Effect and Mechanism of PLSCR4 in The Injury of Human Pulmonary Microvascular Endothelial Cells Induced by LPS


 Background: Previous experiments have revealed that Phospholipid scramblase 4 (PLSCR4) mRNA significantly increased in ARDS model of human pulmonary microvascular endothelial cells (HPMECs) stimulated by lipopolysaccharide (LPS) , but the specific effect and mechanism have not been reported to date.In consideration of the function of PLSCR4 that has been reported, we infer that PLSCR4 may have impact on cell death through pyroptosis. Methods: To investigate the effect and mechanism of PLSCR4 in ARDS in vitro, HPMECs were cultured in 6-well plates and divded into four groups: control group, LPS group ,siRNA+LPS group and transfection reagent group. After 4 h of LPS stimulation, western blot was used to detect the expression of PLSCR4 and pyroptosis – relevant proteins; Using ELISA to exame IL-1β and IL-18; Tracer flux assays was used to evaluate the barrier function of endothelium, and fluorescence assays to observe the distribution of phospholipid and the degree of pyroptosis. At last we used DNA pull-down and protein profiling to study the potential transcription factor of PLSCR4.Results: It was found that when the expression of PLSCR4 was elevated, the concentration of IL-1β and IL-18 decreased, together with barrier damage ( P < 0.05), and HPMECs injury was reduced with more distribution of phospholipid on the external sie of cells, but the pyroptosis-relevant proteins did not change obviously ( P < 0.05); then we found P62280 remarkably increased in the DNA pull-down assay, and it could impact the pyroptosis by regulating the expression of PLSCR4. Conclusion: We found that PLSCR4 alleviated pyroptosis by transporting phosphatidylserine (PS) to the outside of the membrane, blocking the forming of pyroptosis pores composed of gasdermin D, it was further found that the expression level of PLSCR4 during LPS stimulation was regulated by P62280. This study for the first time explains the protective effect of PLSCR4 in the ARDS model, and its possible mechanism, providing a new research idea for the clinical treatment of ARDS.


Introduction
Acute respiratory distress syndrome (ARDS) with in ammatory injury as the main feature is one of the main causes of death in patients with multiple organ dysfunction caused by sepsis, which has high morbidity and mortality (1,2).The main pathological characteristics were the release of in ammatory factors, the in ltration and aggregation of in ammatory cells and the damage of the vascular endothelium that ultimately lead to changes in pulmonary microvascular endothelial permeability and pulmonary edema (3).
Despite the extensive researches on ARDS, presently there are no effective drug treatments (1). In the prior study of our group, high-throughput cell sequencing and bioinformatics analysis were carried out, and the results revealed that the mRNA expression of PLSCR4 protein signi cantly increased, and it was at the central position (4,5) in the co-expression network diagram, suggesting that PLSCR4 may play an important role in the pathological process of the pulmonary microvascular endothelium induced by LPS (Fig. 1). 100 μl of cell lysate (RIPA:PMSF=100:1) was added in each well of the six-well plate, and set the plate on ice for 30 minutes. After the lysis, the cell lysate was collected in a 1.5-ml EP tube. Subseqeuntly, the centrifuge processing of the lysate was carried out at 12,000 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was transferred to another EP tube to obtain the total protein. The total protein concentration was detected using the BCA Protein Assay Kit, and 5× loading buffer was added to the protein samples, which was then heated and denatured at 96°C for ve minutes. The obtained protein samples were subjected to SDS-PAGE gel electrophoresis, and transferred into PVDF membranes. Then the membranes were blocked with 5% skimmed milk for one hour at room temperature, and the sample was washed with TBST for ve minutes for three times at the end of blocking. Incubate the membranes in the primary antibody diluent (1:1,000) overnight at 4°C. On the next day, incubate the membranes in the secondary antibody diluent (1:5,000) at room temperature for one hour, and nally set on the scan machine (10).
ELISA detection of IL-1β and IL-18 After four hours of stimulation by LPS, the supernatant of each group of cells in the six-well plate was collected and added to a EP tube. The supernatant was centrifuged at 1,000 g for 20 minutes at 4°C to remove the impurities and cell debris. The double-antibody sandwich ELISA kit was used for the detection of in ammatory factors IL-1β and IL-18 (11), which were used to assess the pyroptosis. If these could not be detected in time, the sample was frozen at -80°C to avoid repeated freezing and thawing.

