Tranexamic acid and erythropoietin protect the lungs on osteoporosis hip fracture-induced acute lung injury rats by inhibition of inflammatory response and endoplasmic reticulum stress

Tranexamic acid and erythropoietin protect the lungs on osteoporosis hip fracture-induced acute lung injury rats by inhibition of inflammatory response and endoplasmic reticulum stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tranexamic acid and erythropoietin protect the lungs on osteoporosis hip fracture-induced acute lung injury rats by inhibition of inflammatory response and endoplasmic reticulum stress Xi Liu, Dechuan Zhang, Mingjin Li, Huixu Ma, Jinhua Cai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6557105/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in European Journal of Trauma and Emergency Surgery → Version 1 posted 7 You are reading this latest preprint version Abstract The objective of this study was to evaluate the protective effects and elucidate the mechanism underlying Tranexamic acid (TXA) and erythropoietin (EPO) in osteoporosis hip fracture (OHF)-induced acute lung injury (ALI). Sprague-Dawley (SD) rats were randomly divided into the control, OHF, OHF + TXA, and OHF + EPO groups. Lung tissue was analyzed at 12, 24, and 48 h post-treatment to assess injury levels through hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. H&E staining showed that TXA and EPO significantly reduced the OHF-induced lung injury. Wet/dry ratios were identified in the OHF + TXA group and OHF + EPO group than in the OHF group. We found significantly lower levels of inflammatory cytokine production levels and nuclear factor-κB (NF-κB) signaling pathway activity in the lung of the OHF + TXA group and OHF + EPO group than in the hip fracture group. Moreover, TXA and EPO treatments counteracted mitochondrial injury and type II alveolar epithelial cells (AECⅡ) cell apoptosis in OHF rats. The treatment with TXA and EPO also exhibited inhibitory effects on the increase of ER stress-related apoptosis in lung tissue after OHF, as evidenced by reduced expression levels of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), phosphorylated (p)-protein kinase RNA-like endoplasmic reticulum kinase (PERK), p- endoplasmic reticulum-to-nucleus signaling 1 (IRE1)α, Caspase-12, activating transcription factor 4 (ATF4), Bax and induced expression of Bcl-2. Thus, TXA and EPO may have a protective effect on OHF-induced ALI. osteoporosis hip fracture acute lung injury tranexamic acid erythropoietin inflammation endoplasmic reticulum stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hip fractures represent the most common type of fracture among the elderly, predominantly due to reductions in hip bone density and the presence of osteoporosis [ 1 – 3 ]. As the global population continues to age, there has been a steady rise in the incidence of osteoporotic hip fractures [ 4 , 5 ]. Projections indicate that by the year 2050, the worldwide incidence of osteoporosis-related hip fractures will reach approximately 6.3 million, with over 50% occurring in Asia [ 6 , 7 ]. The disability rate and mortality rate of hip fractures in the elderly are very high [ 8 ]. Acute lung injury (ALI) is the main complication and main cause of death of hip fracture [ 9 ]. Prolonged bed rest in patients with hip fractures before and after surgery is considered to be a major risk factor for ALI after fracture [ 10 ]. Recent research indicates that the pathophysiological features of ALI resulting from hip fractures are predominantly characterized by the disruption of the pulmonary microvascular endothelial barrier, apoptosis of lung epithelial cells, and a pronounced inflammatory response [ 11 ]. Endoplasmic reticulum (ER) stress has been associated with a range of pulmonary diseases, including chronic obstructive pulmonary disease, ALI [ 12 ], lung cancer [ 13 ], pulmonary hypertension [ 14 ], and pulmonary fibrosis [ 15 ]. Furthermore, ER stress has been observed in the lung tissues of animals with ALI induced by different factors, suggesting its potential significance in the initiation and progression of ALI [ 16 ]. Tranexamic acid (TXA), a synthetic lysine derivative, demonstrates a strong affinity for plasminogen and the lysine-binding sites of plasminase [ 17 ]. This affinity facilitates its binding to the lysine residues on fibrin, thereby inhibiting the fibrinolytic system and promoting effective hemostasis in postoperative incisions. Research indicates that TXA also mitigates tissue stress following trauma, consequently reducing inflammation [ 18 ]. The combination of carbazochrome sodium sulfonate and TXA has been shown to significantly enhance perioperative hemostasis and reduce inflammation in patients undergoing total hip arthroplasty [ 19 ]. Furthermore, research by Peng et al. revealed that the administration of TXA via enema significantly mitigated pulmonary injury in rats experiencing hemorrhagic shock, primarily through the activation of syndecan-1 [ 20 ]. Erythropoietin (EPO), a hormone-like substance produced by the kidneys and liver, plays a critical role in stimulating the proliferation and differentiation of erythroid progenitor cells [ 21 ]. It is a vital hematopoietic growth factor necessary for the proper growth and development of red blood cells. Studies have demonstrated that EPO is widely distributed in non-hematopoietic tissues and cells, including those of the nervous system, cardiovascular system, kidneys, liver, uterus, endothelial cells, and solid tumors [ 22 ]. Furthermore, it is integral to various non-hematopoietic functions, including anti-apoptotic mechanisms, reduction of oxidative stress responses, suppression of excessive inflammatory reactions, and facilitation of angiogenesis processes [ 23 ]. As a result, researchers have identified the pleiotropic biological functions of EPO, which have been characterized as those of a systemic protective cytokine [ 24 ]. Consequently, EPO has been characterized as a systemic protective cytokine with pleiotropic biological functions. Building on insights from prior research, we propose the hypothesis that treatment with TXA and EPO may mitigate ALI induced by hip fractures through the attenuation of ER stress. The anti-inflammatory properties of TXA [ 25 ], combined with the multifaceted protective mechanisms of EPO—especially its anti-apoptotic and stress-reducing effects—indicate a synergistic potential to alleviate ER stress [ 26 ], thereby potentially reducing the severity of ALI. In the present study, to evaluate our hypothesis, rats were administered intravenous injections of either TXA or EPO prior to the induction of an osteoporosis hip fracture (OHF) model. Subsequently, these rats were assessed for changes in pulmonary injury, levels of inflammation-related factors in serum and lung tissue, and ER stress. Furthermore, we explored the molecular mechanisms through which TXA and EPO attenuate inflammation and ER stress in OHF rats. Methods and materials Animals Sixty-four female Sprague-Dawley (SD) rats, aged 8 months and of specific pathogen-free (SPF) grade, were procured from Dashuo Animal Experiment Co., Ltd., located in Chengdu, Sichuan. The rats were maintained under controlled environmental conditions, with a temperature of 25 ± 1°C, relative humidity between 50% and 60%, and a 12-h light/dark cycle. They were given ad libitum access to food and water. The study protocols received approval from the Ethics Committee of West China Hospital, Sichuan University (approval number: 20230413003) and conducted in accordance with established guidelines and regulations. The study followed ARRIVE guidelines. Model establishment and therapy The control group consisted of fifteen randomly selected rats. In the case of control group rats, ovaries were exposed but not resected. The remaining rats (49 rats in total) were utilized to establish an ovariectomized female rat model with osteoporosis. Specifically, the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg). A 1 cm incision was made at the junction located 1 cm below the ventral costal margin and 1 cm on either side of the spinal cord. Subsequently, bilateral ovaries were ligated and removed. After surgery, 40,000 units of penicillin were injected intramuscularly to prevent infection. Three rats died after ovariectomy. Five weeks post-ovariectomy, the right femurs were scanned with a dual-energy X-ray absorptiometer (DXA; Norland, London, UK) to measure femur bone mineral density (BMD). The specimens were placed in a thin-walled plastic container with a flat bottom and filled with normal saline solution (to ensure complete immersion of the specimen). After a 30-min incubation period, the entire femur segment was subjected to scanning using dual-energy X-ray technology equipped with small animal software, enabling measurement of BMD in g/cm 3 . After the osteoporosis model was successfully constructed, the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg) in the 4th week. After anesthesia, the rat was placed in a supine position on the base plate of a custom-made fracture percussion instrument. The right lower limb was positioned straight and supported by a steel plate to keep it parallel to the horizontal plane. Under real-time imaging from a C-arm, the intertrochanteric line of the right femur was accurately identified, and the percussion instrument was aligned to this target. A 500 g calibrated weight was then dropped from a height of 17 cm, precisely inducing a right hip fracture. Thus, the OHF rat model was successfully established (46 rats in total). Before the establishment of OHF model, rats in TXA group and EPO group were given TXA or EPO intervention. TXA Group: TXA (Tiancheng Pharmaceutical Co., Ltd., Changchun, China) was administered intravenously at a dose of 100 mg/kg, one injection per rat. EPO Group: EPO (Shanghai Kemo Biomedicine Co., LTD, Shanghai, China) was administered intravenously at a dose of 5000 U/kg, one injection per rat. Control Group: Normal saline was administered intravenously in equivalent volume to the control group rats There were 5 rats in each group. Following recovery, rats were housed in cages. Wet/dry (W/D) weight ratio At 12 h, 24 h, and 48 h after modeling, the lower lobe of the right lung was promptly weighed upon collection and subsequently subjected to oven drying at 60°C for a duration of 48 h. The tissue was reweighed post-desiccation to determine the W/D weight ratio, which served as a metric for assessing pulmonary vascular permeability. Hematoxylin and eosin (H&E) staining At 48 h after modeling, the lung tissue of rats was collected and fixed in 4% paraformaldehyde overnight, processed, and embedded in paraffin. The tissue sections, each measuring 5 µm in thickness, were initially placed in a water bath maintained at 49°C and subsequently affixed to slides coated with polylysine. The slides underwent a dewaxing process using toluene, applied twice, followed by dehydration with ethanol. They were then rinsed with distilled water and stained with hematoxylin. Post-washing, the sections were differentiated using 1% hydrochloric acid alcohol, stained with eosin, and subjected to a 10-min wash with distilled water. Finally, the sections were dehydrated using xylene and mounted with a neutral adhesive. The prepared tissue sections were examined under a digital trinocular camera microscope (BA210Digital, Motic, Beijing, China) at magnifications of 100× and 400×. After performing H&E staining, a previously published scoring system was employed to assess lung injury [ 27 ]. The scoring criteria are as follows: 0 points indicate an intact alveolar wall with no thickening, absence of inflammatory infiltrates, and no congestion; 1 point corresponds to mild diffuse infiltration of inflammatory cells in the alveolar wall without thickening; 2 points represent significant and extensive infiltration of inflammatory cells with slight thickening of the alveolar wall (1–2 times); 3 points reflect severe infiltration of inflammatory cells with a 2-3-fold increase in thickness of the alveolar wall in certain areas; and 4 points signify severe infiltration of inflammatory cells with marked thickening. Immunofluorescence (IF) staining IF staining was performed to detect macrophages (F4/80+) and T cells (CD3+) in the lung tissues. Primary antibodies included antibodies against F4/80 (Proteintech, 28463-1-AP) and CD3 (Abcam, ab135372). Nuclei were stained with 4’,6-diamidino-2-phenyl-indole (DAPI, Servicebio, GC305010). Fluorescence images were obtained by fluorescence microscopy (Olympus, Tokyo, Japan). Transmission electron microscopy (TEM) At 48 h after modeling, TEM was employed to examine mitochondrial morphology. Lung tissue samples were first fixed in a 3% glutaraldehyde solution and