Adult male Institute of Cancer Research mice (n=72 animals, 6–8 weeks old, 25±2 g) were purchased from Shanghai Slack Experimental Animal Co., Ltd. [certificate no. SCXK (Hu) 2022–0004]. Mice were housed for 3 days to allow them to acclimate to the environment prior to the experiment. Mice were bred in specific pathogen-free conditions of 23±3°C with 55±15% humidity and a 12 h light/dark cycle, and were allowed free access to sterilized food and water were supplied. All handling procedures used in the present study were approved by the Animal Care and Use Committees at the Zhejiang University School of Medicine (Approved No. ZJU20220294, Hangzhou, China) and were conducted in accordance with the policies of institutional guidelines on the care and use of laboratory animals. Euthanasia was performed with CO₂ at a volume displacement rate of 30% vol/min.

Animals were divided into six groups in a randomized manner with 12 mice in each group. The groups were as follows: i) Control group; ii) LPS group; iii) LPS + TAT-CTM (200 µg/kg) group; iv) LPS + TAT-ATXN1-CTM (50 µg/kg) group; v) LPS + TAT-ATXN1-CTM (200 µg/kg) group; and vi) LPS + TAT-ATXN1-CTM (1,000 µg/kg) group. The mice were anesthetized by nasal inhalation of 4% isoflurane and the depth of anesthesia was maintained with 1.5% isoflurane. Mice in the control group were intratracheally injected with 0.9% sodium chloride injection (2 mg/kg). The murine ALI model was established by intratracheal injection of LPS (2 mg/kg; cat. no. L3129; MilliporeSigma). Treatment groups received LPS (2 mg/kg) and TAT-CTM (200 µg/kg) or TAT-ATXN1-CTM (50, 200, 1,000 µg/kg) administered simultaneously. Mice were euthanized once they reached humane endpoints or the study endpoint. The humane endpoints included signs of loose fur, inactivity or reduced activity, hunched posture, and respiratory distress, and these were not observed in any of the animals. The study endpoint was 8 h after LPS administration. The bronchoalveolar lavage fluid (BALF) was prepared to measure the accumulation of inflammatory cells, lung tissue was harvested for histopathological examination and the degree of pathological injury was scored.

BALF preparation was described previously (25). Briefly, mice were sacrificed, and the trachea of each mouse was surgically exposed and cannulated. The left lung and accessory lobe were ligated to collect the BALF of the right lung. The right lung was lavaged twice with a single volume of warmed 0.5 ml of PBS containing 1% bovine serum albumin (cat. no. A8020; Beijing Solarbio) and 5,000 IU/l heparin. White blood cells were counted under a light microscope. The collected BALF was centrifuged at 400 × g, 4°C for 10 min. The pelleted cells were coated onto microscope slides after drying at room temperature. Wright-Giemsa staining was performed, and the numbers of neutrophils, macrophages and lymphocytes were counted as cells/slide under a light microscope. Briefly, the slides were stained in Wright's stain (cat. no. W100940; Shanghai Aladdin Biochemical Technology Co., Ltd.) for 4 min and then in Giemsa's stain (cat. no. G100959; Shanghai Aladdin Biochemical Technology Co., Ltd.) for 2 min at 25°C. The cells were observed and counted under the light microscope. Stained sections were scanned using the software Multiscan (2.0.0.150) of a digital section scanner (Convergence Technology Co., Ltd.) to obtain screenshots and analyze the processing. Neutrophils have a dark blue multi-lobed nucleus and pale pink cytoplasm; lymphocytes have purple nucleus with sky blue cytoplasm; Macrophages have purple nucleus with blue cytoplasm as lymphocytes but larger than other leukocytes.

The left middle lobe of the lung was fixed in 10% neutral formalin overnight at room temperature and embedded in paraffin, and paraffin sections (5 µm) were prepared. The sections were stained with hematoxylin staining for 3 min and eosin staining for 30 sec at room temperature, and the lung injury and inflammatory cell infiltration were observed under a light microscope (26).

The extent of histological lung damage was quantified by a designated scoring system, which is made up of five grades: 0 points, normal appearing lung; 1 point, mild inflammatory cell infiltration, no tissue injury; 2 points, mild to moderate inflammatory cell infiltration, mild tissue injury; 3 points, moderate inflammatory cell infiltration, mild tissue injury; 4 points, moderate to severe inflammatory cell infiltration with obvious tissue injury; and 5 points, severe inflammatory cell infiltration with significant tissue injury and changes. Slides were assessed by a pathologist blinded to the experimental design.

