Zhuangtongyin Ameliorates Cardiomyocyte Pyroptosis in Rats with Coronary Heart Disease by Regulating Caspase-1 ()
1. Introduction
Coronary atherosclerotic heart disease, referred to as coronary heart disease (CHD), is caused by atherosclerotic changes in the coronary arteries, resulting in narrowing or occlusion of the lumen of the coronary arteries. It ultimately leads to myocardial ischaemia, hypoxia and necrosis if without control [1] [2]. In China, with the improvement of people’s living standards and changes in living habits, the incidence of coronary heart disease population is a younger trend. In the meantime, the incidence of coronary heart disease increases year by year with the accelerated aging process of society [3]. The medical burden of cardiovascular diseases in China is increasing and has become a major public health problem, so the prevention and treatment of cardiovascular disease should not be delayed [4]. Recent studies have demonstrated that pyroptosis has an important impact on the pathological process of many cardiovascular diseases [5] [6], and inhibiting pyroptosis can significantly reduce pathological damages [7] [8].
Compared with drugs for the treatment of coronary heart disease, Chinese medicine has the advantages of fewer toxic side effects and multi-targeted actions, which can be effective through more pathways [9] [10]. Zhuang medicine is the main component of traditional Chinese medicine, and ZTY is a commonly used empirical formula in Guangxi Bourau folklore, which is composed of three flavours of Guangxi characteristic Zhuang medicinal herbs, such as euonymus fortunei, polygala fallax, and pseudo-ginseng. The ZTY has the effect of activating blood circulation and removing blood stasis [11]. It has been proved that ZTY has certain protective effects on the myocardium of rats with coronary heart disease, myocardial ischemia and blood stasis [12], and it can delay the progression of the disease to a certain extent, but there is not enough clarity and depth as to whether ZTY is antiapoptosis and its mechanism of action to protect the cardiomyocytes. Therefore, in this study, based on the Caspase-1 mediated cellular pyroptosis pathway, we investigated the molecular mechanism of ZTY on the expression of cellular pyroptosis pathway factors and explored the molecular mechanism of action on cardiomyocyte pyroptosis [13]-[15]. At the same time, it can provide research ideas for further development of Guangxi characteristic Zhuang medicine in treating coronary heart disease and more research information to deepen and understand the specific mechanism of ZTY in treating cardiovascular diseases [16].
2. Materials
2.1. Instruments
Tissue dehydrator (Thermo Company, USA), KW-100 small animal artificial respirator (Nanjing Calvin Biotechnology Co., Ltd.); digital electrocardiograph (Guangzhou 3ray Electronics Co., Ltd.); ME204E electronic balance (Mettler Toledo Instruments Co., Ltd.); High-speed low-temperature tissue grinder (Wuhan Servicebio Technology Co., Ltd.); RM2245 Slicer (Shanghai Leica Microsystems Co., Ltd.); AD340 enzyme labeller (Beckman Coulter, USA); Transmission electron microscope (HITACHI).
2.2. Drugs and Reagents
Euonymus fortunei, Polygala fallax , pseudo-ginseng (Jiexi Yibai Pharmacy Co., Ltd., batch number: 0000653), Caspase-1 inhibitor Ac-YVAD-cmk (Med Chem Express, batch number: HY-16690), Gluta Fixative ((Beijing Solarbio Science & Technology Co., Ltd., Batch number: P1126), IL-18, IL-1β, CRP ELISA Test Kit (Elabscience Biotechnology Co., Ltd, Batch number: E-EL-R0567C, E-EL-R0012C and E-EL-R0506C); Hematoxylin Eosin Staining Kit (Beijing Solarbio Science Technology Co., Ltd., batch number: G1120), Reactive Oxygen Demonstration Kit (O13, red fluorescent, RXSH Bio-Tech Ltd., lot number: RXSH0845), Universal immunohistochemistry test kit (Proteintech Group, Inc, lot number: PK10006).