subsequently post-fixed in a 1% osmium tetroxide solution for 2 h. The samples underwent dehydration through a graded acetone series and were embedded in Epon812 resin. Semi-thin sections, approximately 1 µm thick, were prepared using an ultramicrotome and stained with toluidine blue for analysis. Ultrathin sections measuring 70 nm in thickness were obtained using a diamond knife. Following staining with uranium acetate and lead citron citrate, the samples were observed using a TEM instrument (JEM-1400Flash, JEOL, Akishima, Japan). The degree of damage to mitochondria was quantitatively evaluated using Flameng scores to assess mitochondrial impairment. Each sample underwent analysis in five fields, with 20 mitochondria selected per field for quantitative assessment. The scoring criteria were as follows: Grade 0 (0 points) indicated normal mitochondrial structures with intact particles. Grade I (1 point) represented normal mitochondrial structures with particle loss, accompanied by mild swelling, reduced matrix density, and ridge separation. Grade II (2 points) exhibited slight mitochondrial swelling, with transparent and clear matrices remaining. Grade III (3 points) entailed disruptions in mitochondrial ridges and solidification of the matrix. Grade IV (4 points) denoted extensive destruction of mitochondrial cristae, along with rupture and incompleteness of both the inner and outer membranes. TdT-mediated dUTP nick-end labeling (TUNEL) assay At 48 h after modeling, apoptosis in lung tissues was assessed using the TUNEL assay kit (Beyotime, Shanghai, China), adhering to the manufacturer's protocol. In summary, paraffin-embedded lung tissue sections underwent dewaxing with xylene for 20 min, followed by permeabilization with 0.1 M sodium citrate (pH 6.0) at 65°C for 1 h. The sections were then fixed in 4% paraformaldehyde for 15 min and subsequently blocked with 3% H 2 O 2 for 10 min. Subsequently, the sections were incubated with the TUNEL reaction buffer at 37°C for 1 h. Visualization of labeled nuclei was then accomplished by staining with DAPI (4',6-diamidino-2-phenylindole). Fluorescent signals from the cells were observed, and images were captured using fluorescence microscopy (IX81, Olympus, Tokyo, Japan). Type II alveolar epithelial (ATII) cell identification by alkaline phosphatase staining Primary cultured and purified alveolar epithelial type II cells (AECⅡs) were utilized in this study. The quantification of alkaline phosphatase activity was performed using the Alkaline Phosphatase Detection Kit (Abcam, MA, USA; Catalog No. ab83369). Following air-drying, the cells were incubated with an alkaline phosphatase (AKP) solution, which included 33 µL of nitro blue tetrazolium (NBT) thoroughly mixed. Subsequently, 16.5 µL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was added. The reaction was halted by rinsing the cells with deionized water after an incubation period of 10–15 min at room temperature. Finally, the samples were counterstained with a nuclear solid red dye and mounted using neutral gum for preservation. Apoptosis detection of type II alveolar epithelial (ATII) cells Primary alveolar epithelial type II cells (AECⅡs) were isolated from Sprague-Dawley (SD) rat lung tissues. Subsequently, the AECⅡ cells were washed with phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA, USA) and adjusted to a concentration of 1.0 × 10 6 cells/mL. The cells were then suspended in a 150 µL buffer solution. A staining procedure was conducted at 4°C using 10 µg/mL Annexin V-FITC and 5 µL propidium iodide (PI) for 20 min in the dark. Apoptotic cells were analyzed using a BD FACSCelesta™ flow cytometer (Becton, Dickinson and Company). Immunohistochemistry (IHC) staining At 12 h, 24 h, and 48 h after modeling, lung tissue sections (4 µm) were routinely dewaxed and rehydrated using a graded ethanol series. The samples underwent antigen retrieval in a solution maintained at 95–99°C for 40 min, followed by cooling at room temperature for 20 min. After three washes, the sections were incubated overnight at 4°C with either glucose-regulated protein 78 (GRP78) antibody (Abcam, MA, USA; Cat. ab21685; 1:100), C/EBP homologous protein (CHOP) antibody (Thermofisher, MA, USA; Cat. PA5-104528; 1:100), or Caspase-12 antibody (Bioss, Beijing, China; BS-1105R; 1:100). The incubation with the appropriate secondary antibody (Servicebio, Wuhan, China; Catalog No. B23303; dilution 1:100) was conducted for 1 h on the subsequent day. Following this, the EnVision detection and color development kit was employed for diaminobenzidine tetrahydrochloride (DAB) chromogenic development, hematoxylin counterstaining, gradient ethanol dehydration, xylene clearing, and subsequent mounting for observation. IHC images were examined using a microscope (BA400Digital, Motic Instruments, Inc., Baltimore, MD, USA). Enzyme-Linked Immunosorbent Assay (ELISA) At 12 h, 24 h, and 48 h after modeling, the levels of interleukin (IL)-1β (ZC-36391, ZCI BIO, Shanghai, China), IL-6 (ZC-36404, ZCI BIO, Shanghai, China), and Tumour necrosis factor (TNF)-α (ZC-37624, ZCI BIO, Shanghai, China) in lung tissue were detected by ELISA. The procedure adhered to the instructions provided in the kit. Briefly, the upper lobe of the left lung was excised, ground, and homogenized in PBS (0.01 M, pH 7.4) with the addition of a proteinase inhibitor cocktail (Roche, Complete). This was followed by centrifugation at 5000 × g for 5–10 min at 4˚C. Subsequently, 25 µL of the cell supernatant was dispensed into each well of the ELISA plate, followed by the addition of 225 µL of sample diluent. The plate was incubated at 37°C for 90 min. Thereafter, 100 µL of a biotin-labeled antibody was added to each well and incubated at 37°C for 60 min. Subsequently, the plate was subjected to three washes utilizing an automated washing apparatus. Thereafter, a colorimetric development solution containing 3,3',5,5'-tetramethylbenzidine (TMB) (90 µL) was introduced and incubated in the absence of light at 37°C for 15 min. To terminate the reaction, a stopping solution (100 µL) was then applied. The absorbance was measured at an optical density of 450 nm using a microplate reader. Measurement of proteins’ RNA levels At 48 h post-ALI induction, total RNA was extracted from lung tissues using the TRIzol® reagent (Thermo Fisher, Massachusetts, USA). Complementary DNA (cDNA) was synthesized utilizing a reverse transcription kit (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR (RT-qPCR) was conducted to determine the relative expression levels of target gene transcripts, employing the SYBR Premix Ex Taq kit (Bao Biological Engineering, Dalian, China). The conditions for the reverse transcription reaction were set as follows: 95˚C for 30 s, 40 cycles of 95˚C for 5 s, and 60˚C for 30 s. The relative gene expression level was determined by applying the 2 −△△Ct method on ABI software, Foster City, CA. Western blot analysis At 12 h, 24 h, and 48 h after modeling, protein extraction from lung tissues was conducted using the RIPA kit (Signaling Technologies, Inc.). Subsequent protein quantification was performed with the BCA kit (Biyuntian Biotechnology Co., Ltd., P0009). Based on the quantification results, a loading volume of 20 µg per well was employed for spot sampling, and the total protein was resolved via 10% SDS-PAGE electrophoresis. Subsequent to the initial procedure, the membrane was transferred onto an Immobilon-PSQ PVDF membrane (Sigma-Aldrich, ISEQ00010). It was then blocked using a 5% skim milk solution and incubated with an appropriate dilution of the primary antibody at 4°C overnight. Following incubation, the membrane was washed with Tris-buffered saline containing 0.1% Tween (TBST). Thereafter, it was incubated with goat anti-rabbit IgG (H + L) HRP (Affbiotech, S0001; 1:5000 dilution) at room temperature for a duration of 2 h. Finally, the ECL color development solution was utilized for chromogenic detection. Antibodies used in Western blot analysis are listed in Table 1 . Table 1 Antibodies used in Western blot analysis. Target Dilution Company Cat. number NF-κB P65 1:2000 Abclonal, Wuhan, China A19653 IKKβ 1:2000 Abclonal, Wuhan, China A19606 IκBα 1:2000 Abcam, MA, USA ab32518 GRP78 1:1000 Abcam, MA, USA ab21685 CHOP 1:2000 Abclonal, Wuhan, China A0221 Caspase-12 1:2000 Abcam, MA, USA ab62484 PERK 1:1000 Abcam, MA, USA ab229912 p-PERK 1:2000 Affinity, OH, USA DF7576 IRE1α 1:2000 Thermofisher, MA, USA PA5-20190 p-IRE1α 1:2000 Thermofisher, MA, USA PA5-117222 ATF4 1:2000 Affinity, OH, USA DF6008 Bcl-2 1:2000 Abclonal, Wuhan, China A0208 Bax 1:2000 Proteintech, Chicago, USA 50599-2-Ig β-actin 1:50000 Abclonal, Wuhan, China AC026 cleaved-Caspase3 1:2000 Affinity, OH, USA AF4005 p38 1:2000 Proteintech, Chicago, USA 14064-1-AP pro-Caspase3 1:2000 Affinity, OH, USA AF6311 T1α 1:2000 Huabio, Hangzhou, China ER1915-23 TTF1 1:500 Huabio, Hangzhou, China ER1902-68 Statistical analysis The experimental data were analyzed using SPSS version 22.0 software (IBM Corp., Armonk, NY, USA). Results are presented as means and standard deviations. A one-way ANOVA was employed for data analysis, and intergroup comparisons were conducted using the least variance method. Statistical significance was established at a threshold of P < 0.05. Results TXA and EPO treatments improved ALI in OHF rats As illustrated in Fig. 1 A and 1 B, the OHF rat model was effectively established. Lung injury was evaluated by measuring the wet-to-dry (W/D) weight ratio. At 12, 24, and 48 h post-induction of the OHF model, the lung W/D ratio in rats demonstrated a progressive increase relative to the control group over time (Fig. 1 C, P < 0.01). Administration of TXA and EPO significantly reduced the lung W/D weight ratio (Fig. 1 C, P < 0.05). Compared with the control group, H&E staining revealed lung injury in the OHF rat model, characterized by alveolar necrosis, lymphocyte and neutrophils, and pulmonary epithelial cell proliferation (Fig. 1 D and 1 E, P < 0.01). The administration of TXA and EPO markedly ameliorated alveolar necrosis, reduced lymphocyte and neutrophil counts, and inhibited pulmonary epithelial cell proliferation in OHF rats (refer to Fig. 1 D and 1 E, P < 0.05 and P < 0.01). In the model mice, a significant elevation in the frequency of CD3 + T cells and F4/80 + macrophages was observed (P < 0.01), which was substantially diminished following TXA and EPO treatment (Fig. 1 F and 1 G, P < 0.05). These results indicated that TXA and EPO exhibited therapeutic potential in alleviating lung injury within the OHF model framework. TXA and EPO treatments reduced lung tissue inflammation in OHF rats The expression of inflammatory mediators in the pulmonary tissue of OHF rats was quantitatively evaluated at various time intervals post-modeling using ELISA. The findings indicated a significant elevation in the levels of IL-6, IL-1β, and TNF-α in the lung tissue of the OHF model group compared to the control group at 12, 24, and 48 h post-modeling (Fig. 2 A- 2 C, P < 0.01). Subsequent administration of TXA and EPO resulted in a marked decrease in the expression levels of these cytokines in the lung tissue of OHF rats at 24 and 48 h post-modeling (Fig. 2 A- 2 C, P < 0.05 and P < 0.01). The transcription factor NF-κB exerts a significant influence on the inflammatory signaling pathway (T. Lawrence. 2009). Subsequent investigations focused on alterations in the NF-κB signaling pathway activity within lung tissue. The results demonstrated a significant upregulation in the expression levels of NF-κB P65 and IKKβ, alongside a marked reduction in IκBα levels in OHF rat lung tissue (Fig. 2 D- 2 G, P < 0.01). Notably, treatment with TXA or EPO effectively mitigated the expression levels of NF-κB P65 and IKKβ, while significantly increasing IκBα levels in OHF lung tissue (Fig. 2 D- 2 G, P < 0.01). TXA and EPO treatments counteracted alveolar epithelial cell damage in OHF rats In comparison to the control group, the type I alveolar epithelial cells (AECIs) and type II alveolar epithelial cells (AECIIs) in the lung tissue of the OHF model group demonstrated necrosis (Fig. 3 A and 3 B). The cytoplasmic structure of these cells appeared disorganized, with a notable loss of ribosomes, and the majority of mitochondria exhibited swelling, disrupted cristae, and dissolution (Fig. 3 A and 3 B). Following treatment with TXA and EPO, the AECIs and AECIIs in the OHF rats remained intact, with only a few mitochondria in the cytoplasm displaying slight swelling (Fig. 3 A and 3 B, P < 0.01). Meanwhile, OHF induces damage to mitochondrial (mt) DNA, leading to a reduction in mtDNA levels ( P < 0.01). This effect was mitigated by treatments with TXA and EPO, as illustrated in Fig. 3 C ( P < 0.01). RT-qPCR and Western blot results showed that the protein levels of T1α and TTF1 were decreased in the lung of OHF rats ( P < 0.05 and P < 0.01), which were reversed by TXA and EPO treatments (Fig. 3 D- 3 H, P < 0.05 and P < 0.01). Subsequently, apoptosis in lung tissues was evaluated using the TUNEL staining assay. The findings indicated an increase in cell apoptosis within the lung tissue of OHF rats (Fig. 3 I and 3 J, P < 0.01). However, TXA and EPO treatments resulted in a reduction of TUNEL-positive cells in lung tissue compared to the model group (Fig. 3 I and 3 J, P < 0.01). Furthermore, primary cultures of AECⅡs over 1–3 days demonstrated that the AECⅡs cells exhibited spindle-shaped or triangular morphologies (Fig. 3 K). AECⅡs appeared bluish-purple under alkaline phosphatase staining (Fig. 3 K). The detection of cell apoptosis was performed using flow cytometry, revealing a significant increase in apoptotic AECⅡs following OHF modeling (Fig. 3 L and 3 M, P < 0.01). TXA and EPO treatments markedly improved the apoptosis of AECⅡs (Fig. 3 L and 3 M, P < 0.01). 