Fu Fang Zhen Zhu Tiao Zhi Capsules Protect against Myocardial Ischemia by Inhibiting Cardiomyocyte Pyroptosis

Background Fu Fang Zhen Zhu Tiao Zhi (FTZ) is a traditional Chinese herbal prescription widely used to treat dyslipidemia, metabolic diseases, and diabetic coronary disorders. Cardiomyocyte death and loss of regenerative ability cause cardiac dysfunction and heart failure. FTZ can effectively treat diabetic cardiomyopathy and macrovascular diseases; however, the mechanism behind the phenomenon is still unclear. Here, we determined the mechanism of action of FTZ in treating myocardial infarction. Methods Male C57BL/6 mice were treated with 2.4 or 1.2 g/kg FTZ, or administered saline by oral gavage daily for four weeks, and a 24-hour ligation was administered to the artery. Echocardiography was used to evaluate cardiac function. Hematoxylin and eosin and Evans blue/triphenyltetrazolium chloride staining were carried out by staining the cardiac tissue, used to evaluate cardiac function and infarct size. Using western blotting and reverse transcriptase-polymerase chain reaction, we determined the relative levels of NOD-like receptor protein (NLRP) 3, ASC, cleaved caspase-l (C-Caspase-1), GSDMD, and GSDMD-N. TUNEL, immunohistochemical, and immunofluorescence staining were used to determine cell death and NLRP3 expression. An enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of interleukin (IL)-1β and IL-18. Results FTZ reduced ischemia-induced cardiomyocyte cell death in vivo and H2O2-induced cell death in vitro by maintaining cardiac architecture and restoring cardiac function. FTZ decreased the NLRP3 expression and inhibited pyroptosis-correlated genes, including NLRP3, ASC, GSDMD, C-Caspase-1, and GSDMD-N. NLRP3 overexpression impaired the efficacy of FTZ by inducing pyroptosis. Conclusion FTZ could preserve cardiac function resulting from ischemic insult by inhibiting pyroptosis, which was partially reversed by NLRP3 overexpression, indicating that NLRP3 could be a potential target of FTZ in treating myocardial infarction.


Introduction
Myocardial infarction (MI) represents a chief cause of chronic heart failure worldwide [1]. Cardiac remodeling after MI results in heart failure, which is responsible for an increase in socioeconomic burden [2]. Finding a lasting and efective agent for the management of these patients is, therefore, a valid approach.
Fu Fang Zhen Zhu Tiao Zhi (FTZ) is a traditional Chinese herbal medicine consists mostly of eight types of herbs [3] and used to treat coronary heart disease [4], nonalcoholic steatohepatitis [5], atherosclerosis [6], diabetes [5], and aging [7,8]. FTZ has been shown to beneft multiple organs and has been used to treat cardiac diseases. We recently discovered that FTZ inhibits infammation and cardiac fbrosis and ameliorates diabetic cardiomyopathy in mice. Moreover, favorable results have been reported in a clinical trial on FTZ [9,10]. However, the mechanism of action of FTZ in treating cardiac disorders remains unclear.
Tere are multiple mechanisms of cardiomyocyte death after MI, including apoptosis, necrosis, pyroptosis, autophagy, and ferroptosis. A diference between apoptosis and pyroptosis is that pyroptosis progresses via the involvement of the infammasome and gasdermin family, whereas apoptosis does not [11]. Caspases 1, 4, 5, and 11 can drive pyroptosis, resulting in large pores [12,13]. Simultaneously, the broken cells release proinfammatory cytokines such as interleukin (IL)-1β, IL-18, and other cellular contents. Among the infammasome proteins, NOD-like receptor protein (NLRP) 3 is well characterized in cardiac ischemia. NLRP3 activates pyroptosis and further aggravates cardiac function by releasing proinfammatory cytokines [14,15]. Several studies have demonstrated that inhibiting NLRP3 preserves functions of the heart, both systolic and diastolic by pyroptotic impairment [16].
Herein, we used multiple methods to determine the efcacy of FTZ in ischemic cardiac disorders and found that FTZ could improve myocardial structure and function by preventing pyroptosis. Tus, this study could broaden our understanding of FTZ in cardiac ischemia.  [17]. FTZ was provided by Guangdong Pharmaceutical University. High-performance liquid chromatography fngerprint analysis was conducted to verify the quality of the FTZ extract ( Figure S1) [18].

