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Introduction

Although significant progress has been made toward the diagnosis and care of acute pulmonary embolism (PE), relevant risk assessment for the disease and proper patient management remains challenging in clinical applications (1). In two previous studies involving large prospective registries, it was demonstrated that, in addition to the manifestations of hemodynamic and clinical instability, acute right ventricular dysfunction is a major prognostic factor in PE patients during their hospitalization (2,3). By monitoring the presence of right ventricular hypokinesis and enlargement in conjunction with echocardiography, patients at a high risk of impending or apparent right ventricular failure can be screened, therefore potentially identifying those patients that may exhibit positive responses to thrombolytic treatments (4,5).

Certain previous studies have demonstrated that 40% of PE patients with disease severity similar to that in a rat model exhibited persistent right ventricular (RV) dysfunction with restricted cardiopulmonary functions, regardless of their state of comorbidity (6,7). In addition, the monophasic response of neutrophils and a potential biphasic response of monocytes were present throughout the progression and resolution of the cardiac inflammation and during the early stage of scar formation in rats. Therefore, the series of events observed in the rat model suggest the involvement of such inflammatory reactions in post-PE cardiac injuries and indicate that the long-term recovery of heart functions may be enhanced by the management of acute inflammation generated during the disease (8). The activation of inflammasomes, as represented by increased NLRP3 expression, has been shown as a key process during inflammation (9).

As a natural antioxidant, resveratrol is extracted from Polygonum cuspidatum and can decrease the viscosity of blood, inhibit vasodilation and the aggregation of platelets, maintain a normal blood flow, and prevent the onset and progression of cancer (10-12). Ji et al (13) reported that resveratrol could downregulate metastasis associated lung adenocarcinoma transcript 1 (MALAT1), therefore decreasing the localization of β-catenin in the nuclei and attenuating the activity of the Wnt/β-catenin signaling pathway, ultimately inhibiting the metastasis of colorectal cancer (13,14).

A total of 10,000s of long noncoding RNAs (lncRNAs), defined as RNA transcripts that have >200 nucleotides in length and have no protein coding capabilities, have been identified and a number of these lncRNAs are uniquely expressed in different types of tissues or specific cancers (15). It has been shown that lncRNA genes outnumber the genes responsible for protein-coding, whereas >90% of genes in the human genome do not encode peptide products (16-18). It has now been widely recognized that compared to the expression of mRNA, the expression of different lncRNAs is under more strict regulation and therefore is limited to specific cell types (19). As a result, lncRNAs are frequently associated with evolutionarily conserved functions, secondary structures and regions of microhomology, although their overall sequence similarity is low (20-22). It was shown that the expression of MALAT1 was increased in patients suffering from unstable angina. On the other hand, the silencing of MALAT1 expression significantly decreased the expression of CXCR2, a target gene of miR-22-3p, by reversing the function of miR-22-3p, therefore leading to aggravated endothelial injury induced by the oxidization of low-density lipoproteins. The protein kinase B pathway was found to be involved in this entire process (23).

Inflammation has been reported to be an important mechanism underlying the cardiac injury associated with pulmonary embolism and inflammasome with a marker of activated NLRP3 is considered a key signaling pathway of the inflammatory process (8,24). Resveratrol has been reported to treat cardiac or lung injury by suppressing the activation of NLRP3 (25,26). Furthermore, it was noted that administration of resveratrol could downregulate the expression of MALAT1 and MALAT1 functions as a sponge of miR-22-3p (14,23). In this study, the involvement of MALAT1/miR-22-3p/NLRP3 in the therapeutic effect of resveratrol in the treatment of cardiac injury associated with pulmonary embolism was explored.