3.4 TXA and EPO treatments alleviated endoplasmic reticulum (ER) stress in the lungs of OHF rats Subsequently, we elucidated the molecular mechanisms by which TXA and EPO interventions attenuate OHF-induced ALI in rat models. Over time, endoplasmic reticulum (ER) stress in the pulmonary tissue of OHF rats showed a significant increase compared to the control group (Fig. 4 A- 4 J). This was evidenced by the progressive upregulation of ER stress-associated proteins GRP78, CHOP, and Caspase-12 (Fig. 4 A- 4 J, P < 0.01). After 48 h of OHF modeling, the protein expression of GRP78, CHOP, and Caspase-12 in lung tissue of the TXA intervention groups exhibited a significant decrease compared to the model group (Fig. 5 A- 5 F, P < 0.05 and P 0.05, it demonstrated a significant inhibitory effect on the expression of Caspase-12 and GRP78 (Fig. 5 A- 5 F, P < 0.05 and P < 0.01). Concurrently, we evaluated the expression levels of several proteins associated with ER stress-induced apoptosis in OHF rat lung tissue (Fig. 6 A- 6 K). As illustrated in Fig. 6 A- 6 K, both TXA and EPO treatments significantly reduced the expression levels of p-PERK, p-IRE1α, p38, ATF4, cleaved-Caspase3, and Bax while elevating the expression level of Bcl-2 ( P < 0.01). Discussion In the present study, we investigated the protective effects of tranexamic acid (TXA) and erythropoietin (EPO) on acute lung injury (ALI) induced by hip fracture through a comprehensive analysis, which included hematoxylin and eosin (H&E) staining of lung tissue, cytological examination, and the evaluation of inflammatory cytokines. Notably, our findings demonstrate that TXA and EPO effectively attenuate endoplasmic reticulum (ER) stress. These results suggest that TXA and EPO have potential therapeutic value in mitigating hip fracture-induced ALI by reducing inflammation and ER stress. The translational relevance of these findings is considerable. In clinical practice, the management of ALI following hip fractures presents a significant challenge. TXA and EPO, as agents with demonstrated anti-inflammatory and ER stress-reducing properties, offer promising new treatment options. The ease of administration and the relatively well-established safety profiles of these drugs in other contexts further enhance their potential as candidates for translation into clinical use. TXA's anti-inflammatory properties may protect against acute lung injury, as shown in a study where TXA reduced lung damage and improved survival and lung permeability in rats with trauma-hemorrhagic shock [ 28 ]. It also inhibited inflammatory cytokines and deactivated the PARP1/NF-κB signaling pathway, reducing pulmonary inflammation [ 29 ]. Meanwhile, EPO has been found to mitigate acute lung injury and multiple organ dysfunction in rats with thermal injury [ 30 ]. Recent research underscores EPO's diverse roles in alleviating ALI through multiple mechanisms. One study demonstrated that EPO could reduce acute lung injury and multiple organ dysfunction in a rat model of thermal injury. The administration of recombinant human EPO (rhEPO) significantly decreased markers of lung injury, inflammation, and apoptosis [ 31 ]. Another investigation explored the protective effects of EPO in ischemia-reperfusion-induced ALI, where EPO was found to alleviate lung injury by blocking the p38 MAPK signaling pathway [ 32 ]. EPO also aids in recruiting endothelial progenitor cells (EPCs) to injury sites, enhancing repair and reducing inflammation, as demonstrated in lipopolysaccharide-induced ALI in mice [ 33 ]. Despite these promising findings, further clinical trials are necessary to assess TXA's and EPO's efficacy and safety fully. The development of ALI is widely recognized to result primarily from an inflammatory response, which is marked by heightened infiltration of inflammatory cells and increased expression of pro-inflammatory cytokines in the pulmonary region [ 34 ]. The activation of the mononuclear phagocytic cell system induces the release of various inflammatory mediators, thereby initiating pulmonary inflammation [ 35 ]. In our study, we observed significant pulmonary injury and inflammatory responses in the cohort of patients with hip fractures. In line with previous studies, our findings indicated that hip fracture could induce inflammatory responses, thereby leading to the development of ALI [ 36 ]. NF-κB signaling pathway is one of the most important inflammatory signaling pathways [ 37 ]. The main downstream effector of NF-κB is a complex of three subunits, Ikkβ, Ikkα, and the NF-κB essential modulator (NEMO) [ 38 ]. Extensive research has identified the role of the NF-κB signaling pathway in ALI. TXA treatment deactivated the poly ADP-ribose polymerase-1 (PARP1)/NF-κB signaling pathway in the lungs of trauma-hemorrhagic shock (T/HS) rats [ 39 ]. The administration of EPO enhanced the production of endothelial progenitor cells while simultaneously reducing inflammatory processes by inhibiting the NF-κB signaling pathway, potentially offering protective effects against ALI and acute respiratory distress syndrome (ALI/ARDS) [ 40 ]. Additionally, pretreatment with EPO alleviated seawater aspiration-induced ALI in rats by modulating NF-κB P65 expression [ 41 ]. While both agents act on the NF-κB pathway, their upstream triggers and downstream effects might differ, suggesting potential synergistic effects when used in combination. In the present study, we also confirmed elevated NF-κB P65 and IKKβ levels and reduced IκBα levels after hip fracture. In agreement with other studies, we showed that TXA and EPO treatments play a protective role in the development of hip fracture-induced ALI through deactivating the NF-κB signaling pathway. In the present study, regarding the model used for ALI induction, which involved ovariectomy in animals, it is important to consider the impact of estradiol removal on the observed inflammatory cytokine increase. Estradiol has known anti-inflammatory effects [ 42 ], and the increase in inflammatory cytokines could be related to the model itself, the removal of estradiol, or a combination of both. Future studies might explore the specific contribution of estradiol removal to the inflammatory response in this model, providing a more nuanced understanding of the interplay between hormonal status and inflammatory pathways in ALI development. This could further elucidate the mechanisms underlying the protective effects of TXA and EPO and guide the development of targeted therapies for ALI. Mitochondria function as the cellular powerhouses and are recognized as the principal sites for ATP synthesis, a process essential for the survival of eukaryotic organisms [ 43 ]. Mitochondrial quality control (QC) encompasses mitochondrial dynamics, including fission and fusion, mitochondrial biogenesis, and mitophagy. These mechanisms facilitate the swift isolation and removal of dysfunctional mitochondria while modulating mitochondrial biological activity [ 44 ]. Preserving the normal structure of mitochondria is critical for numerous cellular processes and the regulation of programmed cell death [ 45 ]. A substantial body of experimental evidence indicated that mitochondrial damage in AECIIs was a critical factor in ALI. AECIIs served as progenitor cells for AECIs, proliferating and differentiating into AECIs during lung injury to maintain alveolar structural integrity and facilitate lung repair. The deletion of MICU1 in alveolar type II (AT2) cells impaired their differentiation into type I alveolar epithelial (AT1) cells during tissue maintenance and alveolar epithelial regeneration following bacterial pneumonia. Furthermore, the regulation of mitochondrial calcium (mCa 2+ ) single-transporter channels by MICU1 has been identified as a pivotal mechanism in the differentiation process from AECIs to AECIIs [ 46 ]. In both in vitro and in vivo contexts, melatonin was observed to alter the dynamic behavior of mitochondria, promoting a transition from fission to fusion. Additionally, melatonin demonstrated inhibitory effects on mitophagy and fatty acid oxidation in lung epithelial cells treated with LPS [ 47 ]. In AECIIs compromised by pneumonia, the activation of mitochondrial quality control was evidenced by elevated levels of citrate synthase, nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), and the ratio of light-chain 3B protein (LC3-I) to LC3II. These alterations contributed to enhanced cell survival, thereby supporting alveolar function [ 48 ]. The present study showed that OHF induced alveolar epithelial cell damage, mitochondrial swelling, and mtDNA loss. TXA and EPO treatments markedly improved the apoptosis of AECIs and AECIIs, reduced mitochondrial swelling, and increased mtDNA levels. Moreover, the synergistic effect of TXA and EPO in mitigating mitochondrial dysfunction warrants further exploration. While TXA has primarily been recognized for its antifibrinolytic properties, emerging evidence suggests its role in modulating mitochondrial dynamics and reducing oxidative stress [ 49 , 50 ]. EPO, on the other hand, has been shown to enhance mitochondrial biogenesis and function, particularly in the context of cell injury [ 51 , 52 ]. Our findings suggested that the combination of TXA and EPO may complement each other by targeting distinct yet interconnected aspects of mitochondrial QC. TXA may exert its protective effects by stabilizing mitochondrial membrane potential and reducing oxidative damage, while EPO stimulates mitochondrial biogenesis and enhances mitochondrial function. This dual mechanism may explain the enhanced efficacy observed in our study, where the combined treatment was more effective than either agent alone in preserving mitochondrial integrity and promoting cell survival. The endoplasmic reticulum (ER) is an essential intracellular organelle that plays a significant role in protein synthesis, modification, and folding [ 53 ]. It is integral to maintaining cellular homeostasis and influencing cell survival [ 54 ]. Numerous extrinsic factors and intracellular processes could compromise the protein-folding capacity of the ER, leading to the induction of ER stress [ 55 ]. Upon reaching a critical threshold, the excessive accumulation of unfolded or misfolded proteins activates the unfolded protein response (UPR). This response functions to effectively remove surplus aberrant proteins, thereby alleviating ER stress and providing an adaptive protective effect on the organism [ 56 ]. The UPR is mediated through three primary pathways, each corresponding to distinct sensors: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 alpha (IRE1α), and protein kinase RNA-like endoplasmic reticulum kinase (PERK) [ 57 ]. Under normal physiological conditions, glucose-regulated protein 78 (GRP78) binds to these sensors, maintaining them in an inactive state. However, under conditions of excessive internal and external stimulation, the UPR becomes overwhelmed in its ability to clear an excess of unfolded/misfolded proteins. These pathways play a crucial role in the pathogenesis of chronic metabolic disorders, including obesity, insulin resistance, and type II diabetes [ 58 ]. Recent research has identified the inhibitory effect of erythropoietin EPO on ER stress. A prior study demonstrated that EPO alleviated hepatic steatosis and obesity by reducing ER stress and enhancing the expression of fibroblast growth factor 21 [ 59 ]. Additionally, intra-arterial administration of EPO was shown to suppress ER stress in the cerebral microvessels of rats experiencing ischemia-reperfusion injury [ 60 ]. Furthermore, EPO activated specific signaling pathways, increased the expression of glucocerebrosidase, decreased the levels of ER stress marker proteins, and enhanced the proliferation rate