Tracer ux assays
In this study, Tracer Flux Assays were performed to detect the changes in endothelial permeability. The Transwell chamber was purchased from Corning, USA, with a lter pore diameter of 0.4 μm and a cell diameter of 12 mm. Next, 1.5 ml of complete medium was added at the bottom chamber, and 0.5 ml of complete culture medium that contained the suspended cells were added to the upper chamber. The number of pulmonary microvascular endothelial cells was 5×10 5 . Afterwards, the Transwell chamber was cultured in an incubator at 37°C. On the next day, the medium was changed, and the cell adhesion was observed under a microscope. After 1-3 days, when the cell con uency reached 90%, the medium in the bottom layer for the experimental group was replaced with fresh medium containing 1 μg/ml of LPS, while the medium in the upper chamber was replaced with fresh culture containing 1 μg/ml of LPS and 1 mg/ml of FITC-dextran. The upper and lower layers of the chamber for the blank control group did not contain LPS, only 1 mg/ml of FITCdextran in the upper layer. Both groups were placed in a cell incubator at 37ºC to continue culturing for four hours. After four hours, 50 μl of the bottom culture medium of each group was taken and added to a 96-well plate. After labeling, the samples were placed in a uorescence microplate reader (BioTek SynergeH1M, USA) to read the data. The excitation light wavelength of the microplate reader was set to 488 nm, and the emission wavelength was set to 520 nm (12). Data analysis was expressed by permeability index PI , that is, the ratio of uorescence intensity of each experimental group to the wells without cells.

Pyroptosis uorescence assays
Fluorescence experiments were performed to detect the degree of pyroptosis of HPMECs (13). The endothelial cells were seeded in a 24-well plate. The medium was changed with 1 ml of fresh and complete culture on the next day, and then cultured for 1-2 days. When the cell con uency reached 80%-90%, LPS (1 μg/ml) was added to the experimental group, and cultured the HPMECs for four hours. After the stimulation, cells in each group were washed twice with DPBS. Next, 96 μl of Annexin V-FITC binding solution, 1 μl of Annexin V-FITC (green uorescence), and 12.5 μl of PI (Propidium iodide, red uorescence) were added to each well. After mixing, the samples were placed on ice, avoiding light for 10 minutes. Finally, an immuno uorescence microscope (Leica dm18, Germany) was used to observe the endothelial cell membrane staining. The Annexin V-FITC uorescence intensity re ects the degree of PS exposure on the cell membrane surface. The PI uorescence intensity represents the degree of cell membrane integrity damage.
DNA pull-down and protein pro ling In order to determine the possible transcription factors of PLSCR4, our study used the DNA pull-down and protein pro ling techniques. T-75 asks were used to cultivate HPMEC cells. These were divided into two groups named the control group and experimental group, and it was ensured that the number of cells in each group was larger than 2×10 7 . The PLSCR4 promoter sequence probe was synthesized and labeled according to the NCBI gene library information, and two sets of cellular proteins were extracted for the DNA pull-down.
After the probe of coupling biotin bonded with the magnetic beads that coupled streptomycin, the probe reacted with the sample, and the protein bound to the probe was obtained. After the one-dimensional or twodimensional electrophoresis, the gel strips and dots of the target protein were obtained, and the in-gel enzymatic hydrolysis method was optimized. If there was a clear difference between the experimental group ( exp group ) and control group ( ctrl group ), mass spectrometry was performed. In this study, LC-MS/MS technology was used to analyze and obtain relevant information, such as the charge and peak map of the protein fragmentation fragments, and the Proteinpilot database software was used to perform bioinformatics analysis on the obtained results. The mass spectrometry analysis result and the silver staining result was analyzed together to obtain the protein information on the target gel point, which might be the transcription factor of PLSCR4.

Statistical analysis
Each group of experiments was repeated for at least three times. The experimental data were expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. All data were analyzed and processed using the SPSS 22.0 statistical software. P<0.05 indicated that the difference was statistically signi cant.

