MAGNESIUM SULFATE AMELIORATES HISTONE-INDUCED COAGULATION DYSFUNCTION AND LUNG DAMAGE IN MICE

ABSTRACT Introduction: Extracellular histones have been determined as significant mediators of sepsis, which can induce endothelial cell injury and promote coagulation activation, and ultimately contribute to multiorgan failure. Evidence suggests that magnesium sulfate (MgSO4) exerts a potential coagulation-modulating activity; however, whether MgSO4 ameliorates histone-induced coagulation dysfunction and organ damage remains unclear. Methods: To measure circulating histone levels, blood specimens were collected from septic patients and mice, and the relationship between circulating histone levels, coagulation parameters, and Mg2+ levels in sepsis was investigated. Furthermore, to explore the possible protective effects of MgSO4, we established a histone-induced coagulation model in mice by intravenous histone injection. The survival rate of mice was assessed, and the histopathological damage of the lungs (including endothelial cell injury and coagulation status) was evaluated using various methods, including hematoxylin and eosin staining, immunohistochemistry, immunofluorescence, electron microscopy, and quantitative polymerase chain reaction. Results: The circulating histone levels in septic patients and mice were significantly associated with several coagulation parameters. In septic patients, histone levels correlated negatively with platelet counts and positively with prothrombin time and D-dimer levels. Similarly, in cecal ligation and puncture mice, histones correlated negatively with platelet counts and positively with D-dimer levels. Interestingly, we also observed a positive link between histones and Mg2+ levels, suggesting that Mg2+ with anticoagulant activity is involved in histone-mediated coagulation alterations in sepsis. Further animal experiments confirmed that MgSO4 administration significantly improved survival and attenuated histone-mediated endothelial cell injury, coagulation dysfunction, and lung damage in mice. Conclusion: These results suggest that therapeutic targeting of histone-mediated endothelial cell injury, coagulation dysfunction, and lung damage, for example, with MgSO4, may be protective in septic individuals with elevated circulating histone levels.


INTRODUCTION
Sepsis is the result of severe infection caused by microbial invasion, thereby leading to a dysregulated systemic host response.Despite advances in defining the pathogenesis of sepsis, it remains a leading cause of death in critically ill patients (1).In sepsis, a dysregulated host immune response causes the acute activation of coagulation, microthrombosis, and platelet (PLT) and coagulation factor depletion, ultimately contributing to disseminated intravascular coagulation (DIC) and multiorgan failure (MOF) with no specific treatment currently available (2).Therefore, we aimed to explore a promising and aggressive therapeutic means of targeting pathological coagulation in sepsis (3).
Recent advances demonstrate that extracellular histones are significant mediators of sepsis, with the ability to induce endothelial cell injury, activate PLTs, and promote thrombin formation, which are responsible for DIC and MOF (4)(5)(6).The mouse model of extracellular histone-induced injury is characterized by endothelial damage and excessive coagulation activation, with increased concentrations of tissue factor (TF) and von Willebrand factor (vWf ), bearing a strong resemblance to sepsis-associated coagulopathy (2,7).In addition, circulating histone levels are higher in septic patients who develop DIC (6); however, the relationship between circulating histones and laboratory markers reflecting coagulation in septic patients and mice has not been reported.Therefore, antagonizing the coagulation dysfunction and MOF mediated by histone toxicity is probably an effective strategy for the treatment of septic patients.
Magnesium (Mg 2+ ) is an essential nutrient that maintains normal cellular physiological activity and homeostasis in the body through more than 300 biochemical reactions (8,9).Previous studies on Mg 2+ and magnesium sulfate (MgSO 4 ) have shown an anticoagulant effect with reduced PLT aggregation, blood clotting, and thrombosis (2,(10)(11)(12).A retrospective observational study has hinted that low Mg 2+ levels are associated with active coagulation and inhibition of fibrinolysis in septic patients; however, whether Mg 2+ is a factor affecting coagulation in sepsis is unclear (13).A randomized controlled trial investigated the impact of MgSO 4 on coagulation properties in gynecological patients undergoing pelvic surgery and observed that maintaining Mg 2+ levels at the upper end of the normal range by MgSO 4 administration attenuated postoperative hypercoagulability (12).
As previously mentioned, MgSO 4 can affect blood coagulability.However, there are no reports concerning whether MgSO 4 affects the coagulation system during histone-mediated lethal coagulation abnormalities.Uncovering the role of MgSO 4 may lead to the development of new therapeutic strategies.Therefore, in this study, we aimed to explore the "MgSO 4 -extracellular histone-coagulation" relationship, preliminarily evaluating the correlation between circulating histone levels, coagulation parameters, and Mg 2+ levels in septic patients and mice through clinical data and animal experiments, and further confirming whether MgSO 4 interferes with histone-mediated coagulation abnormalities and organ damage.

