Protective effect of oxytocin on LPS-induced acute lung injury in mice

Oxytocin (OT), a neurohypophyseal hormone synthesized in the paraventricular and supraoptic nuclei of the hypothalamus, has been reported to have an anti- inflammatory effect. However, its role in acute lung injury (ALI) has never been investigated. The aim of this study was to explore the therapeutic effects and potential mechanism action of OT on lipopolysaccharide (LPS)-induced ALI. Mice were treated with OT 30 min before the intraperitoneal injection of LPS. After 2 h, the effects of OT on lung histopathological changes, lung wet/dry (W/D) ratio, myeloperoxidase (MPO) activity, levels of inflammatory cytokines in the bronchoalveolar lavage fluid (BALF), and expression of inflammation proteins were detected. The results showed that OT significantly reduced LPS-induced pathological injury, W/D ratio, MPO activity, and the levels of interleukin (IL)-1β, IL-18 and IL-6. Further, OT also inhibited LPS-induced Toll-like receptor 4 expression and NLR family pyrin domain containing 3 inflammasome activation. OT receptor antagonist (L-368,899) was given 90 min before injecting OT to further demonstrate the role of OT in LPS-induced ALI. The results showed OT could not alleviate the aforementioned inflammatory reactions after administering L-368,899. In conclusion, the present results indicated that OT could reduce inflammatory responses of LPS-induced ALI.

Pathological changes in lung tissues were observed using HE staining (light microscopy, ×200). The histopathologic scores are presented for the lung tissues (e). The data are presented as the means ± standard error of the mean. ***P < 0.001 vs control. ## P < 0.01 vs LPS. ++ P < 0.01vs OT. Data show means with SEM for six mice/group.

Effects of OT on LPS-induced production of cytokines in BALF.
The production IL-1β, IL-18, IL-6, IL-4 and IL-10 in BALF was analyzed to further assess the anti-inflammatory effects of OT. As shown in Fig. 4, the levels of IL-1β, IL-18 and IL-6 were found to be significantly increased in the LPS group compared with the control group (P < 0.01 and P < 0.001). However, pretreatment with OT apparently decreased the levels of IL-1β, IL-18 and IL-6 in BALF compared with the LPS group (P < 0.01 and P < 0.001). Moreover, L-368,899 administration significantly increased the cytokines levels compared with the OT group (P < 0.01 and P < 0.001). As shown in Fig. 4, IL-4 and IL-10 levels were found to be significantly decreased in the LPS group compared with the control group (P < 0.01, P < 0.001). OT significantly increased IL-4 and IL-10 production compared with the LPS group (P < 0.01 and P < 0.001, respectively). L-368,899 administration significantly decreased IL-4 and IL-10 levels compared with the OT group (P < 0.01 and P < 0.001, respectively).

Effects of OT on LPS-induced gene expression and protein level of cytokines in the lung tissues.
The cytokine gene expression in the lung tissues was determined using qPCR. The expression levels of IL-1β, IL-18, IL-6, IL-4, and IL-10 ( Fig. 5a-e) were in agreement with the cytokines levels in the BALF (Fig. 4). Also, the proteins levels of IL-1β and IL-6 were analyzed using the Western blot analysis ( Fig. 5f-h). As shown in Fig. 5f-h, the result was consistent with the cytokines levels evaluated using qPCR.
Effect of OT on LPS-induced OTR expression. OTR expression was detected in this study to investigate the mechanism underlying the anti-inflammatory effect of OT. As shown in Fig. 6, the protein level of OTR was increased compared with the control group. However, pretreatment with OT significantly decreased the protein expression compared with the LPS group. Moreover, L-368,899 administration significantly increased the protein expression compared with OT group. In addition, the expression of OTR was increased on macrophage in the LPS-induced ALI compared with control group (shown in Supplementary Information Fig. 2).