Results
PLSCR4 is closely correlated to the pyroptosis and permeability of the HPMECs.
In recent years, research on cell death methods has revealed a new type of programmed and in ammatory death-cell pyroptosis (14). The characteristic pathological changes of cell pyroptosis are accompanied by secretion of IL-1β and IL-18 release (15,16), and damage to the integrity of the cell membrane. Hence, cell pyroptosis is also called cell in ammatory necrosis (17,18). In order to verify the change of PLSCR4 expression in vascular endothelial cells and its correlation with the degree of pyroptosis and permeability change, HPMECs were divided into four groups, and seeded into six-well plates. Except that the rst group, which was the control group, the remaining three groups were treated with LPS, PLSCR4 siRNA + LPS, and the transfection reagent, respectively. The PLSCR4 siRNA sequence (5' to 3') was GGAAGUGGAAUGGUUUGUUTT. After four hours of LPS stimulation, western blot was performed to detect the changes in the expression of PLSCR4. At the same time, the supernatant of each group were collected, and ELISA was performed to detect the concentration of IL-1β and IL-18. The Tracer Flux Assay was used to detect the HPMEC permeability changes.
After four hours of LPS stimulation, the expression level of PLSCR4 in the LPS group signi cantly increased, when compared to the control group, which is consistent with the previous high-throughput sequencing results ( Fig. 2A). After the PLSCR4 siRNA silencing, the expression level of PLSCR4 decreased, and the expression of PLSCR4 in the transfection reagent group indicated that the transfection reagent had almost no effect on the HPMECs. The ELISA results revealed that after LPS stimulation, the concentration of IL-1β and IL-18 in the cell culture medium signi cantly increased, compared to the control group. Furthermore, the increase in PLSCR4 siRNA + LPS group was more signi cant ( Fig. 2B and 2C), suggesting that PLSCR4 may have the effect of reducing the release of IL-1β and IL-18 from HPMECs. The Tracer ux assay results revealed that the permeability of HPMECs in the LPS group signi cantly increased compared to the control group, and it increased more signi cantly in the group of PLSCR4 siRNA + LPS (Fig. 2D). These results above showed that with the decrease in PLSCR4 expression, the degree of in ammatory response of pyroptosis in HPMECs after the LPS stimulation, and the permeability of endothelium increased, suggesting that PLSCR4 may plays an important anti-in ammatory role in pyroptosis, and has a function of maintaining the endothelium permeability.
PLSCR4 alleviates pyroptosis through PS, but has no effect on the proteins of the pyroptosis pathway.
In order to verify the mechanism of PLSCR4 upon pyroptosis, HPMECs were grouped and seeded as above. The distribution of PS on the cell surface and the integrity of the cell membrane were observed using the uorescence method. The binding characteristics of Annexin V-FITC (green uorescence) with PS were used to detect the amount of PS on the cell membrane surface. PI (red uorescence) was used to stain the nuclear DNA to re ect the integrity of the cell membrane. Western blot was performed to detect the effects of PLSCR4 on the proteins referring to pyroptosis.
Compared with the LPS group, the green uorescence of Annexin V-FITC on the HPMECs surface of the PLSCR4 siRNA + LPS group decreased, while the red uorescence of PI increased (Fig. 3A). However, there was no obvious change in the control group and transfection agent group, suggesting that when the expression of PLSCR4 was too low to transport PS outside of the cell membrane, the destruction of the cell membrane of HPMECs increased, and N-GSDMD might form more transmembrane pores, which was also consistent with the increase in the concentration of IL-1β and IL-18 in the cell culture medium. The western blot results revealed that when the expression level of PLSCR4 changed, the expression of pyroptosis-related proteins were not affected (Fig. 3B), suggesting that PLSCR4 could reduce the pyroptosis degree of HPMECs.
Hence, it was presumed that PLSCR4 could reduce pyroptosis of HPMECs by transferring PS outside of the cell membrane, with no signi cant effect on the proteins of the pyroptosis pathway.