Materials and methods

Animals

A total of 24 male Sprague-Dawley rats (age, 50-65 days; weight, 300-375 g) were obtained from the animal center of the First Affiliated Hospital of Chengdu Medical College. The rats were housed at room temperature between 20-25°C with a relative humidity between 50-60%. Animals received free access to food and deionized water under a 12 h light/dark cycle. All experiments were carried out with the agreement of Institutional Animal Care and Use Committee of the First Affiliated Hospital of Chengdu Medical College according to Guide for the Care and Use of Laboratory Animals.

PE model

A total of 3 mg⁄kg xylazine and 70 mg⁄kg ketamine was utilized to anaesthetize animals and then 2.0×106⁄100 g polystyrene microspheres (Duke Scientific; Thermo Fisher Scientific, Inc.) was utilized to establish PE by injecting into right jugular vein. Animals treated with 0.01% Tween-20 (0.16 ml⁄100 g) were used as internal control.

In vivo measurements

There are three groups of rats (n=8 per group): Sham groups as the control, animal model of PE group and PE group treated with resveratrol (RS; 30 mg/kg; Santa Cruz Biotechnology, Inc.) solution. A total of 3 mg⁄kg xylazine and 70 mg⁄kg ketamine was utilized to anaesthetize animals and then placed on warming pads at 6 weeks (a preliminary test was performed to determine the best time point for the treatment group; data not shown). The breathing rate of tested animals was counted and the heart rate and systemic pressures were measured using 2-French micromanometer (Millar Instruments, Inc.), which placed in left carotid artery; RV pressures was detected using 2-French bent Millar micro-manometer (Millar Instruments, Inc.) by inserting into jugular vein. Pressure data was acquired using Acknowledge software (ACK100W; Biopac Systems, Inc.). Blood chemistry was analyzed using StatProfile Ultra analyzer (Nova Biomedical) and the blood samples were then stored at -70°C for further analysis.

In vitro RV outflow contractile force measurements

Midline thoracotomy was performed on anaesthetized rats to isolate the heart and then it was stored in ice-cold saline. Krebs-Henseleit bicarbonate buffer was perfused into the aorta for 5 min at 60 mmHg at 37°C in a non-recirculating system. The RV-free walls were isolated and then a strip of muscle was cut longitudinally from the midline of the outflow tract and stored in a tissue bath supplemented with Krebs-Henseleit bicarbonate buffer at 37°C with 95% O2/5% CO2, and then tied to a fixed point using Refined Myograph System (Kent Scientific Corporation, Torrington, CT, USA). Platinum plate electrodes were used to treat strips at 80 V and 0.2 Hz for 10 ms. Acknowledge software (ACK100W; Biopac Systems, Inc.) was used to record data. A total of 0.25 mm increments in length were used to construct length-tension curves to examine contractile force with optimum length induced maximum active force (mN⁄mm2). Finally, components were calculated as follow: mm2 cross-section area = to wet wt/optimum length, then Newton = gm tension ×9.807.

RNA isolation and reverse transcription (RT) PCR

RNeasy total RNA isolation system (Qiagen, Inc.) was used to extract total RNA from HL-1 and AC16 cells and tissue samples. NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.) was used to assess RNA purity and concentration. SuperScript™ III reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) was utilized to synthesize cDNA of NLRP3. FastStart SYBR Green Master (Roche Diagnostics) was used to carry out quantitative PCR on Mx3005P PCR machine (Agilent Technologies). The reaction was carried out as follows: 50°C for 3 min, 95°C for 12 min, then 40 cycles at 94°C for 15 sec and at 60°C for 1 min, then 72°C for 30 sec. Small RNA U6 and GAPDH were served as internal control for MALAT1, miR-22-3P and NLRP3. Comparative 2-ΔΔCq method (27) was utilized to calculate relative expressions of MALAT1, miR-22-3P and NLRP3 mRNA, which normalized to expressions of U6 and GAPDH. Each reaction was repeated three times. The primers pairs used were as follows: MALAT1 forward, 5′-CAG ACC ACC ACA GGT TTA CAG-3′ and reverse, 5′-AGA CCA TCC CAA AAT GCT TCA-3′; miR-22-3P forward, 5′-AAG CTG CCA GTT GAA GAA CTG T-3′ and reverse, 5′-CAG TGC GTG TCG TGG AGT-3′; NLRP3 forward, 5′-CTG CAG ATG GCA AGC ACC CGC TGC-3′ and reverse, 5′-CGT TTG GAT CCC TAC CAA GAA GGC TCA A-3′; U6 forward, 5′-CGC TTC GGC AGC ACA TAT ACTA-3′ and reverse, 5′-CGC TTC ACG AAT TTG CGT GTC A-3′; GAPDH forward, 5′- CTT TGT CAA GCT CAT TTC CTG G-3′ and reverse, 5′-TCT TGC TCA GTG TCC TTG C-3′.

