Estrogen alleviates myocardial ischemia-reperfusion injury by inhibiting NLRP3 inflammasome -mediated pyroptosis

doi:10.21203/rs.3.rs-3319187/v1

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Estrogen alleviates myocardial ischemia-reperfusion injury by inhibiting NLRP3 inflammasome -mediated pyroptosis

https://doi.org/10.21203/rs.3.rs-3319187/v1

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The present study was designed to investigate whether 17β-estrogen (E2) reduces myocardial I/R injury by inhibiting NLRP3 inflammasome mediated pyroptosis. In vivo, four-weeks-old female C57BL/6 mice underwent ovariectomy (Ovx) before E2 replacement therapy. After the establishment of myocardial I/R injury, plasma E2, LDH and CK-MB were detected, and the myocardial infarction (MI) size, TUNEL immunofluorescence, and the expressions of TXNIP, NLRP3, caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC), Gasdermin D protein (GSDMD), IL-1β were compared among each group. In vitro, primary mice cardiomyocytes were isolated to create hypoxia/reoxygenation (H/R) model. The cells viability, the release of LDH, TXNIP, IL-18, IL-1β, caspase-1 viability, level of cardiomyocytes death and the expression of TXNIP, NLRP3, caspase1, ASC, IL-1β, GSDMD were compared among each groups. In vivo, E2 replacement therapy significantly reduced infarct size and pyroptosis compared with mice in OVX + I/R group. In addition, E2 replacement could markedly suppressed expressions of pyroptosis related proteins, including TXNIP, NLRP3, cleaved Caspase-1, ASC, IL-1β as well as the pyroptosis executor GSDMD. In vitro, the effects of E2 on cardiomyocytes injury and pyroptosis related proteins were dramatically reversed when co-administrated with estrogen receptor antagonist ICI 182780. In addition, NLRP3 inhibitor Bay11-7082 could preserve cell viability, reduce pyroptosis as well as expressions of pyroptosis related proteins. Furthermore, pretreatment with Caspase-1 inhibitor AC-YVAD-CMK could significantly limit cell injury and reduce expressions of pyroptosis related proteins. Our study demonstrated that estrogen alleviate myocardial I/R injury by inhibiting the level of pyroptosis via ER/TXNIP/NLRP3/Caspase-1.

Acute myocardial infarction (AMI), also known as a heart attack, remains one of the leading causes of death worldwide. AMI occurs when the blood supply to a part of the heart muscle is interrupted, usually due to thrombosis in the coronary arteries. This interruption of blood flow can cause damage or death to the affected area of the heart. Reperfusion therapy including primary percutaneous intervention and thrombolysis is an effective strategy aimed at restoring blood flow to the affected area of the heart after onset of AMI. However, the process of resuming blood perfusion itself might induce additional damage to the myocardium and paradoxically result in excessive injury even death of cardiomyocytes. This phenomenon, termed as myocardial ischemia/reperfusion (I/R) injury, could worsen the overall prognosis and increase the risk of complications following AMI. Over the past decades, a large number of ischemic and pharmacological cardioprotection strategies discovered in experimental studies, have been examined in the clinical setting of AMI. However, the results from many of the studies have been disappointing[1]. Thus, further understanding of the mechanisms of myocardial I/R injury is necessary to search for effective strategy to prevent the injury and improve clinical outcomes.

The mechanisms of myocardial I/R injury are multifactorial and complex, including oxidative stress, inflammation, calcium overload, mitochondrial dysfunction, production of reactive oxygen species, or their combination. Whatever the underlying mechanisms are, programmed cell death (PCD) has been confirmed to remarkably contribute to the loss of cardiomyocytes in the process of myocardial I/R injury. Apoptosis is probably the most widely recognized type of PCD, and is mechanistically defined by the requirement for particular caspases, including Caspase-3. Apoptotic caspases lead to DNA fragmentation, cytoskeletal breakdown, membrane blebbing, and the formation of apoptotic bodies.