of cells from patients with type II Gaucher disease (GD) [ 61 ]. The present study showed that TXA and EPO treatments reduced the expression of ER stress-related proteins GRP78, CHOP and Caspase-12 in OHF model. Moreover, TXA and EPO treatments inhibited the expression levels of a series of proteins associated with ER stress-induced apoptosis in OHF rat lung tissue. The complementary actions of TXA and EPO in alleviating ER stress provide additional evidence for their synergistic effects. While EPO directly modulates ER stress pathways by enhancing proteostasis [ 62 ], TXA may exert its effects through stabilization of intracellular calcium homeostasis and prevention of oxidative damage, which are critical for maintaining ER function [ 63 ]. The combined treatment appears to amplify the protective effects on the ER, resulting in reduced apoptosis and enhanced cellular resilience. This synergistic interaction between TXA and EPO underscores the potential for their combined use in mitigating multiple facets of cellular injury, including mitochondrial dysfunction and ER stress. While our study has produced intriguing results, it is important to acknowledge several limitations. Firstly, animal models may not fully capture the complexity of human physiology, particularly the intricate nature of inflammatory responses in patients with hip fractures. Secondly, genetic differences and the controlled conditions of animal studies may not reflect the variability encountered in clinical settings, necessitating validation of these findings through human trials. Thirdly, additional research is required to determine the clinical preventive effects of TXA and EPO on hip fracture-induced lung injury, as well as to elucidate the detailed mechanisms underlying their protective effects. Fourthly, the dosages and administration methods of TXA and EPO employed in our study were based on previously established animal protocols, which may differ from those used in clinical practice, potentially impacting the efficacy and safety of these treatments. Lastly, future research will incorporate a combined intervention group of TXA and EPO to explore potential synergistic effects, thereby enhancing our understanding of the mechanisms underlying hip fracture-induced ALI in the elderly. While acknowledging the limitations of our study, we observed that both TXA and EPO exhibited potential protective effects against hip fracture-induced ALI, possibly by suppressing inflammation and ER stress. These findings suggest that TXA and EPO could be considered as promising pharmacological interventions for ALI associated with hip fractures. Furthermore, our results contribute to the understanding of the anti-inflammatory and anti-ER stress properties of TXA and EPO, providing preliminary evidence for their potential therapeutic applications. Further clinical investigations are warranted to validate these observations and fully explore their clinical significance. Declarations Author Contribution XL, HXM and JHC designed experiments; XL and HXM carried out experiments; XL, DCZ, and MJL analyzed experimental results. XL wrote the manuscript; HXM and JHC approved the manuscript. All authors read and approved the final manuscript. References Sui L, Lv Y, Feng KX, Jing FJ. 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Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in European Journal of Trauma and Emergency Surgery → Version 1 posted Editorial decision: Revision requested 13 Jun, 2025 Reviews received at journal 03 Jun, 2025 Reviewers agreed at journal 28 May, 2025 Reviewers invited by journal 06 May, 2025 Editor assigned by journal 04 May, 2025 Submission checks completed at journal 02 May, 2025 First submitted to journal 29 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6557105","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452509492,"identity":"61d8e38f-4ed6-4b67-9bc9-0f9fc1d653d6","order_by":0,"name":"Xi Liu","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Liu","suffix":""},{"id":452509493,"identity":"2ee5e313-d1c9-496d-9095-3503c738db38","order_by":1,"name":"Dechuan Zhang","email":"","orcid":"","institution":"Chongqing Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Dechuan","middleName":"","lastName":"Zhang","suffix":""},{"id":452509494,"identity":"39563df4-6e27-42ae-b6f5-0f9032471609","order_by":2,"name":"Mingjin Li","email":"","orcid":"","institution":"Chongqing General Hospital, Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Mingjin","middleName":"","lastName":"Li","suffix":""},{"id":452509495,"identity":"defacbb1-cab2-4c29-8060-4f67805c00f3","order_by":3,"name":"Huixu Ma","email":"","orcid":"","institution":"Chongqing General Hospital, Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Huixu","middleName":"","lastName":"Ma","suffix":""},{"id":452509496,"identity":"801624ae-4f18-4e18-a9a0-f92e438c63f8","order_by":4,"name":"Jinhua Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYFACxgYGCQOGBAYG5gMHPlSQpoUt8eCMMyTYBdTCY3yYt4UIpfzthxsYLAru5BkcP/PhAG8Dgzy/2AH8WiTOJIIc9qzY4EzuhgOSOxgMZ85OwK/FgAGs5XDihgNALYZnGBIMbhPSwv8QquX8mwcHEtuI0SIBs+VGDsOBg8RokbgBtWXmjWcGBxvOSBD2C39/+gNmiT+HE/vOJz/+/KfCRp5fmoAWIGD/LYFkK0HlYMD4gTh1o2AUjIJRMFIBAKe4THfPY70YAAAAAElFTkSuQmCC","orcid":"","institution":"Children’s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development","correspondingAuthor":true,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2025-04-29 13:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6557105/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6557105/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00068-025-02979-4","type":"published","date":"2025-09-30T15:58:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82342830,"identity":"127a8ccc-24df-482b-a0f0-5ae4a8987ef3","added_by":"auto","created_at":"2025-05-09 09:26:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10005851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXA and EPO treatments improved acute lung injury in OHF rats. \u003c/strong\u003e(A and B) The right femurs were scanned with a dual-energy X-ray absorptiometer to measure femur bone mineral density (BMD). (B) The diagnosis of hip fracture was confirmed by both X-rays. (C) The diagnosis of hip fracture was confirmed by both X-rays. (D) Pathological score of lung injury. The scoring criteria are: 0 points for an intact alveolar wall with no thickening or inflammation; 1 point for mild inflammation without thickening; 2 points for significant inflammation with slight thickening; 3 points for severe inflammation with moderate thickening; and 4 points for severe inflammation with marked thickening. (E) H\u0026amp;E\u0026nbsp;staining was analyzed to evaluate the lung injury. The magnification was ×100 and ×400. Scale, 100 μm. Green arrows, alveolar necrosis, blue arrows, lymphocytes, red arrows, neutrophils, yellow arrows, alveolar epithelial proliferation. (F) Immunofluorescence (IF) staining was performed to detect macrophages (F4/80+) and T cells (CD3+) in the lung tissues. The magnification was ×200. Scale, 50 μm. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Control. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. Model (OHF). n=5.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6557105/v1/9e6461927f76966784b7ef97.png"},{"id":82341299,"identity":"2c9831ef-9903-41c5-9051-a56faed94e6c","added_by":"auto","created_at":"2025-05-09 09:18:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXA and EPO treatments reduced lung tissue inflammation in OHF rats.\u003c/strong\u003e (A-C) The enzyme-linked immunosorbent assay (ELISA) was employed for the detection of inflammatory factors (IL-6, IL-1β, and TNF-α) in the lung tissues. (D) Western blot analysis was used to detect the expression of NF-κB P65, IKKβ, and IκBα in the lung tissues. β-actin was used as the internal reference protein. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Control. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Model (OHF). n=3.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6557105/v1/1bd60cad765b0479dcb191d1.jpg"},{"id":82341308,"identity":"2e8f6bcd-2ffa-4f7c-9489-a06a1c0cb7a4","added_by":"auto","created_at":"2025-05-09 09:18:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8473867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXA and EPO treatments counteract alveolar epithelial cell damage in OHF rats. \u003c/strong\u003e(A) Transmission electron microscopy (TEM) was utilized to observe the morphology of mitochondria. Yellow arrow. Mitochondrial swelling. N, nucleus. Scale, 500 nm. (C-E) The RNA levels of T1α, TTF1, and mtDNA were tested using RT-qPCR. (F-H) The protein levels of T1α and TTF1 were tested using Western blot. β-actin was used as the internal reference protein. (I and J) TdT-mediated dUTP nick-end labeling (TUNEL) staining was performed to detect apoptosis in lung tissue. The magnification was ×400. Scale, 50 μm. (K) Observation of micromorphology of type II alveolar epithelial (ATII) cells and identification by alkaline phosphatase. Scale, 100 μm. (L and M) The apoptosis of AECⅡs was detected by flow cytometry. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Control. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Model (OHF). n=3.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6557105/v1/23a5b95e0258ef40cfbebf6b.png"},{"id":82341300,"identity":"24d55efd-57b5-43d3-8c2d-a1d73c32f46e","added_by":"auto","created_at":"2025-05-09 09:18:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":221074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOHF exacerbated endoplasmic reticulum (ER) stress in lung tissues in a time-dependent manner. \u003c/strong\u003eThe expression of Caspase-12 (A and B), CHOP (C and D), and GRP78 (E and F) in lung tissues was assayed by immunohistochemistry (IHC) staining. The signal of IHC was calculated as the number of positive cells/field of view. The magnification was ×200 and ×400. Scale, 50 μm. (G-J) Western blot analysis was used to detect the expression of Caspase-12, CHOP, and GRP78 in the lung tissues. β-actin was used as the internal reference protein. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Control. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Model (OHF). n=3.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6557105/v1/8cfeeb30fcb25e68623dc8e8.jpg"},{"id":82341304,"identity":"5963a8e5-aaed-4ebc-bac0-0f85832560bb","added_by":"auto","created_at":"2025-05-09 09:18:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":151676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXA and EPO treatments alleviate\u0026nbsp;ER\u0026nbsp;stress in the lungs of OHF rats. \u003c/strong\u003eImmunohistochemistry (IHC) staining was employed to assess the expression of Caspase-12 (A and B), CHOP (C and D), and GRP78 (E and F) in lung tissues. The magnification was ×200 and ×400. Scale, 50 μm. The quantification of the IHC signal was determined by calculating the number of positive cells per field of view. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Control. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Model (OHF). n=3.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6557105/v1/03120e61354b0d1bc5a15268.jpg"},{"id":82343185,"identity":"10224756-4cf6-4866-a9b8-00941761ceaf","added_by":"auto","created_at":"2025-05-09 09:34:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1145163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXA and EPO treatments eliminated the expression of endoplasmic reticulum (ER) stress-apoptosis related proteins in OHF rat lung tissue.\u003c/strong\u003e (A-K) The expression of PERK , p-PERK, IRE1α, p-IRE1α, p38, pro-Caspase3, cleaved-Caspase3, ATF4, Bcl-2, and Bax in the lung tissues was detected using Western blot analysis. β-actin was employed as the internal reference protein. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Control. \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. Model (OHF). n=3.