Taurolidine resists pulmonary artery vasoconstriction induced by influenza virus infection via inhibiting Ca 2+ influx and MLCK/p-MLC signaling pathway

Taurolidine resists pulmonary artery vasoconstriction induced by influenza virus infection via inhibiting Ca 2+ influx and MLCK/p-MLC signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Taurolidine resists pulmonary artery vasoconstriction induced by influenza virus infection via inhibiting Ca 2+ influx and MLCK/p-MLC signaling pathway Chaoxiang Lv, Jin Guo, Rongbo Luo, Yuanguo Li, Bingshuo qian, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4778710/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Influenza virus causes worldwide outbreaks and seasonal epidemics, severely threatening public health and social development. Effective prevention and therapy for influenza infections are a major challenge to global healthcare. Results Taurolidine effectively inhibited the proliferation of several human or animal influenza virus strains and protected mice from lethal-infection. Taurolidine treatment decreased the viral titer in the lungs of infected mice, reduced immune cells infiltration, and alleviated lung pathology. Additionally, influenza virus infection increased blood pressure, pulse wave velocity, and pulmonary aortic thickness in mouse model, as well as promoted the increase of intracellular Ca 2+ concentration and pulmonary artery vasoconstriction. These effects were attenuated by taurolidine treatment through inhibiting the activation of the MLCK/p-MLC pathway. Conclusions These findings confirm the effectiveness of taurolidine as an antiviral agent and highlight its important roles in host immune cell infiltration and vasoconstriction induced by influenza virus infection. Taurolidine vasoconstriction influenza virus calcium influx MLCK/p-MLC signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Influenza virus is the main virus responsible for acute respiratory infections and seasonal influenza, which is easy to cause seasonal outbreaks and pandemics, and pose a significant threat to public health and social development[1-3] . Presently, vaccination and antiviral treatments serve as the main preventive and supportive measures for responding to the influenza pandemic [4]. However, the vaccine is limited to identify pandemic strains and resistance towards an existing class of direct-acting antivirals (DAAs), like adamantane (the M2 inhibitor), oseltamivir (the NA inhibitor) and baloxavir (the PA inhibitor) becomes widespread [5]. Meanwhile, host-targeted therapies, anti-inflammatory drugs, and immunomodulators have also been shown to have broad-spectrum antiviral activity against influenza viruses [2]. In addition, the current ability of vaccines to identify pandemic strains is limited. All these problems highlight the urgent need to develop new antiviral strategies with new mechanisms of action and reduce the possibility of drug resistance [6]. Inflammatory cytokines are markedly elevated following influenza virus infection, leading to the “cytokine storm”, which is hypothesized to be the main cause of mortality [7]. The cytokine storm can accumulate excessive immune cells and viscous secretions in the lung tissues, thereby obstructing gas exchange and ultimately leading to death [8]. A study has showed that the “cytokine storm” effect characterized by over-production and dysfunction of inflammatory cytokines has become a main cause of host death after influenza virus infection [9]. This indicates that severe lung inflammation is related to lung tissue pathology, such as lung tissue edema, infectious pneumonia and alveolar hemorrhage [10]. Consequently, it is essential to maintain the host's antiviral response as the optimal defense mechanism following infection with the influenza virus. The vascular smooth muscle system drives blood flow, cytokine release and immune cell recruitment by regulating the balance between vasoconstriction and vasodilatation [11]. The contraction of vascular smooth muscle is regulated by a voltage- and calcium-dependent process (called excitation-contraction coupling) in response to neuronal stimulation [12]. Additionally, vasomotor control is the basis for the acute and rapid adaptation of blood vessel diameter, which is mainly due to the vasoconstriction caused by the active contraction of vascular smooth muscle cells, and the altered of vascular structure represents the dynamic process of chronic hemodynamic changes [13]. The acute regulation of blood vessel diameter and the consequent vascular resistance depends on the activation state of the contraction mechanism involving actin, that is, the interaction of actin in the vascular smooth muscle cells [14]. The alteration of Ca 2+ flux and membrane potential regulate phosphorylation of myosin-light-chains (MLCs) and actin-myosin cross-bridge cycles, ultimately leading to rapid vasoconstriction [15], which is a critical mechanism for the host to adapt to a rapid immune response. Taurolidine, a derivative of the amino acid taurine, has been found to possess anti-endotoxin, anti-bacterial and anti-adhesion properties [16]. Its mechanism of action involves inhibiting microbial adhesion through chemical reactions with cell walls, including bacterial and fungus [17]. Additionally, previous studies have shown that taurolidine inhibits the synthesis of cytokines (IL-1 and TNF) in human peripheral blood mononuclear cells [18], which indicates that taurolidine may play important roles in cytokine-induced immune responses and related diseases. Despite these findings, the antiviral effectiveness of taurolidine remains unclear. Here, we explored the anti-influenza virus activity of taurolidine in vitro and in vivo . By using genomics and chemical tools, we defined the important role of taurolidine in attenuation of inflammation response. Furthermore, we revealed that the suppression of vasoconstriction signaling pathway through taurolidine treatment can reduce the mortality among mice infected with influenza virus. Therefore, the vasoconstrictor signal provided a mechanism to reduce influenza virus-induced incidence and revealed the unforeseen role of taurolidine as a modulator of inflammatory response and vasoconstriction. Results Administration of taurolidine inhibited the reproduction of influenza virus in vitro The potential anti-influenza virus activity of taurolidine was evaluated using an MDCK cell infection model. Taurolidine efficiently inhibited the IAV/H1N1 replication in cultures exposed to different dose-ranges of drugs for 24h (Fig. 1A, left). The 50% effective concentration (EC 50 value) of taurolidine was observed at 31.63 μg/mL (Fig. 1B, left). Similarly, we also found that taurolidine also inhibited the IBV/S9-MD replication (Fig. 1A, right), with EC 50 of 22.73 μg/mL (Fig. 1B, right). To rule out that the reduction in viral replication was due to an indirect effect on the host cell, MTT experiment was performed to confirm the effect of taurolidine on cytotoxicity. The results showed that taurolidine did not reduce the cellular viability of MDCK (Fig. S1A) and A549 cells (Fig. S1B) in a significant manner at 10, 25, 50 and 100 µg/mL concentrations, suggesting that taurolidine mainly exerts its antiviral effect by inhibiting the proliferation of influenza viruses. The activity of taurolidine against influenza virus was further confirmed using plaque reduction assays. As shown in Fig. 1C, plaque formation in the IAV/H1N1- and IBV/S9-MD-infected cells was reduced significantly after treatment of taurolidine. To explore the potential antiviral effects of taurolidine, oseltamivir (a common anti-influenza drug) was used as a positive control for indirect immunofluorescence assay. As anticipated, oseltamivir significantly inhibited the IAV/H1N1 strain propagation; 38% of MDCK cells nuclei were virus NP-positive in DMEM-treated cells, whereas the percentage of virus NP-positive cells were significantly reduced to 9.8% following taurolidine treatment (Fig. 1D). Comparable results were observed for IBV/S9-MD strain (Fig. 1E). In addition, the protein expression level of virus NP was also significantly decreased after taurolidine treatment in both IAV/H1N1-infected and IBV/S9-MD-infected cells (Fig. 1F). In order to clarify the active stage of taurolidine against influenza virus, we treated A549 cells with three different infection protocols, including pre-treatment, co-treatment and post-treatment. It was observed that post-treatment taurolidine exhibited high inhibition rates in IAV/H1N1-infected (Fig. 2A), IAV/H3N2-infected (Fig. 2B), and IBV/S9-MD-infected cells (Fig. 2C), which indicating that taurolidine had a inhibitory effect on later stages of influenza virus. To further assess the antiviral potential of taurolidine, different influenza virus subtypes (IAV/H1N1-PR8, IAV/H1N1-UI182, IAV/H3N2 and IBV/S9-MD strains) were chosen to infect A549 (Fig. 2D, E, F, G) and MDCK cells (Fig. 2H, I, J, K), respectively. These results indicate that taurolidine showed a significant inhibitory activity against different subtypes of influenza virus. Taurolidine improves the survival rate after influenza virus infection The therapeutic effect of taurolidine was further evaluated in mouse models on account of its obvious anti-influenza activity in vitro above. We found that taurolidine treatment alleviated weight loss in mice infected with influenza viruses, including IAV/H1N1 (Fig. 3A), IAV/H3N2 (Fig. 3B), and IBV/S9-MD (Fig. 3C). Compared to virus-infected mouse, taurolidine treatment significantly increased the overall survival condition of mice in drug-treatment groups, with protection rate of 66.67% for IAV/H1N1 (Fig. 3D), 42.86% for IAV/H3N2 (Fig. 3E), and 66.67% for IBV/S9-MD (Fig. 3F). Subsequently, we examined the viral loads in the lung tissues of mice at 3 dpi and 5 dpi, and found that viral loads were significantly reduced in taurolidine-treated groups (Fig. 3G, H, I). Furthermore, western blot results also confirmed that taurolidine significantly inhibited the replication of influenza viruses in the mouse lung tissues (Fig. 3J, K, L). By observing the lung morphology, we found that both IAV/H1N1 and IBV/S9-MD infection caused extensive bleeding ( black arrow ) in the lung tissue of mice, while taurolidine significantly reversed these results (Fig. 4A). The lung index of mice infected with IAV/H1N1 (Fig. 4B) and IBV/S9-MD (Fig. 4C) also decreased after taurolidine treatment. To investigate the improvement effect of taurolidine on lung pathology caused by influenza virus infection, lung tissues were randomly collected from mice in each group at 5 dpi to perform H&E and IHC assays. The H&E assay showed that the influenza viruses infection caused part of cell necrosis ( red arrow ) and inflammatory cell infiltration ( blue arrow ), but this effect was inhibited by the administration of taurolidine (Fig. 4D). Furthermore, the pathological score also confirmed that taurolidine treatment improved overall lung pathology in mice infected with influenza virus (Fig. 4E, F). In addition, we also found that taurolidine administration significantly inhibited the expression of virus NP in lung tissues (Fig. 4G), and reduced virus NP-positive cells in lung tissues of mice infected with IAV/H1N1 (Fig. 4H) and IBV/S9-MD (Fig. 4I). Moreover, we also observed that taurolidine treatment significantly increased the number of white blood cells (Fig. S2A, B), reduced the number of red blood cells (Fig. S2C), and reduced the number of the number of platelets in mice infected with IAV/H1N1 (Fig. S2D). Similar results were observed in mice infected with IBV/S9-MD (Fig. S3). These results indicated that taurolidine treatment could effectively improve the pathological damage caused by influenza viruses and improve the survival rate of infected mice. Effects of taurolidine on immune cell and cytokine storm induced by influenza virus infection We previously demonstrated that taurolidine treatment improved inflammatory cell infiltration caused by influenza virus infection in lung tissues of infected mice. To further analyze the effects of taurolidine on immune cells of influenza virus-infected mice, peripheral blood was collected from mice in each group at 5 dpi for flow cytometry analysis. The results