Pyroptosis is a form of PCD that is distinct from apoptosis. Pyroptosis occurs in response to certain microbial infections and cellular stressors, including the I/R injury[2, 3]. The defining feature of pyroptosis is the dependence of Caspase-1, which could be initiated by a multi-protein complex, termed as inflammasome. As a protease, activated Caspase-1 could process the inactive precursors of interleukin-1β (IL-1β), IL-18 and pro-Gasdermin D(GSDMD) into mature inflammatory cytokines. Particularly, GSDMD has the property of perforating cell membrane, resulting in cell perforation, lysis and death[4]. The NLRP3 inflammasome is currently the most fully characterized inflammasome consisting of NLRP3(nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3), ASC (apoptosis-associated speck-like protein), and Caspase-1[5, 6]. Once sensed by NLRP, damage signals could promote Caspase-1 cleavage and secretion of the proinflammatory cytokines. Previous study demonstrated that NLRP3 was markedly increased after MI and associated with deteriorated cardiac function in mice subjected to I/R injury[6, 7]. Thioredoxin-interacting protein (TXNIP) belongs to the α-arrestin protein superfamily and directly interacts with NLRP3, hence its essential role for the activation of NLRP3 inflammasome. Deletion of TXNIP has been demonstrated to be protective from I/R injury[8]. Moreover, recent study found that TXNIP-mediated NLRP3 inflammasome activation was a novel mechanism of I/R injury in cardiac microvascular endothelial cells[9]. Thus, inhibiting the activation of NLRP3 inflammasome through blockade of TXNIP/NLRP3 signaling pathway might be a novel therapy for mitigating I/R injury.

It is widely accepted that women have a lower incidence of myocardial infarction and better prognosis than men at a similar age, but this trend reverses after the menopause[10]. The underlying mechanisms of gender disparity on myocardial ischemia injury are poor understood. An early bilateral oophorectomy in females led to increased cardiovascular mortality, suggesting the cardioprotective effect of female sex hormones[11]. Numerous evidence suggested that estrogen replacement therapy could alleviate myocardial I/R injury in female animals suffering from ovariectomy (OVX)[12–16]. Recently, some studies showed that estrogen inhibited NLRP3 inflammasome activation and proinflammatory cytokine production[17–19]. Similarly, our previous work found that 17 beta-estradiol (E2) dramatically ameliorated airway inflammation via inhibiting NLRP3 inflammasome and its downstream products, Caspase-1 and the pro-inflammatory cytokine IL-1β[20]. Moreover, It is reported that E2 could down-regulate the expression level of TXNIP by combining with ER-β[21]. Nevertheless, it remains unclear whether E2 could alleviate myocardial I/R injury through TXNIP/NLRP3 mediated pyroptotic pathway. The present study was designed to explore the effects E2 on TXNIP/NLRP3 mediated pyroptosis in the setting of myocardial I/R injury.

Animals and groups

C57 female mice were purchased from the Animal Experimental Center of Anhui Medical University. All experimental operations were performed in compliance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals (Eighth Edition. Washington, DC: The National Academies Press. https://doi.org/10.17226/12910) and approved by the Ethics Committee of Anhui Medical University (Grant No. LLSC20200498).

Mice were randomly assigned to the following three groups, and there are 14 mice in each group:

1. Ovariectomy (OVX) + Sham heart surgery (Sham)

The protocol of ovariectomy has been described in detail previously[20]. Briefly, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg) after fasting overnight and kept on a heating mat to maintain the body temperature. The bilateral ovaries were removed after abdominal cavity was opened from the back, then the mice were put back into the cage until the resuscitation was completed. All the experimental mice were treat with ovariectomy in the same day.

As for the heart surgery, mice in this group underwent the same procedure including anesthesia, intubation, thoracotomy, stringing, except for no ligation of the anterior descending branch of left coronary artery (LAD).

