Effect of αvβ3 Blockade Against Acute Lung Injury Induced by Inuenza A Virus and Its Mechanism

Integrin αvβ3 is a heterodimer formed by αv and β3 subunits that is expressed in pulmonary endothelial cells, alveolar epithelial cells, interstitial cells and macrophages. Integrin αvβ3 not only has a common role of integrin family molecules in inammation and tissue brosis, but also mediates the adsorption and penetration of various viruses into susceptible cells. Nevertheless, there are few studies on the effect of αvβ3 on acute lung injury (ALI) induced by inuenza virus and its mechanism. Here, the effects of αvβ3 blockade [Cyclo(RGDyK)] against ALI induced by inuenza A virus (IAV) in vitro and in vivo and its possible mechanism were studied. A549 cells and mice were infected with inuenza virus A/FM/1/47 to induce ALI in vitro and in vivo. The results showed that Cyclo(RGDyK) reduced the ALI induced by IAV, alleviated pulmonary edema, improved lung histopathological changes and alleviated the accumulation of inammatory cells in the lung. Cyclo(RGDyK) had inhibitory effect on cells and mice infected by IAV. Cyclo(RGDyK) (150 µg/kg) showed effective antiviral activity in vivo. Cyclo(RGDyK) had 70% protective effect against IAV and effectively reduced virus titer and inammation in lung tissue. Cyclo(RGDyK) exhibited signicantly anti-inammatory and anti-brotic effect on improving the pneumonia and degree of pulmonary brosis and reducing the levels of pulmonary brotic markers (LN, HA, PCIII, IV-C, TGF-β1, and α-SMA). Additionally, Cyclo(RGDyK) inhibited expression of αvβ3,TGF-β1, HIF-1α, NF-κB, and p38 MAPK in the cells and mice lung tissues. The results showed that Cyclo(RGDyK) had a protective effect on ALI in mice infected with IAV and inhibited the progress of lung inammation and brosis, which may be concern with its regulation of αvβ3/TGF-β1/HIF-1α signaling pathway. increased the production of proinammatory cytokines in lung, including IL-1β, IL-6, IFN-γ, MCP-1 and TNF-α, and up-regulated expression of NF-κB and p38 MAPK, which was reversed by Cyclo(RGDyK) treatment. These results suggested that Cyclo(RGDyK) alleviated inuenza-virus-induced pneumonia and brosis in mice by inhibiting NF-κB and p38 MAPK-mediated pro-inammatory signaling cascade. four


Introduction
In uenza is an infectious disease caused by in uenza virus that threatens global public health and sometimes leads to pandemics [1]. In uenza virus often causes acute lung injury (ALI) and promote pulmonary brosis, which can lead to acute respiratory distress syndrome (ARDS) [2,3]. At present, there is still a shortage of effective drugs to curb viral ALI/ARDS. Antiviral drugs may have an effect in the early stage of viral infection, but the effect on severe pneumonia lung injury in the late stage of viral infection is limited, and resistance to antiviral therapy is increasing [4].
Extracellular matrix (ECM) proteins, such as bronectin, collagen, and laminin, interact with integrins, transduce signals to regulate cell growth, differentiation, migration, and other cellular activities [5]. Integrins are cell surface glycoproteins involved in cell-cell and cell-matrix interactions. They are composed of an α and a β subunit [6]. They serve as an entry receptor for various viruses, such as herpesviruses and Epstein-Barr virus [7,8]. Diffuse alveolar injury caused by in ammation is central to the pathophysiology of ALI [9]. Integrins on leukocytes are upregulated in response to various cytokines and chemokines, and affect the progression and prognosis of many in ammatory processes [10]. It is worth noting that integrin αv plays a key role in the formation of in ammatory microenvironment by regulating expression of cytokines and matrix components [11,12].
The activation of transforming growth factor (TGF)-β in epithelial cells is a central pathway in the pathogenesis of pulmonary brosis [13].In uenza A virus (IAV) neuraminidase (NA) can activate TGF-β activation by removing sialic acid motifs from latent TGF-β [14,15]. The latent TGF-β complex contains an arginine-glycine-aspartic acid (RGD) sequence motif, which directly binds to and activates the following integrins: αvβ3, αvβ5, αvβ6 and αvβ8 [16], and leads to the release of TGF-β from the ECM, and biological activation [17]. Integrin and TGF-β play a key role in the process of in ammation by inducing the release of in ammatory mediators [18]. Therefore, intervening in the intergin and TGF-β signaling pathway can reduce in ammation, which is a potential therapeutic strategy for ALI [19].
The integrin family are cell surface receptor glycoproteins that are widely expressed in most cells. Integrin is a heterodimer composed of nine β subunits and 18 α subunits, which constitute ≥ 20 integrins according to different combinations [20]. Studies have shown that integrins can regulate the release of in ammatory cytokines and alveolar capillary permeability, which is a new target for anti-in ammatory therapy [21]. Integrins are also involved in the formation of collagen bers [3,22]. Integrin αvβ3 is a receptor for vitronectin. It is expressed on the lumen and vesicle surface of cultured endothelial cells and in resting rat pulmonary microvessels [23]. Integrin αvβ3 is also found in large blood vessels, human lung airway epithelium, neutrophils and monocytes [24]. Integrin αvβ3 promotes monocyte migration and participates in neutrophil migration on the ECM [25] .
Given the higher expression of integrin αvβ3 in acute lung in ammation [26], we hypothesize that αvβ3 upregulation during in uenza infection is important for pathogenesis of viral ALI. Application of anti-αvβ3 blocking antibodies may play a major role in ALI. The aim of this study was to evaluate the antiviral activity of αvβ3 blockade [Cyclo(RGDyK)] against ALI induced by IAV and its mechanism.

