1. INTRODUCTION
Acute myocardial infarction (AMI) remains a major cause of mortality globally [1]. The coronary recanalization, including percutaneous coronary intervention and thrombolysis, is a standard therapy for AMI, offering life-saving benefits to some extent [2]. However, the reintroduction of fresh blood leads to new, irreversible myocardial damage, which is known as myocardial ischemia/reperfusion (I/R) injury. The irreparable damage reduces the number of cardiomyocytes that could have been saved by reperfusion therapy, severely discounting the effectiveness of the treatment [3]. Therefore, the development of medications to prevent myocardial I/R injury is an urgent and valuable endeavor in clinical treatment.
The NLRP3 inflammasome is a complex containing a sensory receptor (NLRP3), an adaptor apoptosis-related speck-like protein containing a CARD (ASC), and an effector caspase-1 [4]. Growing evidence has confirmed that activation of NLRP3 leads to the formation of an inflammasome with ASC and caspase-1, triggering an inflammatory cascade, which is crucial for the pathogenesis of myocardial I/R injury [5–7]. Our previous study demonstrated that Herba Siegesbeckiae (HS) reduces the expression of NLRP3 and ASC, the activity of caspase-1, and the contents of IL-1β and IL-18 after I/R [8], indicating that HS inhibition of the NLRP3 inflammasome may contribute to the mechanism underlying the alleviation effect on I/R injury. However, the mechanism regulating NLRP3 activation during I/R has not been fully elucidated.
Ubiquitin C-terminal hydrolase L5 (UCHL5), also known as UCH37, is a deubiquitinase [9] that is involved in various pathophysiologic processes, especially disrupting protein ubiquitination by degrading the proteasome [10]. Recent evidence showed that UCHL5 activate NLRP3 in the inflammatory response to infectious diseases, such as Mycobacterium tuberculosis and Salmonella infections, and hepatitis C virus-infected hepatocytes [11, 12]. Interestingly, our previous results demonstrated that UCHL5 expression is remarkably elevated under I/R conditions. Although HS can suppress this elevation, the trend is consistent with elevation of NLRP3 [8]. However, there is no evidence for a relationship between UCHL5 and NLRP3 in myocardial I/R injury.
Recently, increasing preclinical evidence has supported the use of traditional Chinese medicine (TCM) in treating cardiac I/R injuries and has shown significant potential for attenuating reperfusion injury [13, 14]. According to ancient Chinese books (Kai Bao Ben Cao and Jing Yue Quan Shu), HS (the aerial parts of Siegesbeckia pubescens Makino) has detoxifying effects, clearing meridians, and dispelling wind and dampness [15]. According to the TCM text, Lin Zheng Zhi Nan Yi An, HS can be effectively used to treat cardio- and cerebro-vascular diseases. Xinshuning capsules and Xixiantong Shuan capsules, with HS as the main ingredient, have been recorded in the 2020 edition of the Chinese Pharmacopoeia for the treatment of coronary heart disease and stroke and have shown efficacy [16–18]. Experimental studies have confirmed that the cardio- and cerebro-vascular protective effects of HS can be attributed to anti-inflammatory [19], angiogenic [20], and antioxidant activities [21]. Our previous study revealed that HS significantly mitigates myocardial I/R injury, further substantiating the cardioprotective effect of HS [8]. However, the effective subfractions and constituents of HS that protect the myocardium against I/R injury are unclear and the underlying mechanism needs further clarification.
Herein we intend to determine whether UCHL5 can activate NLRP3 to participate in the pathogenesis of myocardial I/R injury and to identify the main active constituents of HS to abate myocardial I/R injury via inhibition of the UCHL5/NLRP3 pathway.
2. MATERIAL AND METHODS
2.1 Plant material
Siegesbeckia orientalis L. WAS purchased FROM San He Pharmaceutical Co., Ltd. (83161101, Beijing, China). The materials were identified by Professor Hai-yu Xu (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China). The voucher specimens have been deposited in Dongzhimen Hospital of Beijing University of Chinese Medicine (no. DZMH-2022-12-10; Beijing, China). The plant name was checked with https://www.worldfloraonline.org on 21 March 2024.
2.2 Myocardial I/R injury model
A rat myocardial I/R injury model was established by ligation and recanalization of the left anterior descending coronary artery (LADCA), as described in our previous study [8]. Male Sprague – Dawley (SD) rats (220 ± 10 g) were purchased from Beijing Vital River Laboratories (Beijing, China). All animals received humane care according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Experimental procedures were approved by the Animal Care Committee of Dongzhimen Hospital Affiliated with the Beijing University of Chinese Medicine (No. 22-23).
