Echinacoside attenuates lipopolysaccharide-induced acute lung injury in newborn mice via inactivation of NF- κB/NLRP3 signaling pathway

Purpose: To investigate the effect of echinacoside (ECH) on acute lung injury (ALI) and the underlying mechanism of action. Methods: The ALI model was established through intranasal instillation of lipopolysaccharide (LPS). Lung tissue damage was determined using hematoxylin and eosin (H&E) staining and lung wet-to-dry– weight ratio. Bronchoalveolar lavage fluid (BALF) protein concentration, cell count, and cytokine level were evaluated. Western blotting was used to determine protein expression level. Results: ECH attenuated lung tissue injury and lung wet-to-dry–weight ratio in the ALI model (p < 0.01). The total protein content and number of total cells, neutrophils, and macrophages increased in BALF of mice treated with LPS, but these increases were reversed by ECH treatment (p < 0.01). The levels of TNF-α and IL-1β increased in BALF and lung tissue of LPS-treated mice; however, ECH treatment decreased these changes (p < 0.01). In addition, ECH inhibited the activation of the nuclear factor-κB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) pathway in LPS-treated mice (p < 0.01). Conclusion: Echinacoside attenuates LPS-induced ALI via inactivation of the NF-κB/NLRP3 pathway, making echinacoside a potential drug for the treatment of ALI.


INTRODUCTION
Pediatric pneumonia, caused by bacterial or viral infection, is common in infants and children, and is an important cause of mortality in children [1]. Studies show that pneumonia is the most common risk factor for acute lung injury (ALI) [2]. In clinical practice, infection-induced ALI is a serious condition requiring intensive care.
Damage to alveolar epithelial integrity, hyaline membrane formation, increased permeability, activated inflammation response, pulmonary edema, hypoxemia, and atelectasis usually occur in ALI patients [3]. Therefore, infectioninduced ALI is a serious threat to infants and children. At present, antiviral drugs and antibiotics are the major therapeutic strategies for ALI [4]. However, the serious side effects of chemical drugs and overuse of antibiotics limit the effectiveness of ALI therapy [1]. Therefore, it is essential to investigate new drugs that show promise for ALI therapy.
Cistanche deserticola is a widely used Chinese herbal medicine with many pharmacological properties, such as anti-inflammatory and antioxidant properties [5,6].
Recently, accumulating evidence revealed that echinacoside (ECH), a phenylethanoid glycoside extracted from C. deserticola [7], exerts neuroprotective and anti-inflammatory effects in vivo and in vitro [7]. For example, ECH attenuated nerve injury and neuroinflammation in Parkinson's disease through modulating the p38MAPK and NF-κB pathways in [8]. Furthermore, ECH alleviated spinal cord injury by reducing activity of the NLRP3 pathway [9]. Moreover, the effects of ECH on ALI have been reported. Zhang et al demonstrated that ECH alleviated oleic acid-induced lung injury [10]. However, the role of ECH in the inflammatory response of ALI is unclear.
Therefore, the aim of the study was to investigate the role of ECH in ALI and its inflammatory response and study the underlying mechanism in these processes.

EXPERIMENTAL Animals and experimental design
Twenty-four neonatal C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). The mice were divided into four groups: control, lipopolysaccharide (LPS), LPS + ECH (10 mg/kg), and LPS + ECH (30 mg/kg). To induce lung injury, 50 μL of LPS (2 mg/kg) (Sigma, St. Louis, MO, USA) was instilled intranasally in mice. In the LPS + ECH (10 mg/kg) and LPS+ECH (30 mg/kg) groups, the mice also received 10 mg/kg ECH and 30 mg /kg ECH, respectively, after the LPS challenge. All animal experiments were conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines [11]. The protocols of all animal experiments were approved by the Ethics Committee of Women and Children's Hospital, School of Medicine, Xiamen University (approval no. 2020-011).

Hematoxylin and eosin (H&E) staining
After receiving the indicated treatments, mice were euthanized and lungs were collected. The middle lobe of the right lung was reserved to determine lung wet-to-dry-weight ratio (W/D).
The remainder of the lung tissue was cut into blocks of approximately 4 mm 3 , fixed in paraformaldehyde, embedded in paraffin, and 5μm sections were prepared. Sections were deparaffinized, rehydrated, stained with H&E, and viewed under a light microscope (Olympus, Tokyo, Japan).

Determination of lung W/D
The middle lobe of the right lung was collected to determine lung wet weight. After the lung was dried for 48 h at 80°C, the dry weight was recorded.

Determination of cell number and protein expression in bronchoalveolar lavage fluid (BALF)
BALF samples were collected and centrifuged, and cell pellets were lysed with red blood cell lysis buffer and resuspended in phosphatebuffered saline (PBS). Numbers of total cells, neutrophils, and macrophages were determined using a hemocytometer. Total protein content in BALF samples was determined using a bicinchoninic acid (BCA) kit (Sigma).

ELISA
BALF and lung tissues of mice were collected to determine the production of tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β). The levels of TNF-α and IL-1β were determined using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's protocols.
The bands were visualized using the ECL detection kit (Thermo Fisher Scientific, Waltham, MA, USA). The band density analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA.