2.3. Animals
Fifty SD rats were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. Animal Qualification Certificate No.: SCXK (Guangdong) 2022-0063. The rats were kept in the SPF Grade Animal Experiment Centre of Youjiang Medical University for Nationalities, and this experiment was approved by the Ethics Committee of Youjiang Medical University for Nationalities.
3. Methods
3.1. Preparation of ZTY Water Extract
In accordance with the requirements of Chinese medicine taking dose and experimental zoology as a reference standard, according to the adult (about 60 kg) ZTY taking dose should be: euonymus fortunei 30 g, polygala fallax 20 g, pseudo-ginseng 15 g as a reference dose [17]. After soaking the above three medicines in distilled water overnight, heat them to boiling and then decoct them with mild fire for 1 h, filtering and removing the dregs. Add distilled water to boiling and then decoct them with mild fire for 1 h. Combine the decoctions, divide them into packages and store them at 4˚C for spare use [18].
3.2. Grouping, Modelling and Drug Administration
The rats were randomly divided into blank group, sham-operation group, model group, ZTY group and Caspase-1 inhibitor group. Except for the blank group, the rats in each group were anaesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg/kg), connected to a small-animal ventilator for mechanically assisted ventilation (respiratory rate: 80 breaths/min, tidal volume: 30 - 40 mL/kg, respiratory time ratio: 1:1), and electrocardiogram monitoring. After the left side of the chest was opened, the heart was exposed, and the rat model of coronary artery disease was established by ligating the left anterior descending branch of the coronary artery [19]. The myocardium below the ligature became greyish in colour (ischemia occurred), and myocardial tissue around the anterior wall of the left ventricle and the apical region of the heart was weakened in movement, and the presence of a 0.2 mm elevation of the S-T segment or the presence of a higher and wider QRS wave on the ECG was considered as the success of constructing the model [20], as shown in Figure 1. After suturing the incision, the rats were kept in cages and injected with 160,000 units of penicillin sodium intramuscularly into the thighs (once a day for 3 d) to prevent infection. In the sham-operation group, only the thorax was opened but not ligated, and the rest of the operation was the same as above [21]. Three days after the operation, the blank group was left untreated, the rats in the sham-operation group and the model group were gavaged with an equal volume of pure water, and the others were gavaged with the corresponding drugs and injected once a day for 4 consecutive weeks [22].
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Figure 1. (A) Electrocardiogram before coronary ligation; (B) Electrocardiogram after coronary ligation (arrow indicates ST segment elevation).
3.3. Transmission Electron Microscopy Observation of Myocardial Tissue Ultrastructure
Sample myocardial tissues in the ischemic area with 2 mm × 2 mm size, as thin as possible. The blade should be sharp to avoid bruising the tissue. Tissues are put into the electron microscope fixative at room temperature avoiding light for 2 h after removal. Then they are transferred to 4˚C preservation and transported with ice packs in time for inspection [23].
3.4. Morphological Observation of Myocardial Tissue
The myocardial tissues in the ischemic area were taken in appropriate quantities, washed with saline, fixed in 4% paraformaldehyde solution for 24 h, routinely dehydrated with ethanol gradient, transparent with xylene, embedded in paraffin, and sliced (thickness of about 5 μm). After that, dewax and hydrate to water, stain nucleus with Hematoxylin Solution for 5 - 20 min and rinse in running tap water. Then differentiate with Differentiation Solution for 3 min, and wash with tap water twice for 2 min each. Re-dyeing with Eosin Y Aqueous Solution for 10 seconds to 2 min and dehydrate in alcohol (75%, 85%, 95%, 100% alcobhol (I)), each for 2 - 3 s, and rinse in 100% alcohol (II) for 1 min. Finally, transparent by xylene and seal with resinene. The morphological characteristics of myocardial tissues of the rats in each group were observed using microscope and photographed [24].
3.5. Detection of IL-1β, IL-18 and CRP Levels in Myocardial Tissue
The levels of IL-1β, IL-18 and CRP in rat myocardial tissues were detected by ELISA method using an enzyme marker. The operation was performed in strict accordance with the instructions of the corresponding reagent kits.
