Combination of ribavirin and reduning protects mice against severe pneumonia induced by H1N1 influenza a virus

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1 Online Sumissions: J Trdit Chin Med 2016 April 1; 36(2): info@journltcm.com ISSN JTCM. All rights reserved. EXPERIMENTAL STUDY TOPIC Comintion of rivirin nd reduning protects mice ginst severe pneumoni induced y H1N1 influenz virus M Yuexi, Zhng Wei, Zho Zhongpeng, Li Min, Liu Jin, Wng Yugung M Yuexi, Deprtment of Internl Medicine, the Trditionl Chinese Medicine, Hospitl of Beijing Huirou, Beijing Hospitl of Trditionl Chinese Medicine ffilited with Cpitl Medicl University, Beijing , Chin Zhng Wei, The Infectious Diseses Dignostic, Therpeutic nd Reserch center, the Synergetic nd Innovtive Centre for the Prevention nd Tretment of Key Infectious Diseses, Cpitl Medicl University, Beijing 10001, Chin Zho Zhongpeng, Deprtment of Immunology, Beijing Institute of Microiology nd Epidemiology, Stte Key Lortory of Pthogen nd Biosecurity, Beijing , Chin Li Min, Lortory Animl Center, Acdemy of Militry Medicl Science, Beijing , Chin Liu Jin, Pneumology Deprtment, Beijing Hospitl of Trditionl Chinese Medicine Affilited with Cpitl Medicl University, Beijing , Chin Supported y the Beijing Key Lortory of Bsic Reserch with Trditionl Chinese Medicine on Infectious Disese, the Specil Fund of Trditionl Chinese Medicine Scientific Reserch Projects in 2009 (No A); nd the Beijing Helth System, Helth nd Technicl Personnel of High level pln (No ) Correspondence to: Wng Yugung, Pneumology Deprtment, Beijing Trditionl Chinese Medicine Hospitl, Beijing , Chin. influenztcm@163.com Telephone: Accepted: Septemer 21, 201 Astrct OBJECTIVE: To investigte the effects of rivirin dministrtion comined with in mouse model of influenz A (H1N1)induced severe pneumoni. METHODS: Influenz A/Beijing/01/2009 (H1N1) infected C7BL/6 mice were rndomly divided into four experimentl groups treted with either mock injection of phosphteuffered sline (), rivirin (66.6 mg/kg dily) or (86.6 mg/ kg dily), or comintion of oth, for 7 dys. Mice were monitored for clinicl signs nd survivl, nd ody weight ws mesured dily for 14 dys. Virus titer, lung wettodry rtios, pthology nd cytokines including interleukin (IL)6, IL10, nd interferon (IFN)γ were ssyed on different dys. RESULTS: In the untreted group injected with phosphte uffer sline, ll the mice died of the infection. The survivl rte of mice treted with ws only 10%, wheres 100% of the rivirin nd the comintiontreted mice survived. Low lung virl lods indicted tht rivirin significntly inhiited virus repliction, wheres did not. Lung wettodry rtios demonstrted tht oth rivirin nd, dministered together or seprtely, reduced cute lung edem compred with results in the untreted group. Pthology nlyses lso showed tht tretment with comintion of oth drugs relieved pthologicl lesions, wheres the single drug tretment did not. Levels of IL6, IL10 nd IFNγ in mice treted with rivirin or the comintion of oth rivirin nd were ll significntly lower thn in the untreted group, especilly in the comintiontreted group. In ddition, dministrtion significntly decresed oth IL6 nd IL10 production ut hd no effect on IFNγ. CONCLUSION: Due to the synergistic effect of ntivirl nd ntiinflmmtion, the comintion of rivirin nd could e n effective tretment for severe H1N1 which ws considered to e significnt to delyed ntivirl nd drug resistnt JTCM. All rights reserved. JTCM www. journltcm. com 181