SD rats were divided into seven groups: (1) sham: LADCA threaded without ligation; (2) I/R; (3) HS-A + I/R group (SD rats received 336 mg/kg of HS-A [equivalent to 4 g/kg of HS] for 1 week before surgery; (4) HS-B + I/R: (SD rats received 16.4 mg/kg of HS-B [equivalent to 4 g/kg of HS] for 1 week before surgery; (5) HS-C + I/R: SD rats received 20 mg/kg of HS-C [equivalent to 4 g/kg of HS) for 1 week before surgery; (6) HS-D + I/R: SD rats received 2.2 mg/kg of HS-D [equivalent to 4 g/kg of HS] for 1 week before surgery; and (7) HS + I/R: SD rats received 4 g/kg of HS as a positive control [the most effective dosage confirmed by a previous study] for 1 week before surgery [8].
2.3 Co-immunoprecipitation (Co-IP)
Tissue lysates were mixed with protein A/G agarose beads (catalog # sc2003; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) with rabbit anti-NLRP3 antibody (catalog # 27458-1-AP, 1:2000; Proteintech, Rosemont, IL, USA) overnight at 4°C on a spinning plate, then Western blotting was performed with mouse anti-UCHL5 antibody (1catalog # sc-271002, 1:2000; Santa Cruz Biotechnology, Inc.).
2.4 Western blotting assay
Left ventricular myocardium around the infarct area was extracted with RIPA lysis buffer (Beyotime, Shanghai, China) and the protein concentration was determined using the BCA method. The expression of UCHL5 (cat# sc-271002, 1:2000, mouse; Santa Cruz Biotechnology, Inc), NLRP3 (catalog # 27458-1-AP, 1:2000, rabbit; Proteintech), ASC (catalog # YT0365, 1:2000, rabbit; ImmunoWay, Beijing, China), caspase-1 (catalog # CY10200, 1:2000, rabbit; Abways, Shanghai, China), and β-actin (catalog # 20536-1-AP, 1:5000, rabbit; Proteintech) was determined by western blotting.
2.5 Hypoxia/re-oxygenation (H/R) model and cell viability
H9c2 cardiomyocytes (National experimental cell resource sharing platform, Beijing, China) were cultured with glucose-free, fetal bovine serum-free DMEM and incubated in 1% O2 and 5% CO2 mixed with 94% N2 for 6 h, followed by re-oxygenation for 18 h to establish the H/R model.
2.6 Cell transfection
UCHL5 overexpression plasmid (Shanghai Genechem, Shanghai, China) was transfected into H9c2 cardiomyocytes with lipo6666 (MF769; Mei5 Biotechnology, Beijing, China) according to the manufacturer’s instructions.
2.7 Preparation of HS subfractions
HS were purchased from Beijing San He Pharmaceutical Co. LTD (83161101; Beijing, China). The plant name was checked with https://www.worldfloraonline.org on 21 March 2024. After soaking 5 kg of HS in 50 L of distilled water for 30 min, the mixture was boiled for 30 min and filtered. This process was repeated with an additional 25 L of water. The filtered liquids were combined and concentrated to 1 L, then subjected to column chromatography over a macroporous adsorption resin (A832686, D101 [0.3 mm–1.25 mm]; Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) and loaded into a glass column (12 × 120 cm) with a bed volume of 6 L. A successive elution of the column with H2O (50 L), 20% ethanol (40 L), 50% ethanol (20 L), and 95% ethanol (10 L) yielded 4 corresponding fractions after removal of solvents (labeled as HS-A, HS-B, HS-C, and HS-D, respectively). The subfractions were stored at 4°C until further use.
2.8 Fingerprint analysis
Fingerprint analysis was performed using a high-performance liquid chromatography-evaporative light-scattering detector [HPLC-ELSD] 1260 Infinity System (Agilent Technologies, Santa Clara, CA, USA) equipped with a Grace C18 column (5 μm, 4.6 × 250 mm; Grace, Columbia, MD, USA). The sample was weighed to the nearest mg, dissolved in 1 mL of 50% water-methanol, vortexed for 5 min, extracted by ultrasonic extraction for 30 min, then allowed to cool to room temperature. The supernatant was filtered through a 0.45-μm filter membrane before analysis on an HPLC-ELSD. The gradient elution used water (A) and acetonitrile (B) as the mobile phase at a flow rate of 1.0 mL/min. The gradient program was as follows: 0–25 min 5% B; and 25–35 min 85% B. The column temperature was maintained at 30°C and the injection volume was 20 μL. The evaporator and the nebulizer temperature for the ELSD were both 65°C.