Effect of OT on LPS-induced TLR4 and NF-κB expression.
In this study, the expression of TLR4 and NF-κB was detected using Western blot analysis, immunohistochemistry (IHC), and immunofluorescence (IF). As shown in Fig. 7, LPS significantly increased TLR4 and NF-κB expression compared with the control group. However, pretreatment with OT significantly decreased the protein expression compared with the LPS group. Moreover, L-368,899 administration significantly increased the protein expression compared with the OT group.
Effect of OT on LPS-induced NLRP3 and caspase-1 expression. The activation of NLRP3 inflammasome was detected in this study to further investigate the mechanism underlying the anti-inflammatory effect of OT. As shown in Fig. 8, LPS significantly increased NLRP3 and caspase-1 expression compared with the control group. However, pretreatment with OT significantly decreased the protein expression compared with the LPS group. Moreover, L-368,899 administration significantly increased the protein expression compared with the OT group.
The level of OT in the lung tissue. In the study, the level of OT in the lung tissue was measured using ELISA. As shown in Fig. 9, LPS significantly deceased OT level compared with control group. However, pretreatment with L-368,899 significantly increased OT level compared with the LPS group and the LPS + OT group. The effect of L-368,899 on inflammatory response. For further illustration the mechanism underlying the anti-inflammatory effect of OT, a better comparator group of LPS + L-368,899 was measured. As shown in Fig. 10, L-368,899 significantly exacerbates inflammation compared LPS group. These results suggested that oxytocin's anti-inflammatory effects were possible due to its binding to OTR, because the anti-inflammatory effects on LPS-induced ALI were effectively blocked by the OTR antagonist L-368,899.