THP-1 and 293T cell lines were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The THP-1 human macrophages were cultured in RPMI-1640 growth medium (cat. no. CGM112.05; CellMax Technologies AB) containing 10% Premium FBS (cat. no. SA101.02; CellMax Technologies AB), 0.05 mM β-mercaptoethanol (cat. no. M6250; Shanghai Macklin Biochemical Co., Ltd.) and 1% penicillin-streptomycin. The 293T cells were cultured in DMEM (cat. no. CGM101.05; CellMax Technologies AB) with 10% FBS (cat. no. 04-001-1ACS; Biological Industries) and 1% penicillin-streptomycin. All cells were cultured in a CO₂ incubator at 37°C with 5% CO₂. THP-1 cells (5×10⁵ cells/ml) were treated with 100 ng/ml phorbol-12-myristate acetate (PMA; cat. no. HY-18739; MedChemExpress) for at 37°C 24 h to induce cell attachment. The cells were further left to rest at 37°C for 24 h in complete RPMI 1640 medium before stimulation with LPS (1 µg/ml; cat. no. L3129; MilliporeSigma) and 10 µM TAT-CTM or TAT-ATXN1-CTM treatments at 37°C for 4, 8 and 24 h.

The human HK2 plasmid containing the HK2 sequences and the pcDNA3.1 vector with three Flag tags were synthesized by Shandong Weizhen Biotechnology Co., Ltd., and empty vector was used as a control. The 293T cells were plated on a 6-well plate to ~80% confluency 1 day before transfection, and then transfected with the plasmid. For transfection, 2 µg plasmid was diluted in 100 µl basal medium (DMEM) before adding to a 6-well plate. Polyethylenimine (PEI; cat. no. 23966; Polysciences, Inc.) at a 1:3 (w/w) cDNA:PEI ratio was added to the diluted plasmid solution and this was gently mixed. The plasmid-PEI mixture was placed in a CO₂ incubator for 20 min and then added to the cells, which were cultured in an incubator with 5% CO₂ at 37°C. After 6 h, the solution in the 6-well plate was replaced with fresh complete medium, and 10 µM TAT-CTM or TAT-ATXN1-CTM (GenScript) for testing was added. Cells were harvested 48 h after transfection for subsequent experiments.

For MTT assays, 5×10³ THP-1 cells were seeded into 96-well plates with complete RPMI 1640 medium and treated with 100 ng/ml PMA at 37°C for 24 h. The cells were further left to rest at 37°C for 24 h in complete RPMI 1640 medium only before treatment with 0, 5, 10 and 20 µM of TAT-ATXN1-CTM or TAT-CTM in RPMI 1640 medium in a total volume of 100 µl at 37°C for 24 h. Subsequently, 10 µl MTT/PBS (5 mg/ml; cat. no. M8180; Beijing Solarbio Science & Technology Co., Ltd.) was added to the medium and cells were incubated in a CO₂ incubator at 37°C for 4 h. Subsequently, 100 µl dimethyl sulfoxide was added and the 96-well plates were shaken for 10 min at room temperature. Finally, optical density of the samples was measured at 490 nm using a multipurpose microplate reader (SpectraMax iD3; Molecular Devices, LLC).

It has been reported that the amino acid sequence 594–610 of ATXN1 interacts with the HK2 protein (24). To increase the membrane permeability of the peptide, a transmembrane domain (TAT) was added to the ATXN1 (594–610) sequence. A CTM sequence was added to degrade the endogenous targeted protein for lysosomal degradation (Table I).

TAT-ATXN1-CTM and TAT-CTM peptides, synthesized by GenScript, were solid-phase synthesized as C-terminal amides and purified using high-performance liquid chromatography (HPLC) using a preparative column, and the final purity was ascertained as 98.1% for TAT-ATXN1-CTM and 98.9% for TAT-CTM using analytical HPLC peak area integration (Figs. S1 and S2). Briefly, The TAT-ATXN1-CTM or TAT-CTM crude product was purified using an HPLC system (LC-20AB, Shimadzu) with the reversed-phase column (Inertsil ODS-3 4.6×250 mm, Shimadzu) at 25 °C. The samples were loaded onto the column (12–15 µl loading volume). The flow rate was set to be 1 ml/min using 0.065% trifluoroacetic in 100% water (v/v) and 0.05% trifluoroacetic in 100% acetonitrile (v/v) as the mobile phase compositions of pump A and pump B, respectively. The wavelength of the detector was set to 220 nm. The purified TAT-ATXN1-CTM or TAT-CTM fractions were collected. The molecular masses of the peptides were determined by electrospray ionization mass spectrometry (ESI-MS, LCMS-2020, Shimadzu). MS parameters for the peptides were: nitrogen gas temperature, 350°C; gas flow, 5 l/min; nebulizer pressure, 15 psi; scan time, 500 msec. The observed mass spectral values for TAT-ATXN1-CTM and TAT-CTM were 5,359.2 and 3,270.0, respectively, which was consistent with the theoretical values (Figs. S3 and S4).