Cell culture and treatment

HL-1 and AC16 cells were obtained from American Type Culture Collection, and incubated with Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 mg/ml streptomycin and 100 U/ml penicillin; Life Technologies; Thermo Fisher Scientific, Inc.) under a humidified atmosphere with 5% CO2 at 37°C in 24-well plates for 24 h. When cells reached 50-80% confluence at a final density of 5×104 per well, HL-1 and AC16 cells treated with 1 or 5 µm RS were transfected with 30 nM miR-22-3p mimic using Lipofectamine RNA iMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following manufacturer's protocol. RT-qPCR assay was performed to evaluate transfection efficiency. Each test was performed three times.

Cell proliferation assay

MTT assay was performed to assess cell viability of HL-1 and AC16 cells transfected with miR-481 mimics. HL-1 and AC16 cells were cultured in 24-well plates at a final density of 1×104 cells/well, and incubated for 48 h, followed by addition with 10 µl MTT (5 mg/ml), and maintained for another 4 h. DMSO was utilized to dissolve the crystals at 37°C from 10 min. A spectrophotometer (NanoDrop Technologies) was used to detect optical density values at 490 nm wavelength. Each experiment was run three times.

Luciferase assay

Using online publicly available algorithms including mirdb.org, miR-22-3p targets were predicted. The full sequence of M'ALAT1 or NLRP3 3′ untranslated region (UTR) containing putative binding site of miR-22-3p was amplified using PCR. The putative binding site of miR-22-3p on MALAT1 or NLRP3 3′UTR was mutated using QuickChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.) in accordance with the manufacturer's protocol and generated mutant MALAT1/NLRP3 3′UTR. Then wild-type or mutant MALAT1 or NLRP3 3′UTR was sub-cloned into pGL3 vector (Promega Corporation) located on immediately down-stream of luciferase gene. A total of 1×105 per well cells were seeded into 48-well plates, Lipofectiamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was utilized to transiently transfect HL-1 and AC16 cells with luciferase construct containing wild-type or mutant MALAT1 or NLRP3 3′UTR and miR-22-3p mimics following manufacturer's protocol. Passive Lysis Buffer (Promega Corporation) was utilized to harvest the cells 36-48 h post-transfection. Dual luciferase assay system (Promega Corporation) was utilized to detect luciferase activities, which was normalized to Renilla luciferase activity. Each test was performed three times.

