Antiviral Therapy 2017; 22: (doi: /IMP3152)

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1 Antivirl Therpy 217; 22: (doi: /IMP3152) Originl rticle Geniposide demonstrtes nti-inflmmtory nd ntivirl ctivity ginst pndemic A/Jingsu/1/29 (H1N1) influenz virus infection in vitro nd in vivo Yunshi Zhng 1, Jing Yo 1, Xin Qi 2, Xing Liu 1, Xieqin Lu 1, Gnzhu Feng 1 * 1 Deprtment of Respirtory Medicine, the Second Affilited Hospitl of Nnjing Medicl University, Nnjing, Chin 2 Deprtment of Acute Infectious Disese Control nd Prevention, Jingsu Province Center for Disese Control nd Prevention, Nnjing, Chin *Corresponding uthor e-mil: zhu @163.com Bckground: Influenz A viruses (IAVs) hve been gret thret to humn helth for centuries, without effective control. Geniposide, min iridoid glycoside compound extrcted from Grdeni jsminoides Ellis fruit, possesses vrious biologicl ctivities including nti-inflmmtion nd nti-virus. Methods: Mdin Drby cnine kidney (MDCK) cells were infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus in vitro. Cytotoxicity nd ntivirl ctivity of geniposide were estimted by MTT ssy. The influenz respirtory trct infection murine model ws estblished by intrnsl instilltion of pndemic A/Jingsu/1/29 (H1N1) influenz virus. One dy fter infection, the mice were dministered with geniposide (5, 1 or 2 mg/kg/dy) or the neurminidse inhibitor (NAI) permivir (3 mg/kg/dy). Body weight, survivl time, virl titre nd lung index of the mice were mesured. The sndwich enzyme-linked immunosorbent ssy (ELISA) ws used to exmine levels of inflmmtory cytokines. Results: The dt showed tht geniposide hd little cytotoxicity on MDCK cells nd protected them from pndemic A/Jingsu/1/29 (H1N1) influenz virus-induced cell injury. In the infected mice, geniposide tretment significntly restored the body weights, decresed the mortlity, llevited virl titres nd virus-induced lung lesions. Geniposide substntilly inhibited the virus-induced lveolr wll chnges, lveolr hemorrhge nd neutrophil-infiltrtion in lung tissues. Levels of inflmmtory meditors, including tumour necrosis fctor- (TNF-), interferon-g (IFN-g), interleukin (IL)-4, IL-6 nd IL-1 were lso mrkedly ltered fter tretment with geniposide. Conclusions: Our investigtion suggested tht geniposide effectively inhibited cell dmge medited by pndemic A/Jingsu/1/29 (H1N1) influenz virus nd mitigted virus-induced cute inflmmtion. Introduction New strins of influenz A viruses (IAVs) emerge periodiclly nd led to pndemics tht pose gret thret to humn helth worldwide [1]. In the 2th century, there were three lrge-scle humn influenz pndemics tht cused high levels of mortlity, including the 1918 Spnish Flu (H1N1) [2], 1957 Asin Flu (H2N2) [3,4] nd 1968 Hong Kong Flu (H3N2) [4]. The 1918 Spnish Flu, the most serious pndemic of the three, ws responsible for more thn 5 million humn deths [5]. By fr, influenz remins n lrming public helth problem for its high morbidity nd mortlity. Influenz A viruses belong to the fmily of Orthomyxoviride influenz virus nd hve negtive-sense segmented RNA genome, which infects the epitheliums of the upper nd lower respirtory trct fter entry through the orl or nsl route. The 29 pndemic influenz A virus subtype, H1N1 (influenz A (H1N1) pdm9) strin, which contins genes from humn, swine nd vin influenz A viruses, is novel influenz virus tht emerged in April 29 [6,7]. It could be circulting widely mong swine nd humns [8]. Dting from August 21, sttisticl dt from the World Helth Orgniztion (WHO) show tht lbortory confirmed cses of influenz A (H1N1) pdm9 hve been reported worldwide, covering more thn 214 countries nd overses territories or communities, leding to over 18,449 deths [9]. Unlike other low pthogenic influenz viruses, the influenz A (H1N1) pdm9 virus cuses severe humn 217 Interntionl Medicl Press (print) (online) 599