2.9 Ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) assay
UPLC-HRMS was performed separately for each subfraction ( Figure 1 ). HS-C (0.2 mg) was dissolved in 1 mL of 50% methanol-water solution, filtered, and stored at 4°C. The UPLC-HRMS method was performed with an Agilent 1260 system. Qualitative determination of HS constituents was achieved using a Q Exactive Plus LC/MS/MS spectrometer (Thermo Scientific, Waltham, MA, USA). A Waters BEH C18 system (Waters, Milford, MA, USA) equipped with an Acquity UPLC HSS T3 chromatographic column (100 × 2.1 mm, 1.7 μm, i.d.; Waters, Milford, MA, USA) at 40°C using a gradient elution at a 0.3 mL/min flow rate for chromatographic separation. The mobile phase consisted of 5% methanol-water (A), methanol (B), and 0.1% formic acid-water (C), and the sample injection volume was set at 3 μL. The gradient program was set as follows: 0–2 min, 0–5% B; 2–4 min, 5%–22% B; 4–18 min, 22%–56% B; 18–45 min; 56%–100% B; and 45–50 min, 100% B. All MS experiments were conducted on the full MS-ddMS2 model with negative and positive scan modes, and the mass scan range was m/z 150–2000.
2.10 Enzyme-linked immunosorbent assay (ELISA)
ELISAs were performed to detect the lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB), cardiac troponin T (cTnT), reactive oxygen (ROS), interleukin-1β (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor-alpha (TNF-α) content in serum according to the manufacturer’s instructions (Dogesce Biotech, Beijing, China).
2.11 Cardiac function evaluation
Electrocardiography was performed prior to surgery and 120 min after reperfusion with ST-segment elevation in thoracic lead II analyzed to assess cardiac function.
2.12 Assessment of myocardial infarct size, apoptosis, and morphology
According to a previous study, myocardial infarct size was assessed using Evans blue (Shanghai Macklin Biochemical Technology Co., Ltd.) and TTC (Solarbio Science Technology Co., Ltd., Beijing, China) staining, while myocardial apoptosis and morphology were evaluated using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit (G1501; Service Biologicals, Wuhan, China) and HE staining [8].
2.13 Identification of main HS-C active compounds
The sample was injected according to chromatographic and mass spectrometry conditions. Combined with the database (mzCloud), Compound Discoverer 3.2 software was used to search for the target compound peaks on the collected raw data and screen for compounds with a score >80. After matching, Xcalibur was used to extract compound peaks to obtain the secondary mass spectra of the compound. Finally, according to the accurate molecular weight, secondary mass spectra, and characteristic cleavage law, the secondary mass spectra were compared with the relevant literature to identify the possible chemical components.
2.14 Determination of compound content
High-performance liquid chromatography-diode array detection (HPLC-DAD) was performed to determine compound content using a 1260 Infinity System equipped with a Grace C18 column (5 μm, 4.6 × 250 mm). The HS-C sample was weighed to the nearest mg, dissolved in 1 mL of 50% water-methanol, vortexed for 5 min, extracted by ultrasonic extraction for 30 min, then allowed to cool. The standard compound was weighed accurately and methanol was added to prepare 1.000, 0.500, 0.250, and 0.125 mg/mL of standard solution. The solution was dissolved by ultrasonic treatment for 30 min, then filtered. The gradient elution used water (A) and acetonitrile (B) as the mobile phase at a flow rate of 1.0 mL/min. The gradient program was as follows: 0–15 min, 30% B. The column temperature was maintained at 30°C and the injection volume was 10 μL. The detection was carried out with a DAD detector at a wavelength of 210 nm. A standard curve was plotted using the mass of the compound reference as the abscissa and the peak area as the ordinate. The linear regression equation (Y = 11337582.92x + 1793788.26) was obtained and the R2 was 0.9991. The peak area of the tested sample was substituted into the linear regression equation to calculate the compound content, as shown in in Figure S29 and Table S2 .