Western blot analysis

Ice-cold PBS (Invitrogen; Thermo Fisher Scientific, Inc.) was used to harvest the cells 2 days after transfection and cold-modified radioimmunoprecipitation buffer (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 15 mM Tris/HCl pH 7.5, 1 mM EDTA, 25 mM KCl, 120 mM NaCl, 0.5% Triton X100, protease inhibitors (Invitrogen; Thermo Fisher Scientific, Inc.) was used to lyse the cells following the manufacturer's protocol. A bicinchoninic acid Protein Assay kit (Bio-Rad Laboratories, Inc.) was used to determine the concentration of the protein based on the manufacturer's protocol. A 4-12% SDS-PAGE was used to separate total protein (35 µg per lane) and then transferred on nitrocellulose membranes (Bio-Rad, Laboratories, Inc.). Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% non-fat dry milk (Merck KGaA) was used to block the membrane for 60 min at room temperature to avoid unspecific binding. Primary rabbit antibodies targeted NLRP3 (cat. no. sc-134306), ASC (cat. no. sc-514414) or Caspase-1 (cat. no. sc-56036; 1;5,000; Santa Cruz Biotechnology, Inc.) or anti-β-actin (cat. no. A1978; 1;10,000; Sigma-Aldrich; Merck KGaA) were incubated with the membrane at 4°C overnight. The secondary diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (cat. no. G-21234; 1:10,000; Thermo Fisher Scientific, Inc.) was incubated with the membrane at 4°C for 12 h. The protein bands were detected using Bio-Rad ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.) with Western Breeze chemilluminescent kit (Invitrogen, Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Three independent tests were performed.

Apoptosis analysis

Annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA) was used to measure cell apoptosis of HL-1 and AC16 cells using a flow cytometer. In brief, 48 h after the HL-1 and AC16 cells were transfected with miR-22-3p mimic, the cells were harvested, then washed twice using PBS. 1X binding buffer was used to resuspend cells at a final density of 4×105 cells per well. A total of 5 µl of PE Annexin V and 5 µl of 7-AAD were used to treat 100 µl of cell suspension, and followed by incubation in darkness for 15 min. FACS MELODY™ (BD Biosciences; Becton, Dickinson and Company) was used to detect cell apoptosis.

ELISA

Levels of IL-1β (cat. no. MLB00C) and IL-18 (cat. no. DY318-05) in serum were tested using ELISA kits (R&D Systems, Inc.) in accordance with manufacturer's protocol.

Immunohistochemistry

Tissue samples were harvested and cold 4% paraformaldehyde was used to fix the samples at 4°C for 12 h, and samples further embedded in paraffin. Then the samples were cut into 5-µm sections. Hematoxylin and eosin staining were performed to detect expression of NLRP3. 3% H2O2 was used to treat sections for 15 min, then 80% carbinol was incubated with sections for 30 min, then the blocking solution was used to treat sections for another 60 min at room temperature. Primary rabbit antibodies targeted NLRP3 (cat. no. sc-134306; 1;500; Santa Cruz Biotechnology, Inc.) were incubated with the sections at 4°C overnight. PBS was used to wash the sections three times. The secondary diluted HRP-conjugated anti-rabbit IgG antibody (cat. no. G-21234; 1:2,000; Thermo Fisher Scientific, Inc.) was used to incubate sections at 37°C for 2 h. 3,3-diaminobenzidine (DAB) was used to stop the reaction. A Nikon ECLPSE 80i fluorescent microscope (Nikon Corporation, Tokyo, Japan) was used to visualize results. All reactions were carried out in triplicate.

TUNEL

One step TUNEL Apoptosis Asssy kit (Roche GmbH, Mannheim, Germany) was used to detect DNA fragmentation based on the manufacturer's protocol. The 5-µm thick sections were deparaffinized and rehydrated. A total of 10 µg/ml proteinase K was utilized to treat sections at 37°C for 30 min and 50 µl of TUNEL inspection fluid was used to incubate sections at 37°C for 60 min, and then washed three times using PBS. DMEM was used as the mounting medium. Nikon ECLIPSE Ti fluorescent microscope (Nikon Corporation) was used to capture the images in three fields of view using 530 nm wavelength emission and 488 nm for excitation. Three independent experiments were performed.

