Seabuckthorn Berries Attenuate Pulmonary Vascular Hyperpermeability in Lipopolysaccharide-induced Acute Lung Injury in Mice

Background Acute lung injury is featured by pulmonary vascular hyperpermeability, resulting in high short-term mortality. Currently pharmacological therapies are still sparse. Methods In the mice model of acute lung injury induced by Lipopolysaccharide, the effect of seabuckthorn berries extract on pulmonary vascular hyperpermeability was evaluated by histopathologic observation and transvascular leakage determination. The key factors involved in alveolar-capillary barrier lesion were assessed. Results The ndings indicated that treatment of seabuckthorn berries alleviated morphological lesion as well as water, Evans blue and total proteins leakage in lung tissue, suppressed the release of TNF-α and IL-6, decreased accumulation of neutrophils, inhibited the activation of NF-κB and down-regulated the expression of ICAM-1 and CD62E. Conclusions These results demonstrated seabuckthorn berries help maintaining alveolar-capillary barrier integrity under endotoxin challenge in mice by suppressing the key factors in the pathogenesis of acute lung injury.


Background
Acute lung injury (ALI) is a clinical syndrome characterized by progressive hypoxemia and respiratory distress, caused by diverse endogenous and exogenous factors that injure lung directly or indirectly. ALI is the source of substantial morbidity and mortality in both adult [1,2] and pediatric [3,4] populations and is a major contributor to intensive care unit (ICU) costs [5]. Sepsis, induced by severe infection of bacteria, virus or fungus, is one of the leading etiologies of ALI [6]. It may cause sequencial functional disorders to multiple organs, among which ALI occurs at the earliest stage and with the highest morbidity [7,8]. Long term clinical observations and experiments have demonstrated that ALI is the distinct manifestation of systemic in ammatory response syndrome (SIRS) in lung. Severe in ammatory response and protein-rich edema uids induced by vascular endothelial injury are presented through the entire process [9]. Under pathogen stress, monocyte/macrophage system is stimulated through Nuclear Factor-kappa B (NF-κB) signal pathway to release pro-in ammatory cytokines such as Tumor Necrosis Factor alpha (TNF-α) and Interleukin-6 (IL-6), which in turn mediate adhesion and combination of neutrophils to endothelial cells in pulmonary microvessels by regulation of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin (CD62E) [10,11]. Activation of neutrophils results in the release of mediators (e.g., oxidants and proteases) that increase vascular permeability by disrupting interendothelial junctions, thereby intravascular uid and macromolecules permeate into alveoli and cause edema formation in pulmonary tissue.
Pharmacological therapies are still sparse for ALI, and supportive treatment of low tidal volume ventilation is the main strategy in clinical practice [12]. However, mechanical tension generated by ventilation itself may introduce in ammatory response and cause further injury [13]. Therefore, searching for alternative therapeutic strategy is of substantial interests.
Seabuckthorn (Hippophae rhamnoides L., Elaegnaceae) berries have been used to treat lung diseases in traditional Tibetan medicine for a long history. Modern research revealed that the berries contain various constituents, including vitamins, fatty acids, free amino acids, carotenoids and phenolic compounds, and present remarkable anti-oxidative and anti-in ammatory activities [14]. In the current work, we investigated the effect of seabuckthorn berries on Lipopolysaccharide (LPS)-induced ALI in mice and explored the possible mechanism.

Plant material and extract preparation
Well-ripened seabuckthorn berries were collected from natural growth site of hilly region in eastern margin The berries were re uxed with deionized water and the supernatant was concentrated under vacuum to yield seabuckthorn berries extract (SBE). We analyzed chemical constituents and established HPLC chromatographic ngerprint spectrum of seabuckthorn berries in previous work [15].

Animals
Since the most common cause of ALI in humans is sepsis, the administration of gram-negative bacteria endotoxin, lipopolysaccharide, has been widely used as an animal model of sepsis-related lung injury in several species [16]. The present study was performed in 6 batches of male KM mice each weighing 18 22 g maintained at 24 ± 0.5℃ with food and water ad libitum. The experimental was approved by the Institute's animal ethical committee and con rmed to national guidelines on the use and care of laboratory animals.
After acclimatization for 2 days, mice were randomly allocated into control group, LPS group and three SBE groups of different dose levels (120, 240 and 480 mg/kg•bw, respectively). Animals in each group received respective treatment (corresponding doses of SBE for SBE groups and saline for control group) once daily through intragastric route for seven consecutive days. On Day 8, mice were administered saline for control group and LPS (O55:B5) for other groups by intraperitoneal injection at a dose of 10 mg/kg. Animals were sacri ced 10h after injection.

