Xuebijing improves inflammation and pyroptosis of acute lung injury by up-regulating miR-181d-5p-mediated SPP1 inactivation

Highlights • XBJ improves LPS-induced lung cell inflammation and pyroptosis.• miR-181d-5p inhibits LPS-induced inflammatory response and pyroptosis of lung epithelial cells.• XBJ elevates miR-181d-5p and improves LPS-induced pyroptosis of lung epithelial cells.• XBJ upregulates miR-181d-5p and inhibits SPP1 to protect lung epithelial cells from LPS-induced injuries.


Introduction
Acute Lung Injury (ALI) refers to a series of lung lesions caused by multiple lung injuries, which can induce serious lung diseases and lead to serious sequelae and high mortality [1,2].ALI is characterized by severe acute inflammatory processes, leading to increased alveolar permeability, protein and white blood cell accumulation, and pulmonary edema [3].There is still a lack of effective drugs to control and treat ALI [4,5].Therefore, there is an urgent need to find new drugs to relieve ALI.
NLRP3-mediated pyroptosis of macrophages can aggravate lung inflammation in patients with ALI [12].NLRP3 inflammasome activation in alveolar macrophages leads to the processing of pro-caspase-1 into two lysed subunits named p10 and p20, which can induce the release of pro-inflammatory cytokines and trigger severe inflammatory responses [2,13].However, the association between XBJ's protection of lung tissue and NLRP3-mediated pyroptosis remains unclear.miRNAs can negatively regulate gene expression at the post-transcriptional level [14] and are substantially implicated in inflammatory lung diseases, including ALI.For example, miR-181a inhibition protects mice from ALI [15].Recent studies have confirmed that miR-181d-5p exerts an antiinflammatory role after renal ischemia-reperfusion injury and can inhibit the expression of inflammatory mediators, thus improving renal function [16].
SPP1, also known as Osteopontin (OPN), is a coding protein located in 4q22.1 and is considered to be a key cytokine involved in immune cell recruitment and expression of type 1 cytokines at inflammatory sites [17,18].Current studies have confirmed that SPP1 is involved in tumor cell progression [19−22].However, its underlying mechanism in inflammation-related ALI has not been fully elucidated.
Here, rat and cellular ALI models were constructed using LPS to explore the potential mechanism of XBJ to improve ALI.At the same time, the effects of XBJ on inflammatory response and pyroptosis in ALI were discussed, as well as the mechanism of miR-181d-5p and SPP1.

ALI animal model
This study was approved by the Animal Protection Professional Committee of Affiliated Danzhou People's Hospital of Hainan Medical University.Male SPF-grade SD rats (180-220g) were purchased from the Animal Experimental Center, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).Rats were kept on a day/night 12/12h cycle with 55% humidity, with free access to water and food.After one week of adaptive feeding, the rats were randomly divided into 3 groups (6 rats/group): Control, ALI, and ALI+XBJ.Rats were first anesthetized by intraperitoneal injection of pentobarbital sodium at 50 mg/kg, followed by intraperitoneal injection of 10 mg/kg LPS (Sigma-Aldrich; USA), while rats in the Control group were injected intraperitoneally with 0.9% normal saline [23].Three days before modeling, rats in the ALI+XBJ group were injected with XBJ (4 mL/kg) twice a day via caudal vein, while rats in the Control and ALI groups were injected with 0.9% normal saline [24].All rats were euthanized 12h after LPS injection.Subsequently, the right lung of 3 rats in each group was ligated, and the left lung was rapidly frozen at -80°C for subsequent RNA, protein, and flow cytometry tests.The remaining right lung was fixed in 4% paraformaldehyde for histological analysis.From the remaining 3 rats in each group, lung tissues were weighed first and then dried at 65°C for 48h to calculate the Wet/Dry (W/D) ratio to estimate pulmonary edema index.