Clinical data
Blood specimens of patients diagnosed with sepsis on the basis of SEPSIS-3 were obtained from Zhujiang Hospital of Southern Medical University (14).Inclusion criteria were patients with life-threatening organ dysfunction arising from an uncontrolled host response to infection, and Sequential Organ Failure Assessment (SOFA) score ≥2.Exclusion criteria were as follows: 1) patients younger than 18 years and older than 80 years, 2) patients with solid organ or bone marrow transplant, and 3) patients taking long-term immunosuppressive drugs or with immunodeficiency.For clinical samples stored in the biobank, all septic patients signed an informed consent form, and the experimental design and research were approved by the Institutional Ethics Committee (2023-KY-153-01).
Circulating histone levels in patients were detected using a Human Histone ELISA assay kit (Jingmei Biotechnology, Jiangsu, China) following the manufacturer's guidelines.Data on coagulation parameters (PLT counts, prothrombin time [PT], activated partial thromboplastin time [aPTT], fibrinogen, and D-dimer) and Mg 2+ levels in the blood of septic patients examined on admission were retrospectively obtained from the laboratory information system.Notably, serum Mg 2+ tests were not performed on admission in five patients, who were thus excluded from the analysis of the relationship between histones and Mg 2+ levels.

Animals
Specific pathogen-free male C57BL/6 mice (aged 8-10 weeks) were kept in ventilated cages at 22°C to 24°C with free access to food and water under normal 12-h/12-h dark/light cycles.Mice were acquired from Guangdong Medical Laboratory Animal Center (Guangzhou, China).Experimental procedures were performed according to international ethical guidelines and authorized by the Institutional Animal Care and Use Committee of Zhujiang Hospital, Southern Medical University (LAEC-2022-071FS).

Histone-induced coagulation model in mice
To explore the possible protective effects of MgSO 4 , we established a histone-induced coagulation model in mice as previously described (5,7).For survival assessment, mice were randomly allocated into the following four groups: the MgSO 4 group intravenously received 50 mg/kg of MgSO 4 (Macklin, Shanghai, China) in the tail vein, the histone group intravenously received 75 mg/kg of histones (Sigma, Santa Clara, CA), the histone + MgSO 4 group intravenously received histones (75 mg/kg) and MgSO 4 (50 mg/kg) simultaneously, and the control group intravenously received equal amounts of sterile saline.Regarding drug dosage, it has been reported that 75 and 50 mg/kg of histones are lethal and sublethal doses, respectively, both of which enable mice to achieve circulating histone H3 levels identical to septic patients and mice (7,15).In addition, the MgSO 4 dose was derived from previous studies, indicating that 50 mg/kg of MgSO 4 could maintain serum Mg 2+ levels at the upper end of the normal range but not at toxic levels (12,16).
To evaluate the histopathological damage of the lungs, mice were randomly assigned into the following three groups: the histone group intravenously received 50 mg/kg of histones, the histone + MgSO 4 group intravenously received histones (50 mg/kg) and MgSO 4 (50 mg/kg) simultaneously, and the control group intravenously received equal amounts of sterile saline.Three hours after histone challenge, mice were euthanized, and the lungs were harvested for further investigation.