Gross and histopathologic observation
After the animal was sacri ced, median sternotomy was performed to expose trachea and pleuroperitoneal cavity, so that lung was excised for visual inspection. Then the lung was xed with an intratracheal instillation of 1 ml buffered formalin (10 %, pH 7.2). The lobe was further xed in 10 % neutral buffered formalin for 48 h at 4 °C. The tissues were embedded in para n wax. Sections approximately 5 μm thick were stained with hematoxylin and eosin using a standard protocol and observed under the light microscope for histopathological changes such as alveolar septum lesion, in ammatory cell in ltration, blood stasis, etc.

Lung water content determination
Wet-to-dry weight ratio was used as an index to estimate the degree of pulmonary edema. After the animal was sacri ced, lungs were excised enbloc, blot dried and placed on pre-weighed glass plates. The wet weight of the tissue was registered immediately. Then the tissue was placed in an incubator at 80 °C for 72 h to obtain a constant weight. After the dry weight of the tissue was registered, the water content of the tissue was calculated as wet weight/dry weight ratio (W/D).

Transvascular leakage analysis
In order to evaluate LPS-induced lung vascular leak, 1% Evans blue dye in saline (10 ml/kg, Sigma, USA) was injected into the tail vein one hour before termination of the experiment. Measurement of Evans blue accumulation in the lung tissue was performed by spectro uorimetric analysis of lung tissue lysates according to the protocol described previously [17,18].
Protein content in bronchoalveolar lavage uid (BALF) re ects macromolecule leakage through impaired endothelia barrier. To analyze BALF, animal's trachea was exposed and an intravenous infusion needle was inserted. The lungs were lavaged three times with 0.5 ml of ice-cold phosphate-buffered saline.
Returned lavage uid was pooled for each animal and centrifuged at 800×g for 5 min at 4°C. The supernatants were harvested for total protein analysis using BCA protein assay kit and the sediments were collected for neutrophils count under light microscope on cytospin slides stained with Wright's solution.

Inflammatory cytokines assay
To evaluate in ammatory response, blood samples from the abdominal aorta of mice were collected before the animals were sacri ced, followed by incubation for 1 h under 37℃ and centrifugation for 5 min at 1500×g. Serum cytokines TNF-α and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.

Immunofluorescent analysis
The transcription factor NF-κB serves as a pivotal mediator of in ammatory response. Activation and nuclear translocation of NF-κB induces various pro-in ammatory cytokines. The expression of NF-κB p65 in lung cells was determined by immuno uorescence technique. Sections of lung tissue were depara nized and rehydrated through submersion in graded alcohols. Antigen retrieval was performed with 10 mM citrate buffer pH 6, for 5 min in a microwave oven. The sections were incubated with a primary antibody against NF-κB p65 (1:400), followed by detection with a fluorescein-conjugated secondary antibody (1:100). The nuclei were counterstained with 2-(4-amidinophenyl)-6indolecarbamidine dihydrochloride (DAPI). The uorescent images were captured using appropriate lters in a Nikon inverted uorescent microscope (Tokoyo, Japan), and IOD (Integrated optical density) and PPA (Percent of positive area) for photomicrographs were calculated using image processing software Image-Pro Plus 6.0 (Media Cybernetics, USA).

Western blot
To evaluate the expression of proteins relative to NF-κB signal pathway, the contents of cytoplasmic Inhibitor of Nuclear Factor-κB Kinase (IKK) and nuclear P65, as well as downstream CD62E and ICAM-1, were determined using western blot technology. Lung tissue homogenate were centrifugation (12,000×g, 10 min, 4 °C) and supernatants were aspirated. Biochemical fractionation of the cells was done using the nuclear extract kit according to the manufacturer's instructions. Proteins were loaded and transferred to a PVDF membrane. After being blocked, membranes were incubated overnight at 4°C with a primary antibody, followed by incubation with secondary antibody for 1 h at room temperature. The membranes were placed into a gel imaging system (Bio-Rad, ChemiDoc XRS, USA) and then exposed. The intensity of blots was quanti ed using the Quantity One Analysis software (Bio-Rad, USA).

