV2 or DENV3 infection. Huh7 cells were transiently transfected with let-7a for 24 h followed by infection with DENV2 or DENV3 for another 48 h. DENV2 NS1 expression was significantly suppressed by let-7a at a concentration of 200 nM; however, DENV3 NS1 gene expression was not affected by let-7a at the same concentration by RT-PCR analysis (Fig. 4A). It is well known that Ras is the target gene of let-7a and its expression could be inhibited by let-7a (Akao et al., 2006, He et al., 2010 and Johnson et al., 2005). Here, a dosage-dependent inhibition of endogenous Ras expression was demonstrated under increased amount of let-7a treatment, indicating that let-7a is functional (Fig. 4B). Under such conditions, our data demonstrated that exogenous let-7a showed dosage dependent inhibition on NS1 protein expression (Fig. 4B). DENV2 negative-stranded NS1 expression was also decreased by let-7a in a dose-dependent manner, indicating that let-7a suppresses viral replication (Fig. 4C). The dosage-dependent inhibition of let-7a on DENV2 NS1 expression in Huh7 cells was further demonstrated by immunofluorescence staining (Fig. 4D). Altogether, overexpression of exogenous let-7a could inhibit DENV2 replication in Huh7 cells through the specific inhibition of NS1 gene expression.
3.4. Honeysuckle treatment before DENV2 infection decreased clinical score, viral replication and prolonged the survival time of infected ICR suckling mice
Because honeysuckle has been widely used against various pathogen infection in China, the prophylactic effect of honeysuckle on DENV2 infection in ICR suckling mice was evaluated. ICR suckling mice were pretreated with honeysuckle at day 4 after birth (twice a day) followed by intracranial (i.c.) injection of DENV2 (PL046 strain, 2.5×105 PFU) on day 7. All the mice continuously drank honeysuckle aqueous extract until day 13 (Fig. 5A).
Our data showed that honeysuckle pretreatment before DENV2 infection significantly decreased the clinical scores of the infected mice at day 6 compared with the DENV2-infected mice without treatment (Fig. 5B). The clinical scores included weight loss, reduced mobility, difficulty moving, forelimb or hind limb weakness, paralysis, and death. Honeysuckle pre-treatment also prolonged the survival time of DENV2-infected mice by one day compared to DENV2-infected group without honeysuckle treatment (Fig. 5C). Pre-treatment of honeysuckle consistently suppressed DENV2 NS1 RNA and protein expression by 20% and 68% in the brain tissue compared with DENV2-infected mice without treatment by RT-PCR and Western blotting, respectively (Fig. 5D and E). Pre-treatment of honeysuckle further decreased viral titer (42%) in the brain tissue of the infected mice compared to the DENV2-infected mice without treatment (Fig. 5F). Altogether, honeysuckle pretreatment alleviated the disease symptoms, prolonged survival time, and was accompanied with inhibition of DENV2 replication and viral titer in the brains of the suckling mice.
3.5. Honeysuckle treatment decreased disease symptoms, prolonged survival time, and suppressed DENV2 replication and viral titer in DENV2-infected suckling mice
We then evaluated the effect of honeysuckle treatment on DENV2 infected ICR mice. Suckling mice were i.c. injected with DENV2 (2.5×105 PFU) at day 6 followed by honeysuckle treatment starting at day 7 twice a day for four days. All the mice were sacrificed at day 12 (Fig. 6A).
Our data showed that honeysuckle treatment after DENV2 infection significantly decreased the clinical scores of the infected mice at day 4, 5 and 6 compared to the DENV2-infected mice without honeysuckle (Fig. 6B). Honeysuckle treatment also prolonged the survival time of the infected mice from day 8 to day 10 (Fig. 6C). Honeysuckle treatment resulted in a 27% reduction of NS1 RNA expression and a 52% reduction of NS1 protein expression (Fig. 6D and E). Further study revealed that honeysuckle treatment decreased over 30% viral titer in the brain tissue of the infected mice (Fig. 6F). In conclusion, our findings demonstrated that honeysuckle treatment partially suppressed the disease symptoms, prolonged survival time, and inhibited DENV2 replication and titer in the mice.