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6557105/v1/732f5c8e5180644867580713.png"},{"id":92883922,"identity":"746af6a1-2891-4a0e-887e-79c1a44b7e3a","added_by":"auto","created_at":"2025-10-06 16:11:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21918077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6557105/v1/09733919-5829-43df-9239-a93bc3883d6d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tranexamic acid and erythropoietin protect the lungs on osteoporosis hip fracture-induced acute lung injury rats by inhibition of inflammatory response and endoplasmic reticulum stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHip fractures represent the most common type of fracture among the elderly, predominantly due to reductions in hip bone density and the presence of osteoporosis [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As the global population continues to age, there has been a steady rise in the incidence of osteoporotic hip fractures [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Projections indicate that by the year 2050, the worldwide incidence of osteoporosis-related hip fractures will reach approximately 6.3\u0026nbsp;million, with over 50% occurring in Asia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The disability rate and mortality rate of hip fractures in the elderly are very high [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Acute lung injury (ALI) is the main complication and main cause of death of hip fracture [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Prolonged bed rest in patients with hip fractures before and after surgery is considered to be a major risk factor for ALI after fracture [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recent research indicates that the pathophysiological features of ALI resulting from hip fractures are predominantly characterized by the disruption of the pulmonary microvascular endothelial barrier, apoptosis of lung epithelial cells, and a pronounced inflammatory response [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Endoplasmic reticulum (ER) stress has been associated with a range of pulmonary diseases, including chronic obstructive pulmonary disease, ALI [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], lung cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], pulmonary hypertension [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and pulmonary fibrosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, ER stress has been observed in the lung tissues of animals with ALI induced by different factors, suggesting its potential significance in the initiation and progression of ALI [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranexamic acid (TXA), a synthetic lysine derivative, demonstrates a strong affinity for plasminogen and the lysine-binding sites of plasminase [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This affinity facilitates its binding to the lysine residues on fibrin, thereby inhibiting the fibrinolytic system and promoting effective hemostasis in postoperative incisions. Research indicates that TXA also mitigates tissue stress following trauma, consequently reducing inflammation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The combination of carbazochrome sodium sulfonate and TXA has been shown to significantly enhance perioperative hemostasis and reduce inflammation in patients undergoing total hip arthroplasty [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, research by Peng et al. revealed that the administration of TXA via enema significantly mitigated pulmonary injury in rats experiencing hemorrhagic shock, primarily through the activation of syndecan-1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eErythropoietin (EPO), a hormone-like substance produced by the kidneys and liver, plays a critical role in stimulating the proliferation and differentiation of erythroid progenitor cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It is a vital hematopoietic growth factor necessary for the proper growth and development of red blood cells. Studies have demonstrated that EPO is widely distributed in non-hematopoietic tissues and cells, including those of the nervous system, cardiovascular system, kidneys, liver, uterus, endothelial cells, and solid tumors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, it is integral to various non-hematopoietic functions, including anti-apoptotic mechanisms, reduction of oxidative stress responses, suppression of excessive inflammatory reactions, and facilitation of angiogenesis processes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. As a result, researchers have identified the pleiotropic biological functions of EPO, which have been characterized as those of a systemic protective cytokine [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, EPO has been characterized as a systemic protective cytokine with pleiotropic biological functions.\u003c/p\u003e \u003cp\u003eBuilding on insights from prior research, we propose the hypothesis that treatment with TXA and EPO may mitigate ALI induced by hip fractures through the attenuation of ER stress. The anti-inflammatory properties of TXA [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], combined with the multifaceted protective mechanisms of EPO\u0026mdash;especially its anti-apoptotic and stress-reducing effects\u0026mdash;indicate a synergistic potential to alleviate ER stress [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], thereby potentially reducing the severity of ALI.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eIn the present study, to evaluate our hypothesis, rats were administered intravenous injections of either TXA or EPO prior to the induction of an osteoporosis hip fracture (OHF) model. Subsequently, these rats were assessed for changes in pulmonary injury, levels of inflammation-related factors in serum and lung tissue, and ER stress. Furthermore, we explored the molecular mechanisms through which TXA and EPO attenuate inflammation and ER stress in OHF rats.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSixty-four female Sprague-Dawley (SD) rats, aged 8 months and of specific pathogen-free (SPF) grade, were procured from Dashuo Animal Experiment Co., Ltd., located in Chengdu, Sichuan. The rats were maintained under controlled environmental conditions, with a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity between 50% and 60%, and a 12-h light/dark cycle. They were given ad libitum access to food and water. The study protocols received approval from the Ethics Committee of West China Hospital, Sichuan University (approval number: 20230413003) and conducted in accordance with established guidelines and regulations. The study followed ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModel establishment and therapy\u003c/h3\u003e\n\u003cp\u003eThe control group consisted of fifteen randomly selected rats. In the case of control group rats, ovaries were exposed but not resected. The remaining rats (49 rats in total) were utilized to establish an ovariectomized female rat model with osteoporosis. Specifically, the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg). A 1 cm incision was made at the junction located 1 cm below the ventral costal margin and 1 cm on either side of the spinal cord. Subsequently, bilateral ovaries were ligated and removed. After surgery, 40,000 units of penicillin were injected intramuscularly to prevent infection. Three rats died after ovariectomy. Five weeks post-ovariectomy, the right femurs were scanned with a dual-energy X-ray absorptiometer (DXA; Norland, London, UK) to measure femur bone mineral density (BMD). The specimens were placed in a thin-walled plastic container with a flat bottom and filled with normal saline solution (to ensure complete immersion of the specimen). After a 30-min incubation period, the entire femur segment was subjected to scanning using dual-energy X-ray technology equipped with small animal software, enabling measurement of BMD in g/cm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter the osteoporosis model was successfully constructed, the rats were anesthetized with 1% sodium pentobarbital (50 mg/kg) in the 4th week. After anesthesia, the rat was placed in a supine position on the base plate of a custom-made fracture percussion instrument. The right lower limb was positioned straight and supported by a steel plate to keep it parallel to the horizontal plane. Under real-time imaging from a C-arm, the intertrochanteric line of the right femur was accurately identified, and the percussion instrument was aligned to this target. A 500 g calibrated weight was then dropped from a height of 17 cm, precisely inducing a right hip fracture. Thus, the OHF rat model was successfully established (46 rats in total). Before the establishment of OHF model, rats in TXA group and EPO group were given TXA or EPO intervention. TXA Group: TXA (Tiancheng Pharmaceutical Co., Ltd., Changchun, China) was administered intravenously at a dose of 100 mg/kg, one injection per rat. EPO Group: EPO (Shanghai Kemo Biomedicine Co., LTD, Shanghai, China) was administered intravenously at a dose of 5000 U/kg, one injection per rat. Control Group: Normal saline was administered intravenously in equivalent volume to the control group rats There were 5 rats in each group. Following recovery, rats were housed in cages.\u003c/p\u003e\n\u003ch3\u003eWet/dry (W/D) weight ratio\u003c/h3\u003e\n\u003cp\u003eAt 12 h, 24 h, and 48 h after modeling, the lower lobe of the right lung was promptly weighed upon collection and subsequently subjected to oven drying at 60\u0026deg;C for a duration of 48 h. The tissue was reweighed post-desiccation to determine the W/D weight ratio, which served as a metric for assessing pulmonary vascular permeability.\u003c/p\u003e\n\u003ch3\u003eHematoxylin and eosin (H\u0026E) staining\u003c/h3\u003e\n\u003cp\u003eAt 48 h after modeling, the lung tissue of rats was collected and fixed in 4% paraformaldehyde overnight, processed, and embedded in paraffin. The tissue sections, each measuring 5 \u0026micro;m in thickness, were initially placed in a water bath maintained at 49\u0026deg;C and subsequently affixed to slides coated with polylysine. The slides underwent a dewaxing process using toluene, applied twice, followed by dehydration with ethanol. They were then rinsed with distilled water and stained with hematoxylin. Post-washing, the sections were differentiated using 1% hydrochloric acid alcohol, stained with eosin, and subjected to a 10-min wash with distilled water. Finally, the sections were dehydrated using xylene and mounted with a neutral adhesive. The prepared tissue sections were examined under a digital trinocular camera microscope (BA210Digital, Motic, Beijing, China) at magnifications of 100\u0026times; and 400\u0026times;. After performing H\u0026amp;E staining, a previously published scoring system was employed to assess lung injury [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The scoring criteria are as follows: 0 points indicate an intact alveolar wall with no thickening, absence of inflammatory infiltrates, and no congestion; 1 point corresponds to mild diffuse infiltration of inflammatory cells in the alveolar wall without thickening; 2 points represent significant and extensive infiltration of inflammatory cells with slight thickening of the alveolar wall (1\u0026ndash;2 times); 3 points reflect severe infiltration of inflammatory cells with a 2-3-fold increase in thickness of the alveolar wall in certain areas; and 4 points signify severe infiltration of inflammatory cells with marked thickening.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence (IF) staining\u003c/h3\u003e\n\u003cp\u003eIF staining was performed to detect macrophages (F4/80+) and T cells (CD3+) in the lung tissues. Primary antibodies included antibodies against F4/80 (Proteintech, 28463-1-AP) and CD3 (Abcam, ab135372). Nuclei were stained with 4\u0026rsquo;,6-diamidino-2-phenyl-indole (DAPI, Servicebio, GC305010). Fluorescence images were obtained by fluorescence microscopy (Olympus, Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eAt 48 h after modeling, TEM was employed to examine mitochondrial morphology. Lung tissue samples were first fixed in a 3% glutaraldehyde solution and subsequently post-fixed in a 1% osmium tetroxide solution for 2 h. The samples underwent dehydration through a graded acetone series and were embedded in Epon812 resin. Semi-thin sections, approximately 1 \u0026micro;m thick, were prepared using an ultramicrotome and stained with toluidine blue for analysis. Ultrathin sections measuring 70 nm in thickness were obtained using a diamond knife. Following staining with uranium acetate and lead citron citrate, the samples were observed using a TEM instrument (JEM-1400Flash, JEOL, Akishima, Japan).