2 Y Zhng et l. illness, which is chrcterized by pneumoni tht develops into cute respirtory distress syndrome (ARDS) nd multiple orgn dysfunction (MOD) [6]. Antivirl compounds re the first-line therpy for pndemic influenz viruses. Sequence nlysis suggests tht the influenz A (H1N1) pdm9 is resistnt to ion chnnel inhibitors, such s mntdine nd rimntdine [1]. The neurminidse inhibitors (NAI), including znmivir, oseltmivir nd permivir, represent n importnt dvnce in the mngement of IAVs. Nevertheless, previous studies hve demonstrted tht the emergence of virus mutnts, like NA-H275Y nd PB1-T296R vrints, develops NAI resistnce [11,12]. Grdeni jsminoides Ellis (Rubicee), lso clled Zhi-Zi in the Chinese phrmcopoeis, is not only of gret nutritionl vlue, but lso used s herbl medicine nd hs long history. Geniposide, the min iridoid component extrcted from Grdeni jsminoides Ellis, hs been shown to possess potent nti-inflmmtory [13,14], ntivirl [15], choleretic [16,17], nti-poptotic [18,19], nti-fibrotic [2,21], nti-llergic [22], nti-nociceptive [23] nd neuroprotective [24,25] properties. In spite of those uncovered biologicl ctivities nd gret therpeutic potentil, geniposide s bility to protect ginst influenz A (H1N1) pdm9 virus-induced ALI remins poorly understood. In the present study, we tested geniposide s protective effect on influenz A (H1N1) pdm9 virus-infected cells in vitro nd investigted whether geniposide could exhibit ntivirl nd nti-inflmmtory effects in influenz A (H1N1) pdm9 virus-infected mice by evluting virl titre, histopthologicl chnges nd cytokine levels. Methods Biosfety sttement All experiments involved with live H1N1 viruses were conducted within the Animl Biosfety Level 2 Lbortory (ABSL2) continment fcilities in Jingsu Province Center for Disese Control nd Prevention, which ws pproved by Chin Ntionl Accredittion Service for Conformity Assessment. All experimentl procedures were pproved by the institutionl niml ethics committee. Animl experiments were crried out in strict ccordnce with the Nnjing Medicl University s guidelines for their cre nd use. Viruses The pndemic A/Jingsu/1/29 (H1N1) influenz virus (NT91) ws isolted from the first humn cse of the 29 influenz pndemic in Jingsu province of Chin. Virus ws cultivted, propgted nd titrted in 9 1-dy-old embryonted chicken eggs for 72 h t 37 C. Hemgglutintion test (Chinese Center for Disese Control nd Prevention. Stndrd operting procedures for the Ntionl Influenz Center: Revised Edition) ws performed using 1% humn red blood cells ccording to the protocol of WHO. Regents Geniposide (purity 97.5%), white powder, ws purchsed from Ntionl Institutes for Food nd Drug Control (Beijing, Chin). Permivir ws purchsed from Nnxin Phrmceuticl Co., Ltd (Gungzhou, Chin). Dulbecco s modified Egle s medium (DMEM), fetl bovine serum (FBS), bovine serum lbumin (BSA), Trypsin-EDTA, Trypsin-TPCK, phosphte-buffered sline (PBS) nd Hnk s blnced slt solution (HBSS) were purchsed from Gibco (Invitrogen Life Technologies, Inc., Crlsbd, CA, USA). Cell culture Mdin Drby cnine kidney (MDCK) cells were generously provided by Jingsu Province Center for Disese Control nd Prevention [26]. The cells were mintined in DMEM supplemented with 1% FBS, 1 IU/ml penicillin nd 1 µg/ml streptomycin nd incubted t 37 C in humidified 5% CO 2 tmosphere. DMEM contining 2 µg/ml trypsin ws used for culture fter virl infection. Cytotoxicity estimtion MDCK cells were incubted in 96-well plte t 37 C in humidified 5% CO 2 tmosphere. Cells were treted with different concentrtions of geniposide (32.5, 65, 13, 26, 52, 1,4 mmol/l) when they were lmost confluent. Concentrtions of geniposide were chosen ccording to the pre-experiment. After 72 h, MTT ssy ws pplied to estimte the cytotoxicity. Cell vibility ws expressed s opticl density. Cell vibility rtio ws clculted s the percentge of opticl density compred with the control group (without geniposide tretment). In vitro ntivirl evlution MDCK cells were seeded in 96-well pltes t density of cells/well nd cultured until 6 7% confluent. Then, the cells infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus t 1 TCID 5 (5% tissue culture infectious dose) were treted with serilly diluted geniposide solutions t -24 h (24 h before virl infection, pre-tretment mode), h (t the sme time s virl infection, simultneous tretment mode) or 24 h (24 h fter virl infection, post-tretment mode). In ech tretment mode, either drugs or virus existing in the superntnts ws wshed off fter 24 h of intervention. Four seril dilutions of geniposide (from 32.5 to 26 mmol/l) were tested in three tretment modes. Permivir is n NAI tht selectively inhibits the neurminidse of humn type A nd B influenz viruses in vitro nd in vivo [1]. It significntly reduces the durtion of sesonl influenz virus infection without sfety Interntionl Medicl Press