Total RNA was extracted from cells using TRIzol^® reagent (cat. no. CW0580S; CoWin Biosciences) according to the manufacturer's instructions. Total RNA was quantified using a micro-spectrophotometer (NAno-300; cat. no. MO00040009; Hangzhou Allsheng Instruments Co., Ltd.), and the optical density 260/280 nm ratio of all samples was 1.8–2.0. Next, cDNA was synthesized on a PCR instrument (SimpliAmp™; cat. no. A24811; Thermo Fisher Scientific, Inc.) using the RNA reverse transcription kit (cat. no. CW2569M; CoWin Biosciences). RT was performed as follows: 42°C for 15 min, incubate at 85°C for 5 min, and keep warm at 4°C. Primers and a fluorescent quantitative PCR kit (cat. no. CW2601H; CoWin Biosciences) containing SYBR Green I fluorescent dye were used to perform fluorescent qPCR on a PCR system (cat. no. 4351106; Thermo Fisher Scientific, Inc.). Primer sequences for human HK2, human GAPDH, mouse HK2 and mouse β-actin (Sangon Biotech Co., Ltd.) are listed in Table II. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The 2^−ΔΔCq method was used for analysis of results and gene levels were normalized to the internal reference gene, GAPDH for human HK2 or β-actin for mouse HK2 (27).

The cells were washed with pre-cooled 1X PBS (cat. no. P1020; Beijing Solarbio Science & Technology Co., Ltd.) and lysed with lysis buffer containing RIPA (cat. no. P0013B; Beyotime Institute of Biotechnology) and PMSF (cat. no. P0100; Beijing Solarbio Science & Technology Co., Ltd.) at a ratio of 1:100 of PMSF:RIPA. The cell lysate was centrifuged at 13,800 × g for 5 min at 4°C to obtain the supernatant. The protein content of the supernatant was determined using a BCA Protein Assay Kit (cat. no. BL521A; Biosharp Life Sciences) and then protein (20 µg/lane) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being electrophoretically transferred onto PVDF membranes (cat. no. 1214429; GVS). The membrane was blocked with ready-to-use blocking solution (cat. no. P0252; Beyotime Institute of Biotechnology) for 1 h at room temperature and then incubated with the indicated primary antibodies overnight at 4°C. The primary antibodies were as follows: Anti-β-actin (1:1,000; cat. no. 3700; Cell Signaling Technology, Inc.), anti-HK1 (1:1,000; cat. no. 2024S; Cell Signaling Technology, Inc.) and anti-HK2 (1:1,000; cat. no. ab209874; Abcam). Subsequently, the following secondary antibodies were added for 1 h at room temperature: Goat anti-rabbit IgG HRP-conjugated (1:12,000; cat. no. 31460; Thermo Fisher Scientific, Inc.) and goat anti-mouse IgG HRP-conjugated (1:12,000; cat. no. 31430; Thermo Fisher Scientific, Inc.). Detection of immunoreactive bands was performed using ECL Western Blotting Detection kit (cat. no. SW2040; Beijing Solarbio Science & Technology Co., Ltd.) on a chemiluminescence gel imaging system (Peiqing JS-1070P; Shanghai Peiqing Science & Technology Co., Ltd.) and data were semi-quantified using ImageJ2× 2.1.4.7 (Rawak Software Development) with β-actin as the loading control.

The lung tissues from mice and supernatants from THP-1 cells were collected. For lung tissues, the protein concentration was assayed using a BCA Protein Assay Kit (cat. no. BL521A; Biosharp Life Sciences). Levels of TNF-α, IL-1β and HK2 were analyzed using commercial ELISA kits according to the manufacturer's instructions. Human TNF-α ELISA Kit (cat. no. CHE0019; Beijing 4A Biotech Co., Ltd.), Human IL-1β ELISA Kit (cat. no. CHE0001; Beijing 4A Biotech Co., Ltd.), Mouse TNF-α ELISA Kit (cat. no. CME0004; Beijing 4A Biotech Co., Ltd.), Mouse IL-1β (cat. no. CME0015; Beijing 4A Biotech Co., Ltd.) and Mouse HK2 ELISA kit (cat. no. ml058727; Mlbio) were used. The absorbance of each well was measured at 450 nm with a multifunctional microplate reader (SpectraMax iD3; Molecular Devices, LLC), and the concentration of TNF-α, IL-1β and HK2 was calculated according to the standard curve.