2. OVX + I/R;

Four weeks after successful OVX, mice in this group underwent coronary artery ligation to establish myocardial I/R model as previously described. In brief, after adequate anesthesia by intraperitoneal injection of sodium pentobarbital (80 mg/kg), a 24-gauge catheter was inserted into the trachea and connected to a ventilator (HX-101E, Techman soft Co., Ltd, Chengdu, China). The heart was exposed by thoracotomy in the fourth intercostal space. The LAD was ligated transiently for 30min with 8 − 0 nylon monofilament (Lingqiao, Ningbo, China). Successful occlusion was confirmed by the whitening of the left ventricular anterior wall and ST-segment elevation on ECG. Reperfusion was initiated by untying the ligation and confirmed by ST-segment recovery.

3. OVX + E2 + I/R

Mice in this group received a subcutaneous supplement of E2 dissolved in sesame oil (5ug/ mL) for four weeks from the next day after OVX. The other two groups were given a subcutaneous injection of the same amount of sesame oil alone.

Serum levels of E2, LDH and CK-MB

At the end of reperfusion, blood samples were collected and serum E2 levels were measured according to the manufacturer's instructions in the E2 kit (Meilian, Shanghai, China). The concentration of LDH and CK-MB in the serum of mice in each group were detected by biochemical analyzer(Siemens, Germany).

Comparison of MI size

MI area was determined by intravenously injection with 1ml of 2% Evans blue solution (Sigma, US) through right internal jugular vein after re-occlusion of LAD.

Then, the heart was frozen at -80℃ for 10min and cut into 2-mm thick slices, which were incubated with 2% TTC solution in dark at 37℃ for 15min. The myocardium unstained with Evans Blue suggested the area at risk (AAR), where viable cardiomyocytes were stained brick red by TTC and infarcted myocardium was white. The Infarct size (IS) was calculated as IS/AAR (%). IS and AAR were digitally quantified using Image J software (1.46r, National institutes of health, USA). IS and AAR were calculated as percentage of the left ventricular area (IS/LV and AAR/LV), respectively.

Primary cardiomyocyte culture

Neonatal ventricular myocytes(NVMs) were isolated from the whole heart of 1ཞ3 days-old C57 mice. In short, heart was incubated with 0.25% trypsin(0.5ml/sample) containing Ethylene Diamine Tetraacetic Acid (EDTA) overnight at 4℃ for preliminary digestion. The next day, Dulbecco's Modified Eagle Media (DMEM) supplied with 10% fatal bovine serum (FBS) was applied to neutralize trypsin. Then, supernatant was discarded and added with 0.1% type Ⅱ collagenase for further digestion (0.6mL/heart) at 37°C several times until no visible tissue. After centrifugation, NVMs were resuspended in DMEM containing 10% FBS and antibiotics. Differential adhesion method was applied to remove myocardial fibroblasts. In the first 24h, bromodeoxyuridine (Brdu, 1mM, sigma) was added into the medium to inhibit fibroblasts.

In vitro experiment groups

NVMs were randomly allocated to the following groups:

1. Control group

NVMs were cultured constantly under normal condition;

2. hypoxia-reoxygenation (H/R) group

NVMs were incubated with mixed gas containing 95%N₂ and 5%CO₂ at 37°C for 6 h, then moved to the incubator with normal condition for 12 h.

3. H/R + E2(10nM);

NVMs in this group were administrated with 17β-estrogen (E2, 10nM) 24h before H/R procedure.

4. H/R + E2 + ICI 182780 (ICI) (100nM);

To explore the underlying mechanism by which E2 confers cardioprotective effects, NVMs in this group were co-administrated with estrogen receptor antagonist ICI 182780 (ICI) at non-toxic concentration(100nM) 24h before H/R procedure.

5. HR + E2 + ICI + Bay11-7082 (5uM)

To demonstrate that E2 inhibits pyroptosis via NLRP3 inflammasome, NVMs in this group were treated with specific NLRP3 inflammasome inhibitor Bay11-7082 at 5uM for 24h before H/R.