3.6. Fluorescent Probe for Detection of Reactive Oxygen Species
First, add 100 µl of fresh serum sample and 10 - 20 µl of O13 probe to a 96-well plate blowing with a pipette to mix well. Be careful to choose a suitable 96-well plate, and the fluorescence detection requires black plate. Secondly, incubate at 37˚C for 15 - 30 minutes, protected from light. Finally, place the plate in a fluorescence photometer or an enzyme labeller to measure the fluorescence intensity at the excitation wavelength of 535 nm and the emission wavelength of 610 nm. Differences in the degree of reactive oxygen species can be compared by using the fluorescence intensity values of the serum samples from the blank group in the experiment as a benchmark, and the fluorescence intensity values of the serum from the model group to be tested as a percentage of the control serum.
3.7. Real-Time Quantitative PCR Method to Detect Myocardial
Tissue ASC, Nlrp3, Caspase-1 and GSDMD mRNA Expression
Cardiac tissue was cut, and total tissue RNA was extracted by Trizol, then total tissue mRNA was used as the template to reverse transcribe cDNA, which was firstly incubated at 50˚C for 15 min, and then at 85˚C for 5 s. After the reaction was finished, it was briefly centrifuged, and then cooled on ice. The PCR reaction system was 20 μL, including 0.4 μL each of upstream and downstream primers, 2 μL of Template, 7.2 μL dd H2O and 10 μL 2x SYBGREEN PCR Master Mix. The expression levels of target genes in each sample group relative to those in the blank group were expressed as 2−ΔΔCt·−ΔΔCt = blank group ΔCt-sample group ΔCt, ΔCt = target gene-reference gene. The ASC, Nlrp3, Caspase-1 and GSDMD gene sequences were synthesised by Shanghai Bioengineering.
3.8. Immunohistochemical Method to Detect the Expression of
Caspase-1 and GSDMD in Myocardium
Paraffin sections were dewaxed to water, microwave heating repair antigen, cold to room temperature. PBS washed 3 times. Add closure buffer closure 30 min and rinse 3 times with PBS. Add appropriate primary antibody diluent in each slice of tissue, 4˚C overnight, and rinse 3 times with PBS. Add the secondary antibody (50 – 100 μL of anti-rabbit/mouse HRP-labelled polymer), incubate at room temperature for 30 min and rinse 3 times with PBS; dropwise addition of DAB chromogenic solution 50 - 100 μL. The sections were incubated at room temperature, reaction time controlled under the microscope, washed with distilled water; hematoxylin re-staining, rinsed with distilled water, returned to the blue by PBS, dehydrated, transparent, blocked and observed under the microscope. The results were analysed semi-quantitatively by Images J for the staining intensity of Caspase-1 and GSDMD.
3.9. Statistical Methods
SPSS 24.0 software was used to statistically analyse the data. One-way ANOVA was used to compare multiple groups when normality and chi-square were satisfied. P < 0.05 was taken as statistically significant difference.
4. Results
4.1. Survival Status of Rats in Each Group
During the modelling process and drug administration, there were no deaths in the sham-operation group, 4 rats died in the model group, 3 rats died in the ZTY group, and 1 rat died in the Caspase-1 inhibitor group. Finally, the blank group (10 rats), the sham-operation group (10 rats), the model group (6 rats), the ZTY group (7 rats), and the caspase-1 inhibitor group (9 rats).
4.2. Ultrastructural Results of Rat Cardiomyocytes in Each Group
Figure 2. Ultrastructural results of rat cardiomyocytes in each group.
In the blank group and the sham-operation group, the cytomembrane of cardiomyocytes was continuous and intact without pore formation. In the model group, the cytomembrane of cardiomyocytes was swollen and damaged, with pore formation, and the mitochondria were severely swollen, with membrane lysis and cristae fracture. In the ZTY group and the Caspase-1 inhibitor group, the cytomembrane of cardiomyocytes was more continuous and intact and the mitochondria were better than model group, as shown in Figure 2.