\u003c/p\u003e \u003cp\u003eThe degree of damage to mitochondria was quantitatively evaluated using Flameng scores to assess mitochondrial impairment. Each sample underwent analysis in five fields, with 20 mitochondria selected per field for quantitative assessment. The scoring criteria were as follows: Grade 0 (0 points) indicated normal mitochondrial structures with intact particles. Grade I (1 point) represented normal mitochondrial structures with particle loss, accompanied by mild swelling, reduced matrix density, and ridge separation. Grade II (2 points) exhibited slight mitochondrial swelling, with transparent and clear matrices remaining. Grade III (3 points) entailed disruptions in mitochondrial ridges and solidification of the matrix. Grade IV (4 points) denoted extensive destruction of mitochondrial cristae, along with rupture and incompleteness of both the inner and outer membranes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTdT-mediated dUTP nick-end labeling (TUNEL) assay\u003c/h3\u003e\n\u003cp\u003eAt 48 h after modeling, apoptosis in lung tissues was assessed using the TUNEL assay kit (Beyotime, Shanghai, China), adhering to the manufacturer's protocol. In summary, paraffin-embedded lung tissue sections underwent dewaxing with xylene for 20 min, followed by permeabilization with 0.1 M sodium citrate (pH 6.0) at 65\u0026deg;C for 1 h. The sections were then fixed in 4% paraformaldehyde for 15 min and subsequently blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 min. Subsequently, the sections were incubated with the TUNEL reaction buffer at 37\u0026deg;C for 1 h. Visualization of labeled nuclei was then accomplished by staining with DAPI (4',6-diamidino-2-phenylindole). Fluorescent signals from the cells were observed, and images were captured using fluorescence microscopy (IX81, Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eType II alveolar epithelial (ATII) cell identification by alkaline phosphatase staining\u003c/h3\u003e\n\u003cp\u003ePrimary cultured and purified alveolar epithelial type II cells (AECⅡs) were utilized in this study. The quantification of alkaline phosphatase activity was performed using the Alkaline Phosphatase Detection Kit (Abcam, MA, USA; Catalog No. ab83369). Following air-drying, the cells were incubated with an alkaline phosphatase (AKP) solution, which included 33 \u0026micro;L of nitro blue tetrazolium (NBT) thoroughly mixed. Subsequently, 16.5 \u0026micro;L of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was added. The reaction was halted by rinsing the cells with deionized water after an incubation period of 10\u0026ndash;15 min at room temperature. Finally, the samples were counterstained with a nuclear solid red dye and mounted using neutral gum for preservation.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis detection of type II alveolar epithelial (ATII) cells\u003c/h2\u003e \u003cp\u003ePrimary alveolar epithelial type II cells (AECⅡs) were isolated from Sprague-Dawley (SD) rat lung tissues. Subsequently, the AECⅡ cells were washed with phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA, USA) and adjusted to a concentration of 1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL. The cells were then suspended in a 150 \u0026micro;L buffer solution. A staining procedure was conducted at 4\u0026deg;C using 10 \u0026micro;g/mL Annexin V-FITC and 5 \u0026micro;L propidium iodide (PI) for 20 min in the dark. Apoptotic cells were analyzed using a BD FACSCelesta\u0026trade; flow cytometer (Becton, Dickinson and Company).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) staining\u003c/h2\u003e \u003cp\u003eAt 12 h, 24 h, and 48 h after modeling, lung tissue sections (4 \u0026micro;m) were routinely dewaxed and rehydrated using a graded ethanol series. The samples underwent antigen retrieval in a solution maintained at 95\u0026ndash;99\u0026deg;C for 40 min, followed by cooling at room temperature for 20 min. After three washes, the sections were incubated overnight at 4\u0026deg;C with either glucose-regulated protein 78 (GRP78) antibody (Abcam, MA, USA; Cat. ab21685; 1:100), C/EBP homologous protein (CHOP) antibody (Thermofisher, MA, USA; Cat. PA5-104528; 1:100), or Caspase-12 antibody (Bioss, Beijing, China; BS-1105R; 1:100). The incubation with the appropriate secondary antibody (Servicebio, Wuhan, China; Catalog No. B23303; dilution 1:100) was conducted for 1 h on the subsequent day. Following this, the EnVision detection and color development kit was employed for diaminobenzidine tetrahydrochloride (DAB) chromogenic development, hematoxylin counterstaining, gradient ethanol dehydration, xylene clearing, and subsequent mounting for observation. IHC images were examined using a microscope (BA400Digital, Motic Instruments, Inc., Baltimore, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eAt 12 h, 24 h, and 48 h after modeling, the levels of interleukin (IL)-1β (ZC-36391, ZCI BIO, Shanghai, China), IL-6 (ZC-36404, ZCI BIO, Shanghai, China), and Tumour necrosis factor (TNF)-α (ZC-37624, ZCI BIO, Shanghai, China) in lung tissue were detected by ELISA. The procedure adhered to the instructions provided in the kit. Briefly, the upper lobe of the left lung was excised, ground, and homogenized in PBS (0.01 M, pH 7.4) with the addition of a proteinase inhibitor cocktail (Roche, Complete). This was followed by centrifugation at 5000 \u0026times; g for 5\u0026ndash;10 min at 4˚C. Subsequently, 25 \u0026micro;L of the cell supernatant was dispensed into each well of the ELISA plate, followed by the addition of 225 \u0026micro;L of sample diluent. The plate was incubated at 37\u0026deg;C for 90 min. Thereafter, 100 \u0026micro;L of a biotin-labeled antibody was added to each well and incubated at 37\u0026deg;C for 60 min. Subsequently, the plate was subjected to three washes utilizing an automated washing apparatus. Thereafter, a colorimetric development solution containing 3,3',5,5'-tetramethylbenzidine (TMB) (90 \u0026micro;L) was introduced and incubated in the absence of light at 37\u0026deg;C for 15 min. To terminate the reaction, a stopping solution (100 \u0026micro;L) was then applied. The absorbance was measured at an optical density of 450 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of proteins\u0026rsquo; RNA levels\u003c/h2\u003e \u003cp\u003eAt 48 h post-ALI induction, total RNA was extracted from lung tissues using the TRIzol\u0026reg; reagent (Thermo Fisher, Massachusetts, USA). Complementary DNA (cDNA) was synthesized utilizing a reverse transcription kit (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR (RT-qPCR) was conducted to determine the relative expression levels of target gene transcripts, employing the SYBR Premix Ex Taq kit (Bao Biological Engineering, Dalian, China). The conditions for the reverse transcription reaction were set as follows: 95˚C for 30 s, 40 cycles of 95˚C for 5 s, and 60˚C for 30 s. The relative gene expression level was determined by applying the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method on ABI software, Foster City, CA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eAt 12 h, 24 h, and 48 h after modeling, protein extraction from lung tissues was conducted using the RIPA kit (Signaling Technologies, Inc.). Subsequent protein quantification was performed with the BCA kit (Biyuntian Biotechnology Co., Ltd., P0009). Based on the quantification results, a loading volume of 20 \u0026micro;g per well was employed for spot sampling, and the total protein was resolved via 10% SDS-PAGE electrophoresis. Subsequent to the initial procedure, the membrane was transferred onto an Immobilon-PSQ PVDF membrane (Sigma-Aldrich, ISEQ00010). It was then blocked using a 5% skim milk solution and incubated with an appropriate dilution of the primary antibody at 4\u0026deg;C overnight. Following incubation, the membrane was washed with Tris-buffered saline containing 0.1% Tween (TBST). Thereafter, it was incubated with goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) HRP (Affbiotech, S0001; 1:5000 dilution) at room temperature for a duration of 2 h. Finally, the ECL color development solution was utilized for chromogenic detection. Antibodies used in Western blot analysis are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibodies used in Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCompany\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat. number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF-κB P65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbclonal, Wuhan, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA19653\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIKKβ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbclonal, Wuhan, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA19606\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIκBα\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam, MA,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eab32518\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGRP78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam, MA,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eab21685\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbclonal, Wuhan, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA0221\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam, MA,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eab62484\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePERK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbcam, MA,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eab229912\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-PERK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAffinity, OH,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDF7576\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIRE1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermofisher, MA,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePA5-20190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-IRE1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermofisher, MA,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePA5-117222\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAffinity, OH,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDF6008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBcl-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbclonal, Wuhan, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA0208\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProteintech, Chicago,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50599-2-Ig\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:50000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbclonal, Wuhan, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAC026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecleaved-Caspase3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAffinity, OH,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAF4005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProteintech, Chicago,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14064-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epro-Caspase3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAffinity, OH,\u0026nbsp;USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAF6311\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuabio, Hangzhou, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eER1915-23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTTF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuabio, Hangzhou, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eER1902-68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were analyzed using SPSS version 22.0 software (IBM Corp., Armonk, NY, USA). Results are presented as means and standard deviations. A one-way ANOVA was employed for data analysis, and intergroup comparisons were conducted using the least variance method. Statistical significance was established at a threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTXA and EPO treatments improved ALI in OHF rats\u003c/h2\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the OHF rat model was effectively established. Lung injury was evaluated by measuring the wet-to-dry (W/D) weight ratio. At 12, 24, and 48 h post-induction of the OHF model, the lung W/D ratio in rats demonstrated a progressive increase relative to the control group over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Administration of TXA and EPO significantly reduced the lung W/D weight ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the control group, H\u0026amp;E staining revealed lung injury in the OHF rat model, characterized by alveolar necrosis, lymphocyte and neutrophils, and pulmonary epithelial cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The administration of TXA and EPO markedly ameliorated alveolar necrosis, reduced lymphocyte and neutrophil counts, and inhibited pulmonary epithelial cell proliferation in OHF rats (refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the model mice, a significant elevation in the frequency of CD3\u0026thinsp;+\u0026thinsp;T cells and F4/80\u0026thinsp;+\u0026thinsp;macrophages was observed (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which was substantially diminished following TXA and EPO treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicated that TXA and EPO exhibited therapeutic potential in alleviating lung injury within the OHF model framework.