Necrostatin-1 attenuates lipopolysaccharide- induced acute lung injury in mice

Enqin Guan, Yue Wang, Caixia Wang, Ruiyun Zhang, Yiming Zhao & Jiang Hong

To cite this article: Enqin Guan, Yue Wang, Caixia Wang, Ruiyun Zhang, Yiming Zhao & Jiang Hong (2017): Necrostatin-1 attenuates lipopolysaccharide-induced acute lung injury in mice, Experimental Lung Research, DOI: 10.1080/01902148.2017.1384083
To link to this article: https://doi.org/10.1080/01902148.2017.1384083

Published online: 04 Dec 2017.

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EXPERIMENTAL LUNG RESEARCH
, VOL. , NO. , – https://doi.org/./..
ORIGINAL ARTICLE

Necrostatin- attenuates lipopolysaccharide-induced acute lung injury in mice
Enqin Guana,b, Yue Wangb, Caixia Wangb, Ruiyun Zhangb, Yiming Zhaob, and Jiang Honga
aDepartment of Pediatrics, the Affiliated Hospital of Qingdao University, Qingdao, Shandong, China; bDepartment of Pediatrics, Qingdao Municipal Hospital, Qingdao, Shandong, China

ARTICLE HISTORY
Received  July  Accepted  September 
KEYWORDS
acute lung injury; LPS; Necrostatin-; RIP-; sepsis

Introduction
Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are inflamma- tory disorders of the lung caused by pneumonia, sepsis, trauma and/or aspiration.[1] ALI and ARDS are charac- terized by pulmonary edema due to increased perme- ability of the alveolar epithelial and endothelial barriers and the subsequent impairment of arterial oxygena- tion.[2,3] Despite improvements in therapy, the mor- bidity and mortality rates of ARDS remain as high as 30–40%.[4–6] Thus, there is a need for innovative phar- macological therapies to improve clinical outcomes.
Lipopolysaccharide (LPS), a component of gram- negative bacterial endotoxin, is recognized as the main cause of ALI.[7] LPS induces ALI in animal models by promoting pulmonary microvascular per- meability and recruiting activated neutrophils and macrophages to the lung.[8,9] These effects damage the

alveolar-capillary membrane, which leads to the deteri- oration of gas exchange.[8,9] It has also been shown that LPS-induced ALI results in an increase in the expres- sion of various pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in bronchoalveolar lavage fluid (BALF).[8] There is also recent clinical evidence that increased TNF-α, IL-1β, and IL-6 levels are associated with poor patient outcome in ALI.[10] Thus, the LPS-induced ALI mice model is widely used for pathogenesis studies and drug development for ALI.[11,12]
The receptor-interacting protein (RIP) kinase family consists of seven members, each of contains a homolo- gous kinase domain (KD).[13] The RIP kinase domain is indispensable for the activation of nuclear factor (NF)- κB as it interacts with its downstream signaling com- ponents such as NF-κB essential modulator (NEMO), the regulatory subunit of the inhibitor of NF-κB kinase

CONTACT Jiang Hong jianghongbs@.com Department of Pediatrics, the Affiliated Hospital of Qingdao University, No  Jiangsu Road, Qingdao, Shandong, , China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/IELU.
©  Taylor & Francis

(IKK) complex and other molecules.[14–16] Moreover, its death domain is required for the association with the upstream signaling component TNF-receptor type 1- associated death domain (TRADD).[14–16] Earlier stud- ies indicate that the RIP kinase family is involved in sev- eral biological processes including tumorigenesis,[17] cell death,[18] necrosis,[19] and inflammation.[20] How- ever, the role of the RIP kinase family in the develop- ment of ALI is still unclear.
Necrostain-1 is an inhibitor of the receptor inter- acting protein 1(RIP1) kinase that has usually been used as a potent and specific inhibitor of necrop- tosis.[21–24] Encouraging studies have demonstrated that necrostain-1 functioned as a protective com- pound in various experimental disease models, such as ischemia-reperfusion injury in brain,[25] spinal cord injury,[26] colitis and colitis associated colon cancer.[27] In the present study, the expression of RIP kinase family members in ALI mice was determined using western blotting and immunohistochemical staining. Necrostatin-1was used to treat LPS-induced ALI mice, followed by survival time recording, histopathological and immunohistochemical staining of lung tissues, western blotting, and ELISA of related cytokines and downstream target expression in BALF and lung tissues.