Reagents, antibodies and animal
Cyclo(RGDyK) was purchased from Selleckchem (Houston, TX,USA), purity ≥ 99%. Oseltamivir carboxylate was purchased from Beyotime (Shanghai, China). These compounds prepared as dimethyl sulfoxide stock solution were diluted in the culture medium before being added to the cells and added at the required concentration during the culture. Anti-HIF-1αprimary antibodies were purchased from Cohesion Biosciences Limited (Suzhou, China).Anti-αvβ3 primary antibodies and anti-TGF-β1 primary antibodie were obtained from Beyotime (Shanghai, China). The neuraminidase Assay Kit was purchased from Beyotime (Shanghai, China). Speci c pathogen free BALB/c mice, weighing 13-15g, were obtained

Cell Culture and Treatment
Human lung adenocarcinoma epithelial cells (A549) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in Dulbecco modi ed eagle's medium (DMEM), supplemented with 10% fetal bovine serum and 1% antibiotic (100 U/ml penicillin, 0.1 mg/ml streptomycin) with L-glutamine, and cultured in a humidi ed atmosphere with 5% CO 2 at 37 ℃. IAV strain A/FM/1/47 (H1N1) was acquired from ATCC. In uenza virus (1MOI) was inoculated on a 6-well plastic plate at 37 ℃ for 2 h, and then washed three times with PBS. After that, a series of concentrations of Cyclo(RGDyK) were carefully added to the cells and cultured at 37 ℃ for 48 hours. DMSO vehicle control was included in these experiments. Then, the supernatants were collected at 72 hpi, and the CPE of virusinfected cells were observed by microscope. Cells viability was assayed by 3-[4,5-dimethyl-thiazol-yl]-2,5diphenyltetrazolium bromide(MTT). Aabsorbance was measured by microplate reader (Bio-Tek ELx800, Winooski, Vermont, USA) at 490 nm.

Reverse transcription and real-time quantitative PCR
Total RNA was reverse transcribed into cDNA using PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Japan) according to the Manufacturer's protocol. M gene of A/FM/1/47 H1N1 was carried out as mentioned before [27]. IFV quantity of PCR products were analyzed in the light of the mode for normalized expression (2 −△△ct ) [28]. Relative quantities (RQ) of in uenza viral load in lung levels were normalized to the results of negative control group as 1.

Analysis of neuraminidase activity
NA enzymatic activity assay was performed as previously described [29] and samples were analyzed according to the neuraminidase assay kit manufacturer's instructions(Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). Brie y, Cells were infected with virus (FM1/H1N1, 1MOI), and Cyclo(RGDyK) for 72 h. Cells were resuspended in 10 mL assay buffer and were mixed with 70 mL detection buffer, 10 mL NA uorogenic substrate and 10 mL water. Cleavage of the uorogenic substrate from NA produces uorescence was measured using a Synergy™ HT Multi-detection microplate reader (BioTek Instruments, Inc, Winooski,Vermont, USA ) at an emission wavelength of 450 nm and an excitation wavelength of 322 nm. The uorescence intensity represents the activity of NA, which is quanti ed as the uorescence above the background values (negative control).