Cecal ligation and puncture + histone-induced injury model in mice
Cecal ligation and puncture (CLP) was performed as previously described to induce polymicrobial sepsis in mice (17,18).Briefly, mice were anesthetized, and the abdominal midline was dissected under aseptic conditions to expose the cecum.The distal half of the cecum was ligated, and the cecum was then perforated with an 18G needle to extrude a small amount of fecal material from the perforated site.Next, the cecum was placed back into the abdominal cavity, and the peritoneum was closed.All mice were resuscitated by subcutaneous injection of 1 mL sterile saline.CLP mice were randomly allocated into the following three groups: the CLP + histone group intravenously received 20 mg/kg of histones in the tail vein after CLP, the CLP + histone + MgSO 4 group intravenously received 20 mg/kg of histones and 50 mg/kg of MgSO 4 simultaneously after CLP, and the CLP group intravenously received equal amounts of sterile saline after CLP.Of note, 20 mg/kg histone can enable mice to achieve circulating histone H3 levels in the range of those found in septic patients and mice (7,15).Blood was collected 24 h after CLP, and laboratory examinations (including coagulation parameters [PLTs, PT, aPTT, fibrinogen, and D-dimer], plasma histone levels, and serum Mg 2+ levels) were performed.PLT values were measured using a blood cell analyzer BC6000 (Mindray, Shenzhen, China).PT, aPTT, and fibrinogen values were detected on a Sysmex CS5100 (Sysmex, Kobe, Japan).Serum Mg 2+ levels were examined on a Cobas c702 analyzer (Roche Diagnostics, Mannheim, Germany).According to the manufacturer's guidelines, circulating histone and D-dimer levels in mice were detected using a Mouse Histone ELISA assay kit and a Mouse D-dimer ELISA assay kit (Jingmei Biotechnology), respectively.

Histopathological analysis
The lower lobe of the left lung in mice was cut and fixed in 4% paraformaldehyde solution for 24 h.The paraffin-embedded tissue was cut into 4 μm thick and stained with hematoxylin and eosin.The severity of lung damage was assessed on the basis of alveolar wall thickening, alveolar congestion, hemorrhage, and inflammatory cell infiltration with a score of 0 to 3, as we previously reported (19).Moreover, freshly obtained samples of the right upper lobe of the lungs were weighed and subsequently put in an oven at 65°C for 24 h and weighed again while they were dried to determine the wet/dry weight ratio (wet/dry weight) and lung water content ([wet − dry weight]/wet [%]).
Fluorescent staining was used for cell injury determination, whereby TUNEL assays were conducted on frozen sections using a commercial kit (Beyotime, Shanghai, China) following the manufacturer's instructions.

Immunohistochemical staining
Immunohistochemical staining was performed as previously described (20).Paraffin-embedded tissue slides were dewaxed and rehydrated.After blocking with goat serum, each slide was incubated with anti-vWF (ProteinTech Group, Chicago, IL) or fibrinogen-β antibody (Affinity Biosciences, Cincinnati, OH) overnight at 4°C, followed by incubation with corresponding secondary antibody and streptavidin-coupled horseradish peroxidase (Fdbio Science, Hangzhou, China).After washing three times, each slide was visualized after diaminobenzidine (ZSGB-BIO, Beijing, China) staining and hematoxylin counterstaining.Images were captured and analyzed using a digital slide scanner and viewer software (Pannoramic MIDI/Case Viewer 2.3; 3D Histech, Budapest, Hungary).

Transmission electron microscopy
The tissue was fixed in transmission electron microscopy (TEM) fixative (4°C), followed by fixation with 1% OsO 4 in 0.1 M phosphate buffer solution for 2 h (20°C-23°C).After gradient dehydration, the tissue was resin embedded, cut into 70 nm thin, and stained with 2% uranium acetate saturated alcohol solution.Subsequently, sections were washed with 70% ethanol and ultrapure water and stained with 2.6% lead citrate.Image acquisition was performed on a transmission electronmicroscope HT7800 (HITACHI, Tokyo, Japan).