showed that influenza (IAV/H1N1) virus infection led to a reduction in the number of CD4 + T cells (Fig. S4A) and CD8 + T cells (Fig. 5A), and the administration of taurolidine reversed this result (Fig. 5B; Fig. S4B). Interestingly, we only observed a reduction in CD8 + T cells (Fig. 5C) and no significant changes in CD4 + T cells in IBV/S9-MD-infected mice (Fig. S4C, D). Importantly, taurolidine treatment also improved the CD8 + T cells reduction caused by IBV/S9-MD infection (Fig. 5D). In addition, we also found that influenza (IAV/H1N1) virus infection increased the number of neutrophils, macrophages, natural killer (NK) cells, and dendritic cells (Fig. 5E). However, the administration of taurolidine significantly reduced the number of neutrophils (Fig. 5F), macrophages (Fig. 5G), NK cells (Fig. 5H), and dendritic cells (Fig. 5I). These findings suggest that taurolidine treatment reduces immune cell infiltration caused by influenza virus infection. Inflammatory cytokines are markedly elevated after influenza virus infection and the cytokine storm being considered the main cause of mortality. To investigate the effect of taurolidine on the inflammatory response induced by influenza (IAV/H1N1) virus infection, we also collected the serum of each group of mice for ELISA detection. The results showed that taurolidine reduced the concentration of key inflammatory cytokines in the serum, such as IL-6 (Fig. 5J), IFN-γ (Fig. 5K), IL-10, TNF-α (Fig. 5L), and IL-1β (Fig. 5M). In addition, we observed that influenza (IAV/H1N1) virus infection leads to up-regulation of cytokine mRNA expression in the lungs of infected mice, and taurolidine-treated significantly reduced the mRNA expression of cytokines and chemokines, including IFN-α , IL-10 , TNF-α , IL-1α , and IFN-γ , as well as CCL5 , CXCL10 , CXCL11 , CCL3 and CCL4 (Fig. S5). These findings support the conclusion that taurolidine has the potential to mitigate the cytokine storm in mouse lungs following influenza virus infection. T aurolidine alleviates vascular pathology in mice infected with influenza virus As a fast-track for immune regulation, blood vessels regulate the transportation and transfer of immune cells during influenza virus infection. In this study, we sought to investigate the effects of taurolidine treatment on blood pressure in mice infected with influenza virus. The results showed that the systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were significantly increased in mice infected with influenza virus, while they were decreased by taurolidine treatment (Fig. 6A, B, C). Two-dimensional ultrasound results showed that influenza virus infection caused thickening of the pulmonary aortic tube wall in IAV/H1N1-infected mice, an effect that was alleviated by taurolidine treatment (Fig. 6D). Compared to the control group, the pluse wave velocity (PWV) of the pulmonary aorta in IAV/H1N1-infected mice was increased at 5 dpi (3.88 ± 0.38 mm/s vs. 2.02 ± 0.18 mm/s), but this was mitigated following taurolidine treatment (2.86 ± 0.28 mm/s vs. 3.88 ± 0.38 mm/s, Fig. 6E). Moreover, the pulmonary aorta in IAV/H1N1-infected mice exhibited greater thickness and smaller diameter in comparison to controls, with these differences being reversed following taurolidine treatment (Fig. 6F, G). Changes in the thickness of the pulmonary aorta were also confirmed by H&E staining (Fig. 6H), and similar results were found in mice infected with IBV/S9-MD (Fig. S6). Taurolidine inhibits vasoconstriction pathways and intracellular calcium elevation induced by influenza virus infection We previously shown that taurolidine mitigated vascular changes in pulmonary artery blood vessels induced by influenza virus infection in mice. To further investigate the effect of taurolidine on the pulmonary artery induced by influenza virus infection, we collected lung tissues from each group of mice to perform transcriptome analysis (Fig. S7A, B). We also conducted classification and enrichment analysis of differential genes (Fig. S7C, D). The results showed that the vascular smooth muscle contraction signaling pathway was significantly enriched after infection with influenza viruses IAV/H1N1 (Fig. S8A) and IBV/S9-MD (Fig. S8B). Subsequently, the expressions of some genes in the vascular smooth muscle contraction signaling pathway were detected. Taurolidine treatment rescued the down-regulation of PKC , ADER5 , MYLK3 , PPP1C and MLC1 expression in lung tissues of mice infected with IAV/H1N1 (Fig. 7A) and IBV/S9-MD (Fig. 7B). Generally, the contraction and relaxation of vascular smooth muscle mainly depend on the state of membrane light chain (MLC) phosphorylation regulated by Ca 2+ /calmodulin complex and by MLC kinase and MLC phosphatase [19,20]. Thus, we examined the concentration of Ca 2+ in VSMCs after influenza virus infection. We found that both IAV/H1N1 and IBV/S9-MD infection increased in intracellular Ca 2+ concentration, which was subsequently attenuated by taurolidine treatment in a time-dependent manner (Fig. 7C, D). By comparing with the intracellular Ca 2+ signaling intensity, we also found taurolidine treatment reduced Ca 2+ concentration in a dose-dependent manner after IAV/H1N1 infection (Fig. 7E, F. G, H). In addition, taurolidine-treated significantly decreased expression level of p-MLC with a dose-dependent manner in VSMCs infected with IAV/H1N1 (Fig. 7I, H) or IBV/S9-MD (Fig. 7J, K), which was not observed after oseltamivir treatment (Fig. S8C, D). The collagen gel-based contraction assay also found taurolidine treatment increased the initial area of IAV/H1N1-infected VSMCs compared to the untreated group (Fig. S9A, B), suggesting that taurolidine controls vascular smooth muscle contraction after influenza virus infection. Taurolidine attenuates the activation of MLCK/p-MLC pathway induced by influenza virus infection To investigate taurolidine regulation in vasoconstriction induced by influenza virus infection, western blot was performed. The results showed that the phosphorylated expression level of MLC (p-MLC) was up-regulated and the protein expression level of MLC was down-regulated in VSMCs after infection with influenza (IAV/H1N1) virus, while it was attenuated by taurolidine treatment in a dose-dependent manner (Fig. 8A). In addition, we found that influenza (IAV/H1N1) virus infection also up-regulated the protein expression level of AT1R (Fig. 8B), CaM (Fig. 8C), and MLCK (Fig. 8D), but this was reversed by taurolidine treatment in a dose-dependent manner. Considering the stability of in vitro environment, we subsequently focused on in vivo experiments. In immunohistochemical (IHC) staining of the pulmonary aorta of mice infected with influenza virus (Fig. 8E), significant up-regulation of p-MLC was observed in the pulmonary aorta of IAV/H1N1-infected mice, and it was reversed by taurolidine treatment (Fig. 8F). Similarly, IAV/H1N1 infection also down-regulated the protein expression level of MLC (Fig. 8G) and up-regulated the protein expression level of AT1R (Fig. 8H), CaM (Fig. 8I), and MLCK (Fig. 8J) in the pulmonary aorta of IAV-infected mice, all of which were reversed by taurolidine treatment. Discussion The continuous replication, recombination and mutation of influenza virus have presented significant challenges for prevention and treatment of influenza diseases [21]. Current antiviral countermeasures primarily consist of preventive vaccines and therapeutic drugs. Despite partial achievements have been obtained, but resistance to existing antiviral drugs has become a serious problem [22]. Thus, repurposed of old drugs is also a selection strategy in combating the virus due to their low-toxicity and high-efficiency in clinical application. In this study, we found that the clinically licensed antibacterial drug taurolidine significantly inhibited the replication of influenza viruses in vitro and improved the survival rate of lethal influenza virus infection in mouse models by suppressing cytokine storms and regulating vasoconstriction. As a derivative of taurine, taurolidine has been proved to be an effective antibacterial agent and used to therapy peritonitis in some countries. Previous studies have shown that it inhibits the synthesis of IL and TNF in human peripheral blood mononuclear cells [18], which indicates that taurolidine is likely to play important roles in IL- and TNF-induced related diseases. Indeed, the application of taurolidine tends to prevent the development of lung metastases [23]. In our study, we found that taurolidine significantly improved the lung damage caused by influenza virus infection in mice. Moreover, the drug treatment significantly reduced the number of neutrophils after influenza virus infection. However, the detailed mechanism behind this requires further studied in future. The clinical outcome after virus infection depended largely on the balance between virus replication and host defense response [24]. The ability of the virus to evade the host's immune response is critical to its pathogenicity [25]. Overexpression of inflammatory factors in the host after influenza virus infection will lead to death, and this effect can be improved by inhibition of cytokine regulating inflammation molecular information [26]. Additionally, cytokines play important roles in activating immune cells, regulating immune response and promoting virus clearance, which suggest that their dynamic balance usually determines the process of host infection [27]. The imbalance of cytokine effect and immune cells recruitment could be used as poor prognostic indicators during highly pathogenic influenza virus infection [28]. Early induction of cytokines (IFN-α, IL-1β, IL-2, IL-6, and TNF-α) and chemokines (CCL2, CCL3, CXCL2, and CXCL10) are related to the symptom’s formation in human [29,30]. TNF-α, IL-1, and IL-6 have multifunctional activities and are related to the morbidity after influenza virus infection [31]. In this study, we found the protein productions of the cytokines (TNF-α, IL-6, and IL-10) in the serum after influenza virus infection were decreased after taurolidine treatment (data not shown). Additionally, chemokines can induce innate immune cells to be recruited to lung tissues, releasing more cytokines to exacerbate the cytokine storm [32]. Importantly, we demonstrate that taurolidine significantly reduces the mortality of mice infected with human pathogenic influenza virus strains by inhibiting cytokines. These findings suggest that at least taurolidine exhibits chemotherapeutic properties in diseases with inflammatory factors as the main pathological component. The tension of blood vessels is regulated by vascular smooth muscle signals. Previous studies suggested that influenza virus infection can lead to pulmonary artery atherosclerosis [33]. Additionally, abnormal vascular smooth muscle signaling often leads to pulmonary hypertension [34], platelet aggregation [35]and atherosclerosis [36], suggesting that the signaling plays a crucial role in the process of anti-influenza virus. Our results showed that taurolidine can inhibit the contraction of vascular smooth and improve survival rate during infection with influenza virus, highlighting the significance of this signal in defending against influenza virus challenge. An interesting question is whether vascular endothelial cells or smooth muscle directly regulate the host immune response. The endothelial cells are involved in anti-inflammatory response [37], but we still need to determine adjustments to taurolidine-mediated endothelial cytokine production. Endothelial cells could regulate the production of cytokines in the lung through complex crosstalk mechanisms with epithelial cells or resident hematopoietic cells [38]. However, the identification of endothelial cells as the central coordinator of immune-mediated inflammation is of fundamental significance and has broad implications for the treatment of many diseases, such as influenza. Furthermore, the etiology of several auto-immune diseases is directly related to inflammation response [39]. Therefore, it is important to understand the biological characteristics of taurolidine in regulating cytokine storm and develop appropriate chemical signaling transduction tools to identify specific molecular targets. This not only provides insights