36 Sprague–Dawley rats (male, 8–9 weeks old, 300–360 g; Hangzhou Medical College, Hangzhou, China) were housed under specific pathogen-free conditions at animal laboratory (20 $\pm$ 2 °C, 12-h circadian rhythm) with unlimited supply of food and sterile tap water. Additionally, research ratification was obtained from Zhejiang Baiyue Biotech Co., Ltd.’s Ethics Committee for Experimental Animals Welfare (ZJBYLA-IACUC-20220427).

Establishment of severe HS rat model referred to an existing study [11]. After being fasted for 12 h, the rats were anesthetized by intraperitoneal injection of 45 mg/kg sodium pentobarbital (P-010, Merck, Darmstadt, Germany), the femoral artery of which was separated and cannulated with polyethylene catheter. Subsequently, their left femoral artery was connected to a multi-channel physiological signal acquisition and processing system (BIOPAC, Goleta, CA, USA) to detect blood pressure (mean arterial pressure, MAP) and heart rate, while the right femoral artery was employed to control hemorrhage. The left jugular vein was cannulated with a PE-50 tube for fluid resuscitation. The total blood loss rate was controlled at 45%, and the bleeding time was controlled within 1 h. The blood flowing out of the left femoral artery was then perfused back into the rats within 40 min.

Based on previous description [14, 15], rats were given subcutaneous injection of phosphate buffer saline (PBS) (A610100, Sangon, Shanghai, China) containing small interfering RNA targeting TUG1 (siTUG1, 5^′-GCAGUAAUUGGAGUGGAUATT-3^′, siBDM0001, Ribobio, Guangzhou, China) or its negative control (siNC, 5^′-UUCUCCGAACGUGUCACGUTT-3^′, siN0000001-4-200, Ribobio, Guangzhou, China) 2 h before surgery. After 6 h of resuscitation, the collected blood samples were centrifuged (3000 g, 15 min), followed by serum storage (–80 °C). The rats were euthanized by an overdose of pentobarbital sodium (200 mg/kg, iv) for heart tissue collection.

Six groups were designed based on experimental animals (Sham, Model, Sham + siNC, Sham + siTUG1, Model + siNC and Model + siTUG1) (6 mice/group). The rats in Sham group experienced anesthesia and surgery, but without bloodletting.

Anticoagulated whole blood samples were collected 2 hours after resuscitation, in which bicarbonate (HCO₃^–) and lactate content were determined employing a Hemogas analyzer (ABL800 FLEX, Radiometer, Copenhagen, Denmark).

The heart was quickly taken out, and repeatedly rinsed with normal saline. Heart tissue was immediately frozen in a –20 °C refrigerator for 20 min, and then cut into 4–5 sections (thickness of 2 mm). Sections were stained with 1.5% TTC (15 min; A610558, Sangon, Shanghai, China), and fixed with 10% neutral formaldehyde solution (E672001, Sangon, Shanghai, China). After the stained sections were imaged under a fluorescence microscope, their infarct area (white area) and non-infarcted region (red area) were analyzed and calculated through AlphaEaseFC image processing software (version 4.0, Alpha Innotech, San Leandro, CA, USA). Infarct size (%) = infarct area/total area $\times$ 100%.

Heart tissues experienced fixation (10% neutral formalin solution, BL-G001, Senbeijia, Nanjing, China), paraffin embedding and sectioning into 5-µm-thick slices. The deparaffinized and hydrated slices underwent color development exploiting H&E kit (BP-DL001, Senbeijia, China), followed by dehydration and transparentization. After sealing with mounting medium (SBJ-0700, Senbeijia, China), the degree of damage to the rat myocardial tissue was observed (microscope, AE2000, Motic, Xiamen, China, 100$\times$).