Materials and methods

Animal model and treatment
Male BALB/c mice aged 6–8 weeks (about 20 g) were purchased from the Experimental Animal Cen- ter of Nanjing University. Animals were free of specific pathogens and were kept on a 12 h light/12 h dark cycle at a room temperature of 22 ± 2°C with free access to food and water. All of the experimental procedures were approved by the Animal Care and Use Committee of Qingdao University (20161028).
According to individual weights, mices were intra- venously injected with 10 mg/kg LPS (E.coli, Sigma) dissolved in 2 ml PBS (GIBCO) with a 20 gauge-needle syringe to establish ALI models. Mice in the control group received a PBS injection without the LPS chal- lenge. Twenty four hours post LPS injection, the lungs were collected for further analysis. For Necrotain-1 treatment, mice received intraperitoneal injection of Necrotain-1 (Selleck, 10 mg/kg), which is dissolved in dimethyl sulfoxide (DMSO), subsequent after LPS injection. The survival time was recorded every 6 hours

and over 72 hours. All of the lung tissues and BALF were collected for further analysis at 24 hours post LPS injection.

Histopathological and immunohistochemical staining
Lungs were instilled with 10% formalin under 15 cm H2O pressure and immersed in the same solu- tion before tissue processing into paraffin-embedded blocks; 4 μm sections were then cut and stained with hematoxylin and eosin (H&E) kit (Beyotime, Beijing China). H&E stained sections were scored (lung injury score) for the presence of leukocytes in the alveolar space, leukocytes in the interstitial space, the existence of hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening, as described previously.[28] A score of 0 represented no damage; l represented mild damage; 2 represented moderate damage; 3 represented severe damage and 4 repre- sented very severe histological damage.[29]
After antigen retrieval for 3 mins in citrate under high pressure, lung tissue sections were incubated with primary antibody against RIP-1 (1:100, Cat. No. ab72139, abcam, UK) for immunohistochemical stain- ing following the instructions of IHC kit (SP9001, Zsbio, Beijing, China) and DAB kit (Maixin, Fuzhou, China). All specimens were evaluated using an Olym- pus BX600 microscope and SPOT Flex camera. The RIP-1 positive cells were scored by counting the num- ber of lung cells expressing the proteins as determined by RIP-1 staining in lungs.

Western blotting analysis
The left lungs were used for protein extracting using T-PER Tissue Protein Extraction Reagent kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Protein concentrations were determined using the BCA protein assay kit (Beyotime, Beijing, China). Western blotting was performed as described previously.[30] The primary antibodies against RIP-1 (1:800, Cat. No. ab72139, abcam, UK), RIP-2 (1:1000, Cat. No. 4142, CST), RIP-3 (1:1000, Cat. No. 95702, CST), IKKα (1:1000, Cat. No. 11930, CST), NF-κB p50 (1:1000, Cat. No. 3035, CST), NF-κB p65 (1:1200, Cat. No. 8242, CST) and GAPDH (1:10000, Cat. No. G9545,
Sigma-Aldrich) were performed to detect the specific protein expression. The relative expression of protein were analyzed.

Enzyme-linked immunosorbent assay (ELISA) analysis and myeloperoxidase (MPO) assay
After treatment, mice were sacrificed to allow collec- tion of bronchoalveolar lavage fluid (BALF) and lung tissue. BALF samples were taken from the right lung, and centrifuged (500 g for 15 min at 4°C). Super- natants were centrifuged again (500 g for 15 min at 4°C) and snap-frozen in liquid nitrogen and stored at
-80°C for ELISA analysis. The left lungs were used for protein extracting using T-PER Tissue Protein Extrac- tion Reagent kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Protein concentra- tions were determined using the BCA protein assay kit (Beyotime, Beijing, China). Concentrations of IL-6, TNF-α, IL-8, COX-2, MCP-1 and IL-1β in BALF and
lung tissues were determined using commercially avail- able ELISA kits for mouse cytokines (Neobioscience, Beijing, China). The protocol was performed accord- ing to the manufacturer’s instructions.
MPO activity in snap frozen mouse lung tissue was determined using aMPO Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions, as previous study indi- cated.[28]