Experimental design of human umbilical vein endothelial cells
We seeded human umbilical vein endothelial cells (HUVECs) at a confluence of approximately 80% per well and stimulated them with histones (100 μg/ mL) + MgSO 4 (0-10 mM) for 1 h to detect the protective effects of MgSO 4 on HUVECs.The cell viability of HUVECs was measured using the cell counting kit-8 (CCK-8) kit (GlpBio, Montclair, CA).The apoptosis analysis of HUVECs was performed using the annexin V-FITC/PI Apoptosis Detection Kit (Dojindo, Kumamoto, Japan), and the percentage of apoptosis in HUVECs was determined on a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA).The extracellular Mg 2+ levels of HUVECs were measured using a Cobas c702 analyzer (Roche Diagnostics, Mannheim, Germany).To detect the intracellular Mg 2+ levels, HUVECs were loaded with 2.5 μM Mag-Fluo-4/AM (the Mg 2+ -specific fluorescent dye; Maokang Biotech, Shanghai, China) for 20 min and then subjected to histones (100 μg/mL) + MgSO 4 (10 mM) for 60 min.The cells were analyzed by a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA).

Quantitative reverse transcription polymerase chain reaction assay
Total RNA was obtained from lung tissue homogenates using AG RNAex Pro Reagent (AG, Changsha, China) following the manufacturer's instructions.Subsequently, cDNA was acquired using Evo M-MLV RT Premix (AG).After mixing the synthesized cDNA, SYBR Green Pro Taq HS Premix (AG), and primers mentioned in Table S1, http://links.lww.com/SHK/B816,quantitative reverse transcription polymerase chain reaction was performed on QuantStudio 3 (Applied Biosystems, Foster City, CA).

Bioinformatics analysis
To evaluate which biological processes of histones and Mg 2+ are involved in the pathogenesis of sepsis, bioinformatic analysis was performed (21).Putative targets associated with MgSO 4 and histones (main pathogenic types: H3 and H4 (5,22)) were harvested from the DrugBank, STITCH, or SuperPred databases.The targets in sepsis were used from web-based GeneCard and DisGeNET databases.The criteria for the selection of sepsis targets in the DisGeNET database were identified as gene-disease association score >0.1, whereas that in the GeneCard database was gene score >1.The correlative targets between MgSO 4 , histones, and sepsis were obtained using Venn diagrams.Subsequently, to explore the underlying biological processes between MgSO 4 , histones, and sepsis, Gene Ontology biological process analysis of the identified correlative targets was performed.

Statistical analysis
All calculations were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA).Data were expressed as means ± SDs.Discrepancies between groups were evaluated using one-way analysis of variance followed by Tukey's post hoc test.Survival studies were explored using the log-rank test.A P value <0.05 was considered statistically significant.

Circulating histone levels in relation to coagulation parameters in septic patients and mice
Circulating histone levels are associated with poor clinical outcomes in septic patients (7,23,24).However, the relationship between circulating histone levels and coagulation parameters in septic patients and mice remains unclear.To analyze the relationship between circulating histones and coagulation function, we collected blood specimens from 56 septic patients in the intensive care unit of Zhujiang Hospital of Southern Medical University.The coagulation function was assessed on the basis of several significant laboratory markers, including PLT counts, PT, aPTT, fibrinogen, and D-dimer.The results indicated a significant correlation between histone levels and coagulation parameters in these septic patients, among which histones were negatively correlated with PLTs and positively with PT and D-dimer (Fig. 1).Furthermore, we made similar observations in CLP mice, a widely used septic model, which indicated that histones were negatively correlated with PLTs and positively with D-dimer (Fig. 2).These outcomes propose that histones may be involved in coagulation changes in septic patients.
MgSO 4 may play a significant role in histone-induced septic injury through coagulation-related biological process regulation Our further analysis showed a positive correlation between circulating histone and Mg 2+ levels in septic patients and mice (Fig. 3, A  and B).In addition, serum Mg 2+ levels were significantly higher in mice injected intravenously with 50 mg/kg histones (Fig. 3C).To evaluate which biological processes of histone and Mg 2+ are involved in the pathogenesis of sepsis.We determined the correlative targets of MgSO 4 , histones (main pathogenic types: H3 and H4 (5,22)), and sepsis through bioinformatics methods.Further Gene Ontology analysis showed that MgSO 4 may play an important part in histone-induced septic injury through coagulation-related biological process regulation (Fig. 3, D and E).