TUNEL Apoptosis Detection Kit (C1086, Beyotime, Shanghai, China) was used for TUNEL staining of heart tissue. After being deparaffinized, hydrated, treated with proteinase K and washed, the section was incubated with TUNEL solution (60 min), and reacted with 4^′,6-Diamidino-2-phenylindole (DAPI) solution (C1002, Beyotime, China) for nuclear staining. Following dehydration, transparentization and mounting, TUNEL-positive cells were finally observed (fluorescence microscope, 200$\times$, DM2500, Leica, Wetzlar, Germany). Relative positive cell rate (%) = positive TUNEL staining/total cell $\times$ 100%. Data were normalized to Sham + siNC group.

MDA content in rat serum was determined by MDA detection kit (SBJ-R0007, Senbeijia, China). Briefly, the standard solution was diluted into different concentrations in preparation for the subsequent standard curve plotting. Afterwards, the diluted serum was added to an antibody-coated 96-well plate, and cultured with different reagents. Following absorbance measurement (450 nm, microplate reader, CMaxPlus, Molecular Devices, San Jose, CA, USA), MDA concentration detection was carried out based on the standard curve.

By means of GSH detection kit (BC1175, Solarbio, Beijing, China), the absorbance (412 nm) of the sample or reagent was detected, and GSH content in rat serum was calculated according to the standard curve.

Using SOD detection kit (BC0170, Solarbio, China), the absorbance (560 nm) of the sample or reagent was quantified, followed by measurement of SOD content in rat serum or cell supernatant.

Rat cardiomyocyte cell line (H9C2, CL-0089, Procell, Wuhan, China) were soaked (37 °C, 5% CO₂) in Dulbecco’s Modified Eagle’s Medium (30-2002, ATCC, Manassas, VA, USA) with 10% fetal bovine serum, 0.1 mg/mL streptomycin and 100 U/mL penicillin. H9C2 cells were tested negative for mycoplasma. H9C2 cells were validated by short tandem repeat (STR) profiling.

Post total RNA extraction (total RNA extraction kit, EZB-RN4, HiFunBio, Shanghai, China) from rat heart tissues or H9C2 cells, reverse transcription and qPCR were completed by One-Step qRT-PCR kit (AE341, TransGen Biotech, Beijing, China) using Real-Time PCR Detection system (CFX Connect, Bio-rad, Hercules, CA, USA). Data were dissected by 2^{-Δ⁢Δ⁢Ct} method [16]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, for lncRNA and mRNA) functioned as an internal control. The details of primers are listed in Table 1.

SiTUG1 (siG151012045709-1-5, 5^′-GCAGUAAUUGGAGUGGAUATT-3^′) and siNC (siN0000002-1-5, 5^′-UUCUCCGAACGUGUCACGUTT-3^′) were purchased from Ribobio (China). The oligonucleotide sequences above were transfected into the cells in a 6-well plate (4 $\times$ 10⁶/well) with the help of transfection reagent (STF02, SinoBiological, Beijing, China). After 24 h, the transfection results were determined by qRT-PCR.

In line with a previous description [17], an oxidative stress injury cell model was set up based on 500 µM H₂O₂ treated H9C2 (24 h). H9C2 were assigned into 4 groups: Control (normal culture), H₂O₂, H₂O₂ + siNC and H₂O₂ + siTUG1 groups (4-h treatment with 400 µM H₂O₂ after siNC/siTUG1 transfection or not).

With 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (SBJ-0191, Senbeijia, China), treated cells (3 $\times$ 10³ per well) were first reacted with 10 µL MTT solution (4 h), and crystal dissolution in each well was realized by 110 µL Formazan solution. Absorbance (490 nm) determination was conducted using multiple detection readers to calculate the relative cell viability. Relative cell viability (%) = [optical density (OD) (experiment) – OD (blank)]/[OD (control) – OD (blank)] $\times$ 100%. Data were normalized to Control group.