Ethics
Statement. C57BL/6 adult male mice (∼8 weeks old, weighing 20 ± 2 g) were purchased from Liaoning Changsheng Biotechnology Corporation, and Guangdong Medical Laboratory Animal Center provided neonatal mice for this study. A temperature-controlled room at 25 ± 2°C was provided with standard solid food and water throughout the experiment. All procedures were approved by the Laboratory Animal Care and Use Committee at Guangdong Pharmaceutical University (permit number: Gdpulacspf2017277).

Mouse Model of MI and Drug
Administration. Te mice were randomly divided into three equal groups: the sham group, the MI group, and the MI + FTZ (1.2 mg·kg −1 ) group.
In the treatment groups, mice were administered an oral medication of FTZ at a dose of 1.2 mg·kg −1 daily for 2 days, whereas those in the other groups, an equivalent volume of solvent was given for 28 days prior to the surgery to induce MI. Mice were anesthetized and then intubated to a rodent ventilator (Shinano, Tokyo, Japan). Tere was a small incision made at the third and fourth intercostal spaces of the left thoracotomy, and a 7-0 prolene suture was applied to ligate the left anterior descending (LAD) coronary artery. Approximately 24 h after MI, mice were sacrifced by euthanasia. Randomization and blinding were adopted for all animal experiments. Surgical procedures for the sham control mice were identical to those used to establish the mouse model of MI, but without the coronary artery ligation step.

Echocardiography.
Before surgery and after 24 h of MI, mice were anesthetized and subjected to echocardiography, using a Vevo 2100 ultrasound machine for echocardiography (Visual Sonics, Toronto, Canada) at a probe frequency of 30 MHz. At the papillary muscles, two-dimensional images were obtained. Te instrument calculated the average of three cardiac cycles for ejection fraction (EF) and fractional shortening (FS) based on M-mode tracings.

Measurement of Myocardial Infarct
Size. Mice were injected with Evans's blue (1%; Solarbio, China) through the abdominal aorta to stain the noninfarcted areas. Hearts were cut into 1 mm-thick sections and stained for 15 min with 2% triphenyltetrazolium chloride (TTC; Solarbio) at 37°C. An optical microscope was used to capture the images (ZEISS, Germany).

Primary Culture of Neonatal Mice Ventricular Cardiomyocytes (NMVCs).
One-to three-day-old neonatal mice were subjected to open-chest surgery to expose the hearts. Heart tissues were digested with 0.25% trypsin (Solarbio, China) to yield single cardiomyocytes. Heart tissues were trypsinized until tissues disappeared, and the cell suspensions were collected by centrifugation at 1000 rpm for 5 min. Isolated cells were resuspended in DMEM (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA) and penicillin/streptomycin (100 U/mL; Beyotime, Shanghai, China). Cardiomyocytes were purifed by centrifuging for diferent time intervals. Te resuspension was plated onto a culture fask for 120 min at 37°C, allowing for the preferential attachment of fbroblasts to the bottom. Cardiomyocytes were removed and seeded into plates, incubated at 37°C with 5% CO 2 and 95% air in a humidifed incubator, and then treatments were administered. In hypoxia treatment, cardiomyocytes were exposed to H 2 O 2 for 24 h, whereas for FTZ interventions, cardiomyocytes were treated with 100 nmol/mL FTZ for 24 h.  Evidence-Based Complementary and Alternative Medicine day, whole hearts were removed from the mice and washed with precooled 10% FBS and 1% penicillin/streptomycin for three times. Ten, discard the liquid and add 3 ml PBS and 2 ml trypsin, put it into a 50 ml centrifuge tube, and shake it at 4°C for 8-12 h. After 8-12 h, add 5 ml of DMEM complete medium to the 50 ml centrifuge tube containing the heart to neutralize the trypsin. Te tissues were digested with type II collagenase. Te cells were pelleted by centrifugation at 1000 rpm for 5 min. Te cell pellets were resuspended in Dulbecco's modifed Eagle's medium with 10% FBS and 1% penicillin/streptomycin, plated onto a T25 fask and incubated at 37°C for least 2 h.

Terminal Deoxynucleotidyl Transferase dUTP Nick End
Labeling (TUNEL) Assay. In order to assess drug-induced pyroptosis in cardiomyocytes, 24-well plates were used. Te cardiomyocytes were fxed and permeabilized with paraformaldehyde and Triton X − 100 at 4% and 1%, respectively. Te cells were incubated with TUNEL staining agents at 37°C in the dark for 1 h and with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. Fluorescence was determined using confocal laser scanning microscopy (FV300, Olympus, Japan).