3 Geniposide demonstrtes ntivirl ctivity ginst influenz virus concerns. The Chinese Food nd Drug Administrtion nnounced the pprovl of permivir for tretment in the H7N9 outbrek in Chin on 6 April 213, with n expecttion to sve ptients with severe symptoms. In ddition, the United Sttes Food nd Drug Administrtion issued n emergency use uthoriztion of intrvenous permivir exclusively for ptients who were hospitlized for influenz A (H1N1) pdm9-ssocited infection on 23 October 29 [27]. In this reserch, permivir ws selected s positive drug. Permivir (.3 µmol/l) ws pplied in the in vitro experiment [28]. Norml cell control, virl infection control, geniposide tretment nd positive drug tretment were included in ll ssys. After incubtion t 37 C for 72 h, the inhibition of virus-induced cytopthogenic effect ws evluted by the MTT ssy. Cell vibility ws expressed by opticl density. The medin effective dose (EC 5 ) ws determined by the Reed Muench method [29]. The cytopthogenic effect reduction ssy ws pplied to evlute the protective effect of geniposide on MDCK cells infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus [3]. Animl infection nd tretment procedures Femle ICR mice (6 to 8 weeks old; Lbortory Animl Reserch Center, Shnghi, Chin) were mintined on stndrd feed nd wter in specific-pthogen-free fcility with controlled environmentl temperture nd humidity. All mice received humn cre in complince with the Animl Experiments Guidelines nd Animl Cre of Chinese Acdemy of Sciences. The following protocol ws conducted in ech experiment. Mice were rndomly divided into six groups (n=1): plcebo control group, virus-infected control group, geniposide tretment group (5, 1 or 2 mg/kg, intrperitonel) nd permivir tretment group (3 mg/kg, intrperitonel). The nimls were nesthetized by intrperitonel dministrtion of 4% chlorl hydrte (Sigm, Munich, Germny) before intrnsl instilltion. On dys post infection (dpi), mice in plcebo control group received 5 µl sterile norml sline without pndemic A/Jingsu/1/29 (H1N1) influenz virus intrnslly, nd mice in the other groups were exposed to 5 µl virl suspension contining 1 medin lethl doses (LD 5 ) of pndemic A/Jingsu/1/9 (H1N1) influenz virus by intrnsl instilltion. On 1 dpi, mice of medicine intervention groups were dministered with geniposide or permivir (s positive control), nd mice of the plcebo control group nd virus-infected group received n equl volume of vehicle (sterile norml sline) insted. The drugs or vehicle were intrperitonelly dministered dily for 14 dys. The body weight chnges nd survivls of mice were observed every dy nd recorded until 14 dpi. The protective effect ws estimted by body weight restortion nd the reduction of mortlity [31]. The surviving mice were then scrificed nd their lungs were plced into sterile centrifuge tube nd stored t -8 C. Determintion of virl titres in the murine lung tissues Mice treted s bove were scrificed nd the lungs were hrvested on 3 dpi or 6 dpi. The lung homogentes were frozen nd thwed once to relese the virus nd centrifuged t 2, rpm t 4 C for 2 min. MDCK cells were used to titrte virl lods present in lung tissues. Virus titrtion ws determined by using hemgglutintion ssy, which identified the presence of certin viruses tht gglutinte red blood cells. The presence of virus would hold the red cells in diffuse mtrix nd prevent them from settling out to the bottom of the well [32]. Mesurement of lung index Mice from ech group were scrificed on 5 dpi. Lung tissues were excised nd weighed. The lung index ws expressed s the rtio of lung weight to body weight nd clculted to ssess tissue oedem [33]. Histologicl immunostining Lung tissues were collected on 6 dpi, fixed with 4% buffered formlin, embedded in prffin nd then stined with hemtoxylin nd eosin (H&E). Virl stining ws performed using nti-h1n1 nucleoprotein (NP) ntibody (bioss, Beijing, Chin). Determintion of TNF-, IFN-g, IL-4, IL-6 nd IL-1 levels The concentrtions of the cytokines, including tumour necrosis fctor- (TNF-), interferon-g (IFN-g), interleukin (IL)-4, IL-6 nd IL-1, in the superntnts of the lung tissue homogentes were mesured using sndwich enzyme-linked immunosorbent ssy (ELISA) kits (Biolegend, Sn Diego, CA, USA) ccording to the mnufcturer s instructions. Sttisticl nlysis All quntittive dt were expressed s mens ± stndrd devition (sd). Sttisticl nlysis ws performed using SPSS 18. softwre. Sttisticl comprisons between groups were crried out using one-wy nlysis of vrince. Survivl curves were nlysed by the log-rnk test. P<.5 is considered s sttisticl difference. Results Geniposide inhibited pndemic A/Jingsu/1/29 (H1N1) influenz virus infection in vitro with little cytotoxicity Cytotoxicity ws detected to estimte the sfety of geniposide tretment. The experimentl results showed tht Antivirl Therpy