MgSO 4 protects mice against histone-induced death
We assessed the significance of MgSO 4 in vivo by challenging the mice with an intravenous injection of histones.Consistent with a previous study (7), mice challenged with histones (75 mg/kg, lethal doses) suffered from oronasal hemorrhage and respiratory distress and subsequently succumbed within 1 h, whereas 50% of mice treated with 50 mg/kg MgSO 4 (histone + MgSO 4 group) remained alive beyond 6 h (Fig. 4A).After histone stimulation (50 mg/kg, sublethal doses), pathological symptoms of mice were observed using a composite clinical scoring method with a double-blind design (25,26) (Table S2, http://links.lww.com/SHK/B816).Moreover, resistance to histone attack was evident 3 h after MgSO 4 administration, with a 12-fold decrease in the composite clinical score in mice of the histone + MgSO 4 group compared with those of the histone group (Fig. 4B).As indicated in Figure 4C and D which were maintained at normal levels during histone stimulation.To investigate the effect of MgSO 4 on histone-induced injury in CLP septic mice, we established a composite model of CLP mice with an intravenous injection of histones.We found that circulating histone levels were significantly higher in CLP mice after intravenous administration of 20 mg/kg histones (Fig. S1A, http://links.lww.com/SHK/B816).In addition, histone stimulation induced decreased survival and abnormal coagulation (decreased PLTs, increased PT and D-dimer) in CLP mice (Fig. 4E; Fig. S1B-F, http://links.lww.com/SHK/B816).As expected, MgSO 4 supplementation improved histone-mediated death and coagulation abnormalities in CLP mice (Fig. 4E; Fig. S1B-F, http://links.lww.com/SHK/B816).Taken together, these findings support the idea that MgSO 4 is potentially an effective adjunct for the treatment of histone-induced death.

MgSO 4 protects mice against histone-mediated histopathological injury in the lungs
Histopathological analysis revealed that the lungs of MgSO 4treated mice had fewer pathological changes, including inflammation, thrombosis, and hemorrhage, than those of mice with only histone stimulation (Fig. 5, A-C).Consistent with these findings, the wet/ dry ratio and water content of the pulmonary lobe were significantly lower after MgSO 4 treatment (Fig. 5, D and E).Furthermore, MgSO 4 treatment dramatically decreased the abundance of histone-mediated apoptotic cells in the lungs (Fig. 5F).These results indicate that MgSO 4 can mitigate histone-induced lung damage.

MgSO 4 reduces histone-induced endothelium injury and thrombogenesis
To identify the impact of MgSO 4 on endothelial cell injury and thrombogenesis, the vWF (a significant marker reflecting endothelial cell injury) expression was detected via immunohistochemistry, and thrombus-associated morphological manifestations were examined using TEM.High vWF expression was observed in the lung tissues of the histone group, whereas vWF expression was remarkably decreased upon MgSO 4 addition (Fig. 6A).Similar to previous studies (7), TEM findings showed that histone stimulation elicited microvascular damage, including reduced endothelial cells, substantial fibrin, and PLT aggregation (Fig. 6B).However, MgSO 4 treatment significantly reduced histone-induced endothelial cell injury and thrombogenesis.To extend the results in vivo, we also explored the protective role of MgSO 4 against histone-induced cellular damage in an endothelial cell model.To examine the viability of HUVECs, CCK-8 tests and flow cytometry were performed and showed that MgSO 4 significantly reduced histone-induced HUVECs death in a dose-dependent manner, with as low as 0.5 mM effective (Fig. 7, A-C).Interestingly, we found that histones induced an increase in extracellular Mg 2 + levels and a decrease in intracellular Mg 2+ levels of HUVECs (Fig. 7, D-E), which is consistent with previous findings that histones stimulated a significant increase in serum Mg 2+ levels in mice.However, MgSO 4 supplementation restored normal intracellular Mg 2+ levels of HUVECs (Fig. 7E).These data illustrate that MgSO 4 plays a protective role in histone-mediated endothelial cell injury and thrombogenesis.
3 h of histone stimulation compared with those of control mice.However, MgSO 4 treatment improved histone-induced coagulation and fibrinolytic imbalances.Immunohistochemical findings of fibrinogen (a significant indicator of thrombus status) in the lung tissues further confirmed gene expression results (Fig. 8E).These results showed that fibrin deposition and thrombus formation were extensive in the lung tissues of mice with histone infusion, and MgSO 4 treatment effectively alleviated fibrin deposition and thrombus formation.Taken together, this study presents a potential therapeutic benefit of MgSO 4 in histone-mediated coagulation dysfunction and lung damage.