Therapeutic e cacy in mice
Mice were anesthetized and intranasal challenged with 5×LD 50 doses of FM1/ H1N1 in sterile phosphatebuffered saline (PBS pre-cooled). On the basis of previous studies, the dosage of Cyclo(RGDyK) in mice as determined according to previous studies [31].After infection, mice were given Cyclo(RGDyK) (150µg/kg, intraperitoneal injection) and monitored for 15 days after infection to observe signs of toxicity and death and to assess survival rate and time. On day 6 post-infection, the mice were killed and lung samples were collected for the detection of relevant indicators. Body weight and wet weight of the lungs were measured. Lung index was calculated as previously described [32].

Hematoxylin and eosin (HE) and Masson Trichrome Staining
Lung specimens were xed in 10% formalin, embedded in para n, cut into 5 µm sections and stained with hematoxylin and eosin (HE) or Masson's trichrome using routine histopathological methods as described previously [33]. 5 µm sections were stained with HE or Masson's trichrome staining and observed under light microscope. Histopathological score was between 0 (normal) and 5 (severe) based on the following pathological conditions: congestion,edema, polymorphonuclear leukocyte and monocyte in ltration, necrosis and hemorrhage as previously described [34]. Randomly selected areas of lung sections were examined by light microscopy at magni cations of ×100, ×200 and ×400 (Chongqing Optech Instruments Co., Chongqing, China). All histological analyses were performed by experienced pathologists who were blinded to the respective treatments. Collagen deposition was also assessed by Masson's trichrome staining. Ten randomly selected images focused on the central vein were analyzed for the extent of brosis using an image analysis system (Image Pro Plus version 6.0, Media Cybernetics, MD, USA). The results were expressed as integral optical density values (IOD).

Electron microscopy examination of lung tissue
Lung was xed in 2.5% glutaraldehyde and 1% osmic acid, dehydrated and dried, and observed by scanning electron microscopy (SEM 1430 VP; LEO Electron Microscopy Ltd., Oberkochen, Germany).

Immunohistochemical analysis
Twelve hours after the last administration at day 5, mice were sacri ced to collect the lungs. Mouse lungs were excised, xed in 10% formaldehyde for at least 24 hours, dehydrated in graded ethanol, and embedded in para n. Five-micrometer sections were stained with hematoxylin-eosin. Expression of αvβ3, HIF-1α and TGF-β1 in tracheal mucosa was determined by immunohistochemistry (IHC). Immunohistochemical staining was performed for αvβ3, HIF-1α and TGF-β1 antibody (Beyotime,Shanghai, China) was performed manually using goat anti-rabbit IgG Antibody Kit (Boster).
Images were captured using the same magni cation (40×/0.75) with an Olympus (HB-2) camera (Olympus Corporation, Tokyo, Japan). Protein labeling intensity measurements were expressed as integrated optical density (IOD) and were performed on six randomly selected sections using the Image-Pro Plus 6.0 image analysis system (Media Cybernetics Inc., Rockville, MD, USA).

Measurement of in ammatory parameters in lung of IAV-infected mice
Levels of MCP-1,TNF-α, IL-6, IL-1β and IFN-γ in lung tissue were detected by ELISA method in the light of the manufacturer's instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd. ,Shanghai, China) by ELISA plate reader (Bio-Tek ELx800, Winooski, Vermont, USA).

Statistical analysis
Data are expressed as the mean ± S.D. and statistically signi cant difference was analyzed by SPSS18.0 software (SPSS Inc., Chicago, IL, USA). The comparison between the two groups was conducted by Student's t-test. Differences were considered statistically signi cant at p < 0.05.

Anti-in uenza abilities of Cyclo (RGDyK) in vitro
MTT assay showed that Cyclo(RGDyK) (0.125-2M) increased cell survival in a dose-dependent manner (Fig. 1A). In addition, Cyclo(RGDyK) at concentrations of 10-40 nM signi cantly inhibited the expression of the M gene (Fig. 1B). In addition, Cyclo(RGDyK) at 10-40 nM almost completely inhibited neuraminidase activity compared to oseltamivir (Fig. 1C). The results suggest that Cyclo(RGDyK) can inhibit neuraminidase activity and viral load and protect cells from viral infection.
These results indicate that Cyclo(RGDyK) can inhibit the expression of p38MAPK and NF-κB in cells induced by in uenza virus.
Compared with NC group, the expression levels of αVβ3, HIF-1α and TGF-β1 in the cells of IAV-C group were signi cantly increased. After administration of Cyclo (RGDyK), the expression of αVβ3, HIF-1α and TGF-β1 was signi cantly reversed in cells (p < 0.01) (Fig. 4A-B,E-F).