Statistical analysis

All results were presented as the mean ± standard deviation. SPSS19.0 software package (IBM Corporation, Armonk, NY, USA) was used to perform statistical analysis using Student's t test. Additionally, one-way analysis of variance was used to examine the difference between more than two groups and a Scheffe's test was utilized as the post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Identification the effect of RS on the promoter efficiency of MALAT1

To explore the underlying mechanism of the therapeutic effect of RS in the treatment of PE associated cardiac injury, constructs containing the MALAT1 promoter were obtained and then sub-cloned upstream of the luciferase gene of pcDNA3 (Fig. 1A). Different doses of RS (1 or 5 µM) were used to treat HL-1 and AC16 cells. Luciferase activity in HL-1 and AC16 cells was significantly dose-dependently downregulated subsequent to transfection with RS compared with the control (P<0.05; Fig. 1B and C), suggesting that RS decreased the promoter efficiency of MALAT1.

Figure 1 RS increased promoter efficiency of MALAT1. (A) Fragment of MALAT1 promoter was inserted into immediately upstream of luciferase gene. (B) Luciferase activity in HL-1 cells transfected with 1 and 5 µm RS compared with the negative controls. *P<0.05 vs. the negative controls. (C) Luciferase activity in AC16 cells transfected with 1 and 5 µm RS compared with the negative controls. *P<0.05 vs. the negative controls. RS, resveratrol; MALAT1, metastasis associated lung adenocarcinoma transcript 1; NC, negative control.

MALAT1 directly targets and represses miR-22-3p expression

To validate the successful transfection of miR-22-3p mimics into HL-1 (Fig. 2A) and AC16 (Fig. 2B) cells, relative expression of miR-22-3p was observed, demonstrating significantly upregulated miR-22-3p level in cells transfected with miR-22-3p mimics compared with the control group (P<0.05). Accordingly, as presented in Fig. 3A and B, a candidate binding site of MALAT1 was determined on miR-22-3p. The potential regulatory relationship between MALAT1 and miR-22-3p was examined using a luciferase assay. miR-22-3p significantly reduced luciferase activity of wild-type MALAT1 but not that of mutant MALAT1 in HL-1 (P<0.05; Fig. 3C) and AC16 (P<0.05; Fig. 3D) cells, suggesting a negative correlation between MALAT1 and miR-22-3p, but this needs to be confirmed.

Figure 2 Transfection of miR-22-3p mimics successfully elevated the relative expression of miR-22-3p. (A) Relative expression level of miR-22-3p with or without the transfection of miR-22-3p mimics in HL-1 cells. *P<0.05 vs. the scramble controls. (B) Relative expression level of miR-22-3p with or without the transfection of miR-22-3p mimics in AC16 cells. *P<0.05 vs. the scramble controls. miR, microRNA.

Figure 3 MALAT1 directly targets and represses miR-22-3p expression. (A) Schematic comparison between MALAT1 and the 'seed sequence' in miR-22-3p. (B) Constructs with Wt/M MALAT1 inserted downstream of the luciferase gene. (C) Luciferase activity of HL-1 cells co-transfected with Wt/M MALAT1 and miR-22-3p compared with the scramble controls. *P<0.05 vs. the scramble controls. (D) Luciferase activity of AC16 cells co-transfected with Wt/M MALAT1 and miR-22-p compared with the scramble controls. *P<0.05 vs. the scramble controls. miR, mircoRNA; MALAT1, metastasis associated lung adenocarcinoma transcript 1; Wt/M, wild-type/mimics; NC, negative control; Mt/M, mutant type/mimics.

NLRP3 is the direct downstream of miR-22-3p

The results from computational analysis demonstrated that a putative binding site of miR-22-3p located within 3′UTR of NLRP3 (Fig. 4A). To further verify the regulatory relationship between miR-22-3p and NLRP3, a luciferase reporter plasmid containing the NLRP3 fragment with 3′UTR or without miR-22-3p binding site was generated, and HL-1 and AC16 cells co-transfected with the above luciferase construct and miR-22-3p mimic or scramble controls. As presented in Fig. 4B and C, the luciferase activity of the NLRP3 fragment with 3′UTR was significantly inhibited by miR-22-3p (P<0.05) but the luciferase activity of the NLRP3 fragment without the 3′UTR demonstrated no obvious difference compared with the scramble control in HL-1 (Fig. 4C) and AC16 (Fig. 4C) cells. These findings validated that miR-22-3p inhibited the expression of NLRP3 by binding to complementary sequences in the 3′UTR of NLRP3.