into the interaction between microorganisms and hosts, but also will reveal other ways to achieve effective immunotherapy in a variety of diseases. We also revealed that the administration of taurolidine reduced the release of Ca 2+ after infection with influenza virus. Indeed, vasoconstriction is regulated by Ca 2+ concentration [40,41]. This suggested that taurolidine may have the potential to treat other diseases that depending on Ca 2+ -related signaling pathways. Importantly, these data indicate that taurolidine might be a negative regulator of cytokine amplification and an activator of vascular smooth muscle signaling, which would give more personality possibility of the host genetic survival advantage or disadvantage. Conclusions In summary, the antiviral ability of taurolidine was demonstrated. Our finding not only reported the inhibitory effect of taurolidine on inflammatory response, but also suggested its important roles of taurolidine in vasoconstriction signaling. In addition, we reveal the potential of taurolidine-mediated mouse models in the treatment of influenza virus infections. Therefore, we provide a theoretical basis for further research on the antiviral mechanism of taurolidine, which providing a promising application for the prevention and treatment of influenza virus infection. Materials and Methods Antibodies, mice, reagents and viruses Antibodies against viral nucleoprotein (virus NP, ab20343), AT1R (cat no. ab124505), CaM (cat no. ab2860), and MLCK (ab236299) were provided by Abcam (Burlingame, CA, USA). The antibodies against MLC (Loc:3672) and p-MLC (Loc:3675) were purchased from Cell Signaling Technology (Beverly, MA, USA). Taurolidine, oseltamivir (PHR1781) and antibody against β-actin were from Sigma-Aldrich (St. Louis, MO, USA). The MLCK inhibitor ML-7 HCL (S8388) and ML-9 HCL (S6847) were purchased from Selleck (Houston, TX, USA). Six- to eight-week-old BALB/c female mice derived from Charles River Laboratory Animal Technology Co., Ltd (Beijing, China). Influenza A viruses (IAV/H1N1-UI182, IAV/H1N1-PR8, IAV/H3N2) originated from the Institute of Changchun Veterinary Research, Chinese Academy of Agricultural Sciences (Changchun, China) [42,43]. Influenza B virus stains (IBV/S9-MD) was rescued according to the sequence of B/Yamagata/16/88 (GenBank accession: CY018765-CY018772) and passaged in mice to get mouse-adapted strains. All experiments with influenza viruses were performed in a biosafety level 2 (BSL-2) laboratory in Changchun Veterinary Research Institute. Cell culture The Madin-Darby canine kidney cell line (MDCK), the human type-II alveolar epithelial cell line (A549), and the vascular smooth muscle cells (VSMCs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS, Gibco, 10091148) with 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were cultured at 37°C and 5% CO 2 in humidified air. Virus infection and drug inhibitory efficacy All virus strains were passaged and titrated on cells at the designated multiplicity of infection (MOI). Three different infection regiments were used to evaluate the active stages of taurolidine against influenza viruses, such as pre-treatment (virus was added to the cells after 15 min of co-incubation with taurolidine), co-treatment (virus was added to the cells after 15 min of co-incubation with taurolidine), and post-treatment (taurolidine was added to the cells for 4 h treatment after virus infection). Taurolidine was solubilized in PBS at a concentration of 4 mg/ml and a series of dilute solutions of taurolidine (0, 5, 10, 25, 50, 100 and 200 μg/mL) was added at 4 hours post-infection. We refer to the previous method to perform drug inhibitory efficacy in vivo [44]. Plaque, hemagglutination (HA) test and cytotoxicity assay MDCK cells were infected with 40 PFU/well of influenza virus. Following adsorption of the virus for 2 h, the inoculum was removed, and the cells were overlaid with DMEM containing 1.5 % agarose with serial dilutions of PG. After 72 h, they were stained and images were captured. For hemagglutination experiment, the MDCK cells infected with influenza virus were divided into different groups, including DMEM-treated group (0 μg/mL), taurolidine-treated groups (5 μg/mL, 25 μg/mL, 50 μg/mL) and oseltamivir-treated groups (5 μg/mL, 25 μg/mL, 50 μg/mL). The cell supernatant (50 μL) of each group was respectively collected and added to the 96-well micro-hemagglutination plate after 48 h of treatment. Subsequently, 50 μL of 1% chicken red blood cell (RBC) suspension was added to each well. After standing at room temperature for 15 min, the results were observed. Cytotoxicity was assayed by MTT assay (Promega). The cells were plated in 96-well plates (48000 cells per well) to culture 48h, and then the cells growth was detected according to the instructions of manufacturer. Briefly, 10µL of MTT solution was added to each well, and each well was measured Spectro-photometrically at 570 nm after incubating for 4h. We conducted three independent cell experiments, and three replicate wells were set for each treatment group in each experiment. Immunofluorescence and immunoblotting analysis The cells were washed three times with phosphate buffer saline (PBS) before 4% paraformaldehyde fixation (30~60min). They were then drilled with 0.2 % triton X-100. Before primary antibodies incubation (overnight at 4°C), the cells were blocked with 2% bovine serum albumin (BSA) for 1h. They were incubated with secondary antibody after washing. The nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI, 10~20min). For immunoblotting analysis, cells or tissues were lysed with radio immunoprecipitation assay (RIPA) buffer. The protein concentration was measured by bicinchoninic acid protein quantitative kit. Then isolated protein lysates were separated by SDS-PAGE and transferred onto PVDF membranes. This was followed by blocking with 5% skin milk in Tris-buffered saline with Tween-20 (TBST) before incubation with the indicated antibodies 1h. Then, these membranes were incubated with secondary antibody (Protein, CA, USA) for 1h. The membranes were washed 3 times in TBST, for 5min each time. Subsequently, membranes were treated for 2min with reagent from an Easysee Western Blot Kit. The results were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD). We conducted three independent cell experiments. Mice infection and quantitative Real-Time PCR (qRT-PCR) analysis BALB / c mice were divided into four different groups, with 13 mice in each group, including control group (Control), virus infection group (Virus), virus + taurolidine treatment group (Virus + taurolidine, i.p.), and virus + oseltamivir treatment group (Virus + oseltamivir, p.o.). Each group conducted two independent experiments. The influenza viruses (10-fold mouse median lethal dose, 10×mLD 50 ) were inoculated intranasally (i.n.) to the mice in virus-infected group, taurolidine-treated group, and oseltamivir-treated group. Taurolidine (400 mg/kg/d) and oseltamivir (25 mg/kg/d) were administrated twice daily (morning and evening), respectively. The body weight and the survival status of mice were recorded daily for two weeks. The lung index is calculated according to the previous method [42-44]. The treatment of all mice was in accordance with the welfare and ethical guidance of Chinese laboratory animals (GB 14925-2001). The agreement was approved by the Animal Welfare and Ethics Committee of the Institute of Chinese Academy of Agricultural Sciences (permit number: SCXK-2012-017).For cytokine transcriptome analysis, total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcription was carried out using ImPromp-II reverse transcriptase (Promega). Semi-quantitative PCR using Ampli Taq polymerase (Applied Biosystems) was performed by including [α-32P]-dCTP in the reactions. The IAV-specific primer sets anneal, and their sequences are listed in Table S1 and TableS2 . Pulmonary pathology and hematoxylin-eosin staining The mice were euthanatized and the lung tissues were quickly placed in polyoxymethylene solution (4% PFA) to fix for 24~72 h. And then, they were embedded in paraffin and randomly cut into 4-8μm slices. After that, the slices were made transparency with xylene and soaked in absolute ethanol for 5-10 minutes. Finally, the slices were stained with hematoxylin-eosin (H&E), dried and observed with an optical microscope. Immunohistochemistry (IHC) assay The immunohistochemistry (IHC) assay is calculated according to the previous method [42]. The pathological severity score of the infected mice was based on the percentage of the inflammation area of each slice collected from each animal, and using the following scoring system: 0, no pathological changes; 1, affected area ≤ 10%; 2, affected area 10%; 3, The affected area is ≥50%. When inflammation, hemorrhage and bronchial epithelial necrosis were observed, the score will be increased by one point. Flow cytometry Peripheral blood of mice in each group was collected into EDTA-anticoagulation tubes. They were mixed with red-blood-cell lysis buffer before centrifugation (3000 rpm, 4 °C, 10 min). They were then incubated with specific antibodies at low temperature for 2 h without light. Antibodies included APC conjugated CD3 (152306, biolgend, USA), FITC conjugated CD4 (100510, biolgend, USA), PE conjugated CD8 (100708, biolgend, USA), PE conjugated CD16 (158004, biolgend, USA), FITC conjugated CD49b (108905, biolgend, USA), APC conjugated CD163 (156705, biolgend, USA), APC conjugated CD (117310, biolgend, USA). Subsequently, the analysis was performed using the BECKMAN CytoFLEX flowmeter (BECKMAN COULTER, USA). The results were visualized using CytExpert 2.3 software. Enzyme-linked immunosorbent assay (ELISA) For ELISA assay, the serum samples of mice in each group were collected randomly. Cytokines (IL1β, IL-6, TNF-α, IFN-γ) were then detected using a custom Mouse cytokine 10-plex kit using V-PLEX (K15048D, MSD, USA) according to the manufacturer's instructions. Discovery Workbench software (v4.0, MSD Corporation, USA) was used for data analysis. Tr anscription sequencing, blood pressure and ultrasound measurements The transcription sequencing was performed by MAGIGENE Technology (Shenzhen, China). Sequencing libraries were constructed using Illumina sequencing platform (Illumina, San Diego, CA). the DESeq method was used to analyze the differential expression of mice in each group. Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for signaling pathway enrichment analysis. The bubble maps were visualized by the R clusterProfiler package (version 4.0.3). For blood pressure and ultrasound measurements, pulmonary artery blood pressure, including systolic blood pressure (SBP) and diastolic blood pressure (DBP), was measured at the 0, 3, 5, 7, 10 and 14 days, respectively. Mean arterial pressure (MAP) = DBP + (1/3×SBP). In brief, mice were anesthetized before pulse wave velocity (PWV) and pulmonary aorta thickness were measured using an ultrasound instrument (FUJIFILM VisualSonics, Toronto, Canada). The visual image processing using Vevo®LAB software. Ca 2+ measurement Cells were treated with or without taurolidine at the designated time after virus-inoculated. For mouse serum samples, serums collected from blood samples of mice were utilized for subsequent determination. Subsequently, the cell suspension and mouse serum were harvested for Ca 2+ determination performed by the kit from Genmed Scientifics Inc. U.S.A (GMS50097.1 v.A). Simply, mixing 100 µL of the test solution (cell suspension or serum) with the reaction solution, incubate at room temperature in the dark for 5 minutes, and read with a spectro-photometer. The corresponding Ca 2+ concentration (mmol/L) of the samples were calculated according to the construction of the standard curve. The cultured cells were washed and incubated the Fluo-4 following AM detection kit (F14202, Invitrogen) before data collection using FDSS/µCELL (Hamamatsu Photonics, C13299, Japan). In calcium imaging analysis, incubated Fluo-4 cells were loaded with fluorescent probe. Subsequently, the calcium imaging was performed using fluorescence microscope (LEICA, DMi8, Italy) after the cells were washed with PBS. We conducted three independent cell experiments, and three replicate wells were set for each treatment