Immunohistochemical (IHC) and Immunofuorescence (IF) Assays.
Heart tissue sections were soaked in EDTA antigen retrieval bufer (pH 8.0). Ten, the sections were incubated with the homologous primary antibody for 30-40 min at 37°C. Te negative control group consisted of normal antirabbit immunoglobulin G horseradish peroxidase polymer conjugated with the second antibody to treat the sections for 30 min, after which diaminobenzidine solution was used for IHC analysis. For IF studies, fuorescein bonded with a secondary antibody was used to treat the sections, which were then visualized using fuorescence microscopy.

Hematoxylin and Eosin (H&E) Staining.
Heart sections were fxed in 4% paraformaldehyde. Ten, the tissue samples were embedded in parafn and each sample was cut into 5 μm-thick sections. H&E staining was performed to evaluate histopathological alterations. Images were captured using microscopy (Olympus Corporation, Tokyo, Japan).

Enzyme-Linked Immunosorbent Assay (ELISA).
IL-1β or IL-18 concentrations in the cell supernatant or serum were determined using an ELISA kit (Elabscience Biotechnology Co., Ltd). In brief, add 100 μL of standard working solution or serum sample to the corresponding plate wells and incubate at 37°C for 90 min. Discarding the liquid, immediately add 100 μL working solution and incubate at 37°C for 60 min. Ten, add 100 μL of HRP enzyme conjugate working solution to each well, and incubate at 37°C for 30 min. Finally, add 90 μL of substrate solution and incubate at 37°C for about 15 min, stop the reaction and immediately read at 450 nm wavelength and process the data.

Lactate Dehydrogenase (LDH) Release
Assay. In the cell-free supernatant, LDH levels refect cellular permeability. LDH was measured with an assay kit at 490 nm from the cell-free supernatant (Beyotime). In brief, inoculate appropriate cells into a 96-well cell culture plate according to the size and growth rate of the cells. After H 2 O 2 stimulation, the cell culture plate was centrifuged at 400 g for 5 min in a multiwell plate centrifuge. Aspirate the supernatant, add 150 μL of the LDH release reagent with PBS, and shake the culture plate in the cell incubator. Ten, the cell culture plate was centrifuged at 400 g for 5 min with a multiwell plate centrifuge. Added 120 μL of the supernatant to a new 96-well culture plate. Finally, add 60 μL LDH detection working solution to each well, put it at room temperature in the dark for 30 min and measure absorbance at 490 nm.
2.14. Western Blotting. Neonatal cardiomyocytes cells were collected, digested with trypsin, and washed twice with phosphate-bufered saline after treatment with H 2 O 2 or FTZ. A lysis bufer containing 1% protease inhibitor is used to extract proteins from the samples (Roche, Switzerland). Protein concentrations were measured using a bicinchoninic acid protein kit (Beyotime Institute of Biotechnology, Shanghai, China). Te proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (PALL, USA). Membranes were incubated with antibodies for 50 min in the dark. Each protein band was quantifed using an infrared fuorescence imaging detector (LI-COR Bioscience) and normalized to β-actin.

RNA Isolation and Real-Time PCR.
TRIzol reagent was used to extract RNA from myocardial tissues or NMVCs (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. A total of 1 μg of RNA was reversetranscribed into complementary DNA under the conditions of 37°C for 15 min, 98°C for 5 min, and 4°C using a reversetranscription kit (Toyobo, Osaka, Japan). SYBR Green (Toyobo) was used for real-time PCR to quantify messenger RNA (mRNA) levels using a 7500 Fast Real-Time PCR System (Biosystems, Singapore). β-actin was used as an internal control. Gene expression was measured using the 2 −ΔΔCT method. Primer sequences are listed in Table 2. 2.16. Statistical Analysis. Te data are presented as the mean ± standard error of the mean. Student's t-test was used for comparisons of the two groups and a one-way analysis of variance was followed by Tukey's postcorrection for comparisons of multiple groups. p < 0.05 was considered to indicate a signifcant diference. GraphPad Prism 5 was used for statistical analysis.