Figure 4 NLRP3 was the direct downstream of miR-22-3p. (A) Schematic comparison between NLRP3 3′UTR and the 'seed sequence' in miR-22-3p. (B) Constructs with wild-type or mutant NLRP3 3′UTR inserted down-stream of the luciferase gene. (C) Luciferase activity of HL-1 cells co-transfected with miR-22-3p/scramble controls and wild-type NLRP3 3′UTR compared with mutant NLRP3 3′UTR. *P<0.05 vs. the mutant NLRP3 3′UTR. (D) Luciferase activity of AC16 cells co-transfected with miR-22-3p/scramble controls and wild-type NLRP3 3′UTR compared with mutant NLRP3 3′UTR. *P<0.05 vs. the mutant NLRP3 3′UTR. miR, microRNA; UTR, untranslated; Con, control; WT, wild-type; Mut, mutant.

MALAT1, miR-22-3p and NLRP3 are differently expressed in different groups

An animal model with PE associated cardiac injury was established and RS was used to treat animal with PE associated cardiac injury. RT-PCR was carried out to detect the expression levels of MALAT1, miR-22-3p and NLRP3 in the sham, PE associated cardiac injury and PE associated cardiac injury plus RS groups. As presented in Fig. 5, the MALAT1 (Fig. 5A) and NLRP3 (Fig. 5C) levels in PE associated cardiac injury group were significantly increased in the sham group (P<0.05), and the administration of RS reduced the upregulated MALAT1 and NLRP3 levels in the PE associated cardiac injury group, although the level remained higher than the sham group. On the contrary, the miR-22-3p (Fig. 5B) level in PE associated cardiac injury group was significantly decreased compared with the PE associated cardiac injury plus RS group and both groups were much lower than the sham group.

Figure 5 MALAT1, miR-22-3p and NLRP3 are differently expressed in different groups. (A) MALAT1 level in PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. P<0.05 vs. the sham group. (B) miR-22-3p level in PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. *P<0.05 vs. the sham group. (C) NLRP3 level in PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. *P<0.05 vs. the sham group. miR, microRNA; PE, pulmonary embolism; RS, resveratrol.

NLRP3, ASC and Caspase-1 is differently expressed in different groups

Western-blot analysis was performed to examine protein levels of NLRP3, ASC and Caspase-1 between the sham, PE associated cardiac injury and PE associated cardiac injury plus RS groups. As presented in Fig. 6, the PE associated cardiac injury group exhibited significantly increased protein levels of NLRP3 (P<0.05; Fig. 6A and B), ASC (Fig. 6A and C) and Caspase-1 (Fig. 6A and D) compared with the sham group, while PE associated cardiac injury plus the RS group displayed significantly decreased protein levels of NLRP3, ASC and Caspase-1 than PE associated cardiac injury group (P<0.05).

Figure 6 NLRP3, ASC and Caspase-1 are differently expressed in different groups. (A) NLRP3, ASC and Caspase-1 levels in PE associated cardiac injury group, PE associated cardiac injury plus RS group, and sham group. *P<0.05 vs. the sham group. (B) NLRP3 level in PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. *P<0.05 vs. the sham group. (C) ASC level in the PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. *P<0.05 vs. the sham group. (D) Caspase-1 level in the PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. *P<0.05 vs. the sham group. PE, pulmonary embolism; RS, resveratrol.