group in each experiment. Collagen gel-based contraction assay Contraction of HUVEC cells was evaluated by collagen gel-based assay according to the method of SakotaY with minor modifications [45]. The cells were suspended in Dulbecco's phosphate buffer solution (DPBS) supplemented with type-I collagen, seeded into a 12-well microplate at a density of 1 × 10 5 cells/mL and then incubated for 30 min at 37°C in a CO 2 incubator for gelation. After addition of the culture medium (equivalent volume of the gel) onto the gel, the embedded cells were cultured for 2 days with changing the medium every day. The gel was detached from the well with a 10µl pipette tip, immersed in the assay medium containing agent for treatment, and then photographed using a digital camera. Contraction was expressed as a percentage of area of the gel to that before treatment. We conducted three independent cell experiments, and three replicate wells were set for each treatment group in each experiment. Hematological analysis Whole blood of mice in each group were collected in sterile EDTA-anticoagulation tubes. After that, they were analyzed by using an automatic hematology analyzer (Mindray Medical, BC-5000, China). Then, hematological indicators were observed and recorded, such as white blood cell counts (WBC), red blood cell counts (RBC), and platelet counts (PLT). Data statistical analysis Statistical comparisons were performed using Graphpad Prism 8.0 software. T-test was used to compare the differences between the two groups. For comparisons between three or more groups, ANOVA analysis was performed. Quantitative data-sets were presented as the means ± standard deviation (SD). The p values less than 0.05 (p<0.05) was regarded as statistically significant, *p<0.05, **p<0.01, ***p<0.001. Declarations Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements We are especially grateful to Wu Zhong who works in the Military Medical Research Institute for kindly providing the technical guidance, and experimental assistance provided by Changchun Veterinary Research Institute (Biosafety Level III laboratory). Fundings This work was supported by the Military Medical Research Institute Foundation project (grant number: AMMS-KYJJ-2022-014), the National Key Research and Development Program of China (2020ZX10001-016-003), and the National Key Research and Development Program of China (ZX10304402-003-006). This work was supported by a grant from National Key R&D Program of China (No. 2021YFC2301700) and the National Key Research and Development Program of China (No. 2020YFC0846100, 2023YFC0871000). Ethics approval and consent to participate The treatment of all mice was in accordance with the welfare and ethical guidance of Chinese laboratory animals (GB 14925-2001). The agreement was approved by the Animal Welfare and Ethics Committee of the Institute of Chinese Academy of Agricultural Sciences (permit number: SCXK-2012-017). Consent for publication All authors consent to submit and publish this article. 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Collagen gel contraction assay using human bronchial smooth muscle cells and its application for evaluation of inhibitory effect of formoterol. Biological & pharmaceutical bulletin. 2014;37(6):1014-20. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4778710","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":340659989,"identity":"1674ff95-ba13-48bc-98a3-f002dee9d316","order_by":0,"name":"Chaoxiang Lv","email":"","orcid":"","institution":"Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chaoxiang","middleName":"","lastName":"Lv","suffix":""},{"id":340659990,"identity":"b30287f8-23d6-40ac-b088-39d592b33a95","order_by":1,"name":"Jin Guo","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Guo","suffix":""},{"id":340659991,"identity":"ef2769a9-9fe3-4e67-bc61-84733751d47c","order_by":2,"name":"Rongbo Luo","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Rongbo","middleName":"","lastName":"Luo","suffix":""},{"id":340659992,"identity":"85e7586d-af61-4b78-be28-4d4f132c3c43","order_by":3,"name":"Yuanguo Li","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuanguo","middleName":"","lastName":"Li","suffix":""},{"id":340659993,"identity":"c44e6d36-62c6-48f4-909f-e7312ef98aa5","order_by":4,"name":"Bingshuo qian","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bingshuo","middleName":"","lastName":"qian","suffix":""},{"id":340659994,"identity":"32c60515-91bf-44fb-bc2a-5e40102fbe29","order_by":5,"name":"Xiaopan Zou","email":"","orcid":"","institution":"Renmin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaopan","middleName":"","lastName":"Zou","suffix":""},{"id":340659995,"identity":"cd5a590f-576c-4aa7-9554-0d0620105640","order_by":6,"name":"Tiecheng Wang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tiecheng","middleName":"","lastName":"Wang","suffix":""},{"id":340659996,"identity":"ab86e3ee-c390-45c2-9dee-3b76e835182e","order_by":7,"name":"Beilei Shen","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Beilei","middleName":"","lastName":"Shen","suffix":""},{"id":340659997,"identity":"207c6e14-0e3f-4800-861d-53566d48b6c5","order_by":8,"name":"Weiyang Sun","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Weiyang","middleName":"","lastName":"Sun","suffix":""},{"id":340659998,"identity":"71a915b2-a338-40f3-8c50-56b25ca6df88","order_by":9,"name":"Yuwei Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIie3RPQrCMBiA4Q8KcQmdEwqtR4gERLCbF2kQ4lLBsWNBiIcQPEZdKwW75AAdrT2AiktHUxyFtqND3imE74H8ANhs/1rEQowmqVntAMg4kkjfxTlAzsYS0AX3STSSsCqe3WpVCEWbq/dkoU9Tp75X/YQzoTZCeVKSnEnuAeI87iXbjAi1NCSeG1KIE2DkDZBzK5RjDqbHkwwiveKI4C85DhGqH2/SPTLCcr3Q5i50P3AXt5Ti1ZqvDA7FpUqS0Cflvm76yDT/2XJ6xruCdGDAZrPZbPABtq9LV3p1MCcAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yuwei","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2024-07-22 02:47:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4778710/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4778710/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62581761,"identity":"b842f145-e7d6-4775-8401-9c459cf55753","added_by":"auto","created_at":"2024-08-16 06:24:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":431907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTaurolidine inhibits the reproduction of influenza virus \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) Dose-response curve of taurolidine against viral titers in MDCK cells respectively infected with IAV/H1N1 and IBV/S9-MD respectively. (B) The semi-logarithmic fitting curve was plotted after converting the concentration of taurolidine into logarithm and the virus titer into percentage, and EC\u003csub\u003e50\u003c/sub\u003e value was calculated according this curve. (C) Plaque experiment in MDCK cells infected respectively infected with IAV/H1N1 and IBV/S9-MD. The experiment was performed independently in triplicate with duplicate plaque assay and representative pictures were shown. (D) Immunofluorescence against viral nucleoprotein (virus NP), the scale bar is 10 µm. After A549 cells infection with influenza virus at an MOI of 0.1, the cells were treated with solvents DMEM and taurolidine (50 μg/mL) for 24 h, and OSTA (50 μg/mL) was used as a positive control. The nucleus was stained with DAPI, and the infected cells were detected by nuclear virus NP staining (scale bar 10 µm). (E) The percentage of virus NP-positive cells in figure 1D was calculated. (F) Western blotting experimental results of virus NP in A549 cells infected with IAV/H1N1 and IBV/S9-MD infection, using β-actin as a loading control.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/309f1ec0e2f8775b3daece96.jpg"},{"id":62581769,"identity":"f496a80a-b7d1-43d3-8432-b1ea4e2e26f4","added_by":"auto","created_at":"2024-08-16 06:24:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":179708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTaurolidineantiviral activity is not specific to virus subtypes or cell lines.\u003c/strong\u003e (A-C) Inhibitory rates of taurolidine against influenza viruses with different infection protocols were evaluated in A549 cells, including IAV/H1N1, IAV/H3N2, and IBV/S9-MD. (D-G) After infecting with influenza viruses (IAV/H1N1-PR8, IAV/H1N1-UI182, IAV/H3N2, and IBV/S9-MD) at an MOI of 0.1, A549 cells were treated with DMEM and taurolidine (50 μg/mL) for 24 h, and oseltamivir (OSTA, 50 μg/mL) was used as a positive control, and virus titer was determined in MDCK cells. (H-K) After infecting with influenza viruses (IAV/H1N1-PR8, IAV/H1N1-UI182, IAV/H3N2, and IBV/S9-MD) at an MOI of 0.1, MDCK cells were treated with DMEM and taurolidine (50 μg/mL) for 24 h, and oseltamivir (OSTA, 50 μg/mL) was used as a positive control, and virus titer was determined in MDCK cells.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/d3d9507fd2ec5ca3529bc59d.jpg"},{"id":62581764,"identity":"20c452ca-806b-4e74-b57f-a1c62f339de0","added_by":"auto","created_at":"2024-08-16 06:24:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":192603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe antiviral effect of taurolidine against influenza viruses in mouse model.\u003c/strong\u003e (A-C) Body weight changes in control group (Control, gray), virus group (Virus, black), taurolidine treatment group (Virus+taurolidine, purple), and oseltamivir treatment group (Virus+oseltamivir, blue), including IAV/H1N1, IAV/H3N2, and IBV/S9-MD. (D-F). Survival rate of infected mice treated or untreated with taurolidine. (G-I) Virus titer in mouse lung tissues at 3 and 5 day post infection (dpi). (J-L) The protein expression level of influenza virus NP in lung tissues of mice at 5 dpi was analyzed by Western blot, and used β-actin as a loading control.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/4516137b7ebff415cfda975b.jpg"},{"id":62582319,"identity":"04a9a60d-d0c3-403b-b38a-217fba8066ef","added_by":"auto","created_at":"2024-08-16 06:32:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":394963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTaurolidine alleviates the pathogenic effect of influenza virus infection. \u003c/strong\u003e(A) Morphological observation of mice at 5 dpi with IAV/H1N1 and IVV/S9-MD, bleeding regions were indicated by black arrows. (B, C) The effect of taurolidine on the lung index of mice was measured at 5 dpi with IAV/H1N1 and IBV/S9-MD infection. (D) Hematoxylin-eosin staining of lung tissues of mice in each group, and the representative images were showed here, the scale bar is 50 μm. Green arrows indicate massive lymphocyte infiltration, and red arrows indicate cell debris. (E, F) Lung pathology score at 5 dpi. The pathology scores are summarized according to (one point each for hemorrhage, alveolar wall thickening, inflammation, and cell necrosis), n = 3. (G) Immunohistochemical analysis of virus NP expression in lung tissues after influenza virus infection. (H, I) Quantification the number of NP-positive cells.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/dd3655fe159354fb81f0deaf.jpg"},{"id":62581768,"identity":"5057c4aa-2930-4c1b-b921-9d515628eb31","added_by":"auto","created_at":"2024-08-16 06:24:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":266716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of taurolidine on immune cell and cytokine storm induced by influenza virus infection. \u003c/strong\u003e(A) CD8\u003csup\u003e+\u003c/sup\u003e T cell amount changes in IAV/H1N1-infected mice. (B) Quantification of CD8\u003csup\u003e+\u003c/sup\u003e T cells seen in Figure 5A. (C) CD8\u003csup\u003e+\u003c/sup\u003e T cell amount changes in IBV/S9-MD-infected mice. (D) Quantification of CD8\u003csup\u003e+\u003c/sup\u003e T cells seen in Figure 5C. (E) The immune cells change in IAV/H1N1-infected mice, including neutrophil, macrophage, NK cell, and dendritic cell. (F) Quantification of neutrophil seen in Figure 5E. (G) Quantification of macrophage seen in Figure 5E. (G) Quantification of NK cell seen in Figure 5E. (G) Quantification of dendritic cell seen in Figure 5E. (J-M) Levels of proinflammatory cytokines were analyzed in serum of IAV/H1N1-infected mice at 5 dpi by ELISA.