Using an Annexin V-FITC Apoptosis Detection Kit (CA1020, Solarbio, China), treated cells (1 $\times$ 10⁶) were first re-suspended with binding buffer, followed by incubation of suspension with 5 µL Annexin V-fluorescein isothiocyanate (room temperature, 10 min, darkness) and 5 µL propidium iodide under the same conditions (5 min). The volume of cell suspension was supplemented to 500 µL with PBS, and then the apoptosis was detected by a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA, USA). Apoptosis rate (%) = Early apoptotic cells (%) + advanced apoptotic cells (%).

LDH release in different groups was evaluated by LDH Cytotoxicity Assay Kit (C0017, Beyotime, China). After different cell treatments, cells in each well were cultivated with 60 µL LDH detection working solution (30 min). Absorbance (490 nm) was detected with a multiple detection reader for the calculation of LDH content. LDH (U/mL) = (sample hole absorbance – background blank control hole absorbance)/(standard tube absorbance – standard blank tube absorbance) $\times$ standard product concentration (U/mL).

Tumor necrosis factor (TNF)-$\alpha$ and Interleukin (IL)-6 in cell supernatant were measured by TNF-$\alpha$ and IL-6 ELISA kits (SBJ-H0038, SBJ-H0465, Senbeijia, China). More precisely, cell supernatant was diluted, and standard dilutions were prepared at the same time. The diluted sample or standard was incubated (90 min) in a 96-well plate, followed by addition of different reagents. Absorbance (450 nm) measurement was performed employing a microplate reader. According to the abscissa (standard concentration) and the ordinate (OD value), corresponding concentration was measured from the standard curve in line with the OD value of the sample and then multiplied by the dilution multiple to obtain the actual concentration of the sample.

Total protein extracted (ExKine™ Total Protein Extraction Kit, KTP3006, Abbkine, Wuhan, China) [18] experienced quantification, separation, electrophoresis, transference to membrane and blocking (5% bovine serum albumin, BSA). The membrane was probed with the primary antibodies (GAPDH as an internal control), rinsed off, and reacted with secondary antibodies. The details of antibodies are exhibited in Table 2. Protein band visualization was conducted by enhanced chemiluminescence (ECL) Western Blotting Substrate (W028-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) on a gel imaging system (610020-9q, QinXiang, Shanghai, China), after which the band intensity was calculated by ImageJ2x V2.1.4.7 (Rawak Software, Stuttgart, Germany). Relative protein expression levels = (gray value of protein band)/(gray value of GAPDH band). Data were normalized to Control group.

Data represented as mean $\pm$ standard deviation were dissected by Graph Prism v8.0 (GraphPad software, San Diego, CA, USA). TUG1 expression between Sham and model groups was analyzed by independent sample t test. One-way analysis of variance (ANOVA) was leveraged for multi-group comparison. p 0.05 was indicative of statistical noteworthy.

TUG1 expression level in rat heart tissue was much higher in model group than Sham group (Fig. 1A, p 0.001). qRT-PCR results showed TUG1 downregulation after transfection with siTUG1, indicating that the transfection was successful (Fig. 1B, p 0.001). As shown in Fig. 1C, the MAP of I/R model rat was decreased significantly at maximum bleed out (MBO), start of resuscitation, and 1 and 2 h after the end of resuscitation, which was improved at the end of resuscitation, 1 and 2 hours after the end of resuscitation following siTUG1 transfection (Fig. 1C, p 0.05). In addition, the decrease of blood actual bicarbonate (HCO3^–) level and the increase of lactate level in rats after HS/R induction were restored to normal levels by siTUG1 treatment (Fig. 1D,E, p 0.001), denoting that siTUG1 improved the HS/R-caused acid-base imbalance.

Fig. 1.