2.15 Nuclear magnetic resonance (NMR) measurement
The 1H NMR sample measurement were performed using a 600 MHz Bruker NMR spectrometer (Bruker, Billericca, MA, USA), functioning at a frequency of 600 MHz and maintained at 26°C.
2.16 Molecular docking
Molecular docking was performed utilizing Autodock software with the genetic algorithm method as the docking mode. The protein structure of UCHL5 (PDB:3RII) was obtained from the Protein Data Bank (www.rcsb.org).
3. RESULTS
3.1 UCHL5 contributes to the activation of NLRP3 in myocardial I/R rats
Our previous research showed that HS effectively restrains upregulation of UCHL5 and NLRP3 caused by myocardial I/R injury. Other studies have confirmed that UCHL5 promotes activation of the NLRP3 inflammasome in infectious diseases [12]. However, the relationship in I/R has not been clarified.
The Co-IP assay showed that NLRP3 interacted with UCHL5 in the sham group and the interaction was significantly enhanced after I/R stimulation ( Figure 1A ).
To further validate the interaction between UCHL5 and the NLRP3 inflammasome, UCHL5 overexpression plasmids were transfected into H9c2 cardiomyocytes. NLRP3 and ASC expression was increased in the UCHL5OE group compared to the UCHL5NC group ( Figure 1B,C ). This finding further verified the regulatory effect of UCHL5 on NLRP3 inflammasion of cardiomyocytes.
3.2 HS subfraction preparation and fingerprints
The extraction and separation processes of HS are shown in Figure 2A . Column chromatography on macroporous adsorption with a gradient elution of a water-ethanol system was applied to separate the HS extract and four subfractions (HS-A, HS-B, HS-C, and HS-D) were obtained. Considering that the chemical components of HS are not easily displayed under UV conditions, HPLC-ELSD was used to establish the fingerprints of HS extraction and the four subfractions, as presented in Figure 2B . However, limited amounts of peaks were observed in the above fingerprints due to the low detection sensitivity of the ELSD detector. Thus, UPLC-HRMS was further applied to resolve this problem and display more peaks, as shown in Figure 3 . The fingerprints derived from the HPLC-ELSD and UPLC-HRMS were reproducible and reliable, which could be used in quality control of the HS subfraction preparation.
3.3. HS-C alleviated I/R-induced myocardial injury
Based on our previous study, HS exhibited the most significant protective effect against I/R injury at a dosage of 4 g/kg. Therefore, the dosages for the four subfractions equivalent to 4 g/kg HS were calculated; the doses for HS-A, HS-B, HS-C, and HS-D to be 336, 16.4, 20, and 2.2 mg/kg, respectively. The LDH content in the I/R group was elevated compared to the sham group, while the LDH content was reduced by HS subfractions to different degrees, as shown in Figure 4A . Notably, the reduction in LDH content by HS-C was statistically significant compared to the I/R group. The results from H9c2 cardiomyocytes showed that HS-C markedly increased cell viability after H/R stimulation, as shown in Figure 4B .

Effect of HS subfractions on LDH content in I/R rats and H9c2 cell viability exposed to H/R.
(A) LDH content in various groups. (B) Effects of HS-C on H/R-induced H9c2 cell viability (n = 6). ** P < 0.001 vs. sham (A) or control (B); 0.001 < # P < 0.05 vs. I/R (A) or H/R (B) group; ## P < 0.001 vs. I/R (A) or H/R (B) group.
3.4. HS-C reduced ST-segment elevation and alleviated I/R injury in rats
Compared to the sham group, the ST-segment was significantly elevated in the I/R group. Conversely, administration of HS-C decreased the ST-segment elevation induced by I/R ( Figure 5A ). This finding was further supported by quantitative results ( Figure 5B ).

Effects of HS-C on electrocardiogram and myocardial injury enzymes in I/R rats.
(A-B) Effect of HS-C on I/R-induced ST segment elevation. Representative ECG of chest lead II in sham (a1), I/R (a2), HS-C + I/R (a3), and HS + I/R groups (a4) with quantitative analysis of ST segment elevation 120 min after reperfusion (B). (C-E) Serum CK-MB (C), cTnT (D), and ROS (E) levels in different groups (n = 6). 0.001 < * P < 0.05 vs. sham group; ** P < 0.001 vs. sham group; 0.001 < # P < 0.05 vs. I/R; ## P < 0.001 vs. I/R group.