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/f7cd7c56019d06a18adaa373.jpg"},{"id":62581762,"identity":"30f2a2aa-bc26-4e29-a3d6-0b3c786e09e4","added_by":"auto","created_at":"2024-08-16 06:24:14","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":308138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTaurolidine alleviates vascular pathology in mice infected with influenza virus. \u003c/strong\u003eThe blood pressure of IAV/H1N1-infected mice were measured, including (A) systolic blood pressure, (B) diastolic blood pressure and (C) mean arterial pressure. (D) Ultrasound assay was used to measure the pulmonary aorta of IAV/H1N1-infected mice at 5 dpi, and the representative ultrasonography images of the pulmonary aorta. (E) Pulse wave velocity of the pulmonary aortic measured by ultrasound. (F) Thickening of the pulmonary aortic wall measured by ultrasound. (G) The pulmonary aortic diameter of the pulmonary aortic wall measured. (H) Representative cross-sections of pulmonary aortic tissues stained with H\u0026amp;E, and Immunohistochemical analysis of virus NP expression intensity in pulmonary aortic after IAV/H1N1-infected.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/8831877ade825c3c1e7ff250.jpg"},{"id":62581765,"identity":"18bfc207-f39f-46ee-aad7-ceedd72871c5","added_by":"auto","created_at":"2024-08-16 06:24:14","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":242543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTaurolidine inhibits vasoconstriction pathways activated and intracellular calcium release by influenza virus infection. \u003c/strong\u003e(A, B) The mRNA expression levels of genes related to vascular smooth muscle contraction signaling. (C, D) The intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration was measured after VSMCs cells infected IAV/H1N1 and IBV/S9-MD, respectively. (E) The Ca\u003csup\u003e2+\u003c/sup\u003e intensity in IAV/H1N1-infected VSMCs (with or without drug treatment) was measured. (F) Quantitative analysis of the relative calcium intensity of each group as in Figure 7E. (G) The Ca\u003csup\u003e2+\u003c/sup\u003e signaling in IAV/H1N1-infected VSMCs (with or without drug treatment) was measured. (H) Quantitative analysis of the relative calcium intensity of each group as in Figure 7G. (I) Western blotting results of p-MLC and MLC in VSMCs after IAV/H1N1 infection. (J) Western blotting results of p-MLC and MLC in VSMCs after IBV/S9-MD infection.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/5096694e120fe6b7ca2a055c.jpg"},{"id":62581767,"identity":"8fb5f4c7-0338-4b24-835e-cd57bd9e8a20","added_by":"auto","created_at":"2024-08-16 06:24:14","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":256303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of taurolidine on the MLCK/p-MLC pathway induced by influenza virus infection.\u003c/strong\u003e Western blotting was used to determine protein levels of (A) p-MLC, (B) AT1R, (C) CaM and (D) MLCK in IAV/H1N1-infected VSMCs post-treated with taurolidine (10, 25, 50 μg/mL) for 24 h. (E) Immunohistochemical staining was used to detect protein levels of p-MLC, MLC, AT1R, CaM and MLCK in pulmonary aorta tissues. (F) Quantification the number of positive cells in p-MLC, MLC, AT1R, CaM, and MLCK.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/35d47bc659bfb1d173bd2f7e.jpg"},{"id":62793928,"identity":"8a425613-97d1-4b8f-a602-c68031dd033a","added_by":"auto","created_at":"2024-08-19 14:45:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3112228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/86c190dc-8edc-4f54-bdec-5a663f4e7515.pdf"},{"id":62582320,"identity":"f982f390-540d-4203-8018-205e0fa9acd5","added_by":"auto","created_at":"2024-08-16 06:32:14","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5694994,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4778710/v1/82b58341546ea90800511f00.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Taurolidine resists pulmonary artery vasoconstriction induced by influenza virus infection via inhibiting Ca 2+ influx and MLCK/p-MLC signaling pathway","fulltext":[{"header":"Background","content":"\u003cp\u003eInfluenza virus is the main virus responsible for acute respiratory infections and seasonal influenza, which is easy to cause seasonal outbreaks and pandemics, and pose a significant threat to public health and social development[1-3]\u0026nbsp;. Presently, vaccination and antiviral treatments serve as the main preventive and supportive measures for responding to the influenza pandemic [4]. However, the vaccine is limited to identify pandemic strains and resistance towards an existing class of direct-acting antivirals (DAAs), like adamantane (the M2 inhibitor), oseltamivir (the NA inhibitor) and baloxavir (the PA inhibitor) becomes widespread [5]. Meanwhile, host-targeted therapies, anti-inflammatory drugs, and immunomodulators have also been shown to have broad-spectrum antiviral activity against influenza viruses [2]. In addition, the current ability of vaccines to identify pandemic strains is limited. All these problems highlight the urgent need to develop new antiviral strategies with new mechanisms of action and reduce the possibility of drug resistance [6].\u003c/p\u003e\n\u003cp\u003eInflammatory cytokines are markedly elevated following influenza virus infection, leading to the \u0026ldquo;cytokine storm\u0026rdquo;, which is hypothesized to be the main cause of mortality [7]. The cytokine storm can accumulate excessive immune cells and viscous secretions in the lung tissues, thereby obstructing gas exchange and ultimately leading to death [8]. A study has showed that the\u0026nbsp;\u0026ldquo;cytokine storm\u0026rdquo; effect characterized by over-production and dysfunction of inflammatory cytokines has become a main cause of host death after influenza virus infection [9]. This indicates that severe lung inflammation is related to lung tissue pathology, such as lung tissue edema, infectious pneumonia and alveolar hemorrhage [10].\u0026nbsp;Consequently, it is essential to maintain the host\u0026apos;s antiviral response as the optimal defense mechanism following infection with the influenza virus.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe vascular smooth muscle system drives blood flow, cytokine release and immune cell recruitment by regulating the balance between vasoconstriction and vasodilatation [11]. The contraction of vascular smooth muscle is regulated by a voltage- and calcium-dependent process (called excitation-contraction coupling) in response to neuronal stimulation [12]. Additionally, vasomotor control is the basis for the acute and rapid adaptation of blood vessel diameter, which is mainly due to the vasoconstriction caused by the active contraction of vascular smooth muscle cells, and the altered of vascular structure represents the dynamic process of chronic hemodynamic changes [13]. The acute regulation of blood vessel diameter and the consequent vascular resistance depends on the activation state of the contraction mechanism involving actin, that is, the interaction of actin in the vascular smooth muscle cells [14]. The alteration of Ca\u003csup\u003e2+\u003c/sup\u003e flux and membrane potential regulate phosphorylation of myosin-light-chains (MLCs) and actin-myosin cross-bridge cycles, ultimately leading to rapid vasoconstriction [15], which is a critical mechanism for the host to adapt to a rapid immune response.\u003c/p\u003e\n\u003cp\u003eTaurolidine, a derivative of the amino acid taurine, has been found to possess anti-endotoxin, anti-bacterial and anti-adhesion properties [16]. Its mechanism of action involves inhibiting microbial adhesion through chemical reactions with cell walls, including bacterial and fungus [17]. Additionally, previous studies have shown that taurolidine inhibits the synthesis of cytokines (IL-1 and TNF) in human peripheral blood mononuclear cells [18], which indicates that taurolidine may play important roles in cytokine-induced immune responses and related diseases. Despite these findings, the antiviral effectiveness of taurolidine remains unclear.\u003c/p\u003e\n\u003cp\u003eHere, we explored the anti-influenza virus activity of taurolidine \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. By using genomics and chemical tools, we defined the important role of taurolidine in attenuation of inflammation response. Furthermore, we revealed that the suppression of vasoconstriction signaling pathway through taurolidine treatment can reduce the mortality among mice infected with influenza virus. Therefore, the vasoconstrictor signal provided a mechanism to reduce influenza virus-induced incidence and revealed the unforeseen role of taurolidine as a modulator of inflammatory response and vasoconstriction.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAdministration of taurolidine inhibited the reproduction of influenza virus in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential anti-influenza virus activity of taurolidine was evaluated using an MDCK cell infection model. Taurolidine efficiently inhibited the IAV/H1N1 replication in cultures exposed to different dose-ranges of drugs for 24h (Fig. 1A, left). The 50% effective concentration (EC\u003csub\u003e50\u003c/sub\u003e value) of taurolidine was observed at 31.63 \u0026mu;g/mL (Fig. 1B, left). Similarly, we also found that taurolidine also inhibited the IBV/S9-MD replication (Fig. 1A, right), with EC\u003csub\u003e50\u003c/sub\u003e of 22.73 \u0026mu;g/mL (Fig. 1B, right). To rule out that the reduction in viral replication was due to an indirect effect on the host cell, MTT experiment was performed to confirm the effect of taurolidine on cytotoxicity. The results showed that taurolidine did not reduce the cellular viability of MDCK (Fig. S1A) and A549 cells (Fig. S1B) in a significant manner at 10, 25, 50 and 100 \u0026micro;g/mL concentrations, suggesting that taurolidine mainly exerts its antiviral effect by inhibiting the proliferation of influenza viruses. The activity of taurolidine against influenza virus was further confirmed using plaque reduction assays. As shown in Fig. 1C, plaque formation in the IAV/H1N1- and IBV/S9-MD-infected cells was reduced significantly after treatment of taurolidine.\u003c/p\u003e\n\u003cp\u003eTo explore the potential antiviral effects of taurolidine, oseltamivir (a common anti-influenza drug) was used as a positive control for indirect immunofluorescence assay. As anticipated, oseltamivir significantly inhibited the IAV/H1N1 strain propagation; 38% of MDCK cells nuclei were virus NP-positive in DMEM-treated cells, whereas the percentage of virus NP-positive cells were significantly reduced to 9.8% following taurolidine treatment (Fig. 1D). Comparable results were observed for IBV/S9-MD strain (Fig. 1E). In addition, the protein expression level of virus NP was also significantly decreased after taurolidine treatment in both IAV/H1N1-infected and IBV/S9-MD-infected cells (Fig. 1F). \u003c/p\u003e\n\u003cp\u003eIn order to clarify the active stage of taurolidine against influenza virus, we treated A549 cells with three different infection protocols, including pre-treatment, co-treatment and post-treatment. It was observed that post-treatment taurolidine exhibited high inhibition rates in IAV/H1N1-infected (Fig. 2A), IAV/H3N2-infected (Fig. 2B), and IBV/S9-MD-infected cells (Fig. 2C), which indicating that taurolidine had a inhibitory effect on later stages of influenza virus. To further assess the antiviral potential of taurolidine, different influenza virus subtypes (IAV/H1N1-PR8, IAV/H1N1-UI182, IAV/H3N2 and IBV/S9-MD strains) were chosen to infect A549 (Fig. 2D, E, F, G) and MDCK cells (Fig. 2H, I, J, K), respectively. These results indicate that taurolidine showed a significant inhibitory activity against different subtypes of influenza virus.