4 Y Zhng et l. geniposide t different concentrtions (32.5, 65, 13, 26, 52, 1,4 mmol/l) hd no inhibitory effect on cell growth fter 72 h tretment (Figure 1A). According to the dt, it is speculted tht geniposide hs no cytotoxicity under 1,4 mmol/l. To investigte the protective effect of geniposide on MDCK cells infected with the influenz virus, three different modes of tretments were evluted s described in Methods. As shown in Figure 1B, 1C, 1D & 1E, pndemic A/Jingsu/1/29 (H1N1) influenz virus ws sensitive to geniposide in different tretment modes. The EC 5 of geniposide dministered for pretretment, simultneous tretment nd posttretment mode were 91.9, nd µmol/l, respectively. It suggested tht even 24 h fter virus dsorption, the protective efficcy ws semblble to the other two modes: the pretretment mode nd simultneous tretment mode. Menwhile, this result ws further confirmed by the inhibition rte curve. The curves lso suggested tht geniposide could block the dmge of pndemic A/Jingsu/1/29 (H1N1) influenz virus on MDCK cells in dose-dependent wy. Geniposide llevited clinicl signs induced by pndemic A/Jingsu/1/29 (H1N1) influenz virus in vivo To ssess nti-influenz virus potency of geniposide in vivo, we estblished murine model of influenz respirtory trct infection. The mice were infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus nd treted with either geniposide or permivir from 1 dpi. Strting from 2 dpi, symptoms of infection grdully ppered in the virus-infected control group mice, including piloerection, gther together nd decrese in rection force. Besides, neurologicl symptoms lso grdully begn to pper, such s slight musculr tremor, hunched posture, hind limb prlysis (Tble 1). Geniposide-treted mice, except for the ones treted with the lowest dose (5 mg/kg), showed lighter clinicl signs nd geniposide tretment restored body weight loss (Figure 2). Moreover, geniposide tretment t ll doses protected virus-infected mice from deth (versus virus-infected control group, P<.5), prticulrly t the highest dose. Wheres, ll virus-infected control mice died from infection by 8 dpi (Figure 3). Geniposide tretment reduced virl repliction in dose-dependent mnner, expressed s the virl titres in the murine lungs detected on 3 dpi nd 6 dpi (Figure 4). The findings indicted tht the pndemic A/Jingsu/1/29 (H1N1) influenz virus ws sensitive to geniposide tretment in vivo. Tken together, geniposide cn effectively improve the clinicl symptoms, reduce the body weight loss, extend the survivl time nd decrese virl titres in virus-infected mice. Menwhile, the efficcy of geniposide tretment in lleviting clinicl signs ws comprble to permivir tretment. Geniposide reduced the severity of virl lung lesions As shown in Figure 5, the lung index ws clculted to ssess tissue oedem. Geniposide significntly decresed the lung index compred with the infected mice (P<.5). H&E stining ws pplied to evlute the histopthologicl chnges. Lung tissues obtined from the plcebo control group showed intct structures nd cler pulmonry lveoli (Figure 6A). The pndemic A/Jingsu/1/29 (H1N1) influenz virus induced severe inflmmtory response nd typicl interstitil pneumoni in lung tissues. The virusinfected lungs were chrcterized by typicl lung injuries, including pulmonry congestion nd oedem, infiltrtions of neutrophils nd mononucler cells into the tissue nd lveoli, thickening of the lveolr wll nd lveolr collpse (Figure 6B). The geniposide (5 mg/kg) tretment group showed similr lung lesions to the virus-infected lungs, but smller portion of interstitil pneumoni tht lmost destroyed the lung tissue structure nd hd severe infiltrtion of inflmmtory cells in bronchil lumen (Figure 6C). Compensting emphysem ws noted in the surrounding lung prenchym. The geniposide (2 mg/kg) tretment group showed pproximte norml pulmonry tissue in comprison with the plcebo control group (Figure 6E). Histologicl nlysis in the permivir tretment group reveled tht most of the lung tissue ws free from inflmmtory cell infiltrtion nd structure dmge (Figure 6F). Virus-infected cells in bronchil epithelium nd lveolr spce were stined with n influenz virus NP ntibody. No infected cells were found in the plcebo control group (Figure 7A). Multiple types of infected cells were observed in the lung tissues of virus-infected group, including epithelil cells from bronchi, terminl bronchioles nd the lveolr lining where type II pneumocytes were the min infected cells (Figure 7B). Infiltrting cells in severe inflmed res lso presented NP-positive stining (Figure 7B). The geniposide (5 mg/kg) tretment group lso showed these chnges, but to lower degree compred with the virus-infected group (Figure 7C). The geniposide (2 mg/kg) tretment group (Figure 7E) nd permivir (3 mg/kg) tretment group (Figure 7F) showed much fewer virus-infected cells, in ccordnce with their slight clinicl signs nd body weight restortion. Our findings indicted tht the degree of virl infection ws directly correlted with pndemic A/ Jingsu/1/29 (H1N1) influenz virus-induced lung pthologicl dmge [34] Interntionl Medicl Press