I/R treatment significantly increased CK-MB and cTnT content compared to the sham group, as shown in Figure 5C and D . However, elevation of the above enzymes was inhibited by HS-C, indicating a protective effect of HS-C on the myocardium. In addition, the ROS content in serum was increased during I/R, but the increase was reversed by HS-C, indicating that HS-C inhibited I/R-induced oxidative stress injury ( Figure 5E ).
3.5 HS-C decreased the myocardial infarction area and myocardial apoptosis, and retained myocardial morphology
The results of Evans blue-TTC staining showed the volume of the myocardial infarction area, in which the blue part represented the normal area, pink denoted the area at risk (AAR), and the white part signified the infarction area. Myocardial infarction was evident in the I/R group compared to the sham group and quantitative analysis showed a significant increase in the AAR/left ventricle (LV) and infarct size/AAR. The AAR/LV ratios in the HS-C and HS treatment groups were similar to that observed in the I/R group but the infarct size/AAR ratios were significantly reduced in the HS-C + I/R and HS + I/R groups compared to the I/R group, indicating that HS-C and HS treatment protected part of the ischemic myocardium and prevented its infarction ( Figure 6A–C ).

Effect of HS-C on I/R-induced myocardial infarct size, myocardial apoptosis, and morphology in I/R rats.
(A) Representative Evans blue-TTC staining of sham, I/R, HS-C + I/R, and HS + I/R. (B-C) Quantitative analysis of AAR/LV (B) and infarct area/AAR (C) in each group. (D) Representative TUNEL and HE staining photographs. Green fluorescence marks apoptotic cell nuclei and blue fluorescence marks normal nuclei. Bar = 50 μm (E) Quantitative analysis of TUNEL-positive cells/field in different groups (n = 6). ** P < 0.001 vs. sham group; 0.001 < # P < 0.05 vs. I/R; ## P < 0.001 vs. I/R group.
TUNEL staining was used to evaluate the apoptosis ratio in all myocardial nuclei. The ratio was significantly increased in the I/R group but the increase was reversed in the HS-C group ( Figure 6D,E ). Additionally, I/R treatment led to disorganized and fractured myocardial fibers, as well as swollen, ruptured, and necrotic myocytes. HS-C treatment significantly ameliorated these morphologic changes ( Figure 6D ).
3.6 HS-C inhibited UCHL5 expression and NLRP3 inflammasome activation
Furthermore, the effect of HS-C on UCHL5/NLRP3 was investigated. UCHL5, NLRP3, ASC, and caspase-1 expression was significantly upregulated in I/R rats. However, HS-C pretreatment inhibited above-mentioned protein expression ( Figure 7A ). Quantitative analysis further confirmed the above results ( Figure 7B–E ).

Effects of HS-C on UCHL5 and NLRP3 inflammasome expression and inflammatory factors contents.
(A) Representative protein bands of UCHL5, NLRP3, ASC, and caspase-1 expression. (B-E) Semi-quantitative analysis of UCHL5 (B), NLRP3 (C), ASC (D), and caspase-1 (E) in each group (n = 4). (F-H) Serum levels of IL-1β (F), IL-18 (G), and TNF-α (H) (n = 6). 0.001 < * P < 0.05 vs. sham; ** P < 0.001 vs. sham group; 0.001 < # P < 0.05 vs. I/R group; ## P < 0.001 vs. I/R group.
As the downstream products of the NLRP3 inflammasome, the IL-1β, IL-18, and TNF-α levels were higher in the I/R group compared to the sham group, while the content of these inflammatory factors was apparently decreased after HS-C treatment ( Figure 7F–H ).
3.7. Kirenol was identified as the major active component of HS-C against myocardial I/R injury
The ion chromatograms of HS-C in both positive and negative ion modes are shown in Figure 8A and B . Except a few smaller peaks, the chemical with a retention time of 18.97 min was the dominant substance in the positive ion flow diagram, while the chemical with a retention time of 19.88 min was the dominant substance in the negative ion flow diagram. Eleven possible components were identified using the mzCloud database and Compound Discoverer 3.2 software, as shown in Table 1 and Supplementary Materials. The primary and secondary mass spectra are shown in Figure S1–28 and Table S1 .
Information of 11 HS-C components determined by UPLC-HRMS.