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DNA pull-down and protein pro ling technology to study the transcription factor of PLSCR4.
In order to improve further understanding of upstream mechanism of PLSCR4, our study investigated the upstream transcription factor of PLSCR4 using DNA pull-down and protein pro ling technology.
The silver staining results for the control group and experimental group revealed that there was a signi cant difference between these two groups at the 18.4 KDa molecular weight (Fig. 4). In the protein pro ling experiment, when the reliability was set to conf ≥ 95% and unique peptides ≥ 1, the number of secondary spectra generated by the sample mass spectrometer were 6,830 and 2,765 respectively, and the number of resolved secondary spectra were 1,685 and 423. Common contaminated proteins and matching peptides were ltered out. The overall situation of the proteins identi ed by the sample from control group are presented in Table 1, and the information of the proteins from experimental group are presented in Table 2 with a molecular weight of 18.4 KDa was obtained ( Table 2). Unused: protein identi cation score; Coverage: conf ≥ 95% protein coverage; Spectrum: total spectrum amount; Uniq spectrum: unique spectrum amout. Unused: protein identi cation score; Coverage: conf ≥ 95% protein coverage; Spectrum: total spectrum amount; Uniq spectrum: unique spectrum amout. * means the protential transcription factor P62280 may be involved as a transcription factor in the protective effect of PLSCR4 on HPMECs during ARDS.
In order to verify the results for the DNA pull-down and protein pro ling, and determine whether P62280 can be used as an upstream transcription factor to exert endothelial protection by regulating the expression of PLSCR4, HPMECs were divided into four groups, named the control group, LPS group, P62280 siRNA + LPS group, and transfection reagent group.The P62280 siRNA sequence (5' to 3') was CCAAACGACTTGGACGATGTT, and the antibody of P62280 was purchased frome Abcam. Western blot was used to detect the expression of PLSCR4 and the pyroptosis-related proteins. ELISA was used to detect the degree of pyroptosis. Tracer Flux Assays were used to detect the permeability of endothelial cells during the pyroptosis. Finally, the uorescence experiment was used to detect the degree of cell membrane integrity and the expression of PS on the cell surface after P62280 silencing.
After silencing P62280 by siRNA transfection, LPS continuously stimulated the HPMECs for four hours. It was found that the expression of PLSCR4 was consistent with the change in P62280. The changes in pyroptosis-related proteins, GSDMD, N-GSDM D, Caspase-1 and Caspaer-1 P20 were not affected by P62280 (Fig. 5A), suggesting that P62280 can regulate the expression level of PLSCR4, but has no signi cant effect on the classical pathway of pyroptosis.
When P62280 was silenced, the concentration of IL-1β and IL-18 in culture medium increased ( Fig. 5B and   5C), and the permeability was also signi cantly higher than that in the LPS group (Fig. 5D), suggesting that when the P62280 expression was reduced, the degree of pyroptosis increased, and the damage on barrier function of the membrane was aggravated. The uorescence experiments revealed that when P62280 was silenced, the PS exposure on the surface of the pyroptosis cell membrane was reduced, compared to the simple LPS group, and the nuclear staining increased (Fig. 5E), suggesting that the integrity of the cell membrane was worse. Based on these results, it can be speculated that PLSCR4 played a protective role when HPMECs were stimulated by LPS, which mainly acted to reduce the degree of in ammation of pyroptosis to protect the integrity of the cell membrane, and maintain the barrier function of HPMECs. This protective effect of PLSCR4 may be conducted by transferring the PS on the inner surface of the cell membrane to the outside, and that reduced the binding of PS to the pyroptosis executive protein N-GSDMD, thereby inhibiting the formation of pyroptosis pores on the cell membrane.