Anti-in uenza activity of Cyclo(RGDyK) in IAV-infected mice.
Evaluation of the protective effect of Cyclo(RGDyK) on in uenza A (H1N1) mice by survival rate and lung index. In this study, Cyclo(RGDyK) (150µg/kg) can reduce the in ammatory response of lung tissue in mice. The lung index of IAV-C group was 1.58 ± 0.18, while that of Cyclo(RGDyK) groups were 1.11 ± 0.15 ( Fig. 5A), which suggested that Cyclo(RGDyK) can effectively reduce the lung index (p < 0.05). Cyclo(RGDyK) can effectively prolong the survival time of infected mice (Fig. 5B) and decline viral load in lung (Fig. 5C).Cyclo(RGDyK) at doses of 150 µg/kg protected 70% of mice from lethal infection, respectively (Fig. 5D). These results demonstrate that Cyclo(RGDyK) exhibited a protective effect on in uenza-infected mice.

Effect of Cyclo(RGDyK) on pathological changes of lung tissue in ALI mice induced by IAV
In the NC group, no evident histological alteration was observed in lung specimens, intact alveoli and lung bronchus structure, no exudates in the bronchial and bronchiolar lumina (Fig. 6A). In IAV-C group, there were obvious ALI, such as a large number of neutrophils in ltration around the lung interstitium, a large amount of exudation in the bronchioles, marked in ltration of neutrophils in the bronchioles, and obvious swelling of the alveolar wall (Fig. 6B). In mice treated with Cyclo(RGDyK) (150µg/kg), these lesions were improved to varying degrees (Fig. 6C-D), suggesting that Cyclo(RGDyK) can protect against ALI induced by IAV.

Ultrastructural changes of lung tissue by electron microscope
Electron microscopic observation showed that the alveolar structure in NC group was normal, no abnormal exudate in the alveolar cavity,and the morphology of type II alveolar epithelial cells was normal (Fig. 7A). In IAV-C group, the lung tissue had different degrees of pathological changes, such as the proliferation and swelling of alveolar epithelial type II cells, a large amount of exudate in the alveolar cavity, including a large number of in ammatory cells,such as monocytes, lymphocytes, etc. A large number of collagen forming cells (such as broblasts, smooth muscle cells, etc.) could be seen in the alveolar septum and a large number of collagen bers and elastic bers proliferated, forming obvious cord like bers crisscross (Fig. 7B). In mice treated with Cyclo(RGDyK) (150µg/kg), these lesions,such as the number and volume of alveolar epithelial cells, exudates and mononuclear phagocytes in alveoli, collagen bers in the alveolar septum, and honeycomb structure of lung were improved to varying degrees (Fig. 7C), indicating that Cyclo(RGDyK) protects against ALI induced by IAV.

Effect of Cyclo(RGDyK) on collagen changes in lung tissue of ALI mice induced by IAV
The histopathological abnormalities of the lung were studied by Masson's trichrome staining. Masson staining showed no collagen deposition, necrosis, in ammation and brosis in the lung tissue of NC group (Fig. 8A). In IAV-C group, in ammatory cell in ltration and collagen deposition were obvious (Fig. 8B). However, 150 µg/kg Cyclo(RGDyK) can signi cantly improve the structure and degree of pulmonary brosis, and signi cantly reduce the area density percentage of collagen deposition (p < 0.05) ( Figure.8C-D).

Effects of Cyclo(RGDyK) on levels of lung brotic markers in ALI mice induced by IAV
The levels of TGF-β1 in serum and α-SMA in lung tissue of IAV-C group were signi cantly higher than those of NC group (p < 0.05 − 0.001, Fig. 9A-B), but Cyclo (RGDyK) treatment (150 µg/kg) signi cantly decreased the levels of these markers (p < 0.05 or 0.01, Fig. 9A-B).