Statistical analyses were performed using GraphPad Prism 7.0 (Dotmatics). All quantitative results are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post hoc test, two-way ANOVA followed by Sidak's post hoc test, Kruskal-Wallis test followed by Dunn's post hoc test or an unpaired two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

TAT-ATXN1-CTM was designed to cross the cell membrane and selectively degrade the HK2 protein. The peptide consists of three functional domains. The first domain (TAT) can induce the peptide to cross the cell membrane. The second domain (PBD) is composed of ATXN1 protein, which can selectively recognize and bind to the HK2 protein. The third domain, CTM, targets lysosomes to degrade proteins (23,24). In the present study, TAT-CTM was used as a control (Fig. 1A).

The effects of TAT-ATXN1-CTM at different concentrations on cell viability were assessed using an MTT assay. The results demonstrated that TAT-ATXN1-CTM treatment for 24 h at 5 and 10 µM had no significant effects on the viability of THP-1 cells, while 20 µM treatment significantly decreased the cell viability compared with the 0 µM treated group [Fig. 1B; 20 µM (TAT-ATXN1-CTM) vs. 0 µM, 78.290±2.099 vs. 100.000±0.000; P<0.001]. Therefore, 10 µM TAT-ATXN1-CTM was used for subsequent experiments to avoid cytotoxicity (Fig. 1B).

Firstly, HK2 was overexpressed and it was confirmed by both western blotting and RT-qPCR that HK2 expression was increased following transfection with the HK2 plasmid compared with the empty vector group (Fig. S5, HK2 plasmid vs. empty vector, for mRNA, 8.257±0.905 vs. 1.016±0.121, P<0.01; Fig. 2A, for protein, 2.955±0.148 vs. 1.000, P<0.001). Subsequently, the degradation of the HK2 protein by TAT-ATXN1-CTM was verified. TAT-ATXN1-CTM treatment significantly reduced HK2 expression following HK2 plasmid-mediated overexpression in 293T cells (Fig. 2A; HK2 plasmid⁺/TAT-ATXN1-CTM⁺ vs. HK2 plasmid⁺, 2.003±0.106 vs. 2.955±0.148; P<0.001). In LPS-induced THP-1 cells, TAT-ATXN1-CTM treatment also markedly reduced HK2 protein expression [Fig. 2B; LPS⁺/control⁺ vs. vehicle (Veh)⁺/control⁺, 1.826±0.076 vs. 1.000; P<0.001; LPS⁺/TAT-ATXN1-CTM⁺ vs. LPS⁺/control⁺, 1.196±0.096 vs. 1.826±0.076; P<0.01]. By contrast, HK2 mRNA levels were significantly increased at 4 and 8 h after LPS treatment, but were not affected at different time points (4, 8 and 24 h) after TAT-ATXN1-CTM administration (Fig. 2C; LPS vs. control, 4 h: 3.611±0.1444 vs. 1.003±0.0561, P<0.05, 8 h: 5.601±0.5804 vs. 0.973±0.0214, P<0.001). In addition, TAT-ATXN1-CTM treatment tended to decrease HK1 protein expression in LPS-induced THP-1 cells (Fig. 2B; LPS⁺/TAT-ATXN1-CTM⁺ vs. LPS⁺/control⁺, 0.717±0.0417 vs. 0.939±0.060; P=0.141), possibly through indirect mechanisms related to inflammation suppression. These results suggested that TAT-ATXN1-CTM was effective in selectively reducing HK2 expression.