6. HR + E2 + ICI + AC-YVAD-CMK (50uM)

To demonstrate that E2 could inhibit pyroptosis, NVMs in this group were treated with Caspase1 inhibitor AC-YVAD-CMK at 50uM for 24h before H/R.

Cell viability assay

Cell viability was measured with CCK-8 kit (Beyotime) according to the manufacture’s instruction. NVMs were cultured in 96-well plates at a density of 5×10⁴ cells per well. Then, NVMs were cultured with 10µL CCK8 for 2h. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices(MD), US). Cell survival rate was calculated using the mean optical density (OD) of each group. The formula was: cell survival rate = OD of the treatment group/OD of the control group ×100%.

The determination of Lactate dehydrogenase (LDH) activity

LDH activity in the medium was measured using a colorimetric assay kit (Nanjing Jiancheng, China) according to the manufacturer's instructions. According to the manufacturer's instructions.

The determination of Caspase-1 activity

Caspase-1 activity is detected by a commercial kit(Solarbio, Beijing, China). The detection kit is based on the caspase-1 catalytic substrate acetyl-tyrosine alanine p-nitroaniline (AC-YVAD-pNA) to generate yellow p-nitroaniline (pNA), with strong absorption at 405nm.Thus, the activity of Caspase-1 can be assessed by measuring the absorbance of pNA using a standard pNA curve.

Il-1, IL-18 and TXNIP tests

Levels of IL-1, IL-18 and TXNIP released in cultured supernatant were determined by enzyme-linked immunosorbent assay (ELISA). IL-1, IL-18, TXNIP levels were measured according to the manufacturer's instructions in the IL-1 (Meilian, Shanghai, China), IL-18 (Meilian, Shanghai, China), TXNIP (Meilian, Shanghai, China), respectively.

TUNEL (Cardiomyocytes immunofluorescence)

After myocardial I/R procedure, mouse hearts were removed, paraffin-embedded and sectioned at the ligation site. DNA fragments were measured using one-step TUNEL apoptotic assay kit (Roche, Germany) according to the kit manufacturing manual. These images were taken using a Nikon Eclipse Ti microscope (Nikon, Japan). Apoptosis detection kit (Yisheng, Shanghai, China) was used to detect the death of cardiomyocytes in each group after corresponding treatment. The principle is that under the action of Terminal Deoxynucleotidyl Transferase (TdT), Alexa Fluor 647-12-DuTP combines with 3´-hydroxyl (3´-OH) terminal which exposed as the genomic DNA broken. Alexa Fluor 640 is a red fluorescent dye with high stability and strong brightness, which can be detected with a fluorescence microscope or flow cytometer. At the same time, DAPI (Beyotime, China) is a blue fluorescent dye that can strongly bind to the DNA in the cardiomyocytes, so we detected the content of cellular DNA based on its fluorescence intensity. The fluorescence intensity of intracellular ROS was recorded using fluorescence microscopy (Olympus BX53, Japan).The apoptotic index (AI) was calculated as AI = (number of apoptotic cells/total number counted) × 100%.

Western Blot Analysis

Western blot analysis was performed as described previously using antibodies against TXNIP (Ab188865, Abcam), NLRP3 (1:1000, Abcam, Ab210491), apoptosis-associated speck-like protein containing a CARD (ASC) (1:200, Santa Cruz, sc-22514-R), caspase-1(1:200, Santa Cruz, sc-514), IL-1β (1 : 1000, Abcam, ab9722), Gasdermin D protein(GSDMD) (1:1000, Abcam, ab219800) and GAPDH (1:1000, CST, D16H11). GAPDH was used as a loading control[22]. The protein bands were detected with an Odyssey color infrared laser scan-imaging instrument (Li-Cor, USA).

Statistical Analysis.