5 Geniposide demonstrtes ntivirl ctivity ginst influenz virus Figure 1. Cytotoxicity of geniposide on MDCK cells nd its protective effect on pndemic A/Jingsu/1/29 (H1N1) influenz virus-infected MDCK cells in vitro A Cell vibility rtio ,4 Drug concentrtion, µmol/l B Pretretment C Simultneous tretment Opticl density (cell survivl) NC VC PER Opticl density (cell survivl) NC VC PER Geniposide, µmol/l Geniposide, µmol/l D Opticl density (cell survivl) NC VC Post-tretment Geniposide, µmol/l PER E Virus inhibition rte, % Drug concentrtion, µmol/l Pretretment Direct dectivtion Tretment (A) Uninfected cells were cultured with geniposide ( 1,4 μmol/l) for 72 h. Three different modes of geniposide tretment on infected Mdin Drby cnine kidney (MDCK) cells (B) pretretment, (C) simultneous tretment, (D) post-tretment in four concentrtions (32.5, 65, 13 nd 26 μmol/l) were conducted. (E) Inhibition rtes by geniposide in the different tretment modes were shown in the form of curves. MTT ssy ws pplied to estimte the cytotoxicity nd the protective effect. Cell vibility ws determined by opticl density. Results re expressed s cell vibility rtio. Dt re showed s mens ± stndrd devition (sd). Versus virus-infected control group, P<.5. b Versus permivir (PER; 3 mg/kg) tretment group, P<.5. NC, norml control; VC, virus control. Antivirl Therpy

6 Y Zhng et l. Tble 1. Resolution of infection signs in pndemic A/Jingsu/1/29 (H1N1) influenz virus-infected nimls fter drug tretment (from 2 dpi to 14 dpi) Number of nimls with infection signs/totl number of nimls Infection signs Virus-infected control Geniposide (5 mg/kg) Geniposide (1 mg/kg) Geniposide (2 mg/kg) Permivir (3 mg/kg) Constitutionl symptoms 1/1 8/1 5/1 3/1 2/1 Neurologicl symptoms b 1/1 /1 /1 /1 /1 Piloerection, gther together, decrese in rection force, etc. b Slight musculr tremor, hunched posture, hind limb prlysis, etc. dpi, dys post-infection. Figure 2. Body weight chnges of mice infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus Chnge in weight, % 1 8 Virus-infected control Geniposide (5 mg/kg) Geniposide (1 mg/kg) Geniposide (2 mg/kg) Permivir (3 mg/kg) Time fter infection, dy Mice (n=1) were infected with influenz virus through the intrnsl route. From 1 dy post-infection (dpi), geniposide (5, 1 or 2 mg/kg) tretment or permivir (3 mg/kg) tretment ws dministered intrperitonelly dily for 14 dys. Virus-infected control group ws treted with sterile norml sline lone under the sme condition. Body weights were monitored until 14 dpi nd expressed s the percentges of the initil body weight on dpi. Geniposide reduced the levels of pndemic A/Jingsu/1/29 (H1N1) influenz virus-ssocited inflmmtory cytokines in vivo A time-course study for the levels of cytokines ws performed to explore whether cytokine production ws ffected by geniposide tretment. The concentrtions of TNF- (Figure 8A), IFN-g (Figure 8B), IL-6 (Figure 8C), IL-4 (Figure 8D) nd IL-1 (Figure 8E) in murine lung tissues incresed significntly fter infection compred with those in the plcebo control group (P<.5). Geniposide (2 mg/kg) effectively inhibited the relese of TNF-, IFN-g nd IL-6 (versus virus-infected group, P<.5), wheres it enhnced the relese of IL-4 nd IL-1 (versus virus-infected group, P<.5) in lung tissues. The permivir tretment ws stronger in inhibiting the relese of TNF-, IFN-g nd IL-6 compred with geniposide (2 mg/kg). The concentrtions of IL-4 nd IL-1 in murine lung tissues from the permivir tretment group were lower thn those from the geniposide (2 mg/kg) tretment group. Discussion Geniposide, the mjor iridoid glycoside ingredient of grdeni herbs, hs emerged s novel multifunctionl tissue-protective gent with nti-edemtous nd nti-inflmmtory ctivities. Lin et l. [35] reported tht geniposide could inhibit both enterovirus 71 (EV71) repliction nd virl IRES (internl ribosome entry site) ctivity through blocking virl protein trnsltion. Yng et l. [15] demonstrted tht geniposide hd n ntivirl effect on the low pthogenic influenz viruses, such s influenz virus strin A/FM/1/47-MA. In view of its suppression of viruses of different species nd pthogenicities, our investigtion further explored the ntivirl property Interntionl Medicl Press

