Abstract Ongoing inflammation in the heart is positively correlated with adverse remodeling, characterized by elevated levels of cytokines that stimulate activation of cardiac fibroblasts. It was found that CaMKIIδ response to Ang II or TAC triggers the accumulation of ROS in cardiomyocytes, which subsequently stimulates NF-κB/NLRP3 and leads to an increase in IL-6, IL-1β, and IL-18. This is an important causative factor in the occurrence of adverse remodeling in heart failure. Sweroside is a biologically active natural iridoids extracted from Lonicerae Japonicae Flos. It shows potent anti-inflammatory and antioxidant activity in various cardiovascular diseases. In this study, we found that sweroside inhibited ROS-mediated NF-κB/NLRP3 in Ang II-treated cardiomyocytes by directly binding to CaMKIIδ. Knockdown of CaMKⅡδ abrogated the effect of sweroside regulation on NF-κB/NLRP3 in cardiomyocytes. AAV-CaMKⅡδ induced high expression of CaMKⅡδ in the myocardium of TAC/Ang II-mice, and the inhibitory effect of sweroside on TAC/Ang Ⅱ-induced elevation of NF-κB/NLRP3 was impeded. Sweroside showed significant inhibitory effects on CaMKIIδ/NF-κB/NLRP3 in cardiomyocytes from TAC/Ang Ⅱ-induced mice. This would be able to mitigate the adverse events of myocardial remodeling and contractile dysfunction at 8 weeks after the onset of the inflammatory response. Taken together, our findings have revealed the direct protein targets and molecular mechanisms by which sweroside improves heart failure, thereby supporting the further development of sweroside as a therapeutic agent for heart failure. Keywords: Sweroside, Inflammation, Cardiac remodeling, Heart failure, Cardiomyocytes Graphical abstract [39]Image 1 [40]Open in a new tab Highlights * • Sweroside inhibits inflammation via regulation of the CaMKⅡδ/NF-κB/NLRP3 pathway in cardiomyocytes. * • CaMKⅡδ is the intervention target for sweroside to alleviate pressure overload-induced heart failure. * • This study provides a new insight into the mechanisms by which sweroside intervene in heart failure. __________________________________________________________________ Abbreviations: AAV adeno-associated virus Ang II angiotensin II CaMKIIδ calcium-CaM-dependent protein kinase IIδ cTnT cardiac troponin-T H3 histone-H3 IL-1β interleukin-1β IL-6 interleukin-6 IL-18 interleukin-18 MDA malondialdehyde NF-κB nuclear factor kappa-B NLRP3 NOD-like receptor thermal protein domain associated protein 3 ROS reactive oxygen species α-SMA α-smooth muscle actin TAC transverse aortic constriction [41]Open in a new tab 1. Introduction Heart failure is a major global health issue characterized by significant morbidity and mortality [[42]1]. Cardiac remodeling, which involves myocardial hypertrophy and fibrosis, is a key factor contributing to ventricular dysfunction in heart failure [[43]2,[44]3]. Numerous recent clinical and experimental studies have emphasized the non-negligible role of abnormal inflammation in the pathogenesis of heart failure. Targeting inflammatory pathways will likely provide valuable therapeutic approaches to managing heart failure [[45]4,[46]5]. Thus, investigating the role of inflammation in heart failure development is crucial for early identification of risk factors and implementing effective treatment strategies to enhance heart failure prognosis. Studies conducted in clinical settings have revealed that individuals suffering from heart failure experience persistent low-grade inflammation within the heart. This inflammatory reaction has been linked to negative clinical outcomes [[47]3]. In heart failure induced by pressure overload, specifically through Ang II or TAC, cardiomyocytes are believed to be the source of the initial inflammatory response [[48]3]. CaMKIIδ within cardiomyocytes plays a role in NF-κB transcription and in regulating the assembly of the NLRP3 inflammasome. This process leads to an increased release of proinflammatory substances like IL-6, IL-1β, and IL-18 [[49][6], [50][7], [51][8]]. Elevated levels of these pro-inflammatory factors trigger the activation of cardiac fibroblasts, ultimately hastening the