2 M YX et l. / Experimentl Study Key words: Influenz, Humn; Pneumoni; Rivirin; Medicine, ChineseTrditionl; injection INTRODUCTION In April 2009, H1N1 swine flu first emerged in Mexico. 1 On August 10th 2010, the World Helth Orgniztion declred influenz A H1N1 (ph1n1) pndemic. During this period, 214 countries reported confirmed cses of influenz A H1N1; nd the disese resulted in deths. Minlnd Chin lone reported totl of 12.8 million cses with 80 deths nd 0.% mortlity rte. Some severely infected ptients developed cute pneumoni, which ws the predominnt cuse of reported deths. Although the mortlity cused y ph1n1 infection ws low, 9%31% of ph1n1 ptients required dmission to intensive cre units, nd 14%46% of these ptients ultimtely died. 2 To this dy, the min tretment strtegy for severe pneumoni relies on different types of djunctive therpies, lthough those tretments re controversil. The clinicl tretment of ph1n1 minly depends on neurminidse inhiitors such s oseltmivir, nd ntivirl compounds such s rivirin. However, ntivirl therpy usully involves delyed response or hs low efficcy. 6 Systemic corticosteroids re often used to tret severe influenz, lthough severl studies hve indicted tht this my increse the risk of mortlity. 7 Chinese medicine hs een widely used in the tretment of ph1n1. Some drugs such s Shufengjiedu cpsules nd Mxingshign decoctions hve shown effectiveness. 8 However, there hve een few studies reporting the effects of Chinese medicine on severe influenz., one clering het nd detoxifying Trditionl Chinese Medicine (TCM) injection, ws mily used for tretment of cute nd severe disese in clinicl. Mny studies hd reported tht could reduce the durtion of influenz illness which ws ttriuted to the role in inhiiting NA nd ntiinflmtion. Compred with ntivirl injections, formultions of cpsules, decoctions, or grnules lone hve een unle to provide immedite relief for severe influenz in clinicl prctice. This study investigted the efficcy of ntivirl dministrtion comined with injections of the Chinese medicine for the tretment of severe influenz. MATERIALS AND METHODS Virus, cells nd nimls Influenz A/Beijing/01/2009 (H1N1) (BJ01) nd Mdin Dry Cnine Kidney cells were otined from the Beijing Institute of Microiology nd Epidemiology, Stte Key Lortory of Pthogen nd Biosecurity. Specific pthogen free (SPF) C7BL/6 femle mice, 46 weeks old (Experimentl Animl Production License No. SCXK ), weighing 1416 g, were provided y the Lortory Animl Center of the Acdemy of Militry Medicl Science (Beijing, Chin). Drugs nd regents Rivirin Injection (Btch No , 0.1 g /2 ml) ws otined from the Shndong Lu Kng Chen Xin Phrmceuticl Co, Ltd. (Shndong, Chin). (Btch No , 6 g /10 ml) ws purchsed from the Kng Yun Phrmceuticl Co, Ltd. (Hunn, Chin). The mouse inflmmtory cytokines cytometric ed rry ws otined from BD Biosciences (Frnklin Lkes, NJ, USA). Therpeutic efficcy of different drugs or comintion ginst ph1n1 in mice To evlute the therpeutic role of rivirin, or oth ginst BJ01 infection, following intrperitonel (i.p.) nesthetiztion with pentoritl sodium, C7BL/6 mice received 2 10 TCID 0 of BJ01 intrnslly (i.n.) on dy 0 s shown previously 910, followed y i.p. injections of (86.6 mg kg 1 d 1 ), rivirin (66.6 mg kg 1 d 1 ), comintion of oth, or mock injection phosphte uffer sline () once dy for 7 dys post infection (in order to chieving therputic drug concentrtion once infection, ech group were dministered 12 h efore infection. Mice in ech group (n = 10) were monitored dily for survivl, clinicl signs nd ody weight. Mice from ech group were euthnized on different dys, nd the lungs were collected s descried elow. The virl lod in the lung tissue of mice Lung tissues from five mice selected from ech group were collected t 4 dys postinfection (DPI). The titers of viruses in the lung tissue were determined y the cell culture infective dose 0% (CCID0) ssy in Mdin Dry cnine kidney cells. 9,10 Acute pulmonry edem (wettodry rtio) Assessment of cute pulmonry edem ws performed t y clculting the lungs' wettodry rtios from the weights of wet lungs nd the dry weight otined fter heting the tissues t 68 for 24 h. Histologicl exmintion Following pentoritl sodium nesthesi, two to four mice from ech group were scrificed t DPI. Lungs were fixed in formlin nd emedded in prffin. Ultrthin sections were otined nd stined with hemtoxylineosin. Lung histopthology ws determined y light microscopy (Olympus, Jpn). Cytokine nd chemokine mesurement For cytokine mesurements, five mice from ech group were euthnized nd roncholveolr lvge fluids (BALFs) were collected t 2 nd. BALFs were ssessed for interleukin (IL)6, IL10 nd interferon (IFN)γ using the cytometric ed rry. Arry nlysis ws performed using FCAP Arry softwre. JTCM www. journltcm. com 182