All values are expressed as mean ± standard deviation, One-Way Anova analysis of variance was used to analyze the differences among each groups, and then Bonferroni post-test, p < 0.05 indicates statistical significance, ImageJ version 1.53 (National Institutes of Health), GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA, USA), and Adobe Illustrator CC 2019 (Adobe Software, San Jose, CA, USA) were used to drawing.

E2 replacement therapy alleviates MI/R injury in vivo

In our previous study, we found that plasma E2 markedly reduced to approximately 250pg/mL after OVX and dramatically increased to 400pg/mL as the result of estrogen replacement therapy. The present study confirms similar plasma E2 level alteration induced by OVX and estrogen replacement (Fig. 1a).

Infarct size was assessed by TTC/Evans blue staining. The typical picture is shown in Fig. 1b. Similarly, as previous studies, we found that ovariectomized (OVX) mice exhibited larger infarction size than sham-operated OVX-mice (Fig. 1c). Meanwhile, the ratio of TUNEL-positive cells in OVX + I/R group was much higher than sham-operated OVX-mice (Fig. 1d and 1e). Consistently, plasma LDH and CK-MB levels were higher in I/R than in the sham group (Fig. 1f). In addition, the effect of estrogen on myocardial I/R injury was also evaluated. We found that E2 replacement therapy significantly reduced infarct size, TUNEL-positive cells as well as plasma LDH and CK-MB levels compared with mice in OVX + I/R group(Fig. 1b-1f).

E2 replacement therapy inhibits NLRP3-mediated pyroptosis during myocardial I/R injury in vivo

We have previously demonstrated that E2 replacement was able to decrease expressions of NLRP3, ASC, and Caspase-1. It well known that pyroptotic cell death can be triggered by Caspase-1 after its activation by various inflammasomes including NLRP3 inflammasome. In the present study, we measured the protein expressions of pyroptosis related genes. We found these proteins, including TXNIP, NLRP3, cleaved Caspase-1, ASC, IL-1β as well as the pyroptosis executor GSDMD, significantly increased in OVX + I/R group. E2 replacement could markedly suppressed expressions of these proteins (Fig. 1g and 1h). The results suggested that E2 replacement therapy might alleviate myocardial I/R injury partly through inhibiting NLRP3-mediated pyroptosis.

E2 treatment alleviated H/R injury via estrogen receptors pathway in vitro

To verify E2 confers cardioprotective effect via receptor or non-receptor pathway, we conducted in vitro studies in primary cardiomyocytes underwent H/R procedure to mimic I/R injury. Compared with cardiomyocytes in the control group, the cell viability decreased significantly after 4-hour hypoxia and 2-hour reoxygenation (Fig. 2a). Both release of LDH and apoptotic index increased dramatically as compared with control group, suggesting evident cell death induced by H/R procedure (Fig. 2b-2d). Consistent with in vivo study, E2 pretreatment significantly preserved cell viability, decreased LDH release and TUNEL positive cells. However, the protective effects of E2 administration on H/R injury could be markedly abolished when simultaneously pretreated with estrogen receptor antagonist ICI 182780, suggesting E2 alleviates H/R inducing cell injury via estrogen receptors pathway (Fig. 2a-2d).

E2 treatment alleviated pyroptosis via estrogen receptors pathway in vitro

In accordance with in vivo study, the viability of Caspase-1 increased significantly after 4 hour hypoxia and 2 hour reoxygenation (Fig. 3a). Release of TXNIP, IL-1β and IL-18 also increased dramatically compared with control group (Fig. 3a-3c). In addition, expressions of TXNIP, NLRP3, ASC, cleaved-Caspase1, GSDMD, and IL-1β significantly increased in H/R group (Fig. 4a-4b). As expected, E2 pretreatment also reduce the viability of Caspase-1, release of TXNIP, IL-1β and IL-18 (Fig. 3a-3c) as well as expressions of TXNIP, NLRP3, ASC, cleaved-Caspase1, GSDMD, and IL-1β(Fig. 4a-4b). Nevertheless, these effects were dramatically reversed when co-administrated with ICI 182780 (Fig. 3a-3c). These results demonstrated substantial pyroptosis induced by H/R procedure and E2 treatment might alleviated pyroptosis via estrogen receptors dependent pathway.