4.3. HE Staining Results of Myocardial Tissue Morphology of Rats in Each Group
The myocardial fibres of rats in the blank group and the sham-operation group were clear in structure, regular in arrangement, well delineated, uniform in staining, and the nuclei of the cells were clearly visible. In the model group, myocardial tissue was unevenly stained, myocardial fibre arrangement was disordered, the gap between myocardial cells was obviously widened, and some myocardial cells were oedematous with vacuolike degeneration. Compared with the model group, the symptoms of myocardial fibrillar lysis, necrosis and exudation were reduced to different degrees in ZTY and Caspase-1 inhibitor groups, as shown in Figure 3.
Figure 3. HE staining results of myocardial tissue morphology of rats in each group.
4.4. Serum IL-1β, IL-18 and CRP Concentrations of Rats in Each Group
Notes: Compared with group A, # indicates P < 0.05, ## indicates P < 0.01; compared with group C, *indicates P < 0.05, **indicates P < 0.01.
Figure 4. Serum IL-1β, IL-18 and CRP concentrations of rats in each group. A: Blank group; B: Sham-operation group; C: Model group; D: ZTY group; E: Caspase-1 inhibitor group.
The levels of IL-1β, IL-18 and CRP in myocardial tissues of rats in the model group were significantly higher than blank group, and the differences were statistically significant (P < 0.05). Compared with the model group, the levels of IL-1β, IL-18 and CRP were significantly reduced in the ZTY group and the Caspase-1 inhibitor group, and the differences were statistically significant (P < 0.05), as shown in Figure 4.
4.5. Serum ROS Concentrations of Rats in Each Group
The levels of ROS in myocardial tissues of rats in the model group were significantly higher than blank group, and the differences were statistically significant (P < 0.01). Compared with the model group, the levels of ROS were significantly reduced in the ZTY group and the Caspase-1 inhibitor group, and the differences were statistically significant (P < 0.01), as shown in Figure 5.
Notes: Compared with group A, ## indicates P < 0.01; compared with group C, **indicates P < 0.01.
Figure 5. Serum ROS concentrations of rats in each group. A: Blank group; B: Sham-operation group; C: Model group; D: ZTY group; E: Caspase-1 inhibitor group.
4.6. ASC, Nlrp3, Caspase-1 and GSDMD mRNA Expression of Rat
Myocardial Tissues in Each Group
Figure 6. ASC, Nlrp3, Caspase-1 and GSDMD mRNA expression of rat myocardial tissues in each group. (Notes as above).
The expression of ASC, Nlrp3, Caspase-1 and GSDMD mRNA was significantly higher in the model group than the blank group (P < 0.05 or P < 0.01); the expression of ASC, Nlrp3, Caspase-1 and GSDMD mRNA were lower in the ZTY group and Caspase-1 inhibitor group in comparison with the model group(P < 0.05 or P < 0.01), as shown in Figure 6.
4.7. Caspase-1 and GSDMD Protein Expression of Rat Myocardial Tissues in Each Group
The expression of Caspase-1 and GSDMD was significantly higher in the model group than in the blank group and the sham-operation group (P < 0.01); the expression of Caspase-1 and GSDMD was lower in the ZTY group and Caspase-1 inhibitor group in comparison with the model group (P < 0.05 or P < 0.01), as shown in Figure 7.
Figure 7. Caspase-1 and GSDMD protein expression of rat myocardial tissues in each group. (Notes as above).
5. Discussion
Coronary heart disease belongs to the category of “thoracic paralysis” and “cardiac pain” in Chinese medicine research [25] [26]. The disease mechanism is usually thought of root deficiency and branch excess, with deficiency of qi as the root deficiency and phlegm and blood stasis as the branch excess [27]. Deficiency of yang qi and qi deficiency makes Qi transformation disordered, resulting in dysfunction of fluid transport, causing phlegm and blood stasis [6]. Stagnation of blood and phlegm in the meridians and blockage of qi, traps the body in a vicious cycle.