Statistical analysis
Data were expressed as mean ± standard deviation. All statistical analysis was performed with Prism 8 software (GraphPad Software, CA, USA). Statistically signi cant differences between groups were determined by ANOVA followed by Tukey's test. Results were considered statistically signi cant if P values were <0.05.

Results
Morphological Changes Figure 1 shows the morphological differences between the lungs of animals from Control, Model and SBE groups. Compared with healthy tissue of Control group, LPS stimulation caused obvious lesion in lungs of model animals, including foamy mucus in some tracheas, enlarged lobes, darker in color with scattered petechiae, and incisions exudation. While SBE treatment provided protection to a certain degree, with alleviated edematous lesion and fewer petechiae observed.

Effects of SBE on vascular permeability
The stimulation of LPS caused edema in lung tissue, re ected in a remarkable increase of Wet-to-dry weight ratio (5.13 ± 0.19 vs. 4.69 ± 0.22). Pretreatment with SBE reversed the increase in a dosedependent manner, with signi cant difference in SBE 480mg/kg group (Figure 2a). The result indicated that SBE alleviated edema in lung tissue induced by LPS.
Evans blue accumulation in lung tissue through transvascular leakage was remarkable higher in LPS group than that in control group (4.58 ± 0.97 vs. 1.68 ± 0.47 μg/g). Protein concentration and neutrophils count in BALF both sharply increased in LPS group, compared to those in control group (1.98 ± 0.43 vs. 0.60 ± 0.37 μg/g, 20.04 ± 5.02 vs. 0.63 ± 0.74, respectively). These results suggested that LPS induced the increase in vascular permeability, resulting in transvascular leakage of dye and proteins. Pretreatment of SBE alleviated the endothelia barrier lesion in a dose-dependent manner, with both 240 and 480 mg/kg groups showing statistic signi cance (Figure 2a, 2b and 2c).

Effects of SBE on inflammatory cytokines
Cytokines TNF-α and IL-6 levels in serum were higher in LPS group than those in control group (489.71 ± 118.99 vs. 279.48 ± 105.90 pg/mL, 5.50 ± 0.16 vs. 4.78 ± 0.11 ng/mL, respectively), which suggested that LPS induced in ammatory response in mice. In SBE treatment groups, TNF-α and IL-6 levels both decreased compared to LPS group, with statistic signi cances in 480 mg/kg group for TNF-α and all three dose groups for IL-6 ( Figure 3).

Effects of SBE on NF-κB activation
The immuno uorescence images showed enrichment of NF-κB P65 protein both in the nuclear and cytoplasmic fraction of lung cells in LPS group, with green uorescence for NF-κB P65 protein and blue uorescence for nuclear. It suggested the increased expression and nuclear translocation of NF-κB upon LPS stimulation. In SBE 480 mg/kg group, uorescence intensity appeared weaker than that of LPS group. Furthermore, IOD and PPA assays both indicated SBE treatment reversed the increased immuno uorescence intensity induced by LPS, with statistic signi cance in 480 mg/kg group (Figure 4).
The results of western blot showed that low basal expression levels of cytoplasmic IKK and nuclear P65 increased signi cantly upon LPS stimulation (0.41 ± 0.02 vs. 0.11 ± 0.02 and 0.50± 0.03 vs. 0.09 ± 0.02, respectively), suggesting the release and nuclear import of NF-κB. By contrast, the expression of IKK and P65 decreased in a dose-dependent manner in SBE groups with statistic signi cance at each dose level ( Figure 5). Similarly, treatment of SBE reserved LPS-induced increased expression of two downstream proteins of NF-κB, ICAM-1 and CD62E ( Figure 6). These results demonstrated that SBE inhibited the activation of NF-κB induced by LPS.