FTZ Improves Cardiac Function after MI in Mice.
To verify the therapeutic efects of FTZ, we induced MI after FTZ treatment. Echocardiography fndings indicated that FTZ signifcantly increased the EF% and FS% in the MI + FTZ group (Figures 1(a) and 1(b)). Moreover, compared with mice in the MI group, Te representative long axis images of the left ventricular internal diameter at the end diastole (LVIDd) and the left ventricular internal diameter at the end systole (LVIDs) of mice treated with FTZ showed a decrease (Figure 1(c)). To determine the expansion of infarct size after myocardial injury, we measured the infarct size in sham and FTZ-treated mice. TTC/Evans blue double staining showed that the infarct size of mice in the MI + FTZ group was dramatically reduced (Figures 1(d) and 1(f)). H&E staining indicated fewer infammatory cells and compact cardiac structure (Figures 1(e) and 1(g)). Tese results suggested that FTZ preserved cardiac function after MI.

FTZ Inhibits Pyroptosis in Acute Ischemic Hearts.
To further explain the precise treatment efect of FTZ in MI, TUNEL staining was performed, compared to the control group, the MI group had signifcantly more TUNEL-positive cells and was considerably blunted after FTZ treatment (Figures 2(a) and 2(b)). To identify whether the apoptotic cells are cardiomyocytes, endothelial cells, or other cells and whether the apoptosis cell is NLRP3 positive. We stained the cardiac tissue with TUNEL and a-actinin or NLRP3 and a-actinin. Te results showed the apoptosis cell was cardiomyocytes, but unfortunately, NLRP3 positivity is not characteristic, but we believe that pyroptosis occurs primarily in cardiomyocytes ( Figures S2(a) and S2(b)). Ten, we measured the protein levels of NLRP3, ASC, cleaved caspase-l (C-Caspase-1), and GSDMD-N in ischemic hearts with or without FTZ treatment. As shown in Figures 2(c)-2(e), the pyroptotic markers including NLRP3, ASC, C-Caspase-1, and GSDMD-N, as well as their transcription, were markedly reduced in FTZ-treated mice. Consistently, FTZ decreased IL-18 and IL-1β levels in mouse serum (Figures 2(f)-2(g)). Besides, IHC staining confrmed that NLRP3 expression increased in mice in the MI group and could be partially reversed by FTZ (Figure 2(h)), suggesting its role in improving cardiac remodeling and function by inhibiting pyroptosis.

FTZ Inhibits Oxidative Stress-Induced Pyroptosis In Vitro.
Ten, we measured cell viability at diferent doses to test the protective efects of FTZ in vitro and selected a dose of 50 μg as optimal ( Figure S3). Oxidative stress plays a critical role in pyroptosis. TUNEL-positive cells signifcantly increased in H 2 O 2 -treated cardiomyocytes and were blunted after treatment with FTZ (Figures 3(a) and 3(b)). In order to determine which cell type undergoes apoptosis, we stained DAPI, α-actinin, and TUNEL. Results showed that cardiomyocytes undergo the most apoptosis ( Figure S4). Moreover, NLRP3, GSDMD, ASC, C-caspase-1, GSDMD-N, and their transcription levels showed a marked decline in the FTZ-treated group (Figures 3(c)-3(e)). Te decrease in LDH release after FTZ treatment refected reduced damage (Figure 3(f )). Infammasome-activatedN-terminal cleavage product (GSDMD-NT) ruptures membrane pores and

FTZ Reduces Cardiomyocyte Pyroptosis by Inhibiting NLRP3.
Several studies have shown that overactivation of NLRP3 can promote infammatory infltrates and inhibit cell viability [20]. To overexpressed NLRP3 in cardiomyocytes to determine whether it was a directed target of FTZ. Terefore, we assessed cardiomyocytes purity and NLRP3 plasmid transfection efciency in primary cardiomyocytes. Te results showed the majority of the isolated cells were cardiomyocytes, and a 0.5 nanomolar concentration was optimal for transfection ( Figures S5(a) and S5(b)). Te TUNEL staining demonstrated that FTZ treatment could decrease cell pyroptosis (Figures 4(a) and 4(b)). FTZ treatment led to a decrease in the expression and transcription of NLRP3, C-caspase-1, GSDMD, ASC, and GSDMD-N (Figures 4(c)-4(e)), which was impaired by NLRP3 overexpression. NLRP3 overexpression also increased LDH levels in vitro (Figure 4(f )). Despite this, too many variables can contribute to myocardial infarction. So, we tried to specifcally inhibit its expression by its specifc inhibitor MCC950 in cardiomyocytes and access the viability. Te results showed both FTZ and MCC950 could improve cell viability ( Figure S6). Tese results suggested that FTZ could protect cardiomyocytes via the NLRP3 infammasome pathway.