Cell culture and treatment
Murine leukemia virus transformed RAW264.7 cells were grown in DMEM containing 10% fetal bovine serum (Gibco, U.S.A). Cells were cultured in a stan- dard humidified incubator at 37°C with 5% CO2. For LPS and Necrostain-1 treatment, RAW264.7 cells were plated in 6-well plate (2 × 105 cells/well in 2 ml medium) and treated with LPS (2 μg/ml) and Necrostain-1 (20 μM) for 24 hours. The cells and supernatant were collected for further western blotting analysis and ELISA analysis.

Cell counting kit-8 (CCK8) assay
For proliferation assays, RAW 264.7 cells were seeded at 2 103 cells per well in 96-well plates as previous study indicated.[31] After necrostatin-1 (0, 20, 40 and 80 μM) treatment for 48 hours, a Cell Counting Kit-8 (Dojindo, Shanghai, China) was used and absorbance was measures at 450 nm for each well a using a micro- plate reader (Thermo fisher scientific, Waltham, MA, USA). The relative cell viability, compared with the cells without necroatin-1, were calculated and analyzed.

Statistical analysis
All of the statistical analysis was performed using GraphPad Prism version 5.0 for Windows (Graph- Pad Software, San Diego, CA, USA). All data were expressed as mean ± S.D. and compared using the stu- dent’s t-test for two-group analysis. A value of p < 0.05 was considered significant.

Results

Pulmonary RIP-1 level was upregulated in LPS-induced ALI mice
To determine the expression levels of RIP family pro- teins in ALI, LPS (Escherichia coli, 10 mg/kg) was intra- venously injected into the mice, and 24 hours later, the lungs were collected for histopathological staining. Typical lung injury, widespread edema-induced alve- olar wall thickness, severe hemorrhage of the alveo- lus, alveolar collapse, obvious inflammatory cell infil- tration, and higher lung injury score were found in the LPS-injected mice than in the untreated mice (Figure 1A).
Furthermore, western blotting indicated that RIP- 1 but not RIP-2 and RIP-3 expression levels signif- icantly increased in the ALI mice (Figure 1B). We next evaluated the pulmonary RIP-1 expression using immunohistochemical staining and found more RIP- 1-positive cells in the ALI mice than in the control mice (Figure 1C), which was consistent with the west- ern blotting results. These data showed that the pul- monary RIP-1 level was upregulated in LPS-induced ALI mice.

Necrostatin-1 inhibited pulmonary RIP-1 expression in LPS-induced ALI mice
We determined the potential role of RIP-1 inhibition in LPS-induced ALI development by intraperitoneally injecting necrostatin-1 (10 mg/kg) after LPS pretreat- ment, and 24 hours later, the lungs were collected for RIP-1 detection using immunohistochemical staining and western blotting. As shown in Figure 2A, fewer RIP-1-positive cells were found in the necrostatin-1– treated mice than in the untreated mice. Furthermore, western blotting demonstrated that necrostatin-1 significantly reversed the LPS-induced RIP-1 upregu- lation in mouse lungs (Figure 2B). Thus, the intraperi- toneal injection of necrostatin-1 efficiently inhibited

Figure . Pulmonary RIP-1 level was up-regulated in LPS-induced ALI mice. A, Histopathological analysis of lungs of PBS injected mice and LPS-induced ALI mice by H&E staining. The lung injury score were analyzed (n , ∗∗,p < ., compared with PBS group). B, Western blotting analysis of pulmonary RIP-, RIP- and RIP- expression level in PBS injected mice and LPS-induced ALI mice. GAPDH was used as a loading control. The relative expression of RIP-, RIP- and RIP- were analyzed (n = , ∗∗,p < ., compared with PBS group). C, Immunohistochemical staining of RIP- expression in lung of PBS injected mice and LPS-induced ALI mice. Scale bar = μm. The RIP- positive cells were analyzed (n = , ∗∗,p < ., compared with PBS group).

the upregulation of pulmonary RIP-1 level in LPS- induced ALI mice.