7 Geniposide demonstrtes ntivirl ctivity ginst influenz virus Figure 3. Survivl time of mice infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus Survivl rte, % Virus-infected control Geniposide (5 mg/kg) Geniposide (1 mg/kg) Geniposide (2 mg/kg) Permivir (3 mg/kg) Time fter infection, dy Mice (n=1) were infected with influenz virus through the intrnsl route. From 1 dy post-infection (dpi), geniposide (5, 1 or 2 mg/kg) tretment or permivir (3 mg/kg) tretment ws dministered intrperitonelly dily for 14 dys. Virus-infected control group ws treted with sterile norml sline lone under the sme condition. Mortlity ws monitored until 14 dpi. Figure 4. Determintion of virl titres in the murine lung tissues t different time points 8 Virl lung titres, log 1 TCID 5 /ml Virus-infected control Geniposide (5 mg/kg) Geniposide (1 mg/kg) Geniposide (2 mg/kg) Permivir (3 mg/kg) Dy 3 post-infection Dy 6 post-infection Mice (n=1) were infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus through the intrnsl route. From 1 dy post-infection (dpi), geniposide (5, 1 or 2 mg/kg) tretment or permivir (3 mg/kg) tretment ws dministered intrperitonelly dily. Virus-infected control group ws treted with sterile norml sline lone under the sme condition. Lung tissues were collected on 3 dpi or 6 dpi nd then Mdin Drby cnine kidney (MDCK) cells were used to titrte virl lods. Dt re shown s mens ± stndrd devition (sd). Versus virus-infected control group, P<.5. b Versus permivir (PER; 3 mg/kg) tretment group, P<.5. TCID 5, 5% tissue culture infectious dose. Antivirl Therpy

8 Y Zhng et l. Figure 5. Determintion of lung index in ech experimentl group 2.5 Plcebo 2. Virus-infected control Lung index, % 1.5 Geniposide (5 mg/kg) Geniposide (1 mg/kg) 1. Geniposide (2 mg/kg) Permivir (3 mg/kg).5. Mice (n=1) were infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus through the intrnsl route. From 1 dy post-infection (dpi), geniposide (5, 1 or 2 mg/kg) tretment or permivir (3 mg/kg) tretment ws dministered intrperitonelly dily. Plcebo control group nd virus-infected control group were treted with sterile norml sline lone under the sme condition. All surviving mice were scrificed t 5 dpi nd the lung tissues were obtined. The lung index ws clculted s lung weight 1%/finl body weight (LW/BW, %). Dt re shown s mens ± stndrd devition (sd). Versus virus-infected control group, P<.5. bversus permivir (3 mg/kg) tretment group, P<.5. Figure 6. Exmintion of histopthologicl chnges in lung tissues (originl mgnifictions, 4) A B C D E F Mice (n=1) were infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus through the intrnsl route. From 1 dy post-infection (dpi), geniposide (5, 1 or 2 mg/kg) tretment or permivir (3 mg/kg) tretment ws dministered intrperitonelly dily. Plcebo control group nd virus-infected control group were treted with sterile norml sline lone under the sme condition. All surviving mice were scrificed t 5 dpi nd lung tissues were obtined for HE stining. Inflmmtory infiltrtions re indicted with rrowheds. (A) Plcebo control group. (B) Virus-infected control group. (C) Geniposide (5 mg/kg) tretment group. (D) Geniposide (1 mg/kg) tretment group. (E) Geniposide (2 mg/kg) tretment group. (F) Permivir (3 mg/kg) tretment group. 66 AVT-17-OA-434_Zhng.indd Interntionl Medicl Press 22/11/217 13:48:31

9 Geniposide demonstrtes ntivirl ctivity ginst influenz virus Figure 7. Immunostining of pndemic A/Jingsu/1/29 (H1N1) influenz virus-infected cells in lung tissues (originl mgnifictions, 1) A B C D E F Mice (n=1) were infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus through the intrnsl route. From 1 dy post-infection (dpi), geniposide (5, 1 or 2 mg/kg) tretment or permivir (3 mg/kg) tretment ws dministered intrperitonelly dily. Plcebo control group nd virus-infected control group were treted with sterile norml sline lone under the sme condition. On 5 dpi, ll surviving mice from ech group were scrificed nd lung tissues were obtined. The nti-h1n1 nucleoprotein (NP) ntibody ws used to perform virl stining. The NP-positive cells, stined drk brown, re indicted with rrows. (A) Plcebo control group. (B) Virus-infected control