3 M YX et l. / Experimentl Study Sttisticl nlyses All dt re shown s mens ± stndrd devition ( xˉ ± s). Survivl dt were nlyzed y KplnMeier survivl nlysis mesurements t single time points nd sttistics performed using nlysis of vrince. If they reched significnce, dt were further nlyzed y twotiled Student's ttest. All sttisticl tests were conducted using GrphPd Prism.0 softwre (Softwre McKiev, Sn Diego, CA, USA). P < 0.0 indictes sttisticl significnce. RESULTS Administrtion of rivirin nd meliorted ph1n1induced cute influenz symptoms At 1, none of the mice tht received the mock injection survived BJ01 infection (0% ). The survivl rte ws significntly improved when mice were given rivirin (100%), or comintion of rivirin nd (100% ). However, dministrtion of only resulted in low survivl rte (10% ) (Figure 1A). Loss in ody weight in the group ws s high s 40% with continuous decline until deth. However, mice treted with rivirin or oth rivirin nd showed weight loss of only 1%, which stopped declining t DPI nd improved t 8 DPI. The therpeutic efficcy of lone ws very low; weight loss in this group ws 2%, with lte recovery in weight tht egn t 10 DPI (Figure 1B). When compred to the mice treted with lone, oth Rivirin nd the comintion Rivirin nd significntly decresed the ody weight loss induced y BJ01 infection, especilly the comintion group (Figure 1C). Acute lung pneumoni, ssessed y lung pthology nd wettodry weight rtio, ws lso improved when mice were treted with either of the single drugs or oth drugs. The pthologicl oservtions showed tht oth drug tretments improved lung pthologicl lesions. At DPI, lungs of injected mice showed congestion, edem nd hemorrhge with diffuse inflmmtory cell infiltrtion, shedding nd necrosis of lveolr epithelil cells s well s ronchil epithelil cell degenertion. The lungs of the mice treted with oth rivirin plus displyed significnt improvement with reduced edem, locl lveolr septl thickening nd inflmmtory cell infiltrtion. The lung lesions of the mice treted with either rivirin or were llevited when compred with the treted group ut not to the extent of oth drugs dministered together (Tle 6 nd Figure 2). Similrly, wettodry rtios of mice treted with rivirin lone or rivirin nd together were significntly reduced compred with the treted group (Tle 1). Notly, the wettodry rtio of mice treted with lone ws lso significntly reduced (Tle 1). Administrtion of oth rivirin nd my induce synergistic ntivirl nd immunomodultory effect Virl lod titers in the lungs of mice treted with rivirin lone or oth rivirin nd were significntly decresed, suggesting tht oth tretment methods efficiently inhiited virus repliction (Tle 1). However, there ws no difference in lung BJ01 titers etween the tretment with lone nd (Tle 1). Compred with the group, dministrtion of ri Survivl rtes (%) Body weight chnge (%) A Rivirin Rivirin plus reduning Dys post infection Dys post infection Body weight chnge (%) Dys post infection C Figure 1 Administrtion of rivirin nd meliorted ph1n1induced cute Rivirin Rivirin influenz symptoms plus reduning A: the survivl rtes of the mice with indicted tretments for 7 successive dys (n = 10); B: Body weight chnges (n = 10); C: Body weight chnges of the rivirin nd rivirin plus groups. Compred with the group, : phosphteuffered sline; P < 0.0 nd P < B Rivirin Rivirin plus reduning JTCM www. journltcm. com 183