Discussion
In this study, we examined the efects of FTZ on ischemic heart diseases and investigated its underlying mechanisms. FTZ reduces the infarct size and exerts a protective efect on the heart. Most importantly, FTZ signifcantly reduced the expression of NLRP3, ASC, and caspase-1in vivo and in vitro. Moreover, FTZ suppressed the proinfammatory cytokines including IL-1β and IL-18, reduced pyroptotic cell death, and preserved cardiac function. Our results indicated that FTZ might improve the contractile function by afecting both the NLRP3 infammasome and pyroptosis.
Acute cardiac injury after MI remains a leading cause of mortality and disability [21]. Long-term ischemia induces an inevitable loss of cardiomyocytes, which further exacerbates cardiac function [22,23]. Previous studies have noted the importance of FTZ in cardiovascular diseases [4,24]. Tis compound contains multiple active ingredients and has been clinically tested for the treatment of metabolic diseases linked to glucose and lipids. It has been reported that FTZ modulates NLRP3 infammasome formation and activation and protects against diabetes-induced coronary damage [25]. FTZ signifcantly suppressed the production of several pyroptosis-related proteins in our study, including NLRP3, ASC, GSDMD-N, and C-Caspase-1. NLRP3 overexpression impaired the benefcial efect of FTZ by inducing pyroptosis,    Evidence-Based Complementary and Alternative Medicine which was consistent with the fndings from another research group [26]. MI triggers striking infammatory responses, and hyperactivated infammation plays a double-sword role in cardiac repair and regeneration. NLRP3 is a member of the infammasome and can be activated upon tissue injury. It is connected with multiple forms of programmed cell death [27]. Tus, targeting NLRP3 and its downstream pathway to prevent pyroptosis has attracted signifcant attention [13,28]. A recent study has shown that the NLRP3 inhibitor MCC950 can deregulate IL-1β [29] and can ofer protection in a mouse model of MI [30] and even in larger animals [31]. Although, FTZ did not directly target NLRP3 in our network pharmacology analysis ( Figure S7). In our study, IHC fndings revealed that FTZ attenuated NLRP3 expression both in vivo and in vitro and reduced infarct size, which was partially reversed by the overexpression of NLRP3. Te underlying mechanism of FTZ can be attributed to the inhibition of C-Caspase-1 and the premature release of IL-1β and IL-18, at least, partly by inhibiting pyroptosis. In spite of this, the exact molecular mechanism by which FTZ interferes with pyroptosis remains unclear. Collectively, our fndings suggest that FTZ could attenuate the proinfammatory response and improve cardiac function.
As we know, reactive oxygen species, mitochondrial damage, infectious diseases, and episodes of MI could    12 Evidence-Based Complementary and Alternative Medicine activate NLRP3 [32,33]. Tus, we treated cardiomyocytes with H 2 O 2 and found that FTZ treatment could mitigate the marker of pyroptosis genes. Several studies have demonstrated that fbroblasts are the primary cell type in pyroptosis and could serve as a target for the therapy. Terefore, we do not rule out the possibility of fbroblasts as targets of FTZ [34].

Conclusion
Te primary goal of this study was to determine the efects of FTZ on MI in a mouse model and determine its potential mechanism of action, as these fndings could have signifcant implications for the heart. A limitation with respect to the use of FTZ is the presence of complex components. FTZ treatment could inhibit cardiac ischemia-induced pyroptosis by downregulating NLRP3 and related cytokines including 1L-1β and 1L-18, which mitigate cardiac dysfunction. Our fndings may promote the use of other traditional Chinese compounds in the management of heart diseases.

Supplementary Materials
Supplement Figure S1: HPLC chromatogram for FTZ. Supplement Figure S2 (a and b) and S4: identify the apoptotic cells type. Supplement Figure S3: dose-response study.
Supplement Figure S5 (a and b): assessed the cardiomyocytes purity and NLRP3 plasmid transfection efciency. Supplement Figure S6: inhibit NLRP3 expression and access cell viability. Supplement Figure S7: analyzing the pharmacology of networks. (Supplementary Materials)