3.6. Let-7a treatment alleviated disease symptoms, prolonged survival time, and suppressed DENV2 replication and viral titer in DENV2 infected suckling mice
The data in Fig. 6 revealed that honeysuckle treatment alleviated clinical scores and increased the survival time of DENV2-infected ICR suckling mice. Let-7a was one of the most up-regulated miRNAs by honeysuckle as demonstrated above both in vitro and in vivo. We further showed that let-7a specifically targets DENV2-NS1, but not DENV3-NS1, accompanied with reduced expression levels of RNA and protein of DENV2-NS1 as well as DENV2 replication ( Fig. 3 and Fig. 4). Here, we further clarified the effect of exogenous let-7a on the pathogenesis of DENV2-infected ICR suckling mice and DENV2 replication. The ICR suckling mice were i.c. injected with DENV2 (2.5×105 PFU) at day 6 followed by i.c. injection of let-7a (7.5 μM and 15 μM in 20 μl of DMEM) at day 7 and day 9, and then sacrificed at day 11 (Fig. 7A). Our data showed that exogenous let-7a treatment (15 μM) significantly alleviated the clinical score of DENV2-infected mice at day 5, 6 and 7 compared to the untreated DENV2-infected mice (Fig. 7B). Furthermore, let-7a treatment prolonged the survival of the infected mice from day 8 to day 9 (Fig. 7C). Further study revealed that let-7a treatment at the concentration of 7.5 μM and 15 μM resulted in a 13–26% reduction of NS1 positive-stranded RNA expression (Fig. 7D) and a 27–52% reduction of NS1 protein expression (Fig. 7E). Finally, let-7a treatment at both 7.5 μM and 15 μM decreased the viral titer in the brain tissue of the infected mice by over 94% (Fig. 7F). In conclusion, let-7a treatment showed a similar result to that of honeysuckle treatment by alleviating the disease symptoms and prolonging the survival time of DENV2-infected mice. Furthermore, let-7 suppressed DENV2 replication and led to decreased virus titer in the brain of the infected mice.
4. Discussion and conclusion
Honeysuckle, a traditional medicine with multiple functions has been used for thousands of years in China. However, the underlying mechanism of its anti-pathogen function remains unclear. Each year over 390 million people were infected by DENV and 96 million showed symptoms (Bhatt et al., 2013). There is currently no effective therapy or vaccine against dengue virus infection. Here, we demonstrate that honeysuckle can alleviate disease symptoms and prolong the survival time of the infected suckling mice when these mice received honeysuckle either before or after DENV2 infection (Fig. 5 and Fig. 6). During DENV replication in the cell, early viral protein is directly translated from its positive-stranded RNA genome followed by replication and synthesis of late mRNAs and proteins. DENV shows the highest translation and replication rate at 10 h and 70 h p.i., respectively (Filomatori et al., 2006). In DENV2-infected human endothelial cells, a large amount of NS1 protein was detected in the membrane and organelles at 12 h p.i. (Poungsawai et al., 2011). Our data showed that the expression levels of DENV2 NS1 RNA and protein were reduced in the mouse brain tissue when suckling mice received honeysuckle either before or after viral infection, suggesting that DENV2 replication was suppressed (Fig. 5D, E, F and Fig. 6D, E, F).