group. (C) Geniposide (5 mg/kg) tretment group. (D) Geniposide (1 mg/kg) tretment group. (E) Geniposide (2 mg/kg) tretment group. (F) Permivir (3 mg/kg) tretment group. of geniposide ginst pndemic A/Jingsu/1/29 (H1N1) influenz virus in vitro nd in vivo. The in vitro experimentl results showed tht geniposide could protect MDCK cells from influenz virusinduced cell dmge, without cytotoxicity t therpeutic doses. Since geniposide delivered in three modes performed similr protective effects, we speculte the cell itself s the drug trget. It hs been proved tht influenz virus infection results in the ctivtion of the mitogenctivted protein (MAP) kinses p38 nd Jun N-terminl kinse (JNK) nd the downstrem trnscription fctors nucler fctor-kb (NF-kB), ctivtor protein 1 (AP-1) nd cyclic AMP response element (CRE) binding fctors [36]. NF-kB s hllmrk of virl infection, plys centrl role in virus-dependent cytokine expression nd pthology [36]. Geniposide hs been reported to ply n nti-inflmmtory role through suppressing the phosphoryltion of p38, JNK nd NF-kB [37,38]. Therefore, we speculte tht geniposide my perform the ntivirl ctivity through ffecting the MAPK NF-kB signlling pthwy in MDCK cells. The influenz A (H1N1) pdm9 virus cuses severe pneumoni tht develops into cute lung injury (ALI), ARDS nd multiple orgn dysfunction syndrome (MODS) if not receiving tretment in time [6]. Hypercytokinemi, lso known s cytokine storm, Antivirl Therpy 22.7 AVT-17-OA-434_Zhng.indd 67 is considered s the mjor pthogenetic mechnism in ALI, ARDS nd MODS [39,4]. A vriety of proinflmmtory cytokines, such s TNF-, IFN-g nd IL-6, emerge in huge quntities rpidly which led to series of bnorml immune responses nd trigger off systemic inflmmtory response syndrome (SIRS) [41,42]. In the present study, to investigte the protection ginst influenz A virus infection, murine model of influenz A (H1N1) pdm9 virus infection ws estblished, nd the virus-induced pulmonry injury nd cytokine chnges were evluted fter drug interventions. In vivo, body weight restortion nd elevted survivl rte reflected geniposide s improvement on mice survivl. The llevitive virus lod nd histologicl observtions in lung tissues indicted tht geniposide could hve ntivirl effects nd protect ginst pndemic A/Jingsu/1/29 (H1N1) influenz virus-induced lung inflmmtion. It hs been reported tht cspse 3 ctivtion, which is induced in n NF-kB-dependent mnner in influenz-infected epithelil cells [43], medites exporting ribonucleoprotein complexes (RNPs) from the nucleus to be pckged into infectious progeny virions t the cell membrne nd is essentil for efficient influenz virus propgtion [44,45]. Moreover, geniposide hs been proved to down-regulte the expression of cleved-cspse 3 nd reduce the ctivtion of 67 22/11/217 13:48:33

10 Y Zhng et l. Figure 8. Determintion of pndemic A/Jingsu/1/29 (H1N1) influenz virus-ssocited inflmmtory cytokines A 1, B 2, TNF-α in lung tissues, pg/ml Dy 3 post-infection Dy 6 post-infection IFN-γ in lung tissues, pg/ml 1,5 1, 5 Dy 3 post-infection Dy 6 post-infection C 15 D 25 IL-6 in lung tissues, pg/ml 1 5 IL-4 in lung tissues, pg/ml Dy 3 post-infection Dy 6 post-infection Dy 3 post-infection Dy 6 post-infection E IL-1 in lung tissues, pg/ml b Plcebo Virus-infected control Geniposide (5 mg/kg) Geniposide (1 mg/kg) Geniposide (2 mg/kg) Permivir (3 mg/kg) Dy 3 post-infection Dy 6 post-infection Mice (n=1) were infected with pndemic A/Jingsu/1/29 (H1N1) influenz virus through the intrnsl route. From 1 dy post-infection (dpi), geniposide (5, 1 or 2 mg/kg) tretment or permivir (3 mg/kg) tretment ws dministered intrperitonelly dily. Plcebo control group nd virus-infected control group were treted with sterile norml sline lone under the sme condition. All surviving mice from ech group were scrificed nd lung tissues were obtined on 3 dpi or 6 dpi. The concentrtions of the cytokines (A) tumour necrosis fctor (TNF)-α, (B) interferon (IFN)-γ, (C) interleukin (IL)-6, (D) IL-4 nd (E) IL-1 in the superntnts of the lung tissue homogentes were mesured with ELISA kits ccording to the mnufcturers instructions. Versus virus-infected control group, P<.5. b Versus permivir (3 mg/kg) tretment group, P< Interntionl Medicl Press