Necrostatin-1 prolonged survival time of mice with ALI
We further determined the biological effects of necrostatin-1 in 10 LPS-induced ALI mice by

first determining their survival time. As shown in Figure 3A, only two mice survived 72 h after LPS injection, and eight mice died 36 h after LPS injec- tion in the dimethyl sulfoxide (DMSO)-treated group (Figure 3A). Notably, only two mice had died 72 h after LPS injection in the necrostatin-1–treated group (Figure 3A). Necrostatin-1 significantly prolonged the survival time of mice with LPS-induced ALI. H&E

Figure . Necrotain-1 inhibited pulmonary RIP-1 level in LPS-induced ALI mice. A, Immunohistochemical staining of RIP- expres- sion in lung of DMSO treated and Necrostain- treated LPS-induced ALI mice. Scale bar μm. The RIP- positive cells were analyzed (n , ∗∗,p < ., compared with DMSO group). B, Western blotting analysis of pulmonary RIP-expression level in DMSO treated and Necrostain- treated LPS-induced ALI mice. GAPDH was used as a loading control. The relative expression of RIP-, RIP- and RIP- were analyzed (n = , ∗∗,p < ., compared with DMSO group).

Figure . Necrotain-1 prolonged the survival time of mice with ALI. A, The survival time of DMSO treated and Necrostain- treated LPS-induced ALI mices were recorded over  h. (n  for each group, p < ., compared with DMSO group. B, Histopathological analysis of lungs of DMSO treated and Necrostain- treated LPS-induced ALI mice by H&E staining. The lung injury score were analyzed (n , ∗∗, p < ., compared with DMSO group).

staining demonstrated LPS-induced lung injury in both the DMSO- and necrostatin-1-treated mice. However, less infiltration of inflammatory cells into the alveoli and lung parenchyma, and less severe hem- orrhage in the alveolus and alveolar collapse were found in the necrostatin-1-treated mice, and this was accompanied by a lower lung injury score (Figure 3B).

Necrostatin-1 protected lungs against inflammation in LPS-induced ALI mice by inhibiting pulmonary
NF-κB activation
To further determine the mechanism mediating the necrostatin-1-induced attenuation of ALI, we mea- sured the expression levels of pro-inflammatory and chemotactic cytokines in the BALF and lung tissue of mice. ELISA indicated that more IL-6, TNF-α, IL-8, COX-2, MCP-1, and IL-1β expression were found both in the BALF and lung tissues of ALI mice. Importantly, the expression of IL-6, TNF-α, IL-8, COX-2, MCP-1, and IL-1β levels was significantly lower in the BALF of necrostatin-1-treated mice than in the untreated mice (Figure 4A). Furthermore, we found that the pul- monary IL-6, TNF-α, IL-8, COX-2, MCP-1, and IL-1β
expression levels were also efficiently decreased by necrostatin-1 in ALI mice (Figure 4B). Next, the MPO activity investigation demonstrated that necrostatin-
1 efficiently inhibited LPS-induced pulmonary inflammation in lung tissues (Figure 4C). Next, the

potential downstream targets of RIP-1 were deter- mined using western blotting. We found that inhibition of RIP-1 by necrostatin-1 dramatically inhibited the activation of NF-κB, which was accompanied by the downregulation of IKKα and NF-κB p50 (Figure 4D). Collectively, the data demonstrated that necrostatin-1 significantly protected the lungs of LPS-induced ALI mice against inflammation by inhibiting pulmonary NF-κB activation.

Necrostatin-1 attenuated LPS-induced inflammation in RAW 264.7 cells
As we known, alveolar macrophages play an impor- tant role during the development of acute inflam- matory lung injury.[32] Thus, we further investigated the molecular mechanism underlying the necrostatin- 1-mediated attenuation of ALI by treating mouse macrophage RAW 264.7 cells with LPS (E. coli) and necrostatin-1. As shown in Figure 5A, the cell via- bility was not influenced by necrostatin-1. Notably, necrostatin-1 efficiently inhibited the effects of LPS on RIP-1 expression and the subsequent IKKα, NF-κB p50, and NF-κB p65 expression (Figure 5B). Further- more, ELISA analysis indicated that necrostatin-1 sig- nificantly inhibited LPS-mediated upregulation of IL- 6, TNF-α, IL-8, COX-2, MCP-1, and IL-1β expression in macrophages (Figure 5C). The data indicate that