Effects of Cyclo(RGDyK) on cytokines production in lung tissue of ALI mice induced by IAV
In uenza virus induced lung in ammation and subsequent ALI are closely related to the over secretion of a variety of proin ammatory cytokines. IL-1β, MCP-1, IFN-γ, IL-6 and TNF-α are key pro-in ammatory cytokines in the process of in ammation. Therefore, we studied the expression of these pro-in ammatory cytokines in lung tissue. As shown in Fig. 10, in uenza virus infection increases the levels of IL-1β, MCP-1, IFN-γ, and TNF-α in the lungs of mice. Cyclo(RGDyK) (150 µg/kg) inhibited the over secretion of proin ammatory cytokines in a dose-dependent manner.

Discussion
Viral lung injury is a kind of pathological lung injury caused by virus infection. Novel coronaviruses, IAV H1N1, severe acute respiratory syndrome coronavirus, IAV H5N1, IAV H7N9 and other viruses can ALI and ARDS [35,36]. In uenza virus is a single-stranded RNA virus that causes acute respiratory infecctions. IAV infection often leads to ALI and pulmonary brosis and ARDS [37,38]. At present, there is still a shortage of effective drugs to control viral ALI/ARDS [4]. Therefore, it is important to develop new targeted drugs for the treatment of these diseases.
Integrin αvβ3 plays a critical role in brosis and in ammation. However, the effect of αvβ3 on ALI induced by IAV is still unclear. This study evaluated the anti-viral activity of αvβ3 blockade against ALI induced by IAV, and its mechanism of action. The results showed that in uenza virus infection led to in ammation and increased levels of brosis and collagen in mice. The αvβ3 blockade with Cyclo(RGDyK) signi cantly inhibited all these in ammatory and brotic changes, suggesting that Cyclo(RGDyK) can reduce lung in ammation and brosis induced by IAV. Cyclo(RGDyK) also signi cantly improved lung tissue morphology, reduced pulmonary brosis score, and inhibited expression of αvβ3, TGF-β1, NF-κB p65, p38MAPK and HIF-1α. These results con rmed the bene cial effect of Cyclo(RGDyK) on ALI and pulmonary brosis induced by in uenza virus.
The exact pathogenesis of ALI induced by IAV has not been fully elucidated [39][39] [39].Cytokine storm is an important cause of uncontrollable in ammation caused by virus infection [40]. Severe types of pneumonia lead to lung ischemia and hypoxia, and eventually to irreversible ARDS [41]. Therefore, viral lung injury (ALI/ARDS) is almost always associated with lung in ammation and brosis [42].Our study showed that the main symptoms of ALI and ARDS in mice infected with IAV were in ammation and collagen deposition. The surviving infected mice developed severe brosis, including typical interstitial and alveolar brosis, alveolar wall thickening, tissue expansion, bronchiolar wall thickening, and broblast proliferation.
The p38MAPK and NF-κB are the key signaling pathways for the expression of in ammatory cytokines and play a key role in the development of ALI [43]. NF-κB is closely related to in ammation and tissue brosis. NF-κB activation can induce the production of in ammatory factors, such as IL-1β, IL-6, TNF-α and TGF-β1, and then lead to lung in ammation and brosis. In this study,we found that in uenza virus infection increased the production of proin ammatory cytokines in lung, including IL-1β, IL-6, IFN-γ, MCP-1 and TNF-α, and up-regulated expression of NF-κB and p38 MAPK, which was reversed by Cyclo(RGDyK) treatment. These results suggested that Cyclo(RGDyK) alleviated in uenza-virus-induced pneumonia and brosis in mice by inhibiting NF-κB and p38 MAPK-mediated pro-in ammatory signaling cascade.
Lung in ammation and brosis were measured in experimental and clinical studies using four indicators: HA, LN, IV-C and PCIII [44]. In this study, the serum levels of all four markers were signi cantly reduced by Cyclo(RGDyK) treatment compared with the IAV-C group, indicating that Cyclo(RGDyK) reduced the serum levels of LN, HA, IV-C and PCIII in mice with pulmonary brosis.
TGF-β is a regulatory factor by controlling the occurrence and regression of in ammatory response and the occurrence and maintenance of brosis [45]. Intervention in TGF-β signaling pathway can reduce in ammation, which is considered as a potential target for the treatment of ALI/ARDS [45]. In uenza virus NA can directly activate latent TGF-β [43]. When mice were infected with in uenza virus, the activity of TGF-β increased. TGF-β1 accelerates the process of in ammation by inducing the release of in ammatory mediators, including IL-6 and IL-17 [46]. Our study also showed that the levels of TGF-β1, TNF-α, IL-6, IL-1 β and TGF-β1 were increased in cells and mice infected with in uenza virus. The Cyclo(RGDyK) could inhibit the levels of in ammatory cytokines induced by in uenza virus.
Hypoxia inducible factor (HIF) is a heterodimer transcription factor, which is composed of HIF-1α (or its analogs, HIF-2α and hif-3α) and HIF-1β subunit, and is the target gene for hypoxia adaptation and in ammation development. The control element of HIF is mainly HIF-1 [47]. HIF-1α is an important therapeutic target to control in ammation and brosis, and plays an important role in ALI [48]. Hypoxia induced HIF-1α gene expression and enhanced expression of α-SMA, TGF-β1 and Smad2 [49]. HIF-1α is involved in the regulation of in ammatory factors such as TNF-α, IL-6 and IL-10 [50], and IL-6 and TNF-α play an important role in the production and development of cytokine storm [51]. The expression of HIF-1α in lung tissue was up-regulated by local hypoxia induced by in uenza virus pneumonia [52]. Our study also showed that the level of HIF-1α in cells and mice infected with in uenza virus increased, while αvβ3 inhibitor Cyclo(RGDyK) effectively reduced expression of HIF-1α in cells and mice.
Integrin αvβ3 is a heterodimer formed by αv and β3 subunits that is expressed in pulmonary endothelial cells, alveolar epithelial cells, interstitial cells and macrophages [21]. Integrin αvβ3 not only has the common role of integrin family molecules in in ammation and tissue brosis, but also can mediate the adsorption and penetration of a variety of viruses into susceptible cells [23,53].
In particular, hypoxia is an important factor to increase the expression of αvβ3. Hypoxia can increase the mRNA production of αvβ3 in endothelial cells, but has no effect on αvβ5 [54].After IAV infection, mice had acute lung in ammation, brosis and other lung injury lesions. Severe pneumonia and other diseases produced local hypoxia in lung tissue. In our previous work, we studied the expression of αvβ3 in mice with in uenza-virus-induced lung injury. We found that in uenza virus induced expression of αvβ3 in cells and mice. Moreover, severe pneumonia caused by in uenza virus infection produced local hypoxic lung environment. Hypoxia increased expression of αvβ3 mRNA in endothelial cells [54].Our work showed that the expression levels of αvβ3, HIF-1α and TGF-β1 protein in lung tissue increased after IAV infection. Cyclo(RGDyK) inhibited viral pneumonia and reduce in ammatory factors and brocytokines in blood and lung tissue. These results indicated that αvβ3, TGF-β1 and HIF-1α and their signaling pathways were involved in the pathochemical process of virus infection, in ammation and tissue brosis. These abnormal functions and pathological changes were reduced by inhibiting αvβ3 signal, suggesting that Cyclo(RGDyK) improved lung in ammation and collagen deposition by inhibiting the expression levels of αvβ3, HIF-1α and TGF-β1.