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTXA and EPO treatments reduced lung tissue inflammation in OHF rats\u003c/h2\u003e \u003cp\u003eThe expression of inflammatory mediators in the pulmonary tissue of OHF rats was quantitatively evaluated at various time intervals post-modeling using ELISA. The findings indicated a significant elevation in the levels of IL-6, IL-1β, and TNF-α in the lung tissue of the OHF model group compared to the control group at 12, 24, and 48 h post-modeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Subsequent administration of TXA and EPO resulted in a marked decrease in the expression levels of these cytokines in the lung tissue of OHF rats at 24 and 48 h post-modeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The transcription factor NF-κB exerts a significant influence on the inflammatory signaling pathway (T. Lawrence. 2009). Subsequent investigations focused on alterations in the NF-κB signaling pathway activity within lung tissue. The results demonstrated a significant upregulation in the expression levels of NF-κB P65 and IKKβ, alongside a marked reduction in IκBα levels in OHF rat lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, treatment with TXA or EPO effectively mitigated the expression levels of NF-κB P65 and IKKβ, while significantly increasing IκBα levels in OHF lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTXA and EPO treatments counteracted alveolar epithelial cell damage in OHF rats\u003c/h2\u003e \u003cp\u003eIn comparison to the control group, the type I alveolar epithelial cells (AECIs) and type II alveolar epithelial cells (AECIIs) in the lung tissue of the OHF model group demonstrated necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The cytoplasmic structure of these cells appeared disorganized, with a notable loss of ribosomes, and the majority of mitochondria exhibited swelling, disrupted cristae, and dissolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Following treatment with TXA and EPO, the AECIs and AECIIs in the OHF rats remained intact, with only a few mitochondria in the cytoplasm displaying slight swelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Meanwhile, OHF induces damage to mitochondrial (mt) DNA, leading to a reduction in mtDNA levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This effect was mitigated by treatments with TXA and EPO, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). RT-qPCR and Western blot results showed that the protein levels of T1α and TTF1 were decreased in the lung of OHF rats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which were reversed by TXA and EPO treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Subsequently, apoptosis in lung tissues was evaluated using the TUNEL staining assay. The findings indicated an increase in cell apoptosis within the lung tissue of OHF rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, TXA and EPO treatments resulted in a reduction of TUNEL-positive cells in lung tissue compared to the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, primary cultures of AECⅡs over 1\u0026ndash;3 days demonstrated that the AECⅡs cells exhibited spindle-shaped or triangular morphologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). AECⅡs appeared bluish-purple under alkaline phosphatase staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). The detection of cell apoptosis was performed using flow cytometry, revealing a significant increase in apoptotic AECⅡs following OHF modeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). TXA and EPO treatments markedly improved the apoptosis of AECⅡs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.4 TXA and EPO treatments alleviated endoplasmic reticulum (ER) stress in the lungs of OHF rats\u003c/p\u003e \u003cp\u003eSubsequently, we elucidated the molecular mechanisms by which TXA and EPO interventions attenuate OHF-induced ALI in rat models. Over time, endoplasmic reticulum (ER) stress in the pulmonary tissue of OHF rats showed a significant increase compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). This was evidenced by the progressive upregulation of ER stress-associated proteins GRP78, CHOP, and Caspase-12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After 48 h of OHF modeling, the protein expression of GRP78, CHOP, and Caspase-12 in lung tissue of the TXA intervention groups exhibited a significant decrease compared to the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). While EPO treatment did not reduce the expression level of CHOP in the lung tissue of OHF rats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, it demonstrated a significant inhibitory effect on the expression of Caspase-12 and GRP78 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Concurrently, we evaluated the expression levels of several proteins associated with ER stress-induced apoptosis in OHF rat lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, both TXA and EPO treatments significantly reduced the expression levels of p-PERK, p-IRE1α, p38, ATF4, cleaved-Caspase3, and Bax while elevating the expression level of Bcl-2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we investigated the protective effects of tranexamic acid (TXA) and erythropoietin (EPO) on acute lung injury (ALI) induced by hip fracture through a comprehensive analysis, which included hematoxylin and eosin (H\u0026amp;E) staining of lung tissue, cytological examination, and the evaluation of inflammatory cytokines. Notably, our findings demonstrate that TXA and EPO effectively attenuate endoplasmic reticulum (ER) stress. These results suggest that TXA and EPO have potential therapeutic value in mitigating hip fracture-induced ALI by reducing inflammation and ER stress. The translational relevance of these findings is considerable. In clinical practice, the management of ALI following hip fractures presents a significant challenge. TXA and EPO, as agents with demonstrated anti-inflammatory and ER stress-reducing properties, offer promising new treatment options. The ease of administration and the relatively well-established safety profiles of these drugs in other contexts further enhance their potential as candidates for translation into clinical use.\u003c/p\u003e \u003cp\u003eTXA's anti-inflammatory properties may protect against acute lung injury, as shown in a study where TXA reduced lung damage and improved survival and lung permeability in rats with trauma-hemorrhagic shock [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It also inhibited inflammatory cytokines and deactivated the PARP1/NF-κB signaling pathway, reducing pulmonary inflammation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Meanwhile, EPO has been found to mitigate acute lung injury and multiple organ dysfunction in rats with thermal injury [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Recent research underscores EPO's diverse roles in alleviating ALI through multiple mechanisms. One study demonstrated that EPO could reduce acute lung injury and multiple organ dysfunction in a rat model of thermal injury. The administration of recombinant human EPO (rhEPO) significantly decreased markers of lung injury, inflammation, and apoptosis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Another investigation explored the protective effects of EPO in ischemia-reperfusion-induced ALI, where EPO was found to alleviate lung injury by blocking the p38 MAPK signaling pathway [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. EPO also aids in recruiting endothelial progenitor cells (EPCs) to injury sites, enhancing repair and reducing inflammation, as demonstrated in lipopolysaccharide-induced ALI in mice [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Despite these promising findings, further clinical trials are necessary to assess TXA's and EPO's efficacy and safety fully.\u003c/p\u003e \u003cp\u003eThe development of ALI is widely recognized to result primarily from an inflammatory response, which is marked by heightened infiltration of inflammatory cells and increased expression of pro-inflammatory cytokines in the pulmonary region [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The activation of the mononuclear phagocytic cell system induces the release of various inflammatory mediators, thereby initiating pulmonary inflammation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In our study, we observed significant pulmonary injury and inflammatory responses in the cohort of patients with hip fractures. In line with previous studies, our findings indicated that hip fracture could induce inflammatory responses, thereby leading to the development of ALI [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. NF-κB signaling pathway is one of the most important inflammatory signaling pathways [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The main downstream effector of NF-κB is a complex of three subunits, Ikkβ, Ikkα, and the NF-κB essential modulator (NEMO) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Extensive research has identified the role of the NF-κB signaling pathway in ALI. TXA treatment deactivated the poly ADP-ribose polymerase-1 (PARP1)/NF-κB signaling pathway in the lungs of trauma-hemorrhagic shock (T/HS) rats [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The administration of EPO enhanced the production of endothelial progenitor cells while simultaneously reducing inflammatory processes by inhibiting the NF-κB signaling pathway, potentially offering protective effects against ALI and acute respiratory distress syndrome (ALI/ARDS) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, pretreatment with EPO alleviated seawater aspiration-induced ALI in rats by modulating NF-κB P65 expression [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. While both agents act on the NF-κB pathway, their upstream triggers and downstream effects might differ, suggesting potential synergistic effects when used in combination. In the present study, we also confirmed elevated NF-κB P65 and IKKβ levels and reduced IκBα levels after hip fracture. In agreement with other studies, we showed that TXA and EPO treatments play a protective role in the development of hip fracture-induced ALI through deactivating the NF-κB signaling pathway.\u003c/p\u003e \u003cp\u003eIn the present study, regarding the model used for ALI induction, which involved ovariectomy in animals, it is important to consider the impact of estradiol removal on the observed inflammatory cytokine increase. Estradiol has known anti-inflammatory effects [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and the increase in inflammatory cytokines could be related to the model itself, the removal of estradiol, or a combination of both. Future studies might explore the specific contribution of estradiol removal to the inflammatory response in this model, providing a more nuanced understanding of the interplay between hormonal status and inflammatory pathways in ALI development. This could further elucidate the mechanisms underlying the protective effects of TXA and EPO and guide the development of targeted therapies for ALI.\u003c/p\u003e \u003cp\u003eMitochondria function as the cellular powerhouses and are recognized as the principal sites for ATP synthesis, a process essential for the survival of eukaryotic organisms [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Mitochondrial quality control (QC) encompasses mitochondrial dynamics, including fission and fusion, mitochondrial biogenesis, and mitophagy. These mechanisms facilitate the swift isolation and removal of dysfunctional mitochondria while modulating mitochondrial biological activity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Preserving the normal structure of mitochondria is critical for numerous cellular processes and the regulation of programmed cell death [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A substantial body of experimental evidence indicated that mitochondrial damage in AECIIs was a critical factor in ALI. AECIIs served as progenitor cells for AECIs, proliferating and differentiating into AECIs during lung injury to maintain alveolar structural integrity and facilitate lung repair. The deletion of MICU1 in alveolar type II (AT2) cells impaired their differentiation into type I alveolar epithelial (AT1) cells during tissue maintenance and alveolar epithelial regeneration following bacterial pneumonia. Furthermore, the regulation of mitochondrial calcium (mCa\u003csup\u003e2+\u003c/sup\u003e) single-transporter channels by MICU1 has been identified as a pivotal mechanism in the differentiation process from AECIs to AECIIs [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In both in vitro and in vivo contexts, melatonin was observed to alter the dynamic behavior of mitochondria, promoting a transition from fission to fusion. Additionally, melatonin demonstrated inhibitory effects on mitophagy and fatty acid oxidation in lung epithelial cells treated with LPS [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In AECIIs compromised by pneumonia, the activation of mitochondrial quality control was evidenced by elevated levels of citrate synthase, nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), and the ratio of light-chain 3B protein (LC3-I) to LC3II. These alterations contributed to enhanced cell survival, thereby supporting alveolar function [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The present study showed that OHF induced alveolar epithelial cell damage, mitochondrial swelling, and mtDNA loss. TXA and EPO treatments markedly improved the apoptosis of AECIs and AECIIs, reduced mitochondrial swelling, and increased mtDNA levels.