To clarify how honeysuckle suppresses DENV2 replication through miRNA, deep sequencing analysis was conducted to identify the overexpressed miRNAs in the blood of mice and human volunteers after honeysuckle treatment. We further analyzed overexpressed miRNAs in human blood samples and found that some miRNAs are predicted to be able to target the DENV genome sequences at NS1 and 3′UTR regions (Table 1). We also demonstrated that the overexpressed miRNA let-7a could target the DENV NS1 sequence in the human cell line and the mouse model. Moreover, our analysis reveal that the DENV2 NS1 sequence region 3313-3333 could be recognized and targeted by honeysuckle induced miRNAs (Fig. 1). In humans, six of the twenty-two DENV2-targeting miRNAs can target NS1 sequence at the 3302-3333 region among 80 of the honeysuckle up-regulated miRNAs (Supplementary Table 1). Similar phenomenon was seen in the mice after honeysuckle ingestion (data not shown). Above results suggest that the NS1 sequence 3302-3333 is a hotspot targeted by honeysuckle-induced miRNAs. Interestingly, NS1 3313-3333 region corresponding to amino acid 297-303 with the peptide sequence “SLRTTT” (AA 297-302; Fig. 1) is within the most-conserved region 267-312 of DENV (Chen et al., 2010 and Masrinoul et al., 2011). This unique region has 98–100% identity in four serotypes of DENVs, and can’t induce the strong immune response compared to other conserved regions (Chen et al., 2010). This feature warrants the development of universal miRNAs against four serotypes of DENVs. NCBI BLAST analysis indicates that the peptide “SLRTTT” presents in both JEV and DENV and shows 100% alignment with human MHC binding protein-2 (MBP-2), c-Myc intron-binding protein 1, transcription factor HIVEP2, intestinal mucin, transcription factor ATRX, zinc finger helicase, X-linked nuclear protein, neuroblastoma-amplified protein, and ral GTPase-activating protein subunit beta. Because this conserved peptide sequence region was detected in diverse human proteins, therefore, it is difficult to induce strong immune response through the immune selection mechanism. On the other hand, the C-terminal domain of DENV NS1 (amino acid 271–352, nucleotide 3234–3477) is an epitope of anti-NS1 autoantibody, which cross-reacts with human endothelial cells and platelets and causes pathogenesis of platelet dysfunction (Stevens et al., 2009 and Wan et al., 2014). Because let-7a is a ubiquitously expressed miRNA in the cells, it may suppress DENV2 replication by targeting the NS1 (from 3313 to 3333) region (Fig. 1) in addition to the conventional antibody suppression. Therefore, to prevent anti-NS1 autoantibody-related auto-immune diseases, honeysuckle-induced let-7a provides an alternative approach to suppress DENV replication in vivo. It is possible that the host innate miRNA immunity was generated by a competitive process during the co-evolution of DENV and its host. In summary, the conserved sequence in DENV NS1 region lacks of effective epitope for antibody recognition. However, this conserved region can be targeted and inhibited by specific miRNAs such as let-7a.
Let-7a expression was significantly increased in the blood of both human volunteers and mice after honeysuckle treatment (Fig. 2, Table 1). The water extraction of honeysuckle increased let-7a expression in human hepatoma Huh7 cells (Supplementary data 1). These findings support our speculation that honeysuckle can induce let-7a expression both in vitro and in vivo. Further studies reveal that let-7a specifically targets DENV2 NS1 (region 3313-3333) to reduce the levels of DENV2 NS1 ( Fig. 3 and Fig. 4), suggesting that let-7a indeed targets the DENV2 NS1 sequence to reduce DENV activity. The specific targeting of let-7a at the DENV2 NS1 sequence was further validated by its preferential suppression of DENV2 but not DENV3 (Fig. 4A). Our findings confirmed the prediction of the ViTa in silico analysis. We further demonstrated that intracranial inoculation of let-7a after DENV2 infection of the suckling mice attenuated the disease symptoms, and prolonged the survival time of the mice ( Fig. 7B and C). Both positive as well as negative RNA and protein levels of DENV2 NS1 were suppressed by let-7a treatment (Fig. 4A, B, C and 4D), suggesting that let-7a suppresses DENV2 activity. Moreover, decreased viral load in the brain tissue of mice after let-7a treatment with statistical significance was detected compared to that of the untreated group (Fig. 7F).
In addition to the involvement of humoral and cellular immune responses in DENV-related DHF and DSS development, viral load also plays an important role in disease progression and symptom development (Pawitan, 2011). DENV infection could up-regulate cytokines including IFN-γ, MCP-1, and RANTES in the infected mice (Tuiskunen et al., 2011), which affect the inflammatory response and cause the cytokine storm and result in plasma leakage and death. In summary, viral load and cytokine production affect the severity of clinical symptoms in DENV patients (Rothman, 2011). In our mouse model, although both honeysuckle and let-7a could suppress DENV2 NS1 expression and viral load to various degrees, the improvement in disease symptoms and survival rate of the infected mice was limited and transient (Figs. 5C, 6C and 7C). This discrepancy may be explained at least in part by the presence of residual viruses and/or overexpression of cytokines. Nevertheless, our findings suggest that an optimal therapeutic approach for the treatment of DENV2 patients may be a combined therapy of honeysuckle and immunopathogenesis-related drugs.