Discussion
In the present study, we investigated the effect of seabuckthorn berries on ALI in mice, inspired by its therapeutic use in traditional Tibetan medicine for treating various pulmonary diseases and relieving hypoxic respiratory distress. Since sepsis is the most common clinical setting in which ALI develops and bacterial endotoxin is implicated as an important toxin precipitating lung injury, the widely-accepted sepsis-related lung injury model by LPS administration was used. Anotomical ndings revealed severe pulmonary tissue injuries induced by LPS, including foamy mucus and scattered petechiae, were alleviated to some extent by SBE treatment.
Airway vascular endothelial injury is a major pathological feature of ALI. Endotoxin induces in ammatory response, with accumulation of in ammatory mediators in lung tissue, causing alveolar-capillary barrier lesion, which is associated with increased vascular permeability and accumulation of protein-rich interstitial and alveolar uid. We determined lung water content by wet-to-dry weight ratio, which increased obviously in LPS group, indicating pulmonary edema in model animals. Albumin leakage was determined by Evans blue assay. Intravenously administrated Evans blue binds to serum albumin with high a nity and serves as a probe to trace albumin leakage [19]. Elevated level of Evans blue concentration in lung tissue of LPS group showed pulmonary vascular hyperpermeability. This is veri ed by increased total protein concentration in BALF of LPS group. These ndings demonstrated the integrity of alveolar-capillary barrier was impaired in LPS-induced ALI mice, consistent with other reports [12,16,17]. By contrast, data in SBE groups showed alleviation of transvascular leakage in a dose-dependent manner. These results suggest that seabuckthorn berries can protect alveolar-capillary barrier integrity upon endotoxin challenge.
Activation of neutrophils sequestered in pulmonary microvessels is an important factor in the pathogenesis of increased lung vascular permeability and tissue injury [20]. We found in this study that elevated level of neutrophils in BALF induced by LPS was reversed by SBE treatment, which is consistent with the endothelial permeability results.
In ammation is associated with the pathological process of ALI. Pro-in ammatory cytokines TNF-α and IL-6 have been strongly implicated in the pathogenesis of ALI in human and animal models [21]. Our results con rmed that SBE curtailed TNF-α and IL-6 release induced by LPS, showing signi cant antiin ammatory activity.
Since NF-κB signal pathway plays a key role in in ammatory response, we assume it may be the effect target of SBE. In a quiescent state, NF-κB dimers are anchored in the cytoplasm by inhibitor IκB. When activated by signals, IKK degrades IκB through phosphorylation and ubiquitination, and NF-κB is then freed to enter the nucleus where it can turn on the expression of relative genes. We investigated NF-κB expression in lung tissue by immuno uorescent analysis, as well as cytoplasmic IKK and nuclear NF-κB P65 expression by Western blot. The results demonstrated that SBE suppressed the expression of IKK and the nuclear translocation of NF-κB stimulated by LPS. The downstream proteins of NF-κB relative to neutrophil activation include ICAM-1 and CD62E, which mediate neutrophil interaction with endothelial cells. We also evaluated the expression levels of the two proteins and con rmed the similar suppression by SBE. These ndings provide consistent evidences supporting the inhibition activity of SBE on NF-κB signal pathway.
The notable alveolar-capillary barrier protection and anti-in ammtory activity of seabuckthorn berries may attribute to multiple constituents. The main active ingredients include avonoids, a kind of natural anti-oxidative and anti-in ammtory agents. Total avonoids from seabuckthorn have been used in treating cardiovascular disease. Also, the high content of vitamins (B, C, E and K) makes seabuckthorn a popular nutritional supplement. Oil from seabukchtorn berries containing fatty acids and carotenoids facilitates the wound healing in burning and ulcer. Therefore, SBE may serve as a natural complex preparation to provide bene cial effect in ALI mice. The speci c contribution for individual component merits further investigation.

Conclusion
In the present research, we demonstrated that seabuckthorn berries can protect alveolar-capillary barrier from hyperpermeability in LPS-induced ALI mice by suppressing the key factors in the pathogenesis of ALI, including the release of cytokines TNF-α and IL-6, the activation of NF-κB signal pathway, the expression of ICAM-1 and CD62E, and the adhesion of neutrophil to endothelial cell. The effective constituents need further research and development, in order to provide a supplemental preventive and therapeutic strategy for ALI.

Declarations
Ethics approval and consent to participate Not applicable.

Consent to publication
Not applicable.

Availability of data and materials
The research data generated from this study is included within the article.

Competing interests
The authors declare that they do not have any con icts of interest.  Effects of SBE on vascular permeability, evaluated by (a) wet-to-dry ratio, (b) pulmonary vascular leakage, and (c) total protein and (d) neutrophils concentration in BALF. The results were presented as mean ± SD (n = 10). *p < 0.05 or **p < 0.01, vs. LPS group.