Differential levels of inflammatory factors in various groups

ELISA was carried out to determine protein levels of IL-1β and IL-18 among sham, PE associated cardiac injury and PE associated cardiac injury plus RS groups. As presented in Fig. 7, IL-1β (Fig. 7A) and IL-18 (Fig. 7B) levels in PE associated cardiac injury group were significantly increased compared with the sham group (P<0.05), and significantly decreased in PE associated cardiac injury plus RS group compared with the PE associated cardiac injury group (P<0.05).

Figure 7 Differential levels of inflammatory factors including IL-1β and IL-18 in various groups. (A) IL-1β level in the PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. *P<0.05 vs. the sham group. (B) IL-18 level in PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group. *P<0.05 vs. the sham group. IL, interleukin; PE, pulmonary embolism; RS, resveratrol.

Differential NLRP3 protein level in different groups

Immunohistochemistry was performed to examine the protein levels of NLRP3 in the sham, PE associated cardiac injury and PE associated cardiac injury plus RS groups. As presented in Fig. 8, the PE associated cardiac injury group exhibited higher protein levels of NLRP3 (Fig. 8) compared with the sham group, while PE associated cardiac injury plus RS group displayed a slightly reduced protein levels of NLRP3 (Fig. 8) compared with the PE associated cardiac injury group

Figure 8 NLRP3 protein in PE associated cardiac injury group, PE associated cardiac injury plus RS group and sham group (magnification, ×40). PE, pulmonary embolism; RS, resveratrol.

Discussion

Since the incidence of massive PE is quite low, no individual physician or hospital can rely on their own experiences to achieve optimal patient management. Despite the application of anticoagulation treatment, the mortality rate of submassive PE doubles in patients suffering from RV dysfunction and preserved systemic arterial pressure (28). Three major factors have been demonstrated to play a role in the clinical manifestation and progression of acute pulmonary embolism: The severity of pre-existing dysfunctions in the cardiopulmonary system, the extent of embolic obstruction in the pulmonary artery and the presence of thrombosis in peripheral venous vessels, which can potentially induce the recurrence of throm-boembolic events (29,30). The interactions between these factors can lead to afterload stress in the RV and the onset of hypertension in the pulmonary artery (29). The presence of above clinical manifestations can be accurately detected using ultrasound imaging (31). It has been demonstrated in previous studies that when the afterload stress in the RV resulted in clinically evident right heart failure (especially when it was also accompanied by symptoms of cardiogenic shock), the prognosis in the patients was poor (32,33).

RS usually exists in the form of cis- and trans- isomers. As the preferred steric form, trans-RS is more stable if protected from light and high pH. The production of trans-RS in plants can be triggered by UV radiation, microbial infection, or ozone exposure (34-36). A recent study has demonstrated that RS could improve the ability of spatial learning as well as memory capacity in septic mice. These conclusions were drawn based on the observed inhibition of neuronal apoptosis and the activation of microglia after the administration of RS. In addition, RS was demonstrated to inhibit the NLRP3/IL-1β axis both in vivo and in vitro (37). Several underlying mechanisms are believed to participate in the downregulation of NLRP3 expression and the maturation of IL-1β after the administration of RS. Sirt1 was demonstrated to deacetylate nuclear factor-κB, therefore preventing the nuclear translocation of p65 subunit (38,39). In this study, the underlying mechanism of the therapeutic effect of RS in the treatment of PE associated cardiac injury was explored and it was found that RS upregulated promoter efficiency of MALAT1. Furthermore, in-silico analysis was performed to investigate the regulatory relationship between MALAT1 and miR-22-3p, and it was revealed that MALAT1 directly targeted and repressed miR-22-3p expression. In addition, the regulatory relationship between miR-22-3p and NLRP3 was investigated using online publicly available algorithms, and it was found that miR-22-3p inhibited the expression of NLRP3 by binding to complementary sequences in the 3′UTR of NLRP3.