4 M YX et l. / Experimentl Study A B C D virin,, or oth drugs comined significntly reduced the levels of IL6, IL10 nd IFNγ in BALF t oth 2 nd, lthough the effect on IFNγ ws much less drmtic in the only group (Tle 2). Levels of ll three cytokines in BALF were similr in the mice treted with either rivirin lone or the comintion of rivirin nd t, which were significntly lower thn in the group. Furthermore, IL10 levels were only significntly decresed t 2 DPI in the mice tht received the comined tretment (Tle 2). DISCUSSION Figure 2 Histologicl findings from lung tissues. Representtive pthologicl imges from the lungs t dys postinfection. The lungs of two to four mice from ech group were fixed in formlin nd emedded in prffin. Ultrthin sections were otined nd stined with hemtoxylineosin ( 100) A: mice treted with showing inflmmtory infiltrte nd lveolr dmge. B: Mice treted with for 7 dys exhiited no difference from the group in terms of histopthology. C: Mice treted with rivirin for 7 dys showed noticele reduction in inflmmtion nd tissue dmge compred with the group. D: Mice treted with rivirin nd together for 7 dysexhiited the lightest dmge compred with nygroup else. : phosphteuffered sline There is n urgent need for n effective strtegy for tretment of severe influenz. Current recommendtions for influenz tretment primrily focus on lleviting clinicl symptoms nd restoring respirtory function.11 Previous utopsy cse reports hve indicted tht lung injury in severe influenz ptients included serious interstitil lung injury, hyline memrne formtion, lveolr septl edem, lveolr epithelil cell prolifertion, intrvsculr firin thromosis, nd fine ronchil wll necrosis.12 Some ph1n1 cses in teengers over 18 yers of ge without ny other diseses hve displyed histologicl chnges similr to those in Tle 1 Wettodry rtios nd virl lods of lungs t 4 dys postinfection ( xˉ ± s) Group n Virus titer (logccid 0 /ml) Wettodry rtios P vlue P vlue 7.3± ± ± ± Rivirin 4.7± ±0.3 Rivirin plus 4.6± ±0.3 Notes: group: infected mice treted with (1 ml) y i.p. injection for 7 dys; group: infected mice treted with (86.6 mg kg 1 d 1) y i.p. injection for 7 dys; rivirin group: infected mice treted with rivirin (66.6 mg kg 1 d 1, 1 ml) for 7 dys; rivirin plus : infected mice treted with oth rivirin (66.6 mg kg 1 d 1, 1 ml) nd (86.6 mg kg 1 d 1, 1 ml) for 7 dys. Virl lods in lungs were determined in Mdin Dry Cnine Kidney cells. DPI: dys postinfection; : phosphteuffered sline. All tretment groups compred with group, P < 0.0. Tle 2 Concentrtions of IL6, IL10 nd IFNγ t 2 nd 4 dys post infection (pg/ml, ± s) Group IL10 IL6 IFNγ 2 DPI Nive 18.6± ± ± ± ± ± ± ± ± ± ± ± ± ±66.4 Rivirin 22.1± ± ± ± ± ±27.4 Rivirin plus 18.4± ± ± ± ± ± DPI 2 DPI 22.± ± ± ±79.8 Notes: Nive group: mice treted with (1 ml) i.p. for 7 dys; : infected mice treted with (1 ml) i.p. once dy for 7 dys; : infected mice treted with (86.6 mg kg 1 d 1) once dy for 7 dys i.p.; rivirin: infected mice treted with rivirin (66.6 mg kg 1 d 1) once dy for 7 dys i.p.; rivirin plus : infected mice treted with oth rivirin (86.6 mg kg 1 d 1) nd (66.6 mg kg 1 d 1) once dy for 7 dys i.p. Concentrtions of IL6, IL10, IFNγ in roncholveolr lvge fluids smples were determined y cytometric ed rry. IL: interleukin; IFNγ: interferonγ; DPI: dys postinfection; : phosphteuffered sline. group compred with nïve group, P < 0.0 nd ll tretment groups compred with the group, P < 0.0. JTCM www. journltcm. com 184 April 1, 2016 Volume 36 Issue 2

5 M YX et l. / Experimentl Study Tle 3 Lung Histopthology Group Rivirin Rivirin plus Inflmmtory cell infiltrtion HN1 infection cses. 13 Thus, the prognosis for severe influenz ptients is prticulrly ssocited with the severity of lung pthologicl injuries. Some niml models hve vlidted the fct tht virus repliction nd immune dysregultion with higher levels of proinflmmtory cytokines nd chemokines ply importnt roles in the pthogenesis of severe influenz. 14 Nowdys, ecuse of the slow progress in developing ntivirl gents for influenz, comining ville drugs provides n opportunity to generte more effective tretment thn ntivirl drugs lone. 1 Adjunctive tretments for influenz, prticulrly those modulting excessive proinflmmtory host responses to infection, hve een shown to e effective. 