E2 treatment therapy alleviated H/R injury partly through suppressing pyroptosis in vitro

The above results demonstrated that E2 treatment alleviated pyroptosis and H/R injury via estrogen receptors pathway. To further demonstrate that inhibiting pyroptosis is involved in the cardioprotective effect of E2, primary cardiomyocytes were administrated either NLRP3 inflammasome inhibitor Bay11-7082 or Caspase1 inhibitor AC-YVAD-CMK with E2 before H/R procedure. Compared with H/R + E2 + ICI group, pretreatment with Bay11-7082 could significantly preserve cell viability, reduce LDH release and apoptotic index (Fig. 2). In addition, Bay11-7082 reduced viability of Caspase-1, release of TXNIP, IL-1β and IL-18 (Fig. 3) as well as expressions of TXNIP, NLRP3, ASC, cleaved-Caspase1, GSDMD, and IL-1β(Fig. 4).

Similarly, pretreatment with AC-YVAD-CMK could significantly preserve cell viability, reduce LDH release and apoptotic index (Fig. 2). Furthermore, AC-YVAD-CMK also reduced viability of Caspase-1, release of TXNIP, IL-1β and IL-18 (Fig. 3) as well as expressions of TXNIP, NLRP3, ASC, cleaved-Caspase1, GSDMD, and IL-1β (Fig. 4).

Myocardial I/R injury is an pathological process that ischemia induced cardiomyocytes injury paradoxically aggravated in spite of the recovery of blood flow and oxygen supply. Previous studies demonstrated that estrogen replacement therapy could alleviate myocardial I/R injury. However, the exact mechanism remains unclear thoroughly. To explore the effects of estrogen on TXNIP/NLRP3 mediated pyroptosis in the setting of myocardial I/R injury, we simulated the myocardial I/R injury by ligating the left coronary artery of OVX mice in vivo. We found that pyroptosis related proteins, including TXNIP, NLRP3, cleaved Caspase-1, ASC, IL-1β as well as the pyroptosis executor GSDMD, significantly increased in OVX + I/R group. E2 pretreatment could significantly alleviate myocardial I/R injury alongside with decreased expression of the above proteins. These results from in vivo study suggest NLRP3 activation-mediated pyroptosis might play an important role in the myocardial I/R injury. To further confirm this novel mechanism for the cardioprotective effect of E2, we established H/R model to imitate the myocardial I/R injury in vitro. Similarly with in vivo study, E2 obviously alleviated H/R injury. We also verified that E2 alleviates H/R inducing cell injury via estrogen receptors pathway. Moreover, we found that E2 treatment alleviated pyroptosis via estrogen receptors pathway and E2 alleviated H/R injury partly through suppressing pyroptosis. These results suggest that estrogen alleviates myocardial I/R injury in female mice by suppressing NLRP3 mediated pyroptosis.

Women usually develop less severe atherosclerosis in coronary arteries and later myocardial infarction (MI) than men at a similar age. However, the incidence shows a dramatic increase after the permanent cessation of menstruation[10, 11, 23]. Thus, female hormone estrogen has been proved to partly contribute to the protection of women against coronary artery disease[24]. Nevertheless, previous investigations indicated that male was susceptible to myocardial I/R injury than female, indicating the beneficial role of estrogen in the setting of myocardial I/R injury[25]. In mice subjected to I/R injury using a Langendorff system, Bae reported better recovery of left ventricular function and smaller infarct size in female than in male[25]. 17 beta-estradiol (E2) is the most patent and active form of estrogen. In OVX female rabbits, intravenously administration of E2 30min prior to LAD ligation resulted in a significant decrease in infarct size[26].Similarly, Anderson reported that acute E2 treatment markedly limited the changes in cytosolic PH, Na⁺, and Ca²⁺ in Krebs-Henseleit (KH)-perfused hearts from OVX rats. Furthermore, E2 improved recovery of Left ventricular developed pressure (LVDP) and diminished release of LDH during reperfusion, thereby limiting I/R injury[27]. Consistent with these previous studies, the present study found that E2 replacement therapy significantly reduced infarct size, TUNEL-positive cells as well as plasma LDH and CK-MB levels. Similar to in vivo study, we also found that E2 pretreatment significantly preserved cell viability, decreased LDH release and TUNEL positive cardiomyocytes in the H/R injury model.