DISCUSSION
Here, we report that circulating histones in septic patients and mice may be associated with the coagulation process.Intriguingly, we also observed a positive correlation between circulating histones and Mg 2+ levels in septic patients and mice.Studies have reported an increase in serum Mg 2+ levels upon exposure to acute stress stimuli (including poisoning, hypotension, and vomiting) (29,30).Therefore, we speculate that serum Mg 2+ may be related to histone-mediated stress injury in sepsis.Bioinformatic analysis of the "Mg 2+ -histonesepsis" triad suggests that Mg 2+ may influence histone-mediated injury in sepsis through coagulation-related biological process regulation.Our further animal studies demonstrate that histones can induce coagulation activation and widespread thrombosis, and MgSO 4 might offer excellent protection against histone-mediated coagulation dysfunction and lung damage, offering an alternative rationale for treating septic patients with MgSO 4 .
The dysregulated immune response in septic patients can cause coagulation activation, thrombosis, and massive PLT and coagulation factor depletion, which manifest as low PLT counts, prolonged PT, and increased D-dimer concentrations, ultimately leading to DIC and MOF (2,31,32).Extracellular histones have been proposed as significant mediators in sepsis pathogenesis, with several experiments demonstrating that extracellular histones can induce endothelial cell injury and impaired coagulation (5,33).In mouse models, intravenous histone administration provokes perturbations in the coagulation system similar to sepsis-induced consumptive coagulopathy and DIC, including endothelial damage, increased concentrations of TF and vWf, subsequently leading to PLT activation, extensive thrombosis, and coagulation factor depletion, thereby presenting as low PLT counts and prolonged PT and aPTT (2,24,(34)(35)(36).One study reported that circulating histone levels were higher in septic patients who developed DIC (6); however, the relationship between histones and various significant coagulation parameters (e.g., PLT counts, PT, aPTT, D-dimer, and fibrinogen) remains unclear.In our study, clinical data showed that histone concentrations in septic patients were related to the coagulation process, with histone concentrations negatively correlating with PLT counts and positively with PT and D-dimer values.Furthermore, animal experiments indicated that histone concentrations in CLP mice were also related to the coagulation process, with histone concentrations negatively correlating with PLT counts and positively with D-dimer values.These data suggest that histones may play a significant role in sepsis-induced coagulopathy.
Animal studies have shown that increased serum Mg 2+ levels occur after acute stress stimulus exposure (20,30).We observed a positive correlation between circulating histones and Mg 2+ levels in septic patients and mice, and a significant increase in serum Mg 2+ levels in mice injected intravenously with histones, which might be related to histone-mediated stress damage.Our animal experiments confirm that histones can induce procoagulant reactions and widespread thrombosis, and MgSO 4 treatment can significantly alleviate histone-induced coagulation dysfunction and lung damage.The possible protective mechanisms of MgSO 4 are summarized as follows: 1. Excessive endothelial cell injury and subsequent exposed TFs can induce the onset of coagulation dysfunction (24,37,38).Our data showed that serum Mg 2+ levels were significantly higher in mice injected intravenously with histones.Further experiments confirmed that histones induced a decrease in intracellular Mg 2+ levels and an increase in extracellular Mg 2+ levels of endothelial cells, which might then be damaged due to the lack of intracellular Mg 2+ .Studies have shown that exogenous Mg 2+ supplementation promotes Mg 2+ influx and increases intracellular Mg 2+ levels (39-41).Similarly, we observed that MgSO 4 supplementation increased intracellular Mg 2+ levels in histone-stimulated endothelial cells.Therefore, increased