development of myocardial fibrosis in heart failure [[52]3,[53]8]. Natural products offer a valuable reservoir for drug development to address complex diseases, including heart failure [[54][9], [55][10], [56][11]]. Pharmacokinetic studies of sweroside in rats showed that its absolute bioavailability was 11.90 % on average. Tissue distribution results showed that sweroside was mainly distributed in tissues with abundant blood-supply (kidneys, liver, spleen and lungs), and was also distributed in heart and brain tissues [[57]12]. We have previously demonstrated that sweroside, one of the main active components of Lonicerae Japonicae Flos, distributed in the heart tissue as original type and had biological properties that ameliorate cardiovascular disease [[58]13]. Prior research has shown that sweroside primarily exhibits antioxidant and anti-inflammatory properties, making it commonly utilized in China and other Asian nations for various purposes such as treating myocardial injury and hepatic steatosis [[59]14,[60]15]. A recent study found that sweroside is anti-inflammatory to attenuate myocardial ischaemia-reperfusion injury, and the mechanism of its effect may be the inhibition of NLRP3 inflammasome [[61]16]. Sweroside has also demonstrated the ability to shield cardiomyocytes from aconitine-induced harm by preventing calcium overload and decreasing reactive oxygen species (ROS) levels [[62]17]. Despite these encouraging findings, the specific mechanisms underlying sweroside's actions remain unknown. Moreover, its effectiveness in addressing cardiac remodeling due to pressure overload remains to be explored. In this research, our hypothesis was that sweroside could suppress the CaMKIIδ/NF-κB/NLRP3 in cardiomyocytes under pressure overload, resulting in a notable decrease in the levels of IL-6, IL-1β, and IL-18. We explored the impact of sweroside on inflammatory responses in cardiomyocytes stimulated by Ang II. We created myocardial high-expressing CaMKIIδ mice using adeno-associated virus (AAV) and observed the effects of sweroside on CaMKIIδ/NF-κB/NLRP3 following TAC/Ang II induction. Furthermore, we examined the potential direct interaction between sweroside and CaMKIIδ, as well as the cardioprotective effects of sweroside on cardiac remodeling in TAC-induced mice. 2. Materials and methods 2.1. Antibodies and reagents Sweroside was purchased from MREDA (purity ≥98 %, Beijing, China). Its chemical structure is illustrated in [63]Fig. 1A. Ang Ⅱ was obtained from Solarbio (Beijing, China). Primary antibodies: NLRP3, CaMKⅡδ, MDA, NF-κB p65, IL-18, collagen Ⅰ, α-SMA, cTnT, β-actin, H3, pro-caspase-1/p20, ASC, IL-1β, and IL-6. The catalog numbers of the primary antibodies utilized are outlined in [64]Table S1. HRP-conjugated antibodies were purchased from Proteintech (Wuhan, China). Alexa 488-labeled antibody, Alexa 647-labeled antibody, and Alexa 594-labeled antibody were purchased from Abcam (Shanghai, China). Fig. 1. [65]Fig. 1 [66]Open in a new tab Sweroside inhibits cardiomyocyte hypertrophy and ameliorates heart failure. (A) Structural information for sweroside; (B) Cell viability was assessed using a CCK-8 assay (n = 6); (C) qRT-PCR results of ANP and BNP in H9c2 (n = 6); (D) qRT-PCR results of ANP and BNP in AC16 (n = 6); (E) qRT-PCR results of ANP and BNP in NRCM (n = 6); (F) Cytoskeleton staining of H9c2 (n = 6). Scale bar = 130 μm; (G) Representative image of echocardiography in difference group mice. The levels of EF and FS were recorded by echocardiography (n = 6); (H) KEGG enrichment analysis by RNA sequencing in the hearts of sweroside intervention mice; (I) GO enrichment analysis by RNA sequencing in the hearts of sweroside intervention mice. The results are expressed as the mean ± SEM. Comparisons among multiple groups were made by ANOVA, followed by Tukey's post hoc analysis. *P < 0.05, **P < 0.01. 