16 As gunosine nlogue, rivirin is rodspectrum ntivirl drug tht cn inhiit oth DNA nd RNA viruses. There hs een no report out the influenzrelted virl resistnce of rivirin, which my e relted to its unique mechnism of ction A recent study hs indicted tht the comintion of oseltmivir, mntdine nd rivirin hs high degree of synergy in inhiiting virl repliction. Also, this regime gretly reduces side effects ecuse of the low dose required for ech drug. 20 is composed of Artemisi crvifoli, Lonicer jponic Thun, nd Grdeni jsminoides Ellis. Artemisi crvifoli is spicy, itter nd cold, with the function of clering het nd removing toxins ccording to the theory of Trditionl Chinese Medicine. exhiits strong neurminidse inhiitory ctivity in vitro neurminidse inhiitor model. In ddition, cn lso significntly reduce the levels of IL8, TNFα s well s other proinflmmtory cytokines in the plsm nd BALFs, which my hve role in decresing the severity of pulmonry edem nd improving the pthologicl chnges of cute lung injury. 21 Our study showed tht llevited pulmonry edem nd suppressed expression of IL6 nd IL10 in the BALFs of mice infected with BJ01. However, it did not decrese virus titer nor prevent mortlity, suggesting limited protection ginst severe influenz. In contrst, rivirin s prophylctic ntivirl drug significntly decresed virus titer nd reduced lung edem Fluid lekge Inflmmtion of lveolr sept ut filed to prevent lung injury. It ws found tht, even though virus repliction hd een suppressed y rivirin, infection remined. This remining infection could trigger the cytokine storm nd drive immunopthologicl progression. When comined, rivirin nd showed primrily dditive interctions. Mice treted with oth drugs hd miniml weight loss nd lung injury nd significnt suppression of IL6 nd IL10 in BALF compred with rivirin lone. The synergistic effect of the two drugs ws clerly more effective thn either one dministered seprtely. This study suggests tht might enhnce the ctivity of rivirin with n ntiinflmmtory mechnism tht decreses the production of IL6 nd IL10 during the erly stges of ph1n1(2009) infection. The current dt provide n importnt reference for the comined ppliction of trditionl Chinese compounds nd ntivirl tretments in clinicl prctice which is helpful for prevention of drug resistnt vrints in clinicl investigtions. REFERENCES 1 Dwood FS, Jin S, Finelli L, et l. Emergence of novel swineorigin influenz A (H1N1) virus in humns. N Engl J Med 2009; 360(2): Butist E, Chotpitysunondh T, Go Z, et l. Clinicl spects of pndemic 2009 influenz A (H1N1) virus infection. N Engl J Med 2010; 362(18): Kumr A, Zrychnski R, Pinto R, et l. Criticlly ill ptients with 2009 influenz A (H1N1) infection in Cnd. JAMA 2009; 302(17): We SA, Pettil V, Seppelt I, et l. Criticl cre services nd 2009 H1N1 influenz in Austrli nd New Zelnd. N Engl J Med 2009; 361(20): Rello J, Rodriguez A, Inez P, et l. Intensive cre dult ptients with severe respirtory filure cused y Influenz A (H1N1)v in Spin. Crit Cre 2009; 13(): R Zheng BJ, Chn KW, Lin YP, et l. Delyed ntivirl plus immunomodultor tretment still reduces mortlity in mice infected y high inoculum of influenz A/HN1 virus. Proc Ntl Acd Sci USA 2008; 10(23): Hui DS, Lee N. Adjunctive therpies nd immunomodulting gents for severe influenz. Influenz Other Respir Viruses 2013; 7 Suppl 3: 29. JTCM www. journltcm. com 18 Alveolr consolidtion Pulmonry hemorrhge Notes: : infected mice treted with (1 ml) i.p. once dy for 7 dys; : infected mice treted with (86.6 mg kg 1 d 1 ) once dy for 7 dys i.p.; rivirin: infected mice treted with rivirin (66.6 mg kg 1 d 1 ) once dy for 7 dys i.p.; rivirin plus : infected mice treted with oth rivirin (86.6 mg kg 1 d 1 ) nd (66.6 mg kg 1 d 1 ) once dy for 7 dys i.p. Histopthology chnges including inflmmtory infiltrtion, lveolr sept dmge nd thickening s well s pulmonry hemorrhge in the lung tissue were oserved. : phosphteuffered sline; : No Injury; : Mild Injury; : Moderte Injury; : Mteril Injury.

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