Zhou et al. reported that honeysuckle per se encoded the miR2911 can be uptaken by the host to target the influenza A viruses genome sequence and suppress its infection ( Zhou et al., 2015). However, above finding has been subverted by miRBase (a biological database), which announces that miR2911 is a fragment of rRNA not microRNA.
MiRNAs play diverse roles under different conditions. Thus, in order to develop a drug capable of inducing endogenous miRNAs which can target the viral genome and inhibit viral replication, we also need to consider its side effects on the host. Some miRNAs show a suppressive effect on viral replication but are also toxic to the host when they are highly expressed. It is possible that some plants can also induce DENV-targeting miRNAs which have side effects in patients. Honeysuckle is a traditional herb which has long been used to treat flu-like symptoms in China, suggesting that the possible side effects induced by honeysuckle are negligible. However, the toxicity study in human of the extract of honeysuckle needs further investigation. Our data showed that honeysuckle induces multiple DENV2-targeting miRNAs simultaneously. These different miRNAs have different target sequences on the DENV2 genome (Table 1). Therefore, honeysuckle offers a unique advantage in that it can prevent drug resistance that caused by viral RNA sequence mutation, a phenomenon seen with many antiviral drugs.
Various active ingredients of honeysuckle have been reported with anti-microbial activities; however the functional component in honeysuckle responsible for let-7a overexpression and the anti-DENV activity remains undetermined. In the present study, we demonstrate for the first time that honeysuckle uptake enhances innate miRNA let-7a to suppress DENV2 activity both in vitro and in vivo. In addition, this study opens a new avenue for exploring the antiviral effects through the regulation of the endogenous miRNAs.
We have read the Journal's policy and the authors of this manuscript have no competing interests.
Conceived and designed the experiments: DDG, HSL, SFY. Analyzed the data: YRL, SFY, HSL, DDG. Wrote the first draft of the manuscript: YRL, HSL, DDG. Agreed with manuscript results and conclusions: XMR, HZ, SDH, YSL, TMY, HDH, CCH. ICMJE criteria for authorship read and met: YRL, SFY, XMR, HZ, SDH, HDH, CCH, YSL, TMY, HSL and DDG.
We thank Ms. Lingling Yu, Xiaohong Xu, Maoshan Chen, Shuwen He (BGI-Shenzhen), Ms. Yaping Zhang and Mr. Zhen Bian (NJU, China) for their technical support. We also thank Dr. Maobin Dn (IBJPCAS), Dr. Guanghai Liu (LHI), Mr. Faqiang Wang (LJP Co), Dr. Fenyong Liu (UC Berkeley), Dr. Yie Liu (NIH, USA), and members of the State Key Laboratory of Pharmaceutical Biotechnology (NJU, China) for their assistance.
This work was supported by “Project 985” and “Project 211” grants provided by the Ministry of Education of the People's Republic of China, and the Open Fund of the State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, China (Grant number# KF-GN-200906) to DDG. It was also supported by grants from the National Science Council of Taiwan (NSC 101-2314-B-705-003-MY3) and Chiayi Christian Hospital.
Appendix A. Supplementary material
- Supplementary material Supplementary Data 1. Honeysuckle extract induced let-7a expression in Huh7 cells. Honeysuckle (200 g) was extracted from 4 liter of double distilled water and the extract was quantified after frozen drying. Huh7 cells were treated with honeysuckle for 36 h and 48 h and the expression of let-7a was determined by real-time PCR. Double distilled water was used as the mock control.
- Supplementary material Supplementary Table 1. Differential expression levels of miRNAs in human blood after honeysuckle treatment. Human drank honeysuckle extract, and the differentially expressed miRNAs were determined by Solexa® deep sequencing. Among them, the miRNAs targeting DENV2-NS1 region were identified. Supplementary Table 2. The spectra of the compounds in honeysuckle aqueous extract determined by UPLC-Q/TOF MS.
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