2.2. Cell culture and treatment H9c2 and AC16 cardiomyocytes were purchased from the Cell Resource Center, Peking Union Medical College. Cells were cultured in DMEM (BI, Shanghai, China) containing 10 % FBS (BI, Shanghai, China) and 1 % penicillin-streptomycin (BI, Shanghai, China). In preparation for Ang II or sweroside treatment, the cardiomyocytes underwent a 10-h incubation in serum-free DMEM medium. After serum-free, cells were placed in DMEM containing 1 μmol/L Ang II and various concentrations of sweroside for 24 h. 2.3. Primary neonatal rat cardiomyocytes (NRCM) isolation The hearts were extracted from 2- to 3-day-old Sprague-Dawley rat pups of both genders (SIPEIFU, Beijing, China) following previously established protocols [[67]18]. The extracted hearts were rinsed in HBSS (Solarbio, Beijing, China) and subjected to multiple digestions using 0.05 % trypsin (BI, Shanghai, China) and 0.15 % type II collagenase (Worthington, NJ, USA) in HBSS. NRCMs were isolated through a 90-min differential adherent culture at 37 °C. The centrifuged cardiomyocytes were harvested and seeded on culture plates containing 5-BrdU (Sigma, MO, USA). Isolated cardiomyocytes were identified by immunofluorescence labeling of cTnT, as shown in [68]Fig. S1. Animal experimental protocols were approved by the Committee on the Ethics of Medical Laboratory Animals of Beijing University of Chinese Medicine (approved NO. BUCM-4-20220107005-1128). 2.4. Animal experiments Male C57BL/6 N mice aged 8 weeks (Vital River, Beijing, China) were housed in a SPF facility (12-h light/dark cycle), had free access to food and water, and underwent a 7-day acclimatisation period. All research procedures adhered to institutional guidelines and ethical standards. To explore the protective properties of sweroside, we utilized a TAC murine model and an Ang II infusion murine model following established protocols, as outlined in the subsequent sections [[69]19,[70]20]. Mice were randomly divided into the following groups (6 mice per group): (i) sham; (ii) TAC; (iii) TAC + captopril (15 mg/kg/day, Sigma, MO, USA); (iv) TAC+15 mg/kg/day sweroside; (v) TAC+30 mg/kg/day sweroside; (vi) TAC+60 mg/kg/day sweroside. Studies have shown that the absolute bioavailability of sweroside when taken orally is poor [[71]12]. Intraperitoneal injection of drugs will have higher drug absorption and bioavailability [[72]21]. Either sweroside or captopril dissolved in normal saline was given intraperitoneally, with the dosage based on previous research [[73]22,[74]23]. The treatment duration for all groups was 8 weeks. When performing TAC, 1.5 % tribromoethanol (MACKLIN, Shanghai, China) was used for anesthesia. All surgeries were performed under aseptic protocol. A limited median sternotomy was performed under a surgical microscope from the suprasternal incision to the second rib in mice. A 26-gauge blunt needle was placed along the aortic arch after isolation of the aorta. Around the puncture needle, the aorta between the innominate and the left common carotid artery was knotted with 6-0 nylon thread, and the puncture needle was withdrawn and subsequently secured with sutures. In the sham procedure, the steps were similar except for not tying the knot around the aorta. Animal experimental protocols were approved by the Ethics Committee of Beijing Academy of Science and Technology (approved NO. 2022026). We overexpressed CaMKⅡδ in C57BL/6 N murine hearts to examine the mechanism of sweroside on TAC or Ang II infusion. For experiments, mice were infected with AAV9 encoding CaMKⅡδ (AAV-CaMKⅡδ) or an empty vector (AAV-NC). Mouse CaMKⅡδ gene was cloned into AAV9 by GenePharma (Suzhou, China). 24 mice were randomly divided into 2 groups: AAV-NC and AAV-CaMKⅡδ. Mice were given one-time injection of AAV-NC or AAV-CaMKⅡδ (100 μL vehicle containing 1.0 × 10^13 AAV9 vector particles, intravenous injections). For intravenous injections, we delivered AAV9 via tail vein of mice. After intravenous injection, the returning blood will first carry the AAV9 to the right ventricle and lungs. However, the results of the study showed that no pathway-related differences were seen in all tissues/organs tested after AAV9 injection, proving that the route of administration is not a major problem for AAV9 [[75]24]. The mice in the AAV-NC or AAV-CaMKⅡδ condition were randomly divided into 4 groups: (i) Sham/Control; (ii) TAC/Ang Ⅱ; (iii) TAC/Ang Ⅱ+30 mg/kg/day sweroside; (iv) TAC/Ang Ⅱ+60 mg/kg/day sweroside. The murine TAC model operation protocol was described above. Ang II was infused using osmotic mini-pumps (RWD, Shenzhen, China) for a duration of 7 days (1001W, 1000 ng/kg/min), following the previously outlined procedures [[76]20]. Mice in each group were treated with sweroside for 7 days. Animal experimental protocols were approved by the Committee on the Ethics of Medical Laboratory Animals of Beijing University of Chinese Medicine (approved NO. BUCM-2023032306-1137). 