Figure . Necrotain-1 protected system and pulmonary against inflammation in LPS-induced ALI mice. A, The BALF from Control (Ctrl) and DMSO treated and Necrostain- treated LPS-induced ALI mices were collected for IL-, TNF-α, IL-, COX-, MCP- and IL-β expression detection by ELISA (n , ∗∗,p < ., compared with Ctrl group; ##, p < ., compared with DMSO group). B, The lungs from Control (Ctrl) and DMSO treated and Necrostain- treated LPS-induced ALI mices were collected for protein extracting. The pulmonary IL-, TNF-α, IL-, COX-, MCP- and IL-β expression were detected by ELISA (n , , p<., compared with Ctrl group; ##, p < ., compared with DMSO group). C, The lungs from Control (Ctrl) and DMSO treated and Necrostain- treated LPS-induced ALI mices were collected for MPO assay. (n , ∗∗,p < ., compared with Ctrl group; ##, p < ., compared with DMSO group). D, The lungs from Control (Ctrl) and DMSO treated and Necrostain- treated LPS-induced ALI mices were collected for protein extracting. The pulmonary IKKα, NF-κB p and NF-κB p expression were detected by western blotting. GAPDH was used as loading control (n , ∗∗,p < ., compared with Ctrl group; ##, p < ., compared with DMSO group).

necrostatin-1 attenuated LPS-induced inflammation in RAW 264.7 cells.

Discussion
Necrostain-1 has usually been used as a potent and specific inhibitor of necroptosis by inhibiting RIP1 kinase. Necstatin-1 restrained TNF-α-induced osteo- cyte necroptosis in rats with E2 deficiency-induced osteoporosis and represented a novel therapeu- tic strategy for the treatment of postmenopausal

osteoporosis.[24] Previous study by Jie et al have shown that necrostatin-1 enhanced the resolution of estab- lished inflammation and may have potential roles for the treatment of diseases with increased or persis- tent inflammatory responses.[33] They indicated that necrostatin-1 is not only an inhibitor of necroptosis, but also a promoter of apoptosis, of neutrophils.[33] In our study, we also demonstrated that protected against LPS-induced ALI in mice by inhibiting inflammation and pulmonary NF-κB activation. Further mechanism investigation indicated that necrostatin-1 attenuated

Figure . Necrotain-1 attenuated LPS-induced inflammation in RAW 264.7 cells. A, CCK assay was performed to detect the cell viability of RAW . cells that were treated with necrotain- (, ,  and  μM) for  hours. (n , ns, no significant difference). B, LPS and Necrostain- were employed to treat RAW . cells for  hours and the cells were collected for protein extracting. The cells without LPS treatment was collected as control (Ctrl) group. The RIP-, IKKα, NF-κB p and NF-κB p expression were detected by western blotting. GAPDH was used as loading control (n , ∗∗,p < ., compared with Ctrl group; ##, p < ., compared with DMSO group). C, LPS and Necrostain- were employed to treat RAW . cells for  hours and the supernatant were collected for IL-, TNF-α, IL-, COX-, MCP- and IL-β expression detection by ELISA analysis. The supernatant from the cells without LPS treatment was collected as control (Ctrl) group. (n = , ∗∗,p < ., compared with Ctrl group; ##, p < ., compared with DMSO group).