ELISA
Cell culture supernatant or rat lung tissues were centrifuged at 500 × g at 4°C for 10-min for ELISA.Inflammatory cytokines were detected using commercial ELISA kits for IL-1β, IL-18, and TNF-α (R&D System, USA).
Cell culture and treatment BEAS-2B Cell line (ATCC) was cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a 37°C incubator with 5% CO 2 .XBJ group was pretreated with 2 g/L XBJ (Tianjin Datong New Pharmaceutical Co., LTD., China) in BEAS-2B cells for 24h [25] and treated with 1 μg/mL LPS (Sigma-Aldrich) for 4h and then with 5 mM ATP (A6559, Sigma-Aldrich) for 30 min to induce pyroptosis, while Control and LPS groups were added with equal amounts of PBS [26].The remaining groups were transfected before treatment with XBJ, LPS, and ATP.

Cell transfection
miR-181d-5p mimic/inhibitor and oe-SPP1, and their corresponding controls (mimic NC, inhibitor NC, and vector) were synthesized in Gene-Pharma (Shanghai, China) and transfected into BEAS-2B cells using Lipofectamine 2000 (Invitrogen).The medium was replaced at 8h, and cells were harvested at 48h to evaluate the transfection efficiency by RT-qPCR or immunoblotting.

RT-qPCR
Total RNA was extracted from rat lung tissue and BEAS-2B cells by Trizol Reagent (Invitrogen), and RNA quality and concentration were determined by Nanodrop 2000.Reverse transcription of miRNA was implemented by Taqman® MicroRNA Reverse Transcription kit (Invitrogen), while that of mRNA was done by cDNA synthesis kit (Thermo Fisher Scientific).RT-qPCR assay was performed using the SYBR Premix Ex Taq TM II kit (RR820A, Takara) and analyzed by Biosystems 7900 thermocycler (Thermo Fisher Scientific).With U6 and GAPDH as the reference genes, respectively, expression was calculated by the 2 −ΔΔCt method.The primer sequence is shown in Table 1.

HE-staining
The lung tissues were prepared into slices with 5 μm thickness using a microtome (RM2235, Leica, Germany) after fixation in 4% paraformaldehyde for 24h and embedment in paraffin.Then, the tissues were reacted with hematoxylin (Beyotime, China) for 5-10 min, rinsed with running water for 3-min, dyed with eosin (Beyotime) for 1-2 min, and viewed using an optical microscope (Olympus).

TUNEL staining
To measure apoptosis, a TUNEL assay was carried out using a commercial kit (Beyotime).Paraffin sections (5 μm) were incubated with 45 μL labeled buffer and 5 μL TdT enzyme solution for 60-min, rinsed 3 times with PBS, stained with DAPI for 5-min, and observed under a fluorescence microscope (Olympus).Quantification of images was performed with ImageJ software.

RIP assay
Magna RIP Kit (Millipore) was purchased to conduct RIP assay.Cell lysates were harvested using RIP lysis buffer and combined with magnetic beads with Ago2 or IgG antibodies at 4°C for 6h.After elution, the immunoprecipitates were collected for RT-qPCR analysis.

Statistical analysis
To analyze the data, GraphPad Prism software v8.0 was utilized.
Data were expressed as mean ± Standard Deviation (SD) and collected from each experiment in replicates.Student's t-test compared the difference between the two groups, and one-way ANOVA analyzed that among multiple groups; p < 0.05 was considered statistically significant.

XBJ improves ALI
LPS inducer was used to establish ALI rat models.Subsequently, HEstaining evaluated the morphological changes in the lung tissue.ALI rats showed obvious pathological injury, thickened alveolar wall diaphragm, shrunk alveolar cavity and inflammatory cells infiltrated in the lung tissue.However, the lung tissue structure of ALI rats pretreated with XBJ was intact, and the degree of inflammatory cell infiltration was low (Fig. 1A).Meanwhile, TUNEL staining showed that XBJ reduced the number of lung tissue apoptosis induced by LPS (Fig. 1B).The lung W/D ratio was increased in ALI rats, and XBJ improved LPS-induced lung injury (Fig. 1C).ELISA results demonstrated that XBJ preconditioning inhibited LPS promotion of inflammatory cytokines IL-1β, IL-18 and TNF-α in rat lung tissue (Fig. 1D).Meanwhile, immunoblotting assayed that cellular inflammation (p-p65) and pyroptosis-related proteins (NLRP3, ASC, caspase-1 p20, and GSDMD-N) were elevated in the lung tissues of ALI rats, while XBJ pretreatment could inhibit these proteins (Fig. 1 E, F).