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTaurolidine improves the survival rate after influenza virus infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe therapeutic effect of taurolidine was further evaluated in mouse models on account of its obvious anti-influenza activity \u003cem\u003ein vitro\u003c/em\u003e above. We found that taurolidine treatment alleviated weight loss in mice infected with influenza viruses, including IAV/H1N1 (Fig. 3A), IAV/H3N2 (Fig. 3B), and IBV/S9-MD (Fig. 3C). Compared to virus-infected mouse, taurolidine treatment significantly increased the overall survival condition of mice in drug-treatment groups, with protection rate of 66.67% for IAV/H1N1 (Fig. 3D), 42.86% for IAV/H3N2 (Fig. 3E), and 66.67% for IBV/S9-MD (Fig. 3F). Subsequently, we examined the viral loads in the lung tissues of mice at 3 dpi and 5 dpi, and found that viral loads were significantly reduced in taurolidine-treated groups (Fig. 3G, H, I). Furthermore, western blot results also confirmed that taurolidine significantly inhibited the replication of influenza viruses in the mouse lung tissues (Fig. 3J, K, L). By observing the lung morphology, we found that both IAV/H1N1 and IBV/S9-MD infection caused extensive bleeding (\u003cstrong\u003eblack arrow\u003c/strong\u003e) in the lung tissue of mice, while taurolidine significantly reversed these results (Fig. 4A). The lung index of mice infected with IAV/H1N1 (Fig. 4B) and IBV/S9-MD (Fig. 4C) also decreased after taurolidine treatment.\u003c/p\u003e\n\u003cp\u003eTo investigate the improvement effect of taurolidine on lung pathology caused by influenza virus infection, lung tissues were randomly collected from mice in each group at 5 dpi to perform H\u0026amp;E and IHC assays. The H\u0026amp;E assay showed that the influenza viruses infection caused part of cell necrosis (\u003cstrong\u003ered arrow\u003c/strong\u003e) and inflammatory cell infiltration (\u003cstrong\u003eblue arrow\u003c/strong\u003e), but this effect was inhibited by the administration of taurolidine (Fig. 4D). Furthermore, the pathological score also confirmed that taurolidine treatment improved overall lung pathology in mice infected with influenza virus (Fig. 4E, F). In addition, we also found that taurolidine administration significantly inhibited the expression of virus NP in lung tissues (Fig. 4G), and reduced virus NP-positive cells in lung tissues of mice infected with IAV/H1N1 (Fig. 4H) and IBV/S9-MD (Fig. 4I). Moreover, we also observed that taurolidine treatment significantly increased the number of white blood cells (Fig. S2A, B), reduced the number of red blood cells (Fig. S2C), and reduced the number of the number of platelets in mice infected with IAV/H1N1 (Fig. S2D). Similar results were observed in mice infected with IBV/S9-MD (Fig. S3). These results indicated that taurolidine treatment could effectively improve the pathological damage caused by influenza viruses and improve the survival rate of infected mice.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEffects of taurolidine on immune cell and cytokine storm induced by influenza virus infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe previously demonstrated that taurolidine treatment improved inflammatory cell infiltration caused by influenza virus infection in lung tissues of infected mice. To further analyze the effects of taurolidine on immune cells of influenza virus-infected mice, peripheral blood was collected from mice in each group at 5 dpi for flow cytometry analysis. The results showed that influenza (IAV/H1N1) virus infection led to a reduction in the number of CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig. S4A) and CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig. 5A), and the administration of taurolidine reversed this result (Fig. 5B; Fig. S4B). Interestingly, we only observed a reduction in CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig. 5C) and no significant changes in CD4\u003csup\u003e+\u003c/sup\u003e T cells in IBV/S9-MD-infected mice (Fig. S4C, D). Importantly, taurolidine treatment also improved the CD8\u003csup\u003e+\u003c/sup\u003e T cells reduction caused by IBV/S9-MD infection (Fig. 5D). In addition, we also found that influenza (IAV/H1N1) virus infection increased the number of neutrophils, macrophages, natural killer (NK) cells, and dendritic cells (Fig. 5E). However, the administration of taurolidine significantly reduced the number of neutrophils (Fig. 5F), macrophages (Fig. 5G), NK cells (Fig. 5H), and dendritic cells (Fig. 5I). These findings suggest that taurolidine treatment reduces immune cell infiltration caused by influenza virus infection.\u003c/p\u003e\n\u003cp\u003eInflammatory cytokines are markedly elevated after influenza virus infection and the cytokine storm being considered the main cause of mortality. To investigate the effect of taurolidine on the inflammatory response induced by influenza (IAV/H1N1) virus infection, we also collected the serum of each group of mice for ELISA detection. The results showed that taurolidine reduced the concentration of key inflammatory cytokines in the serum, such as IL-6 (Fig. 5J), IFN-\u0026gamma; (Fig. 5K), IL-10, TNF-\u0026alpha; (Fig. 5L), and IL-1\u0026beta; (Fig. 5M). In addition, we observed that influenza (IAV/H1N1) virus infection leads to up-regulation of cytokine mRNA expression in the lungs of infected mice, and taurolidine-treated significantly reduced the mRNA expression of cytokines and chemokines, including \u003cem\u003eIFN-\u0026alpha;\u003c/em\u003e, \u003cem\u003eIL-10\u003c/em\u003e, \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e, \u003cem\u003eIL-1\u0026alpha;\u003c/em\u003e, and \u003cem\u003eIFN-\u0026gamma;\u003c/em\u003e, as well as \u003cem\u003eCCL5\u003c/em\u003e,\u003cem\u003e CXCL10\u003c/em\u003e,\u003cem\u003e CXCL11\u003c/em\u003e, \u003cem\u003eCCL3\u003c/em\u003e and \u003cem\u003eCCL4\u003c/em\u003e (Fig. S5). These findings support the conclusion that taurolidine has the potential to mitigate the cytokine storm in mouse lungs following influenza virus infection.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003cstrong\u003eaurolidine alleviates vascular pathology in mice infected with influenza virus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a fast-track for immune regulation, blood vessels regulate the transportation and transfer of immune cells during influenza virus infection. In this study, we sought to investigate the effects of taurolidine treatment on blood pressure in mice infected with influenza virus. The results showed that the systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were significantly increased in mice infected with influenza virus, while they were decreased by taurolidine treatment (Fig. 6A, B, C). Two-dimensional ultrasound results showed that influenza virus infection caused thickening of the pulmonary aortic tube wall in IAV/H1N1-infected mice, an effect that was alleviated by taurolidine treatment (Fig. 6D). Compared to the control group, the pluse wave velocity (PWV) of the pulmonary aorta in IAV/H1N1-infected mice was increased at 5 dpi (3.88 \u0026plusmn; 0.38\u0026thinsp;mm/s vs. 2.02 \u0026plusmn; 0.18\u0026thinsp;mm/s), but this was mitigated following taurolidine treatment (2.86 \u0026plusmn; 0.28\u0026thinsp;mm/s vs. 3.88 \u0026plusmn; 0.38\u0026thinsp;mm/s, Fig. 6E). Moreover, the pulmonary aorta in IAV/H1N1-infected mice exhibited greater thickness and smaller diameter in comparison to controls, with these differences being reversed following taurolidine treatment (Fig. 6F, G). Changes in the thickness of the pulmonary aorta were also confirmed by H\u0026amp;E staining (Fig. 6H), and similar results were found in mice infected with IBV/S9-MD (Fig. S6).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTaurolidine inhibits vasoconstriction pathways and intracellular calcium elevation induced by influenza virus infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe previously shown that taurolidine mitigated vascular changes in pulmonary artery blood vessels induced by influenza virus infection in mice. To further investigate the effect of taurolidine on the pulmonary artery induced by influenza virus infection, we collected lung tissues from each group of mice to perform transcriptome analysis (Fig. S7A, B). We also conducted classification and enrichment analysis of differential genes (Fig. S7C, D). The results showed that the vascular smooth muscle contraction signaling pathway was significantly enriched after infection with influenza viruses IAV/H1N1 (Fig. S8A) and IBV/S9-MD (Fig. S8B). Subsequently, the expressions of some genes in the vascular smooth muscle contraction signaling pathway were detected. Taurolidine treatment rescued the down-regulation of \u003cem\u003ePKC\u003c/em\u003e, \u003cem\u003eADER5\u003c/em\u003e, \u003cem\u003eMYLK3\u003c/em\u003e, \u003cem\u003ePPP1C\u003c/em\u003e and \u003cem\u003eMLC1\u003c/em\u003e expression in lung tissues of mice infected with IAV/H1N1 (Fig. 7A) and IBV/S9-MD (Fig. 7B).\u003c/p\u003e\n\u003cp\u003eGenerally, the contraction and relaxation of vascular smooth muscle mainly depend on the state of membrane light chain (MLC) phosphorylation regulated by Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin complex and by MLC kinase and MLC phosphatase [19,20]. Thus, we examined the concentration of Ca\u003csup\u003e2+\u003c/sup\u003e in VSMCs after influenza virus infection. We found that both IAV/H1N1 and IBV/S9-MD infection increased in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration, which was subsequently attenuated by taurolidine treatment in a time-dependent manner (Fig. 7C, D). By comparing with the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e signaling intensity, we also found taurolidine treatment reduced Ca\u003csup\u003e2+\u003c/sup\u003e concentration in a dose-dependent manner after IAV/H1N1 infection (Fig. 7E, F. G, H). In addition, taurolidine-treated significantly decreased expression level of p-MLC with a dose-dependent manner in VSMCs infected with IAV/H1N1 (Fig. 7I, H) or IBV/S9-MD (Fig. 7J, K), which was not observed after oseltamivir treatment (Fig. S8C, D). The collagen gel-based contraction assay also found taurolidine treatment increased the initial area of IAV/H1N1-infected VSMCs compared to the untreated group (Fig. S9A, B), suggesting that taurolidine controls vascular smooth muscle contraction after influenza virus infection.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTaurolidine attenuates the activation of MLCK/p-MLC pathway induced by influenza virus infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate taurolidine regulation in vasoconstriction induced by influenza virus infection, western blot was performed. The results showed that the phosphorylated expression level of MLC (p-MLC) was up-regulated and the protein expression level of MLC was down-regulated in VSMCs after infection with influenza (IAV/H1N1) virus, while it was attenuated by taurolidine treatment in a dose-dependent manner (Fig. 8A). In addition, we found that influenza (IAV/H1N1) virus infection also up-regulated the protein expression level of AT1R (Fig. 8B), CaM (Fig. 8C), and MLCK (Fig. 8D), but this was reversed by taurolidine treatment in a dose-dependent manner. Considering the stability of \u003cem\u003ein vitro\u003c/em\u003e environment, we subsequently focused on \u003cem\u003ein vivo\u003c/em\u003e experiments. In immunohistochemical (IHC) staining of the pulmonary aorta of mice infected with influenza virus (Fig. 8E), significant up-regulation of p-MLC was observed in the pulmonary aorta of IAV/H1N1-infected mice, and it was reversed by taurolidine treatment (Fig. 8F). Similarly, IAV/H1N1 infection also down-regulated the protein expression level of MLC (Fig. 8G) and up-regulated the protein expression level of AT1R (Fig. 8H), CaM (Fig. 8I), and MLCK (Fig. 8J) in the pulmonary aorta of IAV-infected mice, all of which were reversed by taurolidine treatment. \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe continuous replication, recombination and mutation of influenza virus have presented significant challenges for prevention and treatment of influenza diseases [21]. Current antiviral countermeasures primarily consist of preventive vaccines and therapeutic drugs. Despite partial achievements have been obtained, but resistance to existing antiviral drugs has become a serious problem [22]. Thus, repurposed of old drugs is also a selection strategy in combating the virus due to their low-toxicity and high-efficiency in clinical application. In this study, we found that the clinically licensed antibacterial drug taurolidine significantly inhibited the replication of influenza viruses\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e and improved the survival rate of lethal influenza virus infection in mouse models by suppressing cytokine storms and regulating vasoconstriction.