LPS-induced pro-inflammatory cytokine expression and NF-κB activation in RAW 264.7 cells, but had no significant effect on the cell viability of RAW 264.7 cells. It’s indicated that necrostatin-1 induced necrop- tosis independent downregulation of NF-κB activation in macrophages, which is consistent with the regula- tion role of RIP on NF-κB activation.[34] These novel findings would provide solid evidenced for better understanding the mechanism under necrostatin-1 mediated ALI attenuation.
Recent studies have provided solid evidence to enhance the understanding of ALI/ARDS and demon- strated that sepsis is a leading factor for ALI/ARDS development.[35,36] Moreover, sepsis may be directly induced by LPS on the outer membrane of various gram-negative bacteria.[35,36] Thus, LPS is an efficient agent for establishing ALI animal models through intravenous or intraperitoneal injection, or intratra- cheal instillation.[7,11,37] In the present study, 10 mg/kg LPS was intravenously injected into mice to establish the ALI model. Typical lung injury, widespread alveo- lar wall thickness caused by edema, severe hemorrhage

in the alveolus, alveolar collapse, obvious inflamma- tory cell infiltration, and higher lung injury score were found in the mice after LPS injected for 24 hours. Intravenous injection of LPS caused a typical lung injury phenotype and enabled the establishment of a suitable model for further research.
In our model, we found that RIP-1 was significantly increased in the lung tissue from mice treated with LPS for 24 hours. Furthermore, the inhibition of RIP-1 by the specific inhibitor, necrostatin-1, prolonged the survival time of mice and alleviated lung injury. This observation indicates that RIP-1 may promote LPS-induced ALI. This finding was consistent with the pro-inflammatory role of RIP-1 demonstrated in pre- vious studies.[13,20] We also observed that necrostatin-1 had anti-inflammatory effects. This beneficial effect could contribute to prolonging the survival time of mice and attenuation of lung injury. We found that necrostatin-1 inhibited the expression of the pro- inflammatory cytokines IL-6, TNF-α, IL-8, COX-2, MCP-1, and IL-1β in the BAFL of mice with LPS- induced ALI. Similarly, pulmonary IL-6, TNF-α, IL-8,

COX-2, MCP-1, and IL-1β expression levels were also decreased by necrostatin-1 in mice with LPS-induced ALI. These results confirm that the potent, protective effect of necrostatin-1 against LPS-induced ALI is related to its attenuation of lung inflammation. The anti-inflammatory effect of necrostatin-1 observed in this study is consistent with the results of previous reports.[27] Liu et al[27] found that necrostatin-1 treat- ment reduced the production of pro-inflammatory cytokines and extracellular high-mobility group box 1 (HMGB1) release in HT-29 cells in active necroptosis, accompanied by the suppression of tumor growth and development in a colitis-associated cancer model. These results provide solid evidence of the anti- inflammatory effect of necrostatin-1 in ALI mice and indicate it could also be a novel therapeutic strategy for sepsis and ALI.
NF-κB transcription factors bind as dimers to κB
sites in promoters and enhancers of a variety of genes and induce or repress transcription.[38] Various stud- ies have demonstrated that the dysregulation of NF-κB activity is linked to inflammatory disorders, autoim- mune and metabolic diseases, as well as cancer.[38] In LPS-induced ALI, NF-κB was abnormally activated and subsequently mediated the LPS-induced inflam- matory cytokine expression.[39–41] Huang et al.[27] reported that the NEMO-binding domain peptide pro- tects against LPS-induced ALI in mice by attenuating LPS-induced IκB-α and NF-κB p65 activation. The cyclin-dependent kinase (CDK) interacting protein (C1P) also inhibits LPS-induced NF-κB phosphoryla- tion and IL-8 production in human neutrophils and, thereby, attenuates LPS-induced acute lung inflam- mation, which suggests that C1P could be a valu- able candidate for the treatment of ALI.[39] In our study, we demonstrated that necrostatin-1 is an effi- cient agent for inhibiting LPS-induced NF-κB activa- tion in ALI mice and LPS-induced macrophages. This would be the molecular mechanism likely underlying the necrostatin-1-induced attenuation of LPS-induced ALI in mice.
In conclusion, the present study revealed that the inhibition of RIP-1 by necrostatin-1 attenuated LPS- induced ALI through inhibiting LPS-induced pro- inflammatory NF-κB activation in macrophages. These results further provided evidence that the administra- tion of necrostatin-1 prolonged the survival time of LPS-induced ALI mice, and inhibited the LPS-induced RIP-1 upregulation and pulmonary pro-inflammatory

cytokine expression. An enhanced understanding of the role and mechanism of RIP-1 in inflammation may facilitate the future development of new treatments for lung diseases.

Conflict of interest
All authors declare that there are no conflicts of interest.

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