Name | Molecular formula | RT [minute] | Chemical structure | Reference ion | m/z | |
---|---|---|---|---|---|---|
1 | ent-2β,15,16-trihydroxypimar-8(14)-en-18-oic acid | C20H32O5 | 14.03 |
![]() | [M−H]− | 351.2177 |
2 | ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene | C20H32O4 | 18.59 |
![]() | [M+H]+ | 337.2369 |
3 | ent-15,16-dihydroxypimar-1,8(14)-dien-3-one | C20H30O3 | 18.63 |
![]() | [M+H]+ | 319.2266 |
4 | Kirenol | C20H34O4 | 18.97 |
![]() | [M+H]+ | 339.2523 |
5 | Siegesbeckic acid | C20H32O4 | 19.23 |
![]() | [M−H]− | 335.2228 |
6 | Eicosapentaenoic acid | C20H30O2 | 19.63 |
![]() | [M+H]+ | 303.2314 |
7 | Quercetin | C20H34O3 | 19.88 |
![]() | [M+H]+ | 323.2574 |
8 | Oxydipropyl dibenzoate | C20H22O5 | 27.26 |
![]() | [M+H]+ | 343.1539 |
9 | Dodecyl hydrogen sulphate | C12H26O4S | 32.27 |
![]() | [M−H]− | 265.1480 |
10 | 4-dodecylbenzenesulfonic acid | C18H30O3S | 37.69 |
![]() | [M−H]− | 325.1843 |
11 | Monopalmitin | C19H38O4 | 38.38 |
![]() | [M+H]+ | 331.2848 |
The molecular formula of compound 1, C20H32O5, was established by the negative HRESIMS at m/z 351.2177 [M−H]− (Calcd. for C20H31O5, 351.2177), which is designated ent-2β,15,16-trihydroxypimar-8(14)-en-18-oic acid. The molecular formula of compound 2, C20H32O4, was established by the positive HRESIMS at m/z 337.2373 [M+H]+ (Calcd. for C20H33O4, 337.2369), which is designated ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene. The molecular formula of compound 3, C20H30O3, was established by the positive HRESIMS at m/z 319.2268 [M+H]+ (Calcd. for C20H31O3, 319.2266), which is designated ent-15,16-dihydroxypimar-1,8(14)-dien-3-one. The molecular formula of compound 4, C20H34O4, was established by the positive HRESIMS at m/z 339.2530 [M+H]+ (Calcd. for C20H35O4, 339.2523), which is designated kirenol. The molecular formula of compound 5, C20H32O4, was established by the negative HRESIMS at m/z 335.2228 [M−H]− (Calcd. for C20H31O4, 335.2228), which is designated siegesbeckic acid. The molecular formula of compound 6, C20H30O2, was established by the positive HRESIMS at m/z 303.2318 [M+H]+ (Calcd. for C20H31O2, 303.2314), which is designated eicosapentaenoic acid. The molecular formula of compound 7, C20H34O3, was established by the positive HRESIMS at m/z 323.2581 [M+H]+ (Calcd. for C20H35O3, 323.2574), which is designated quercetin. The molecular formula of compound 8, C20H22O5, was established by the positive HRESIMS at m/z 343.1540 [M+H]+ (Calcd. for C20H23O5, 343.1539), which is designated oxydipropyl dibenzoate. The molecular formula of compound 9, C12H26O4S, was established by the negative HRESIMS at m/z 265.1479 [M−H]− (Calcd. for C12H25O4S, 265.1480), which is designated dodecyl hydrogen sulphate. The molecular formula of compound 10, C18H30O3S, was established by the negative HRESIMS at m/z 325.1843 [M−H]− (Calcd. for C18H29O3S, 325.1843), which is designated 4-dodecylbenzenesulfonic acid. The molecular formula of compound 11, C19H38O4, was established by the positive HRESIMS at m/z 331.2843 [M+H]+ (Calcd. for C19H39O4, 331.2848), which is designated monopalmitin.
Previous pharmacologic studies indicated that diterpenoids and flavonoids are effective constituents of HS [22]. Therefore, H/R-induced H9c2 cardiomyocyte injury in vitro was adopted to determine the cardioprotective effect of these diterpenoid and flavonoid compounds. The half-maximal effective concentration (EC50) against H/R injury of ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene, kirenol, siegesbeckic acid and quercetin was 6.88 ± 0.27, 0.79 ± 0.39, 4.47 ± 1.38, and 2.15 ± 1.24 μM, respectively ( Figure 9A–D ; Table 2 ). These findings suggest that kirenol possesses the most significant cardioprotective activity among HS-C.

A–D. The EC50 curve of ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene (A), kirenol (B), siegesbeckic acid (C), and quercetin (D) in elevating H9c2 cardiomyocyte viability. (E) 1H NMR results of HS-C and kirenol.