Conclusions
In conclusion, Cyclo(RGDyK) had a protective effect on ALI in mice infected with in uenza virus, and inhibited the progress of lung in ammation and brosis, which may be related to its regulation of the αvβ3/TGF-β1/HIF-1α signaling pathway. This nding provided a new perspective for understanding the effect of αvβ3 on ALI induced by IAV. More studies are needed to study the pathogenesis of viral ALI and the mechanism of αvβ3 on viral ALI, which may provide evidence for the treatment of viral diseases.

Declarations Declaration of Competing Interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper. Ethical

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.  Cells(1×105/well) were treated with various amounts of Cyclo (RGDyK) at the same time as inoculation with FM1/H1N1 suspension (100 TCID50/well). After incubation for 72h at 37℃ and 5% CO2, the levels of cytokines were measured by ELISA assay. Data were presented as mean ± SD. Asterisks denote the signi cance levels: * p <0.01; ** p <0.01; *** p <0.001, compared with IAV-C group.  Cells(1×105/well) were treated with various amounts of Cyclo (RGDyK) at the same time as inoculation with FM1/H1N1 suspension (100 TCID50/well). After incubation for 72h at 37℃ and 5% CO2, the expression level of proeteins were measured by western blot assay.GAPDH protein was used as a loading control.Values are mean ± SD. Asterisks denote the signi cance levels: * p <0.05; ** p <0.01; *** p <0.001, compared with IAV-C group.    PCIII, procollagen type III; IV-C, collagen type IV; TGF-β1, Transforming growth factor beta 1; α-SMA, alpha smooth muscle actin.