XBJ upregulates miR-181d-5p and inhibits SPP1 to protect lung epithelial cells from LPS-induced injuries
The oe-SPP1 was transfected into BEAS-2B cells, which successfully overexpressed SPP1 in cells (Fig. 6 A, B).Cells transfected with oe-SPP1 were treated with XBJ and LPS.Functional experiments noted that overexpressing SPP1 weakened the inhibition of XBJ on LPS-induced inflammation (Fig. 6 C, D).Meanwhile, increased expression of SPP1 antagonized the inhibition of XBJ on LPS-and ATP-induced pyroptosis (Fig. 6 E, F).

Discussion
ALI is a respiratory disease caused by multiple factors, which may develop into acute respiratory distress syndrome [28].A previous double-blind randomized trial demonstrated that XBJ protects lung injury by reducing neutrophil infiltration by downregulating inflammatory mediators [29].However, the protective effect of XBJ on lung function impairment caused by ALI remains largely unknown.This study sought to explore the mechanism of XBJ in ALI and eventually confirmed that XBJ could improve ALI by mediating the miR-181d-5p/SPP1 axis to inhibit ALI inflammation and pyroptosis.
XBJ can ameliorate increased lung permeability and inflammatory response caused by sepsis [30,31].This study constructed rat and cellular ALI models using LPS to investigate XBJ function.XBJ has been reported to protect septic ALI by inhibiting inflammation and apoptosis [32].This is consistent with the present findings that XBJ can inhibit  In addition, since the discovery of miRNA in 1993, changes in miRNA expression have been associated with the pathogenesis of inflammatory lung diseases, making them biomarkers for novel diagnosis and treatment [33,34].miR-181d family suggests importance in inflammation and cell growth [35].miR-181d can promote TNF-α expression [36].This work explored the role of miR-181d-5p in ALI inflammation and found that miR-181d-5p was abnormally downregulated in ALI, and restoring miR-181d-5p could inhibit inflammation caused by ALI.In addition, Liu's study confirmed that miR-223 specifically targets NLRP3, thereby inhibiting NLRP3 translation expression and affecting pyroptosis [37].Notably, our study also confirmed that miR-181d-5p inhibited LPS-induced pyroptosis.Subsequently, the study explored and revealed that silencing miR-181d-5p could counteract the improvement effect of XBJ on inflammation and pyroptosis of ALI lung epithelial cells.
MiRNAs negatively regulate gene expression by binding to the 3′UTR of target genes [38].Therefore, the study confirmed that miR-181d-5p targeted SPP1.SPP1 is a multifunctional protein expressed at the site of inflammation.For example, SPP1 is involved in acute and chronic neuritis [39].and can mediate transfusion-related ALI by stimulating pulmonary neutrophilic accumulation [40].This study also confirmed that SPP1 expression was elevated in ALI, and promoting SPP1 could block the protective effect of XBJ on ALI.That is, SPP1 overexpression enhanced inflammation and pyroptosis.
Some limitations exist in this study.For example, whether XBJ mediates miR-181d-5p/SPP1 axis to improve ALI was not explored in animal models of ALI, and the molecular mechanism of SPP1 regulating inflammatory response and pyroptosis has not yet been studied.

Conclusion
XBJ improves LPS-induced ALI and inhibits LPS-induced inflammation and pyroptosis.XBJ is protective for lung cells by upregulating miR-181d-5p, thereby inhibiting SPP1.Generally speaking, XBJ provides a new therapeutic target and strategy for the clinical treatment of ALI.

Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.
apoptosis and inflammation induced by ALI.But more importantly, the study also found that XBJ inhibited NLRP3, ASC, caspase-1, p20, and GSDMD-N levels, and could reverse LPS promotion of the proportion of Caspase-1 and PI-positive cells in BEAS-2B cells.Further experiments confirmed that XBJ could improve ALI by mediating the miR-181d-5p/ SPP1 axis to inhibit ALI inflammatory response and pyroptosis.