Binding energy with UCHL5 and EC50 value of four HS-C core active constituents.
Compound | Binding energy (kJ/mol) | EC50 (μM) |
---|---|---|
ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene | −6.37 | 6.88 ± 0.27 |
Kirenol | −6.54 | 0.79 ± 0.39 |
Siegesbeckic acid | −5.34 | 4.47 ± 1.38 |
Quercetin | −6.44 | 2.15 ± 1.24 |
Kirenol is one of the major constituents of HS [23], with a retention time of 16.73 min in HPLC-ELSD chromatograms and a retention time of 18.97 min in the UPLC-HRMS positive ion chromatograms. Calibration curves were constructed for kirenol using a series of standard working solutions to determine the content of kirenol in the HS-C. All the results are presented in Table 3 . Kirenol accounted for 62.4% of the HS-C according to the linear regression equation, indicating that kirenol was the major constituent of HS-C. This finding was also supported by the 1H NMR spectrum, which displays the main NMR signals attributed to kirenol, as shown in Figure 9E . However, the content of the other 10 constituents was too low to calculate.
3.8. Molecular docking suggested that the active components bound to UCHL5
The binding degree of the active constituent interaction in HS-C with UCHL5 was assessed through molecular docking. A binding energy <−5.0 kJ/mol was considered spontaneous and stable interactions, as observed in the bindings between UCHL5 and ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene, kirenol, siegesbeckic acid, and quercetin ( Figure 10A–D and Table 2 ).

Molecular docking of core active constituents of HS-C with UCHL5.
Representative molecular docking diagrams of four core active components.
Hydrogen bonds are the predominant factor in the binding process. Hydrophobic interactions also have a prominent role in stabilizing the binding conformation of ligands [24]. Ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene interact with UCHL5 through three hydrogen bonds with GLY-24 and LEU-181. Ent-15-oxo-2β,16,19-trihydroxypimar-8(14)-ene had a good interaction with UCHL5 through hydrophobic interactions with residues [VAL-39, GLN-138, GLY-61, GLU-63, SER-142, GLU-62, TRP-58, and PRO-60] ( Figure 10A ). Kirenol binds to UCHL5 through four hydrogen bonds with SER-142, LEU-181, and LYS-81 residues. Kirenol was able to establish hydrophobic interactions with PHE-143, GLY-61, PRO-60, TRP-58, GLY-24, LYS-23, and GLU-20, as shown in Figure 10B . Siegesbeckic acid forms three interacting hydrogen bonds with ALA-65, LYS-206, and GLU-177 of UCHL5. Hydrophobic interactions were formed via residues GLU-63, PRO-64, ARG-207, GLU-62, and LYS-210 ( Figure 10C ). Quercetin interacts with UCHL5 through four hydrogen bonds with GLU-205, GLU-39, LEU-220, and ILE-35. Hydrophobic interactions were formed via residues SER-37, ILE-208, PHE-218, ASN-219, LEU-38, and TRP-36 ( Figure 10D ).
4. DISCUSSION
The present study demonstrated that I/R fosters the interaction between UCHL5 and NLRP3, and overexpression of UCHL5 significantly activates NLRP3. Interestingly, 50% ethanol eluted a subfraction of HS (HS-C) had an apparent protective effect against I/R injury, which significantly reduced ST-segment elevation and the myocardial injury enzyme levels, inhibited cardiac infarct size and myocardial apoptosis, and restored myocardial morphology in I/R rats. HS-C notably restrained the expression of UCHL5, then inhibited the activation of NLRP3 inflammasome, thereby blocking I/R-induced inflammatory cascade, suggesting that the UCHL5/NLRP3 pathway may be part of the mechanisms by which HS-C mitigates myocardial I/R injury. The mass spectrometry analysis showed that kirenol could be the main effective component of HS-C, which bound well to UCHL5 protein.
Inflammation is a vital pathophysiologic process of myocardial I/R injury and is closely related to infarct size after reperfusion [25]. The contribution of the inflammatory response induced by NLRP3 inflammasome activation in myocardial I/R injury has been supported by collective evidence [26]. The NLRP3 inflammasome is a complex composed of NLRP3, ASC, and inflammatory caspase-1 [27]. Activated NLRP3 further activates caspase-1 by integrating with ASC under myocardial I/R conditions, stimulating the production of IL-1β and IL-18, release of multiple inflammatory factors, infiltration of various inflammatory cells, and the occurrence of pyroptosis [28].