11 Geniposide demonstrtes ntivirl ctivity ginst influenz virus cspse 3 [46,47], which is the possible mechnism of geniposide s inhibition on virl propgtion. Permebility oedem is life-thretening compliction of ALI nd ARDS [48]. In this study, lung index ws clculted to quntify the mgnitude of pulmonry oedem nd the results indicted tht geniposide tretment ttenuted the development of pulmonry oedem. Clinicl nd experimentl evidence hs confirmed tht complicted network of inflmmtory cytokines plys mjor role in mediting, mplifying nd perpetuting the lung injury process. The cytokines TNF-, IFN-g [49] nd IL-6 cn strt n inflmmtory cscde nd led to wide rnge of tissue dmge [5]. Simultneously, TNF- induces lveolr epithelil cells to produce other cytokines nd chemotctic fctors, such s IL-6, nd ctivte neutrophils, which contribute to the severity of lung injury [51]. These proinflmmtory cytokines were detected in our investigtion, nd found to be up-regulted fter infection. The symptoms of typicl influenz virus infection including fever, mylgi nd cough hve been reported to strt between 1 nd 4 dys post-infection nd persist for 3 to 5 dys [36]. Animl investigtions show tht TNF- is significntly elevted from 4 to 14 dpi in pigs infected with H1N1 intrnslly, without significnt difference between 4 dpi nd 7 dpi, t which time points TNF- is the highest [52]. IFN-g hs been reported to trnsiently increse t 6 dpi [53] or rech the highest levels on 4 nd 7 dpi during H1N1 infection [54]. Moreover, our experimentl results showed tht the untreted mice died dting from 4 dpi nd none survived until 8 dpi, which indictes the ftl lung injury occurred erliest on 4 dpi. Proinflmmtory cytokines re presumed to rech the pek or sty t flt pek before deth. Therefore, combined with our dt, TNF- nd IFN-g re speculted to keep flt pek on 3 nd 6 dpi nd to continuously induce nd ggrvte pulmonry dmge. Inhibition on the production of TNF-, IFN-g by geniposide keeps in line with its protective effect on infection. The expressions of IL-4 [55] nd IL-1 [56] re relted to TNF- level. IL-4, Th2 signture cytokine, is produced by TC2 cells [57] nd is proved to be produced by IL-4+ secreting cells in the lte recovery phse [53]. IL-1, considered s n importnt nti-inflmmtory cytokine, cts s negtive regultor of the response of both innte nd dptive immune cells nd is produced in the infected lungs t high level by ntivirl CD8+ nd CD4+ effector T-cells during cute influenz infection [58]. As reported, IL-4 nd IL-1 levels re both elevted in H1N1-infected ptients [59], which is in line with our experimentl results. We speculte tht IL-4 nd IL-1 re up-regulted due to the ctivtion of immune cells by H1N1 nd further perform nti-inflmmtory nd immunoregultory effect during infection. It hs been reported tht geniposide is ble to exert its ntiinflmmtory nd immunoregultory effect through enhncing the expression of IL-4 nd IL-1 in djuvnt rthritis [6,61]. So, further up-regultion of IL-4 nd IL-1 by geniposide is considered s one of its protection mechnism. It is worth noting tht the drug dose of geniposide ffected cell vibility nd therpeutic efficcy. Wei et l. [62] provided n insight into geniposide-induced heptotoxicity in rt model. It is reported tht high-dosge (3 mg/kg) nd medium-dosge (1 mg/kg) geniposide could induce different levels of liver injury while low-dose (3 mg/kg) geniposide did not exhibit heptotoxicity. Previous reserches showed tht the sfe dose of geniposide ws less thn 8 mg/kg [48,63]. Although these findings suggest tht geniposide my be promising gent for preventing pndemic A/ Jingsu/1/29 (H1N1) influenz virus-induced ALI, further studies re still required to investigte its clinicl relevnce nd the precise ntivirl mechnism relted. Acknowledgements This study ws supported by the Project of Jingsu Provincil Trditionl Chinese medicine Administrtion Bureu (LZ13228); the Medicl Science nd Technology Development id Project of Nnjing Helth Bureu (YKK13176) nd the Ntionl Nturl Science Foundtion of Chin (Generl Progrm Project, ). Disclosure sttement The uthors declre no competing interests. References 1. Novel Swine-Origin Influenz A (H1N1) Virus Investigtion Tem, Dwood FS, Jin S, et l. Emergence of novel swine-origin influenz A (H1N1) virus in humns. N Engl J Med 29; 36: Worobey M, Hn GZ, Rmbut A. Genesis nd pthogenesis of the 1918 pndemic H1N1 influenz A virus. Proc Ntl Acd Sci U S A 214; 111: Joseph U, Linster M, Suzuki Y, et l. Adpttion of pndemic H2N2 influenz A viruses in humns. J Virol 215; 89: Wendel I, Rubbenstroth D, Doedt J, et l. The vin-origin PB1 gene segment fcilitted repliction nd trnsmissibility of the H3N2/1968 pndemic influenz virus. J Virol 215; 89: Zhou Z, Li X, Liu J, et l. 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