\u003c/p\u003e \u003cp\u003eMoreover, the synergistic effect of TXA and EPO in mitigating mitochondrial dysfunction warrants further exploration. While TXA has primarily been recognized for its antifibrinolytic properties, emerging evidence suggests its role in modulating mitochondrial dynamics and reducing oxidative stress [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. EPO, on the other hand, has been shown to enhance mitochondrial biogenesis and function, particularly in the context of cell injury [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Our findings suggested that the combination of TXA and EPO may complement each other by targeting distinct yet interconnected aspects of mitochondrial QC. TXA may exert its protective effects by stabilizing mitochondrial membrane potential and reducing oxidative damage, while EPO stimulates mitochondrial biogenesis and enhances mitochondrial function. This dual mechanism may explain the enhanced efficacy observed in our study, where the combined treatment was more effective than either agent alone in preserving mitochondrial integrity and promoting cell survival.\u003c/p\u003e \u003cp\u003eThe endoplasmic reticulum (ER) is an essential intracellular organelle that plays a significant role in protein synthesis, modification, and folding [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. It is integral to maintaining cellular homeostasis and influencing cell survival [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Numerous extrinsic factors and intracellular processes could compromise the protein-folding capacity of the ER, leading to the induction of ER stress [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Upon reaching a critical threshold, the excessive accumulation of unfolded or misfolded proteins activates the unfolded protein response (UPR). This response functions to effectively remove surplus aberrant proteins, thereby alleviating ER stress and providing an adaptive protective effect on the organism [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The UPR is mediated through three primary pathways, each corresponding to distinct sensors: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 alpha (IRE1α), and protein kinase RNA-like endoplasmic reticulum kinase (PERK) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Under normal physiological conditions, glucose-regulated protein 78 (GRP78) binds to these sensors, maintaining them in an inactive state. However, under conditions of excessive internal and external stimulation, the UPR becomes overwhelmed in its ability to clear an excess of unfolded/misfolded proteins. These pathways play a crucial role in the pathogenesis of chronic metabolic disorders, including obesity, insulin resistance, and type II diabetes [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent research has identified the inhibitory effect of erythropoietin EPO on ER stress. A prior study demonstrated that EPO alleviated hepatic steatosis and obesity by reducing ER stress and enhancing the expression of fibroblast growth factor 21 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Additionally, intra-arterial administration of EPO was shown to suppress ER stress in the cerebral microvessels of rats experiencing ischemia-reperfusion injury [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Furthermore, EPO activated specific signaling pathways, increased the expression of glucocerebrosidase, decreased the levels of ER stress marker proteins, and enhanced the proliferation rate of cells from patients with type II Gaucher disease (GD) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The present study showed that TXA and EPO treatments reduced the expression of ER stress-related proteins GRP78, CHOP and Caspase-12 in OHF model. Moreover, TXA and EPO treatments inhibited the expression levels of a series of proteins associated with ER stress-induced apoptosis in OHF rat lung tissue.\u003c/p\u003e \u003cp\u003eThe complementary actions of TXA and EPO in alleviating ER stress provide additional evidence for their synergistic effects. While EPO directly modulates ER stress pathways by enhancing proteostasis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], TXA may exert its effects through stabilization of intracellular calcium homeostasis and prevention of oxidative damage, which are critical for maintaining ER function [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The combined treatment appears to amplify the protective effects on the ER, resulting in reduced apoptosis and enhanced cellular resilience. This synergistic interaction between TXA and EPO underscores the potential for their combined use in mitigating multiple facets of cellular injury, including mitochondrial dysfunction and ER stress.\u003c/p\u003e \u003cp\u003eWhile our study has produced intriguing results, it is important to acknowledge several limitations. Firstly, animal models may not fully capture the complexity of human physiology, particularly the intricate nature of inflammatory responses in patients with hip fractures. Secondly, genetic differences and the controlled conditions of animal studies may not reflect the variability encountered in clinical settings, necessitating validation of these findings through human trials. Thirdly, additional research is required to determine the clinical preventive effects of TXA and EPO on hip fracture-induced lung injury, as well as to elucidate the detailed mechanisms underlying their protective effects. Fourthly, the dosages and administration methods of TXA and EPO employed in our study were based on previously established animal protocols, which may differ from those used in clinical practice, potentially impacting the efficacy and safety of these treatments. Lastly, future research will incorporate a combined intervention group of TXA and EPO to explore potential synergistic effects, thereby enhancing our understanding of the mechanisms underlying hip fracture-induced ALI in the elderly.\u003c/p\u003e \u003cp\u003eWhile acknowledging the limitations of our study, we observed that both TXA and EPO exhibited potential protective effects against hip fracture-induced ALI, possibly by suppressing inflammation and ER stress. These findings suggest that TXA and EPO could be considered as promising pharmacological interventions for ALI associated with hip fractures. Furthermore, our results contribute to the understanding of the anti-inflammatory and anti-ER stress properties of TXA and EPO, providing preliminary evidence for their potential therapeutic applications. Further clinical investigations are warranted to validate these observations and fully explore their clinical significance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXL, HXM and JHC designed experiments; XL and HXM carried out experiments; XL, DCZ, and MJL analyzed experimental results. XL wrote the manuscript; HXM and JHC approved the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSui L, Lv Y, Feng KX, Jing FJ. Burden of falls in China, 1992\u0026ndash;2021 and projections to 2030: a systematic analysis for the global burden of disease study 2021. 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Arthroscopy. 2023;39(12):2529\u0026ndash;e25462521. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1016/j.arthro.2023.08.019\u003c/span\u003e\u003cspan address=\"10.1016/j.arthro.2023.08.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-trauma-and-emergency-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejot","sideBox":"Learn more about [European Journal of Trauma and Emergency Surgery](http://link.springer.com/journal/68)","snPcode":"68","submissionUrl":"https://submission.nature.com/new-submission/68/3","title":"European Journal of Trauma and Emergency Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"osteoporosis hip fracture, acute lung injury, tranexamic acid, erythropoietin, inflammation, endoplasmic reticulum stress","lastPublishedDoi":"10.21203/rs.3.rs-6557105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6557105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to evaluate the protective effects and elucidate the mechanism underlying Tranexamic acid (TXA) and erythropoietin (EPO) in osteoporosis hip fracture (OHF)-induced acute lung injury (ALI). Sprague-Dawley (SD) rats were randomly divided into the control, OHF, OHF\u0026thinsp;+\u0026thinsp;TXA, and OHF\u0026thinsp;+\u0026thinsp;EPO groups. Lung tissue was analyzed at 12, 24, and 48 h post-treatment to assess injury levels through hematoxylin and eosin (H\u0026amp;E) staining, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. H\u0026amp;E staining showed that TXA and EPO significantly reduced the OHF-induced lung injury. Wet/dry ratios were identified in the OHF\u0026thinsp;+\u0026thinsp;TXA group and OHF\u0026thinsp;+\u0026thinsp;EPO group than in the OHF group. We found significantly lower levels of inflammatory cytokine production levels and nuclear factor-κB (NF-κB) signaling pathway activity in the lung of the OHF\u0026thinsp;+\u0026thinsp;TXA group and OHF\u0026thinsp;+\u0026thinsp;EPO group than in the hip fracture group. Moreover, TXA and EPO treatments counteracted mitochondrial injury and type II alveolar epithelial cells (AECⅡ) cell apoptosis in OHF rats. The treatment with TXA and EPO also exhibited inhibitory effects on the increase of ER stress-related apoptosis in lung tissue after OHF, as evidenced by reduced expression levels of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), phosphorylated (p)-protein kinase RNA-like endoplasmic reticulum kinase (PERK), p- endoplasmic reticulum-to-nucleus signaling 1 (IRE1)α, Caspase-12, activating transcription factor 4 (ATF4), Bax and induced expression of Bcl-2. Thus, TXA and EPO may have a protective effect on OHF-induced ALI.\u003c/p\u003e","manuscriptTitle":"Tranexamic acid and erythropoietin protect the lungs on osteoporosis hip fracture-induced acute lung injury rats by inhibition of inflammatory response and endoplasmic reticulum stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 09:18:48","doi":"10.21203/rs.3.rs-6557105/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-13T07:37:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-04T03:56:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146978343953469082615849619911328734637","date":"2025-05-28T14:52:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-06T08:08:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-04T20:25:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-02T04:12:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Trauma and Emergency Surgery","date":"2025-04-29T13:38:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-trauma-and-emergency-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejot","sideBox":"Learn more about [European Journal of Trauma and Emergency Surgery](http://link.springer.com/journal/68)","snPcode":"68","submissionUrl":"https://submission.nature.com/new-submission/68/3","title":"European Journal of Trauma and Emergency Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3e728540-5eb0-4d4a-b248-f3e497ef99fa","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:04:40+00:00","versionOfRecord":{"articleIdentity":"rs-6557105","link":"https://doi.org/10.1007/s00068-025-02979-4","journal":{"identity":"european-journal-of-trauma-and-emergency-surgery","isVorOnly":false,"title":"European Journal of Trauma and Emergency Surgery"},"publishedOn":"2025-09-30 15:58:07","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-05-09 09:18:48","video":"","vorDoi":"10.1007/s00068-025-02979-4","vorDoiUrl":"https://doi.org/10.1007/s00068-025-02979-4","workflowStages":[]},"version":"v1","identity":"rs-6557105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6557105","identity":"rs-6557105","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}