As one of the natural estrogens, E2 exerts its biological effects by binding to its specific estrogen receptors. There are two types of classic estrogen receptors including estrogen receptor-α(ERα), ERβ[28]. Both receptors exhibited similar binding affinity to E2[13] and were reported to mediate protective effect of estrogen on myocardial I/R injury[14, 15, 29, 30]. ICI 182780, also known as fulvestrant, is a non-selective ER antagonist that downregulates the ER and has no agonist effects[26]. In order to explore whether ER were involved in the mechanism that E2 decreased cardiomyocytes damage, we treated cardiomyocytes with ICI 182780 24h before H/R procedure. The results revealed that the protective effects of E2 on H/R injury could be markedly abolished when simultaneously pretreated with ICI 182780. The above data demonstrated that estrogen replacement therapy could significantly alleviate myocardial I/R injury via estrogen receptors dependent pathway.

Apoptotic cell death is the most widely recognized type of programmed cell death during I/R injury. Previous studies have reported that E2 reduced I/R injury via inhibiting apoptosis [16, 31]. In addition to apoptosis, recent studies have demonstrated that a new type of programmed cell death, pyroptosis, plays an important role in the I/R injury[32, 33]. However, it has not been reported that whether pyroptosis were involved into the cardioprotective effect of E2 on myocardial I/R injury. TXNIP is a vital factor in maintaining cellular redox balance and interacted with NLRP3 to trigger pyroptosis signaling pathway, which is termed as the TXNIP-NLRP3 axis[9, 34, 35]. As far as we know, no study has reported whether E2 could modulate pyroptosis by regulating this axis. Since TUNEL-positive cell rate and the level of caspase-1 is widely used to detect pyroptosis[36, 37], we continued to use this method to verify the occurrence of pyroptosis. Both in I/R and H/R injury model, we found that pyroptosis was induced as evident by increased TUNEL-positive cell and up-regulation of cleaved Caspase-1. Furthermore, expression of TXNIP/NLRP3, ASC, inflammatory cytokine IL-1β, as well as the pyroptosis executor GSDMD were significantly augmented. In addition to mitigating I/R injury, E2 replacement therapy could also markedly inhibit pyroptosis, accompanied by reduced expression of TXNIP-NLRP3, ASC, IL-1β, as well as GSDMD. These cardioprotective effects of E2 could be partly abolished when administrated with ER antagonist ICI 182780. The above data suggested TXNIP-NLRP3 mediated pyroptosis might be involved in the protective effect of E2 replacement therapy on myocardial I/R injury.

Caspase-1 activation plays a central role in initiating pyroptosis[2]. Both in vivo and in vitro studies provided substantial evidence that Caspase-1 inhibitor administration at reperfusion could significantly decrease infarction and preserve ventricular function[3, 38]. Therefore, selective Caspase-1 inhibitor AC-YVAD-CMK was applied in the following experiments to demonstrate that pyroptosis was directly involved in the protective role of E2 on myocardial I/R injury. The results showed that E2 could remarkably suppress cleaved Caspase-1, which could be partly repealed by ER antagonist ICI 182780.As expected, AC-YVAD-CMK “add-on” regained the suppression of E2 on cell injury, TUNEL positive cells, inflammatory cytokine, as well as pyroptotic death executor GSDMD. The above data demonstrated that E2 treatment alleviated H/R injury partly through suppressing Caspase-1-mediated pyroptosis in vitro.