Discussion
ALI has high morbidity and mortality among patients, with no effective drugs available in the clinic 16,17 . LPS-induced ALI is characterized by the release of inflammatory cytokines and expression of inflammatory proteins in the lung tissue. Previous studies demonstrated that the OTR gene contained response elements for acute-phase reactants and ILs 18,19 , and the expression of OTR was increased in LPS-activated macrophage 10 . OT is also involved in the modulation of immune and inflammatory response. Specifically, OT could decrease the release of IL-1β and IL-6 in vitro 20 and in vivo 21,22 . Moreover, OT could also regulate the immune response by www.nature.com/scientificreports www.nature.com/scientificreports/ increasing levels of anti-inflammatory cytokines, such as IL-4 and IL-10 22 . These data indicated that OT might be a potential therapeutic agent for alleviating LPS-induced ALI.
The results of the present study showed that OT could attenuate LPS-induced ALI in mice. The study also found that OT inhibited the production of inflammatory cytokines in BALF. Besides, Western blot analysis, IHC, and IF all showed that OT significantly inhibited NLRP3 and NF-κB expression. Moreover, the upregulation of expression OTR on the alveolar macrophages (shown in Supplementary Information Fig. 2) may contribute to the anti-inflammatory properties of OT. But compared to control group, endogenous OT level decreased in response to LPS, it is likely that the anti-inflammatory effects mostly were from exogenous OT. These results indicated the positive effects of exogenous OT on LPS-induced ALI.
In ALI, neutrophils are the earliest immune cells recruited to the site of injury, which generate cytotoxic products 23 . A previous study showed that the elimination of neutrophils could relieve the severity of ALI 3 . MPO activity, a marker of neutrophil influx into tissues, is measured for quantifying neutrophil accumulation in tissues 24 . This study found that OT significantly inhibited MPO activity. In addition, LPS-induced lung histopathological injury was also inhibited by OT. Pulmonary edema is a characteristic of ALI. The lung W/D ratio was measured to assess the magnitude of pulmonary edema. The study showed that OT significantly decreased the lung W/D ratio, indicating a significant inhibition of edema in lung tissue. However, L-368,899 could obviously weaken these effects of OT to increase MPO activity and W/D ratio, and increased the level of OTR and OT. Meanwhile, our experiment showed that L-368,899 significantly exacerbates inflammation compared LPS group. These data suggested that intraperitoneal oxytocin's anti-inflammatory effects were possible due to its binding to OTR, because the anti-inflammatory effects on LPS-induced ALI were effectively blocked by the OTR antagonist L-368,899.
A complex network of cytokines, including IL-1β, IL-6, and IL-18, mediates the inflammatory response in LPS-induced ALI 25 . These cytokines are released mainly from LPS-stimulated monocytes and macrophages, and recruit neutrophils into lung tissues, which is vital for host defense and contributes to the development of ALI 26 . Furthermore, IL-4 and IL-10, which are potent anti-inflammatory cytokines, could suppress the activity  ACTIN (g and h). The values are presented as means ± standard error of the mean. *P < 0.05, **P < 0.01 vs control. # P < 0.05, ## P < 0.01 vs LPS. + P < 0.05, ++ P < 0.01 vs OT. Data show means with SEM for four to eight mice/group. www.nature.com/scientificreports www.nature.com/scientificreports/ of many inflammatory molecules 27,28 . Two signaling pathways are required for the maturation and secretion of IL-1β and IL-18: NF-κB and NLRP3 inflammasome 29 . NF-κB, an important transcription factor, is vital in regulating the inflammatory mediators 30 . Upon exposure to LPS, activated NF-κB could regulate the expression of pro-IL-1β and pro-IL-18. NLRP3 inflammasome, an important factor in innate immunity, regulates the maturation and secretion of IL-1β and IL-18. Activated NLRP3 inflammasome leads to the activation of caspase-1, which induces the secretion and maturation of IL-1β and IL-18 31 . In addition, previous studies showed that inhibiting the NLRP3 signaling pathway could attenuate LPS-induced ALI 31,32 . The effects of OT on NF-κB expression and NLRP3 inflammasome activation were detected to investigate the mechanism underlying the anti-inflammatory effect of OT. OT dramatically decreased the expression of these cytokines and inhibited NF-κB expression and NLRP3 inflammasome activation. In contrast, OT increased the expression of anti-inflammatory cytokines, such as IL-4 and IL-10. However, L-368,899 could obviously weaken the aforementioned effects of OT. These observations further confirmed the protective and therapeutic effects of OT combined with OTR against LPS-induced ALI.
TLR4, a pattern recognition receptor, could detect LPS from Gram-negative bacteria 33 . A previous study showed that LPS-TLR4 signaling not only activated NLRP3 inflammasome and release of IL-1β, but also upregulated IL-1RI expression through NF-κB-dependent signaling 34 . The present study showed that OT inhibited TLR4 expression; however, L-368,899 increased TLR4 expression.
In conclusion, the present study demonstrated that OT combined with OTR had protective effects against LPS-induced ALI by inhibiting the inflammatory response in mouse models of LPS-induced ALI. The mechanism might involve blocking the activation of TLR4/NLRP3/NF-κB signaling pathway. These data provided evidence supporting the idea that OT represented as a novel therapeutic strategy against ALI.
Animals. Male C57BL/6 mice (aged 6-8 weeks, weighing 20-22 g) were purchased from the Experimental Animals Center of Shandong University (Jinan, China). The experimental protocol for all mice was approved by the Medical Ethics Committee for Experimental Animals of Shandong University (Number ECAESDUSM 2012029). We also confirmed that all methods were performed in accordance with the relevant guidelines and regulations. The animals were housed in a temperature-controlled room with a 12-h day/night cycle at 25 °C, and had free access to food and water.  IF (a,b). The Western blot analysis, IHC, and IF of OTR were quantified respectively (c-e). *P < 0.05, ***P < 0.001 vs control. # P < 0.05, ## P < 0.01, ### P < 0.001 vs LPS. + P < 0.05, +++ P < 0.001 vs OT. Data show means with SEM for five mice/group. www.nature.com/scientificreports www.nature.com/scientificreports/ After adaption for a week, all mice were randomly divided into four groups (n = 9 in each group), and all drugs were injected intraperitoneally (ip): control group (90 min saline +30 min saline + saline), LPS group [90 min saline +30 min saline + LPS (10 mg/kg, 2 mg/mL)], LPS + OT group [90 min saline +30 min OT (0.1 mg/kg, 10 µg/mL) + LPS (10 mg/kg, 2 mg/mL)], and LPS + OT + L-368,899 group [90 min L-368,899 (5 mg/kg, 1 mg/mL) +30 min OT (0.1 mg/kg, 10 µg/mL) + LPS (10 mg/kg, 2 mg/mL)]. The doses of L-368,899 used in this study were based on a previous study 35 . Two hours after LPS treatment, the mice were anesthetized by an intraperitoneal (ip) injection of 50 mg/kg sodium pentobarbital. Then, the BALF and lung tissues were collected for further analyses.
Histopathological assessment with hematoxylin and eosin staining. The right upper lung was collected and fixed in 4% paraformaldehyde. Then, the tissues were embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin and eosin (HE). Finally, the pathological changes in lung tissues were observed using an optical microscope. The histological scoring parameters included edema, intra-alveolar cell infiltration, congestion and alveolar hemorrhage. The score of each item was recorded as 1 of the following 4 grades: normal(0), mild 1 , moderate 2 ; and severe 3 .