MALAT1, an lncRNA, was first discovered in invasive non-small cell carcinoma and its overexpression has been found in a number of other cancerous tissues, suggesting that MALAT1 plays a role in the invasion and metastasis of cancer (40-43). In the present study, it has been shown that the expression of MALAT1 in 60 colorectal cancer (CRC) tissues was increased compared with in the adjacent normal tissues and the upregulation in MALAT1 expression was correlated with the invasion and metastasis capabilities of CRC. In the screening experiments, the monomers extracted from two Chinese traditional medicines were identified as having a direct effect on the expression of MALAT1. Among these two monomers, the potency of RS was higher. It was demonstrated in a previous study that the inhibition of MALAT1 expression by shRNA significantly reduced the invasion and migration of CRC LoVo and HCT116 cells (43). Similar to the outcome of RS treatment, the downregulation of MALAT1 expression also suppressed the nuclear translocation of β-catenin, therefore leading to a decreased level of c-Myc and matrix metalloproteinase-7. On the other hand, the upregulation of MALAT1 expression achieved the opposite results (14). Therefore, one key MALAT1 question is to determine the correct conditions of cellular stress and pathological environments so that the presence of MALAT1 in vivo becomes essential (44). A recent study has demonstrated the essential role of MALAT1 in the tight regulation of LPS-induced inflammatory responses, therefore presenting the first piece of evidence supporting the role of MALAT1 in the regulation of inflammation and innate immunity (45). In addition, another study demonstrated that between the livers of wild-type and MALAT1 knockout mice, the only gene with significantly altered expression was serum amyloid A3, an inflammatory mediator (37). In the present study, it was found that MALAT1 and NLRP3 levels were increased, while miR-22-3p level was decreased in the PE associated cardiac injury plus RS group compared with the sham group. In addition, MALAT1 and NLRP3 levels were increased, and miR-22-3p level was decreased in PE associated cardiac injury group compared with the PE associated cardiac injury plus RS group. Furthermore, the present study demonstrated that NLRP3, ASC and Caspase-1 levels in the PE associated cardiac injury plus RS group was increased compared with the sham group, while a greater increase was demonstrated in the PE associated cardiac injury group compared with the PE associated cardiac injury plus RS group.

The activation of inflammasomes, as represented by increased NLRP3 expression, has been shown to be a key process during inflammation (9). Several endogenous signals, such as extracellular adenosine triphosphate, monosodium urate, crystals of calcium phosphates crystals of cholesterols, amyloid-β, hyaluronan and islet amyloid polypeptide (also known as amylin) can trigger the activation of NLRP3 inflammasomes, whereas exogenous signals such as silica and asbestos can also activate NLRP3 inflammasomes (46-53). The activation of the NLRP3 inflammasomes by these signals of danger can induce the release of IL-1β and subsequent inflammatory responses, therefore leading to the damage in human organs or tissues (9). In this study, it was also revealed that PE associated cardiac injury plus RS group exhibited higher levels of IL-1β and IL-18 compared with the sham group, and the PE associated cardiac injury group displayed higher levels of IL-1β and IL-18 compared with the PE associated cardiac injury plus RS group. Meanwhile, it was demonstrated that NLRP3 was increased in PE associated cardiac injury group compared with the PE associated cardiac injury plus RS group, both of them were increased compared with the sham group.

In conclusion, it was demonstrated that RS has been shown to prevent the development of cardiac injury induced by PE via modulating the expression of MALAT1 and further affect miR-22-3p and NLRP3.

Acknowledgments

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

KY and WL designed the study. WD and YJ collected the literature. KY, WL, WD, YJ, NH, YL, BR and JS collected and analyzed the data, KY and WL wrote the manuscript and the remaining authors read and approved the final manuscript.

Ethics approval and consent to participate

All experiments were carried out with the agreement of Institutional Animal Care and Use Committee of the First Affiliated Hospital of Chengdu Medical College according to Guide for the Care and Use of Laboratory Animals.

Patient consent for publication

Not applicable.

Competing interests

The authors declare they have no competing interests.

References