To further confirm that E2 could inhibit pyroptosis via downregulating NLRP3, BAY11-7082 was used in the subsequent in vitro study due to its good ability in inhibiting NLRP3 activation[37, 39]. We found that E2 could dramatically suppress NLRP3 and pyroptosis, thus alleviating H/R injury in cardiomyocytes, which could be partly reversed by ER antagonist ICI 182780. Co-administration of BAY11-7082 significantly inhibited pyroptosis, along with preserved cell viability and reduced LDH in cardiomyocytes. In addition, expression IL-1β and GSDMD were also downregulated in vitro experiments. Taken together, these results indicated that E2 could alleviate myocardial I/R injury by suppressing TXNIP-NLRP3 mediated pyroptosis.

We demonstrated that E2 play an important role in reducing myocardial I/R injury by inhibit NLRP3-mediated pyroptosis. In addition, we reported the mechanism that E2 alleviated myocardial I/R injury via its receptor ER to down-regulate TXNIP-NLRP3 and further inhibit Caspase-1, thereby suppressing ASC, and IL-18, IL-1β, GSDMD. In conclusion, these results suggested that E2 alleviates myocardial I/R injury by inhibiting NLRP3 inflammasome -mediated pyroptosis (Fig. 5).

Our study demonstrated that estrogen alleviate myocardial I/R injury by inhibiting the level of pyroptosis via ER/TXNIP/NLRP3/Caspase-1. However, there are some limitations in our study. Firstly, ER antagonist and pyroptosis inhibitors were administrated in vitro only. Secondly, we didn’t further explore the molecular mechanisms between ER and TXNIP because of the restriction of laboratory condition. Thus, ER or TXNIP gene knockout mice should be applied to further explore the interaction between ER and TXNIP.

Data Availability

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

Conflict of Interest

The authors declare no conflict of interest.

Funding Statement

This work was supported Foundation for University Excellent Talents by the Education Department of Anhui Province (Grant No.gxyq2019013, gxgwfx2020023 to He Fei), Science and Technology New Star Program of Second affiliated Hospital of Anhui Medical University(Grant No.2018KA06 to He Fei), Natural Science Foundation of China (Grant No.81903833 to Cheng Jing, Grant No.82102308 to Cheng Cheng).

Acknowledgements

The experiments of present study were performed partly in Key Laboratory of Xin’an Medical Education Ministry, Anhui University of Traditional Chinese Medicine. All authors of the present study wish to express deepest gratitude to the staff of the laboratory.

Author Contributions

He Fei, Cheng Jing and Cheng Cheng performed study concept and design; He Fei, Shen Shichun, and Cheng Jing performed development of methodology and writing, review and revision of the paper; He Fei and Shen Shichun provided acquisition, analysis and interpretation of data, and statistical analysis; Sheng Jianlong, Xu Banglong, and Wang Xiaochen provided technical and material support. All authors read and approved the final manuscript.

Ethics Statement and Consent to Participate

All animals used in the present study received ethical and humane care. Experimental procedures were conducted in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Anhui Medical University (Grant No. LLSC20200498).

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No competing interests reported.

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Estrogen alleviates myocardial ischemia-reperfusion injury by inhibiting NLRP3 inflammasome -mediated pyroptosis

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Abstract

Figures

Introduction

Materials and methods

Animals and groups

3. OVX + E2 + I/R

Serum levels of E2, LDH and CK-MB

Comparison of MI size

Primary cardiomyocyte culture

Cell viability assay

The determination of Lactate dehydrogenase (LDH) activity

The determination of Caspase-1 activity

Il-1, IL-18 and TXNIP tests

TUNEL (Cardiomyocytes immunofluorescence)

Western Blot Analysis

Statistical Analysis.

Results

Discussion

Conclusions

Declarations

References

Additional Declarations

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