\u003c/p\u003e\n\u003cp\u003eAs a derivative of taurine, taurolidine has been proved to be an effective antibacterial agent and used to therapy peritonitis in some countries. Previous studies have shown that it inhibits the synthesis of IL and TNF in human peripheral blood mononuclear cells [18], which indicates that taurolidine is likely to play important roles in IL- and TNF-induced related diseases. Indeed, the application of taurolidine tends to prevent the development of lung metastases [23]. In our study, we found that taurolidine significantly improved the lung damage caused by influenza virus infection in mice. Moreover, the drug treatment significantly reduced the number of neutrophils after influenza virus infection. However, the detailed mechanism behind this requires further studied in future.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe clinical outcome after virus infection depended largely on the balance between virus replication and host defense response [24]. The ability of the virus to evade the host\u0026apos;s immune response is critical to its pathogenicity [25]. Overexpression of inflammatory factors in the host after influenza virus infection will lead to death, and this effect can be improved by inhibition of cytokine regulating inflammation molecular information [26]. Additionally, cytokines play important roles in activating immune cells, regulating immune response and promoting virus clearance, which suggest that their dynamic balance usually determines the process of host infection [27]. The imbalance of cytokine effect and immune cells recruitment could be used as poor prognostic indicators during highly pathogenic influenza virus infection [28]. Early induction of cytokines (IFN-\u0026alpha;, IL-1\u0026beta;, IL-2, IL-6, and TNF-\u0026alpha;) and chemokines (CCL2, CCL3, CXCL2, and CXCL10) are related to the symptom\u0026rsquo;s formation in human [29,30]. TNF-\u0026alpha;, IL-1, and IL-6 have multifunctional activities and are related to the morbidity after influenza virus infection [31]. In this study, we found the protein productions of the cytokines (TNF-\u0026alpha;, IL-6, and IL-10) in the serum after influenza virus infection were decreased after taurolidine\u0026nbsp;treatment (data not shown). Additionally, chemokines can induce innate immune cells to be recruited to lung tissues, releasing more cytokines to exacerbate the cytokine storm [32]. Importantly, we demonstrate that taurolidine significantly reduces the mortality of mice infected with human pathogenic influenza virus strains by inhibiting cytokines. These findings suggest that at least taurolidine exhibits chemotherapeutic properties in diseases with inflammatory factors as the main pathological component.\u003c/p\u003e\n\u003cp\u003eThe tension of blood vessels is regulated by vascular smooth muscle signals. Previous studies suggested that influenza virus infection can lead to pulmonary artery atherosclerosis [33]. Additionally, abnormal vascular smooth muscle signaling often leads to pulmonary hypertension [34], platelet aggregation [35]and atherosclerosis [36],\u003csup\u003e\u0026nbsp;\u003c/sup\u003esuggesting that the signaling plays a crucial role in the process of anti-influenza virus. Our results showed that taurolidine can inhibit the contraction of vascular smooth and improve survival rate during infection with influenza virus, highlighting the significance of this signal in defending against influenza virus\u0026nbsp;challenge. An interesting question is whether vascular endothelial cells or smooth muscle directly regulate the host immune response. The endothelial cells are involved in anti-inflammatory response [37], but we still need to determine adjustments to taurolidine-mediated endothelial cytokine production. Endothelial cells could regulate the production of cytokines in the lung through complex crosstalk mechanisms with epithelial cells or resident hematopoietic cells [38]. However, the identification of endothelial cells as the central coordinator of immune-mediated inflammation is of fundamental significance and has broad implications for the treatment of many diseases, such as influenza.\u003c/p\u003e\n\u003cp\u003eFurthermore, the etiology of several auto-immune diseases is directly related to inflammation response [39]. Therefore, it is important to understand the biological characteristics of taurolidine in regulating cytokine storm and develop appropriate chemical signaling transduction tools to identify specific molecular targets. This not only provides insights into the interaction between microorganisms and hosts, but also will reveal other ways to achieve effective immunotherapy in a variety of diseases. We also revealed that the administration of taurolidine reduced the release of Ca\u003csup\u003e2+\u003c/sup\u003e after infection with influenza virus. Indeed, vasoconstriction is regulated by Ca\u003csup\u003e2+\u003c/sup\u003econcentration [40,41]. This suggested that taurolidine may have the potential to treat other diseases that depending on Ca\u003csup\u003e2+\u003c/sup\u003e-related signaling pathways. Importantly, these data indicate that taurolidine might be a negative regulator of cytokine amplification and an activator of vascular smooth muscle signaling, which would give more personality possibility of the host genetic survival advantage or disadvantage.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the antiviral ability of taurolidine was demonstrated. Our finding not only reported the inhibitory effect of taurolidine on inflammatory response, but also suggested its important roles of taurolidine in vasoconstriction signaling. In addition, we reveal the potential of taurolidine-mediated mouse models in the treatment of influenza virus infections. Therefore, we provide a theoretical basis for further research on the antiviral mechanism of taurolidine, which providing a promising application for the prevention and treatment of influenza virus infection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAntibodies, mice, reagents and viruses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibodies against viral nucleoprotein (virus NP, ab20343), AT1R (cat no. ab124505), CaM (cat no. ab2860), and MLCK (ab236299) were provided by Abcam (Burlingame, CA, USA). The antibodies against MLC (Loc:3672) and p-MLC (Loc:3675) were purchased from Cell Signaling Technology (Beverly, MA, USA).\u0026nbsp;Taurolidine, oseltamivir (PHR1781)\u0026nbsp;and antibody against β-actin were from Sigma-Aldrich (St. Louis, MO, USA). The MLCK inhibitor ML-7 HCL (S8388) and ML-9 HCL (S6847) were purchased from Selleck (Houston, TX, USA). Six- to eight-week-old BALB/c female mice derived from Charles River Laboratory Animal Technology Co., Ltd (Beijing, China). Influenza A viruses (IAV/H1N1-UI182, IAV/H1N1-PR8, IAV/H3N2) originated from the Institute of Changchun Veterinary Research, Chinese Academy of Agricultural Sciences (Changchun, China)\u0026nbsp;[42,43]. Influenza B virus stains (IBV/S9-MD) was rescued according to the sequence of B/Yamagata/16/88 (GenBank accession: CY018765-CY018772) and passaged in mice to get mouse-adapted strains. All experiments with influenza viruses were performed in a biosafety level 2 (BSL-2) laboratory in Changchun Veterinary Research Institute.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;Madin-Darby canine kidney cell line\u0026nbsp;(MDCK),\u0026nbsp;the human type-II alveolar epithelial cell line (A549), and the vascular smooth muscle cells (VSMCs) were cultured in\u0026nbsp;Dulbecco’s modified Eagle’s medium\u0026nbsp;(DMEM) supplemented with 10 % fetal bovine serum (FBS, Gibco, 10091148) with 100\u0026nbsp;U/mL penicillin and 100\u0026nbsp;μg/mL streptomycin.\u0026nbsp;All cells were cultured at 37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e in humidified air.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVirus infection and drug\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einhibitory efficacy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll virus strains were passaged and titrated on cells at the designated multiplicity of infection (MOI). Three different infection regiments were used to evaluate the active stages of taurolidine against influenza viruses, such as pre-treatment (virus was added to the cells after 15 min of co-incubation with taurolidine), co-treatment (virus was added to the cells after 15 min of co-incubation with taurolidine), and post-treatment (taurolidine was added to the cells for 4 h treatment after virus infection). Taurolidine was solubilized in PBS at a concentration of 4 mg/ml and a series of dilute solutions of taurolidine (0, 5, 10, 25, 50, 100 and 200 μg/mL) was added at 4 hours post-infection. We refer to the previous method to perform drug inhibitory efficacy \u003cem\u003ein vivo\u003c/em\u003e [44].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlaque, hemagglutination (HA) test and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDCK cells were infected with 40 PFU/well of influenza virus. Following adsorption of the virus for 2 h, the inoculum was removed, and the cells were overlaid with DMEM containing 1.5 % agarose with serial dilutions of PG. After 72 h, they were stained and images were captured.\u0026nbsp;For hemagglutination\u0026nbsp;experiment, the MDCK cells infected with influenza virus were divided into different groups, including DMEM-treated group (0 μg/mL),\u0026nbsp;taurolidine-treated groups (5 μg/mL, 25 μg/mL, 50 μg/mL) and oseltamivir-treated groups (5 μg/mL, 25 μg/mL, 50 μg/mL). The cell supernatant (50 μL) of each group was respectively collected and added to the 96-well micro-hemagglutination plate after 48 h of treatment. Subsequently, 50 μL of 1% chicken red blood cell (RBC) suspension was added to each well. After standing at room temperature for 15 min, the results were observed. Cytotoxicity was assayed by MTT assay (Promega). The cells were plated in 96-well plates (48000 cells per well) to culture 48h, and then the cells growth was detected according to the instructions of manufacturer. Briefly, 10µL\u0026nbsp;of MTT solution was added to each well, and each well was measured Spectro-photometrically at 570 nm after incubating for 4h. We conducted three independent cell experiments, and three replicate wells were set for each treatment group in each experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence and immunoblotting analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were washed three times with phosphate buffer saline (PBS) before 4% paraformaldehyde fixation (30~60min). They were then drilled with 0.2 % triton X-100. Before primary antibodies incubation (overnight at 4°C), the cells were blocked with 2% bovine serum albumin (BSA) for 1h. They were incubated with secondary antibody after washing. The nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI, 10~20min). For immunoblotting analysis, cells or tissues were lysed with radio immunoprecipitation assay (RIPA) buffer. The protein concentration was measured by bicinchoninic acid protein quantitative kit. Then isolated protein lysates\u0026nbsp;were separated by SDS-PAGE and transferred onto PVDF membranes. This was followed by blocking with 5% skin milk in Tris-buffered saline with Tween-20 (TBST) before incubation with the indicated antibodies 1h. Then, these membranes were incubated with secondary antibody (Protein, CA, USA) for 1h. The membranes were washed 3 times in TBST, for 5min each time. Subsequently, membranes were treated for 2min with reagent from an Easysee Western Blot Kit. The results were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD). We conducted three independent cell experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice infection\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and quantitative\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eReal-Time PCR (qRT-PCR) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALB / c mice were divided into four different groups, with 13 mice in each group, including control group (Control), virus infection group (Virus), virus + taurolidine treatment group (Virus + taurolidine, i.p.), and virus + oseltamivir treatment group (Virus + oseltamivir, p.o.). Each group conducted two independent experiments. The influenza viruses (10-fold mouse median lethal dose, 10×mLD\u003csub\u003e50\u003c/sub\u003e) were inoculated intranasally (i.n.) to the mice in virus-infected group,\u0026nbsp;taurolidine-treated\u0026nbsp;group, and\u0026nbsp;oseltamivir-treated\u0026nbsp;group. Taurolidine\u0026nbsp;(400 mg/kg/d) and\u0026nbsp;oseltamivir\u0026nbsp;(25 mg/kg/d) were administrated twice daily (morning and evening), respectively. The body weight and the survival status of mice were recorded daily for two weeks. The lung index is calculated according to the previous method\u0026nbsp;[42-44].