Measurement of MPO activity in BALF.
MPO activity, a biochemical marker for the infiltration of neutrophils and macrophages into the lungs, was measured using test kits purchased from Wuhan Huamei Biotechnology Institute, China. MPO activity in BALF was assayed using the MPO assay kit according to the manufacturer's protocol. The enzymatic activity was assessed at 450 nm using a Varioskan Flash multifunction plate reader (Thermo Scientific, IL, USA).
Measurement of lung wet/dry ratio. The severity of pulmonary edema was assessed by the wet/dry ratio (W/D ratio). The wet weight of right lower lungs was measured, and then the lungs were incubated in an oven (60 °C for 72 h) to obtain a dry weight. Finally, the W/D ratio was calculated using the wet and dry weights.

Measurement of cytokines levels in BALF.
Mouse BALF was used to measure the levels of inflammatory cytokines, such as IL-1β, IL-18, IL-6, IL-4, and IL-10. These cytokines were measured using ELISA kits following Figure 7. Effects of OT on the expression of NF-κB and TLR4 in the lung tissues from mice with LPS-induced ALI. The expression levels of these proteins were evaluated using the Western blot analysis, IHC, and IF (a,d,g). The Western blot analysis, IHC, and IF of OTR were quantified respectively (b,c,e,f,h,i). *P < 0.05, **P < 0.001,***P < 0.001 vs control. # P < 0.05, ## P < 0.01, ### P < 0.001 vs LPS. + P < 0.05, +++ P < 0.001 vs OT. Data show means with SEM for five mice/group. www.nature.com/scientificreports www.nature.com/scientificreports/ the manufacturer's protocol. The optical density of the microplate was read at 450 nm using a Varioskan Flash multifunction plate reader (Thermo Scientific).
Real-time quantitative polymerase chain reaction analysis. The levels of relative genes were measured by quantitative polymerase chain reaction (qPCR). The total RNA from lung tissues was extracted using kits according to the manufacturer's protocol (Aidlab Biotech, China). The cDNA was synthesized using the SYBR Green PCR Kit (Toyobo, Japan). Subsequently, the fluorescent quantification of relative genes was performed using the Bioer Real-Time qPCR System (Bioer Technology, China) with a 10-µL reaction system Figure 8. Effects of OT on the expression of NLRP3 and Caspase-1 in the lung tissues from mice with LPSinduced ALI. The expression levels of these proteins were evaluated using the Western blot analysis (A), IHC, and IF (a,d,g). The Western blot analysis, IHC, and IF of OTR were quantified respectively (b,c,e,f,h,i). **P < 0.01,***P < 0.001 vs control. # P < 0.05, ## P < 0.01, ### P < 0.001 vs LPS. + P < 0.05, ++ P < 0.01, +++ P < 0.001 vs OT. Data show means with SEM for five mice/group. www.nature.com/scientificreports www.nature.com/scientificreports/ comprising the following: cDNA, 1 µL; forward primers, 0.1 µL; reverse primers, 0.1 µL; mix (TaKaRa, Japan), µL; and nuclease-free water, 3.8 µL. The specific sequences of primers used in the present study for gene amplification are shown in Table 1. The primers were obtained from Beijing Genomics Institute (Beijing, China). The cycling conditions were as follows: 95 °C for 30 s, 95 °C for 10 s, 56 °C for 10 s, and 72 °C for 30 s. The expression levels of targeting mRNA levels were normalized to actin.
Measurement of OT levels in lung tissue. The total protein of lung tissue was extracted and concentrations were assessed in the same manner as described as Western blot. OT was measured using ELISA kits following the manufacturer's protocol. The optical density of the microplate was read at 450 nm using a Varioskan Flash multifunction plate reader (Thermo Scientific).
Statistical analysis. Student's t-test was performed for paired samples. For data that were not normally distributed (such as cytokines), multiple comparisons were carried out using Kruskal-Wallis test. All results were presented as the means ± standard error of the mean and analyzed using Sigmaplot software (version 20.0 for Windows; SPSS Inc. IL, USA). A P value < 0.05 was considered to indicate a statistically significant difference.

Data Availability
The data set supporting the results of this article are included within the article.