intracellular Mg 2+ levels in endothelial cells may maintain cell stability, reduce endothelial cell injury, and ultimately improve coagulation function.2. Studies have confirmed the role of inflammation in promoting coagulation (13,42).MgSO 4 , as an anti-inflammatory agent (43)(44)(45), may modulate coagulation dysfunction by inhibiting inflammation.3. Calcium (coagulation factor IV) functions as a cofactor throughout the coagulation cascade (13).As a calcium antagonist, Mg 2+ may regulate coagulation activation by controlling calcium activity (12).As previously mentioned, MgSO 4 may alleviate histone-mediated coagulation dysfunction and lung damage through several biological activities.However, the underlying mechanisms by which MgSO 4 ameliorates histone-induced injury have not been well understood and need further investigation.
Previous studies have demonstrated that heparin and recombinant human-activated protein C have antagonistic effects on histone toxicity and can alleviate histone-induced coagulation activation and organ damage (5,7).However, the use of heparin for sepsis may carry a risk of bleeding (24,46).Furthermore, activated protein C is ineffective in the treatment of sepsis and presents a significant risk of bleeding; therefore, it is not recommended for use in septic patients (42,47).Our animal studies have indicated that MgSO 4 alleviates histone-induced lethal thrombosis and lung damage, and no bleeding adverse effects were observed throughout the treatment.Notably, it has been reported in the literature that Mg 2+ may regulate coagulation dysfunction and play a protective role in certain bleeding disorders, including intracerebral hemorrhage (48,49).Owing to its versatility, cheapness, and established safety profile as a commonly used clinical drug, MgSO 4 could be used as a therapy for sepsis (sepsis-induced coagulopathy).A randomized controlled study suggested that MgSO 4 accelerates lactate clearance in septic patients and may help improve the outcome of sepsis management (50).However, whether MgSO 4 improves sepsis prognosis and exerts a protective effect by inhibiting histone-induced coagulation dysfunction remains to be further demonstrated.
This study had some limitations.First, it was not designed to investigate the molecular mechanisms by which MgSO 4 affects histone-induced coagulation dysfunction.Second, we only focused on the short-term effects of MgSO 4 ; therefore, long-term effects remain unclear.Third, although MgSO 4 is effective in improving coagulation dysfunction in histone-induced and CLP + histone-induced injury model mice, its protection against coagulation in other sepsis-related models, such as the endotoxemia model, needs to be further estimated.Finally, although no bleeding was observed during the MgSO 4 treatment, which was not assessed by laboratory coagulation indicators, MgSO 4related adverse effects remain to be further evaluated.

CONCLUSIONS
These studies are the first to examine the protective effects of MgSO 4 against histone-mediated injury.Our findings show that MgSO 4 attenuates histone-mediated coagulation dysfunction and lung damage, suggesting that MgSO 4 may affect the coagulation system to reduce host susceptibility to histone-mediated death in sepsis.To verify whether proper MgSO 4 supplementation

FIG. 6 .
FIG.6.MgSO 4 reduces histone-induced endothelium injury and thrombogenesis.A, Immunohistochemical staining of the von Willebrand factor showed endothelial cell injury (n = 6).B, Morphological manifestations associated with thrombus examined using TEM (n = 2-3).TEM findings of the histone group showed reduced endothelial cells in the lumen of the microvessels, with large amounts of fibrin and PLTs aggregating and obstructing the vessels.The rough endoplasmic reticulum was dilated, and the membrane was broken.BM indicates basement membrane; BP, blood platelet; EC indicates endothelial cell; Fib, fibrocyte; FP, fibrin deposition; M, mitochondrion; PLT, platelet; RER, rough endoplasmic reticulum; TEM, transmission electron microscopy; TJ, tight junction.