Discussion
The acute lung injury or ARDS caused by sepsis appears as diffuse lung in ammation and pulmonary interstitial edema. The key step is the microvascular barrier dysfunction caused by the lung microvascular endothelial cell injury (19,20). In the past, the mechanism of sepsis-induced pulmonary microvascular endothelial cell damage mainly focused on the endothelial cell death including necrosis, apoptosis, and cytoskeletal remodeling (21,22). However, the exact mechanism remains inconclusive. Using high-throughput sequencing in the preliminary experiments of the research group, it was found that the PLSCR4 mRNA expression signi cantly increased after LPS stimulated HPMECs for four hours. However, the speci c role and mechanism of PLSCR4 have not been reported yet.
PLSCRs are a class of single-pass transmembrane proteins that can transfer PS from the inside to the outside of the cell membrane in a Ca 2+ -dependent manner, increasing the exposure of PS. This process is involved in many pathophysiologies, such as cell phagocytosis, coagulation and in ammation, and plays an importang role (23)(24)(25). The research conducted by Wiedmer et al. con rmed that the expression levels of PLSCRs in humans and mice are different (6). Furthermore, there are more studies on PLSCR1-3 in the PLSCR family, while less research has been conducted on the speci c effect of PLSCR4 in humans. Our study revealed that when LPS stimulated the HPMECs, the expression of PLSCR4, the concentration of the in ammatory factors IL-1β and IL-18, and the endothelial permeability all increased. After the silencing of PLSCR4, HPMEC in ammation and permeability more signi cantly increased, suggesting that PLSCR4 in endothelial cells have a protective effect. Then the next work was to study the mechanism of the protective effect. Studies have reported that the expression level of PLSCRs is consistent with the exposure level of PS (26)(27)(28).
PS is the most abundant negatively charged membrane phospholipid in the cell membrane. When cell is infected, PS is transported outside of the cell membrane by PLSCRs. The increased exposure of the outside of the PS membrane is correlated to various pathophysiological processes, such as apoptosis, pyroptosis and necroptosis. Among which, studies have revealed that PS is involved in the anchor of the pyroptosis executive protein GSDMD in the cell membrane. Hence, we inferred that PLSCR4 might play an important effect in the pyroptosis (29)(30)(31), especially in the early stage of infection (32). Pyroptosis is a new type of programmed cell death characterized with in ammation after necrosis and apoptosis (14). The main mechanism is that in the presence of noxious stimuli, caspase-1 is activated into its active form caspase-1 P20 (33).,Afterwards, caspase-1 P20 activates the proin ammatory IL-1β and IL-18 to their active forms.
Secondly, caspase-1 P20 cleaves the GSDMD into an active N-terminal fragment N-GSDMD, which speci cally binds to the inner PS(30) of the membrane and forms a homopolymer, a hydrophilic pores with a diameter of approximately 10-15 nm through the plasma membrane, destroying the integrity of the cell membrane. IL-1β and IL-18 are released to recruit other in ammatory cells, expanding the in ammatory response (34). Hence, pyroptosis is also called in ammatory necrosis of the cell, which can be detected for positive Annxin V-FITC staining. However, propidium iodide (PI) can stain the cell nucleus through the pores formed by N-GSDMD during pyroptosis, together with signi cant in ammatory response, which did not normally happen in the apoptosis. At the same time, unlike necrosis, the cell morphology usually remains normal in the early stage of pyroptosis (35), referring to the cellular defense response (36). Recent studies have reported that when stimulated by LPS, pulmonary vascular endothelial cells undergo extensive cell pyroptosis (37)(38)(39). Therefore, determining whether PLSCR4 exerts a protective effect on endothelial cells by regulating the distribution of PS has become the main concern of this study. In the results, it was found that when LPS stimulates HPMECs, with the increase in PLSCR4 expression, the Annexin V-FITC staining intensity of PS increased, and so did the nucleus staining with PI. When PLSCR4 was silenced, the PS exposure decreased, and the PI staining of the nucleus signi cantly increased, which is consistent with the change in in ammatory factors in the cell culture medium. This shows that when the expression of PLSCR4 decreases and PS could not be transported to the outside, the degree of pyroptosis of HPMECs increases. When the experiment used DNA pull-down and protein pro ling experiments to further improve the mechanism of PLSCR4, it was found that PLSCR4 may be regulated by P62280 when stimulated by LPS.
In conclusion, our study is the rst to reveal the protective effect of PLSCR4 and its mechanism. It was presumed that by transporting PS to the outside of the cell membrane, PLSCR4 could reduce the binding of the pyroptosis executive protein N-GSDMD to PS and the formation of pyroptosis pores, thereby reducing the degree of pyroptosis. The further discovery revealed that the expression of PLSCR4 mainly regulates by P62280. It is hoped that the experimental results of the present study can provide a new focus for studying endothelial cell damage, and new ideas for nding a solution for the clinical treatment of ARDS.   The SDS-PAGE silver staining results for the two groups of protein samples after DNA pull-down. The ctrl group is a blank control group. The exp group is the experimental group. The bead group is the blank magnetic bead control group without the DNA probe. The results revealed that at a molecular weight of 18.4

Abbreviations
KDa, the exp group was signi cantly different from the other groups. Pyrodeath test revealed that the P62280 siRNA group had a higher degree of pyrolysis than the LPS group,