\u0026nbsp;The treatment of all mice was in accordance with the welfare and ethical guidance of Chinese laboratory animals (GB 14925-2001). The agreement was approved by the Animal Welfare and Ethics Committee of the Institute of Chinese Academy of Agricultural Sciences (permit number: SCXK-2012-017).For\u0026nbsp;cytokine transcriptome analysis,\u0026nbsp;total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcription was carried out using ImPromp-II reverse transcriptase (Promega). Semi-quantitative PCR using Ampli Taq polymerase (Applied Biosystems) was performed by including [α-32P]-dCTP in the reactions. The IAV-specific primer sets anneal, and their sequences are listed in\u0026nbsp;\u003cstrong\u003eTable S1\u003c/strong\u003eand\u003cstrong\u003eTableS2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePulmonary pathology and hematoxylin-eosin staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice were euthanatized and the lung tissues were quickly placed in polyoxymethylene solution (4% PFA) to fix for 24~72 h. And then, they were embedded in paraffin and randomly cut into 4-8μm slices. After that, the slices were made transparency with xylene and soaked in absolute ethanol for 5-10 minutes. Finally, the slices were stained with hematoxylin-eosin (H\u0026amp;E), dried and observed with an optical microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunohistochemistry (IHC) assay is calculated according to the previous method\u0026nbsp;[42]. The pathological severity score of the infected mice was based on the percentage of the inflammation area of each slice collected from each animal, and using the following scoring system: 0, no pathological changes; 1, affected area ≤ 10%; 2, affected area \u0026lt;50% and \u0026gt;10%; 3, The affected area is ≥50%. When inflammation, hemorrhage and bronchial epithelial necrosis were observed, the score will be increased by one point.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral blood of mice in each group was collected into\u0026nbsp;EDTA-anticoagulation tubes. They were mixed with red-blood-cell lysis buffer before centrifugation (3000 rpm, 4 °C, 10 min). They were then incubated with specific antibodies at low temperature for 2 h without light. Antibodies included APC conjugated CD3 (152306, biolgend, USA), FITC conjugated CD4 (100510, biolgend, USA), PE conjugated CD8 (100708, biolgend, USA), PE conjugated CD16 (158004, biolgend, USA), FITC conjugated CD49b (108905, biolgend, USA), APC conjugated CD163 (156705, biolgend, USA), APC conjugated CD (117310, biolgend, USA).\u0026nbsp;Subsequently, the analysis was performed using the BECKMAN CytoFLEX flowmeter (BECKMAN COULTER, USA).\u0026nbsp;The results were visualized using CytExpert 2.3 software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor ELISA assay, the serum samples of mice in each group were collected randomly. Cytokines (IL1β, IL-6, TNF-α, IFN-γ) were then detected using a custom Mouse cytokine 10-plex kit using V-PLEX (K15048D, MSD, USA) according to the manufacturer's instructions. Discovery Workbench software (v4.0, MSD Corporation, USA) was used for data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTr\u003c/strong\u003e\u003cstrong\u003eanscription sequencing,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;blood pressure and ultrasound measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcription\u0026nbsp;sequencing was performed by MAGIGENE Technology (Shenzhen, China). Sequencing libraries were constructed using Illumina sequencing platform (Illumina, San Diego, CA). the DESeq method was used to analyze the differential expression of mice in each group. Kyoto Encyclopedia of Genes and Genomes (KEGG)\u0026nbsp;was used for signaling pathway enrichment analysis.\u0026nbsp;The bubble maps were visualized by the R clusterProfiler package (version 4.0.3). For blood pressure and ultrasound measurements, pulmonary artery blood pressure, including systolic blood pressure (SBP) and diastolic blood pressure (DBP), was measured at the 0, 3, 5, 7, 10 and 14 days, respectively. Mean arterial pressure (MAP) = DBP + (1/3×SBP). In brief, mice were anesthetized before pulse wave velocity (PWV) and pulmonary aorta thickness were measured using an ultrasound instrument (FUJIFILM VisualSonics, Toronto, Canada). The visual image processing using Vevo®LAB software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCa\u003csup\u003e2+\u003c/sup\u003e measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were treated with or without taurolidine at the designated time after virus-inoculated. For mouse serum samples, serums collected from blood samples of mice were utilized for subsequent determination. Subsequently, the cell suspension and mouse serum were harvested for Ca\u003csup\u003e2+\u003c/sup\u003e determination performed by the kit from Genmed Scientifics Inc. U.S.A (GMS50097.1 v.A). Simply, mixing 100 µL of the test solution (cell suspension or serum) with the reaction solution, incubate at room temperature in the dark for 5 minutes, and read with a spectro-photometer. The\u0026nbsp;corresponding Ca\u003csup\u003e2+\u003c/sup\u003e concentration (mmol/L) of the samples were calculated according to the construction of the standard curve.\u0026nbsp;The cultured cells were washed and incubated the Fluo-4 following AM detection kit (F14202, Invitrogen) before data collection using FDSS/µCELL (Hamamatsu Photonics, C13299, Japan). In calcium imaging analysis, incubated Fluo-4 cells were loaded with fluorescent probe. Subsequently, the calcium imaging was performed using fluorescence microscope (LEICA, DMi8, Italy) after the cells were washed with PBS. We conducted three independent cell experiments, and three replicate wells were set for each treatment group in each experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollagen gel-based contraction assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContraction of HUVEC cells was evaluated by collagen gel-based assay according to the method of SakotaY with minor modifications\u0026nbsp;[45]. The cells were suspended in Dulbecco's phosphate buffer solution (DPBS) supplemented with type-I collagen, seeded into a 12-well microplate at a density of 1 × 10\u003csup\u003e5\u003c/sup\u003ecells/mL and then incubated for 30 min at 37°C in a CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eincubator for gelation. After addition of the culture medium (equivalent volume of the gel) onto the gel, the embedded cells were cultured for 2 days with changing the medium every day. The gel was detached from the well with a 10µl pipette tip, immersed in the assay medium containing agent for treatment, and then photographed using a digital camera. Contraction was expressed as a percentage of area of the gel to that before treatment. We conducted three independent cell experiments, and three replicate wells were set for each treatment group in each experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood\u0026nbsp;of mice in each group\u0026nbsp;were collected in\u0026nbsp;sterile\u0026nbsp;EDTA-anticoagulation tubes. After that, they were analyzed by using an automatic hematology analyzer (Mindray Medical, BC-5000, China). Then, hematological indicators were observed and recorded, such as white blood cell counts (WBC), red blood cell counts (RBC), and platelet counts (PLT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical comparisons were performed using Graphpad Prism 8.0 software. T-test was used to compare the differences between the two groups. For comparisons between three or more groups, ANOVA analysis was performed. Quantitative data-sets were presented as the means ± standard deviation (SD). The p values less than 0.05 (p\u0026lt;0.05) was regarded as statistically significant, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are especially grateful to Wu Zhong who works in the Military Medical Research Institute for kindly providing the technical guidance, and experimental assistance provided by Changchun Veterinary Research Institute (Biosafety Level III laboratory).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Military Medical Research Institute Foundation project (grant number: AMMS-KYJJ-2022-014), the National Key Research and Development Program of China (2020ZX10001-016-003), and the National Key Research and Development Program of China (ZX10304402-003-006). This work was supported by a grant from National Key R\u0026amp;D Program of China (No. 2021YFC2301700) and the National Key Research and Development Program of China (No. 2020YFC0846100, 2023YFC0871000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe treatment of all mice was in accordance with the welfare and ethical guidance of Chinese laboratory animals (GB 14925-2001). The agreement was approved by the Animal Welfare and Ethics Committee of the Institute of Chinese Academy of Agricultural Sciences (permit number: SCXK-2012-017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to submit and publish this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGarc\u0026iacute;a-Sastre A. Influenza virus receptor specificity: disease and transmission. The American journal of pathology. 2010;176(4):1584-5.\u003c/li\u003e\n\u003cli\u003eTeijaro J.R., et al. 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Biological \u0026amp; pharmaceutical bulletin. 2014;37(6):1014-20.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Taurolidine, vasoconstriction, influenza virus, calcium influx, MLCK/p-MLC signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-4778710/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4778710/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfluenza virus causes worldwide outbreaks and seasonal epidemics, severely threatening public health and social development. Effective prevention and therapy for influenza infections are a major challenge to global healthcare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTaurolidine effectively inhibited the proliferation of several human or animal influenza virus strains and protected mice from lethal-infection. Taurolidine treatment decreased the viral titer in the lungs of infected mice, reduced immune cells infiltration, and alleviated lung pathology. Additionally, influenza virus infection increased blood pressure, pulse wave velocity, and pulmonary aortic thickness in mouse model, as well as promoted the increase of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration and pulmonary artery vasoconstriction. These effects were attenuated by taurolidine treatment through inhibiting the activation of the MLCK/p-MLC pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings confirm the effectiveness of taurolidine as an antiviral agent and highlight its important roles in host immune cell infiltration and vasoconstriction induced by influenza virus infection.\u003c/p\u003e","manuscriptTitle":"Taurolidine resists pulmonary artery vasoconstriction induced by influenza virus infection via inhibiting Ca 2+ influx and MLCK/p-MLC signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-16 06:24:09","doi":"10.21203/rs.3.rs-4778710/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f7dcd71-cd6e-4b68-8059-a6b7a215c801","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-19T14:37:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-16 06:24:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4778710","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4778710","identity":"rs-4778710","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}