HK2 expression is increased during inflammatory processes (28) and whether TAT-ATXN1-CTM exerted anti-inflammatory effects by degrading the HK2 protein was next investigated. Treatment with 10 µM TAT-ATXN1-CTM significantly reduced the levels of TNF-α and IL-1β in the supernatant of THP-1 cells induced by LPS (Fig. 3; LPS⁺/control⁺ vs. Veh⁺/control⁺, TNF-α: 25.670±1.015 vs. 0.194±0.009, P<0.001, IL-1β: 12.840±0.699 vs. 0.637±0.0554, P<0.001; LPS⁺/TAT-ATXN1-CTM⁺ vs. LPS⁺/control⁺, TNF-α: 17.860±0.184 vs. 25.670±1.015, P<0.001, IL-1β: 8.739±0.291 vs. 12.840±0.699, P<0.001). Notably, TAT-CTM, as a control treatment, also decreased IL-1β protein expression (Fig. 3B; LPS⁺/TAT-CTM⁺ vs. LPS⁺/control⁺, IL-1β: 9.458±0.425 vs. 12.840±0.699; P<0.001). These data imply that TAT-ATXN1-CTM could suppress proinflammatory cytokines in LPS-induced THP-1 cells.

The anti-inflammatory effect of TAT-ATXN1-CTM was investigated in a murine model of ALI (Fig. 4A). TAT-ATXN1-CTM (1,000 µg/kg) treatment significantly reduced the number of total cells, neutrophils, macrophages and lymphocytes in the BALF compared to the control group [Fig. 4B-F; LPS⁺ vs. control, total cells: 47.050±3.004 vs. 9.545±0.796, P<0.001, neutrophils: 15.900±2.424 vs. 0.05±0.017, P<0.001, macrophages: 5.330±1.396 vs. 0.036±0.010, P<0.001 and lymphocytes: 25.820±2.746 vs. 9.460±0.784, P<0.001; LPS⁺/TAT-ATXN1-CTM (1,000 µg/kg)⁺ vs. LPS⁺, total cells: 20.000±1.676 vs. 47.050±3.004, P<0.001, neutrophils: 5.985±1.044 vs. 15.900±2.424, P<0.01, macrophages: 1.570±0.227 vs. 5.330±1.396, P<0.001 and lymphocytes: 12.450±1.303 vs. 25.820±2.746, P<0.001]. Notably, TAT-CTM also slightly decreased the number of total cells (Fig. 4C; total cells: 36.130±2.010 vs. 47.050±3.004; P<0.01), macrophages (Fig. 4E; 2.560±0.392 vs. 5.330±1.396; P<0.05) and lymphocytes (Fig. 4F; 18.220±1.655 vs. 25.820±2.746; P<0.01) in BALF. In conclusion, these data suggested that TAT-ATXN1-CTM treatment inhibited the accumulation of inflammatory cells in the lungs of LPS-induced mice.

Based on lung histological examination, LPS induced a pulmonary inflammatory response, including notable alveolar wall thickening, congestion with inflammatory cell infiltration around the alveoli or airways, which was significantly attenuated by 1,000 µg/kg TAT-ATXN1-CTM treatment [Fig. 5; LPS⁺ vs. control, 2.727±0.333 vs. 0.083±0.083, P<0.001; LPS⁺/TAT-ATXN1-CTM (1,000 µg/kg)⁺ vs. LPS⁺, 1.364±0.152 vs. 2.727±0.333, P<0.05]. These results indicated that inhibition of HK2 attenuated lung histopathological damage in LPS-induced ALI in mice.

Based on the aforementioned findings, TAT-ATXN1-CTM (1,000 µg/kg) ameliorated ALI in vivo, whereas other low concentrations of TAT-ATXN1-CTM had limited effect in treating ALI. Consequently, the effects of TAT-ATXN1-CTM (1,000 µg/kg) on the expression of HK2 and proinflammatory factors in LPS-challenged mice only were investigated. TAT-ATXN1-CTM (1,000 µg/kg) treatment reduced the protein expression levels of HK2, TNF-α and IL-1β without affecting HK2 mRNA levels in the lung tissues of ALI mice [Fig. 6; LPS⁺ vs. control, HK2: 0.744±0.045 vs. 0.320±0.004, P<0.001, TNF-α: 420.600±32.250 vs. 158.800±4.788, P<0.001 and IL-1β: 129.300±12.040 vs. 17.630±0.921, P<0.001; LPS⁺/TAT-ATXN1-CTM⁺ (1,000 µg/kg) vs. LPS⁺, HK2: 0.461±0.021 vs. 0.744±0.045, P<0.001, TNF-α: 262.500±15.750 vs. 420.600±32.250, P<0.001 and IL-1β: 59.040±5.229 vs. 129.300±12.040, P<0.001]. Collectively, these data suggested that 1,000 µg/kg TAT-ATXN1-CTM reduced the protein expression levels of HK2 and proinflammatory factors in ALI mice induced by LPS.