While in the theory of Zhuang medicine, coronary heart disease belongs to the Zhuang medicine “a boring”. “A boring” refers to the “dragon road” blockage [28] and it can lead to sudden pain in the anterior region of the heart. Even the pain can lead to the back of the shoulder, wheezing, and can not lie down, etc.
Cellular pyroptosis is a pro-inflammatory programmed death mediated by inflammatory cysteine asparaginase, accompanied by the release of large amounts of pro-inflammatory factors that cause inflammatory injury [29].
Inflammatory mediators belong to the category of “toxicity” in traditional Chinese medicine. “Toxicity, the evil gas is not resolved”. It is assumed that inflammatory mediators (toxicity) can cause visceral dysfunction and disorder of fluid metabolism [30]. The toxicity is transformed into phlegm turbidity, stagnation of phlegm turbidity transforming into heat, and it violates the blood to form stasis, phlegm and stasis paralysis in the blood, which leads to a series of pathological changes such as qi and yin deficiencies, phlegm and stasis in the blood, and so on [31] [32]. Hence, it is proposed that cellular pyroptosis may be a reflection of the microscopic pathology that phlegm, stasis and toxicity are obstructions in the development process of blood vessels [33].
Figure 8. The pathogenesis of pyroptosis in CHD.
Zhuangtongyin is an empirical formula that Zhuang folk doctors based on “Three routes and two roads are not accessible” theory of the disease mechanism [34]. The whole formula is combined with the common Guangxi herbs Euonymus fortunei, Polygala fallax and pseudo-ginseng, which has fewer herbs but more refined. There is the function of blood circulation, dissipate blood stasis and supplement the deficiency, turbid lipid lowering, dredging the three routes and two roads [35].
Caspase-1 is a member of the cysteoaspartic enzyme family and makes a difference to the pyroptosis pathway by promoting the maturation of inflammatory factors such as IL-1β and IL-18, as well as cleaving the pyroptosis substrate GSDMD [6] [36]-[38]. It results in the formation of micropores in the cell membrane, the outflow of intracellular ions and the inward flow of extra-membranous water, which leads to cell swelling and lysis, further leading to the release of numerous inflammatory factors, such as IL-1β and IL-18, and ultimately inducing cellular pyrolysis [39]-[42], as shown in Figure 8.
6. Conclusion
The results of transmission electron microscopy showed that the cell membranes of cardiomyocytes in the ZTY group were more complete without pore formation, and mitochondrial swelling was relieved in comparison with the model group. Staining results showed that fibrillar lysis was reduced in the ZTY group. These results showed that ZTY could protect cardiomyocytes, reduce mitochondrial swelling, relieve inflammation and reduce the death of cardiomyocytes. It was further found that ZTY could inhibit the expression of ASC, Nlrp3, Caspase-1 and GSDMD mRNA in Caspase-1 mediated proptosis pathway. At the transcriptional level, ZTY can also inhibit the expression of Caspase-1 and GSDMD proteins. Pyroptosis is often accompanied by the release of inflammatory factors, while ZTY can reduce the content of inflammatory factors IL-1β, IL-18 and ROS in the serum of rats with coronary heart disease model. Therefore, the above experimental results indicate that Zhuangtongyin can inhibit the caspase-1 mediated pyroptosis by reducing the expression of ROS, relieving myocardial inflammation and protecting cardiomyocytes. This study can provide more research information for deepening and understanding the specific mechanism of ZTY in treating cardiovascular diseases. At the same time, it also provides research ideas for further development of Guangxi’s characteristic Zhuang medicine for treating coronary heart disease.
Authors’ Contributions
HQT and SMZ conceived and designed the experiments. SMZ, XFH, and WCW performed the experiments. SMZ and WZW performed data analysis. XQZ and JYW provided experimental guidance. SMZ wrote the manuscript. HQT revised the manuscript. All authors have read and approved the final manuscript.
Ethics Approval and Consent to Participate
All animal care and use procedures were approved by the Ethics Committee of Youjiang Medical University for Nationalities.
Funding
This work is supported by the National Natural Science Foundation of China (project No.8246151084).