NLRP3 inflammasome acts as an initial danger sensor for I/R injury. Inhibiting activation of the NLRP3 inflammasome is an important intervention to alleviate myocardial I/R injury. The current study provided evidence that HS-C downregulates the expression of NLRP3, ASC, and caspase-1 in myocardium exposed to I/R, as well as reducing the levels of inflammatory cytokines (IL-1β, IL-18, and TNF-α). These results confirm that the inhibitory effect on the NLRP3 inflammasome is one of the mechanisms by which HS prevents myocardial I/R injury. However, the regulatory mechanisms of NLRP3 inflammasome activation in myocardial I/R require further exploration.
NLRP3 activation is tightly regulated at levels of transcription, translation, and post-translational modification (PTM). PTM is the main regulatory mechanism underlying NLRP3 activation. Ubiquitination/deubiquitination is one of the most common PTM features [29]. NLRP3 is often ubiquitinated in resting cells and deubiquitinated during initiation and activation [30].
Previous studies have revealed that UCHL5 deubiquitinates and activates the NLRP3 inflammasome. UCHL5 is a highly conserved deubiquitination enzyme that catalyzes removal of the ubiquitin portion from the target protein or polyubiquitin chain and inhibits ubiquitination of the substrate [31]. Recent evidence has shown that UCHL5 regulates a variety of diseases by inhibiting ubiquitination of the NLRP3 inflammasome, while promoting inflammation and apoptosis. UCHL5 inhibits the ubiquitination of NLRP3, promotes NLRP3 inflammasome assembly and activation and IL-1β maturation, which exacerbates the chronic infectious inflammatory response induced by hepatitis C virus [12]. UCHL5 also reduces inflammation and vascular remodeling in atherosclerosis by activating the NLRP3 inflammasome [32]. In addition, UCHL5 directly binds to NLRP3 in mesenchymal stem cells to regulate mandibular healing [33]. The above studies confirm the importance of UCHL5 in the PTM of NLRP3. Consistent with this finding, the present study demonstrates that there is direct binding between NLRP3 and UCHL5, and the interaction is more significant under I/R stimulation, further expanding the current knowledge on NLRP3 PTM by UCHL5.
HS is a traditional herb widely used in the treatment of rheumatic arthralgia caused by waist and knee pain and numbness of limbs [34]. Growing evidence has confirmed the multiple pharmacologic effects of HS, including anti-inflammation, antioxidation, immune regulation, and anti-thrombus formation, promoting its application in cardiovascular and cerebrovascular diseases [15]. Our previous study verified the effect of HS against myocardial I/R injury [8], while it remained unclear which specific components contributed to the effect. The present study has provided a preliminary answer to this question, showing that the 50% ethanol part of HS (HS-C) significantly alleviates myocardial I/R injury. We enriched a component of HS into HS-C and further identified it as kirenol, which was highly abundant and active in HS-C. As the main active components of HS, kirenol belongs to the diterpenoid family [35]. Interestingly, in addition to its anti-arthritic effects, kirenol has also been shown to have a good myocardial protective effect, ameliorating diabetic cardiomyopathy through its anti-inflammatory and anti-fibrosis effects, and inhibiting doxorubicin-induced cardiotoxicity through its antioxidant effect [36, 37]. In addition, the present study confirmed the anti-myocardial I/R effect of HS-C with kirenol as its main component. Therefore, we speculate that kirenol might exert a therapeutic role against I/R injury through anti-inflammatory and antioxidant effects. However, the specific underlying mechanism warrants further elucidation. It is noteworthy and gratifying that we found that HS-C downregulated the expression of UCHL5 and inhibited activation of the NLRP3 inflammasome, suggesting HS-C protects the myocardium against I/R injury through the UCHL5/NLRP3 pathway, and the key active compound may be kirenol.
5. CONCLUSION
In summary, we found that the 50% ethanol part of Herba Siegesbeckiae ameliorated myocardial I/R injury in rats and demonstrated that UCHL5 induced inflammation by affecting the NLRP3 inflammasome. UCHL5/NLRP3 inflammasome pathway is inhibited by HS-C. We reasoned that kirenol might be the main active component of HS-C in improving I/R injury. However, further investigation of the therapeutic effect and underlying mechanisms of kirenol on I/R injury is still lacking in this study, which will be explored in future experiments.