Effects of TUG1 silencing on MAP and metabolic acidosis in rat model with HS/R. Construction of 6 groups based on experimental animals (6 mice/group): Sham (receiving anesthesia and surgery without bloodletting); Model (receiving anesthesia, surgery and bloodletting); Sham + siNC (receiving subcutaneous injection of siNC 2 h before anesthesia and surgery without bloodletting); Sham + siTUG1 (receiving subcutaneous injection of siTUG1 2 h before anesthesia and surgery without bloodletting); Model + siNC (receiving subcutaneous injection of siNC 2 h before anesthesia, surgery and bloodletting); Model + siTUG1groups (receiving subcutaneous injection of siTUG1 2 h before surgery anesthesia, surgery and bloodletting). (A) TUG1 expression in myocardial tissue of rats in Sham and Model group (n = 6) (qRT-PCR, GAPDH as internal control). (B) TUG1 expression in myocardial tissue after transfected with siTUG1 (qRT-PCR, GAPDH as internal control). (C) MAP at MBO, at the start of resuscitation, at the end of resuscitation, and 1 and 2 h post resuscitation. (D,E) Actual bicarbonate (HCO₃^–) and lactate levels in rat blood samples (commercially available assay kits). ^*p 0.05, ^**p 0.01, ^*⁣**p 0.001. The experiments were independently repeated at least thrice. n = 3. siTUG1, small interfering RNA targeting TUG1; HS, hemorrhagic shock; HS/R, hemorrhagic shock and fluid resuscitation; qRT-PCR, quantitative reverse transcription polymerase chain reaction; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; MAP, mean arterial pressure; MBO, maximum bleed out.

TTC staining results suggested the area of myocardial infarction in the Model + siNC group was increased relative to Sham + siNC group, which however was then reduced by silencing TUG1 (Fig. 2A,B, p 0.001). Meanwhile, H&E staining data were presented at Fig. 2C, based on which we could observe that cardiomyocytes were irregularly arranged, the myocardial gap was widened, the muscle fibers were broken in Model + siNC group, as compared with Sham + siNC group. Besides, TUG1 silencing improved the myocardial injury of the model group, cardiomyocytes were irregularly arranged, however, the space between cardiomyocytes was narrowed. As shown in Fig. 3A,B, TUNEL-positive cell rate in Model + siNC group was significantly increased, which was reversed by siTUG1 (Fig. 3A,B, p 0.001). Afterwards, we assessed MDA, GSH and SOD levels in rat serum, and discovered that MDA level was higher but GSH and SOD levels were lower in Model + siNC group than Sham + siNC group (Fig. 3C,D, p 0.001). However, TUG1 silencing offset the effect of modeling on MDA, GSH and SOD levels (Fig. 3C,D, p 0.01). Similarly, the upregulation in inflammatory factors TNF-$\alpha$ and IL-6 in model rats was also suppressed by siTUG1 (Fig. 3E, p 0.001).

Fig. 2.

Effect of TUG1 silencing on myocardial injury in rat model with HS/R. Construction of 4 groups based on experimental animals (6 mice/group) (Sham + siNC, Sham + siTUG1, Model + siNC and Model + siTUG1). The rats were given subcutaneous injection of phosphate buffer saline containing siTUG1 or siNC 2 h before surgery and then underwent Sham or HS/R operation. (A,B) Myocardial infarction size of rats (TTC staining). (C) The myocardial injury of rats (H&E staining, 100$\times$ magnification). Scale bars, 50 µm. Arrow in Model + siNC group: cardiomyocytes were irregularly arranged, the myocardial gap was widened, the muscle fibers were broken; arrow in Model + siTUG1 group: cardiomyocytes were irregularly arranged, the space between cardiomyocytes was narrowed. ^*⁣**p 0.001. The experiments were independently repeated at least thrice. n = 3. TTC, Triphenyl-2H-Tetrazolium Chloride; H&E, Hematoxylin and eosin.

Fig. 3.