Statistical analysis
SPSS version 22.0 (Chicago, IL, USA) was used to analyze the data. All data are shown as mean ± standard deviation (SD). Differences among groups were compared using one-way ANOVA with LSD post-hoc test. P < 0.05 was considered statistically significant.

ECH alleviates LPS-induced ALI
The LPS-treated mice presented with marked lung damage characterized by lung tissue congestion and alveolar collapse, whereas ECH attenuated these changes ( Figure 1A). Further, the W/D ratio of lung tissue in LPS-treated mice was significantly increased, but was decreased by treatment with 10 mg/kg and 30 mg/kg ECH (p < 0.01, Figure 1B). These results show that ECH alleviates LPS-induced ALI.

ECH alleviates LPS-induced inflammatory response
The total protein content of BALF was increased in LPS-treated mice, but ECH decreased the total protein content (p < 0.01; Figure 2A). Further, the number of total cells in BALF was increased in LPS-treated mice (p < 0.01; Figure  2B). However, ECH decreased the number of total cells in BALF induced by LPS (p < 0.01, Figure 2B). Similarly, the numbers of neutrophils and macrophages in BALF were significantly increased in LPS-treated mice, and this effect was reversed by ECH (p < 0.01, Figure 2C and D). These results indicate the protective role of ECH against LPS-induced inflammatory response.

ECH alleviates LPS-induced NF-κB/NLRP3 pathway activity
Phosphorylation of p65 to form p-p65, and NLRP3 expression in LPS-treated mice were increased (p < 0.01, Figure 4). ECH decreased both p65 phosphorylation and NLRP3 expression (p < 0.01, Figure 4). Thus, ECH inhibits activation of the NF-κB/NLRP3 pathway in LPSinduced ALI. Figure 4: Effect of ECH on NF-κB/NLRP3 pathway in LPS-induced ALI. Western blotting was used to determine the levels of p-p65, p65, and NLRP3 in mice treated with LPS and ECH (10 and 30 mg/kg); **p < 0.01 compared to control group; ##p < 0.01 compared to LPS group DISCUSSION Pediatric pneumonia is a common disease in infants and children, and it is a major risk factor for ALI [1,2]. ALI induced by bacterial or viral infection is a serious condition with severe lung damage. Therefore, it is essential to identify effective therapeutic strategies for ALI. In this study, the effect of ECH on ALI was investigated, and results demonstrated that ECH attenuated LPS-induced ALI in newborn mice through inhibiting activation of NF-κB/NLRP3 signaling.
To determine the effect of ECH on infectioninduced ALI, LPS was used to establish the ALI model. In the LPS-treated mice, obvious lung damage with lung tissue congestion, alveolar collapse, and increased W/D ratio were observed, and were reversed by ECH administration. These results are in accordance with the study performed by Zhang et al [10], who found that ECH attenuated ALI induced by oleic acid in rats [10]. Thus, the results of this study indicate that ECH exerted a protective role in LPS-induced ALI.
In LPS-induced ALI, the inflammatory response has a critical role, and the excessive activation of neutrophils and macrophages aggravated ALI [12]. The previous study revealed that neutrophils could be recruited to the lungs and cause endothelial injury, thereby inducing lung edema during ALI [13]. Macrophages regulate the production of proinflammatory mediators in different pathological stages of ALI [14]. In this study, neutrophils and macrophages in BALF were increased in LPS-treated mice, and ECH decreased these changes. Further, the total protein content and total cell number in BALF were increased, and ECH also decreased these changes. In addition to immune cells, cytokines participate in ALI [13]. In this study, TNF-α and IL-1β were increased in BALF and lung tissue of mice treated with LPS, and ECH decreased these changes. Therefore, these results reveal that ECH alleviated LPS-induced inflammatory response and cytokine production.
NF-κB and NLRP3 signaling are important inflammatory signaling pathways and participate in regulation of ALI [15]. The level of NF-κB activation determines the severity of ALI [15]. Liu et al showed that NLRP3 was essential for paraquat-induced ALI [16]. Accumulating evidence has revealed that inactivation of the NF-κB and NLRP3 pathways reduced ALI [17,18]. In this study, the level of p65 phosphorylation and NLRP3 expression increased in mice treated with LPS, and ECH decreased these changes. In other words, ECH reversed LPS-induced changes in the NF-κB/NLRP3 pathway. The influence of ECH on the NF-κB/NLRP3 pathway was in agreement with previous studies [8,9]. Zhang et al revealed that ECH protected against neuroinflammation via modulation of the NF-κB pathway in Parkinson's disease [8]. Gao et al showed that ECH alleviated spinal cord injury through suppression of the NLRP3 signaling pathway [9]. Taken together, these findings suggest that ECH may attenuate LPS-induced ALI through inactivation of the NF-κB/NLRP3 signaling pathway.

CONCLUSION
Echinacoside attenuates LPS-induced ALI via inactivation of the NF-κB/NLRP3 pathway. Thus, echinacoside is a promising drug for ALI therapy.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Meijiao Fu and Tong Shen designed the study, supervised data collection, and analyzed the data. Ying