Effects of TUG1 silencing on apoptosis, oxidative stress and inflammatory factor expressions of myocardial tissue in rat model with HS/R. Construction of 4 groups based on experimental animals (6 mice/group) (Sham + siNC, Sham + siTUG1, Model + siNC and Model + siTUG1). (A,B) The apoptosis of myocardial tissue (TUNEL staining) (magnification, 200$\times$). Scale bars, 50 µm. (C,D) MDA, GSH and SOD levels in rat serum (corresponding kits). (E) TNF-$\alpha$ and IL-6 levels in rat myocardial tissue (qRT-PCR). ^**p 0.01, ^*⁣**p 0.001. The experiments were independently repeated at least thrice. n = 3. TUNEL, Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; TNF-$\alpha$, Tumor necrosis factor-$\alpha$; IL-6, Interleukin-6.

To explore whether TUG1 mediated oxidative stress of H9C2, we transfected siTUG1 into H9C2 cells and determined the transfection efficiency by qRT-PCR (Fig. 4A, p 0.001). In H₂O₂-treated H9C2, TUG1 level was increased greatly (Fig. 4B, p 0.001). Interestingly, the H₂O₂-induced upregulation of TUG1 can be abrogated by siTUG1 (Fig. 4B, p 0.001).

Fig. 4.

TUG1 silencing counteracted the influences of H₂O₂ on viability and apoptosis of H9C2 cells. H9C2 cells were assigned into 6 groups: Control (normal culture); siNC (transfection with siNC); siTUG1 (transfection with siTUG1); H₂O₂ (4-h 400 µM H₂O₂ treatment); H₂O₂ + siNC (transfection with siNC and 4-h 400 µM H₂O₂ treatment); H₂O₂ + siTUG1 (transfection with siTUG1 and 4-h 400 µM H₂O₂ treatment). (A) TUG1 expression in H9C2 cells transfected with siTUG1 or siNC (qRT-PCR, GAPDH as internal control). ^*⁣**p 0.001. (B) TUG1expression in H9C2 cells treated with H₂O₂ and transfected with siTUG1/siNC (qRT-PCR, GAPDH as internal control). (C) viability of H9C2 cells treated with H₂O₂ and transfected with siTUG1/siNC (MTT). (D,E) Apoptosis rates of H9C2 cells treated with H₂O₂ and transfected with siTUG1/siNC (flow cytometry). ^**p 0.01, ^*⁣**p 0.001. The experiments were independently repeated at least thrice. n = 3. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

H₂O₂ treatment attenuated cell viability (Fig. 4C, p 0.001) and facilitated apoptosis (Fig. 4D,E, p 0.001). Further, H₂O₂treatment promoted the secretion of LDH (Fig. 5A, p 0.001), decreased SOD level (Fig. 5B, p 0.001), and elevated TNF-$\alpha$ and IL-6 levels (Fig. 5C,D, p 0.001). However, these effects were reversed by siTUG1 (Fig. 4C–E and Fig. 5A–D, p 0.01). We also found that H₂O₂ can induce NF-$\kappa$B/p65 expression in H9C2 cells, which can be counteracted by TUG1 silencing (Fig. 5E,F, p 0.001). The same results also were observed in p-NF-$\kappa$B/NF-$\kappa$B ratio (Fig. 5G, p 0.001).

Fig. 5.

TUG1 silencing reversed the influences of H₂O₂ on oxidative stress, inflammation and NF-$\kappa$B pathway of H9C2 cells. H9C2 cells were divided into 4 groups (Control; H₂O₂; H₂O₂ + siNC; H₂O₂ + siTUG1). (A,B) LDH and SOD levels in cell supernatant (corresponding kits). (C,D) TNF-$\alpha$ and IL-6 contents in cell supernatant (ELISA). (E–G) p-NF-$\kappa$B/p65 and NF-$\kappa$B/p65 protein levels in H9C2 cells treated with H₂O₂ and transfected with siTUG1/siNC (Western blot, GAPDH as internal control). ^**p 0.01, ^*⁣**p 0.001. The experiments were independently repeated at least thrice. n = 3. LDH, lactate dehydrogenase.