2.5. Echocardiography As described previously [[77]25], transthoracic echocardiograms were performed on conscious mice using a Vevo 2100 system (Visual Sonics, Canada). Left ventricular ejection fraction (EF) and fractional shortening (FS) were calculated. The operator and data analyzer were both blinded to sample allocation and group information. 2.6. Histological assessment on hearts Paraffin sections of cardiac tissue were assessed for myocardial structure by H&E, Masson's, and Sirius red staining. To analysis cardiomyocyte hypertrophy, cardiac tissue was stained with wheat germ agglutinin (WGA; Sigma, USA). Myocardial collagen levels and the size of the cardiomyocytes were measured using Image J. 2.7. Immunofluorescence In the process of immunofluorescence, the sections were subjected to overnight incubation with WGA, as well as primary antibodies (anti-NLRP3 1:800, anti-α-SMA 1:800). Subsequently, the sections were incubated with secondary antibodies (Alexa 647-labeled antibody 1:1000; Alexa 594-labeled antibody, 1:1000). Slides were mounted using antifade mounting medium (Solarbio, China). H9c2 cardiomyocytes (2 mL, 0.5 × 10^4 cells) were seeded in confocal culture dishes. Following various treatments, cells were incubated with primary antibodies (anti–NF–κB p65 1:800 or anti-NLRP3 1:800, anti-ASC 1:500). Next, cells were exposed to secondary antibodies (Alexa 488-labeled antibody 1:1000, Alexa 594-labeled antibody 1:1000). Fluorescence intensity or colocalization analysis of cardiomyocytes was performed using Image J. 2.8. mRNA sequencing experimental Purity and quantity were measured after extraction of total RNA from cardiac tissue, and RNA integrity was assessed. Library construction, transcriptome sequencing and analysis were carried out by OE Biotech Co., Ltd. (Shanghai, China). Differential expressed genes underwent GO and KEGG pathway enrichment analysis based on the hypergeometric distribution. Using R (v 3.2.0) enriched terms were screened for significance. Furthermore, R (v 3.2.0) was utilized to create chord diagrams and bubble diagrams representing the significant enrichment terms. 2.9. Immunoprecipitation Cardiac tissue immunoprecipitation experiments were performed using Classic Magnetic Protein A/G IP Kits (Epizyme, Shanghai, China). Protein solutions (1000 μg) were incubated with anti-NLRP3 (5 μg) for 1 h. The cocktail was incubated with magnetic beads for another 1 h. After magnetic separation for 1 min, the beads underwent four washes with lysis buffer. Finally, immunoprecipitated proteins were separated by 12.5 % SDS-PAGE and probed with anti-pro-caspase-1 or anti-ASC. 2.10. Cell viability Following various treatments, 10 μL of CCK-8 (LABLEAD, Beijing, China) was added to the medium. Absorbance was measured at 450 nm with a microplate reader (BioTek, USA) to assess cell viability. 2.11. Cytoskeleton staining Cytoskeletal staining of H9c2 cardiomyocytes by phalloidin. After 4 % paraformaldehyde fixation, cardiomyocytes were incubated with 100 nmol/L phalloidin (LABLEAD, Beijing, China). Fluorescence inverted microscopy (Echo Revolve, USA) was used to record multiple fields of view, and cell surface was quantified by Image J. 2.12. ROS detection Intracellular ROS levels were determined with DCFH-DA (LABLEAD, Beijing, China). According to the doses provided by previous methods [[78]26], 200 μmol/L Tempol (TargetMol Chemicals, Shanghai, China) was used as a negative control in Ang II-induced H9c2 cardiomyocytes. Following the designated treatment, H9c2 cardiomyocytes were treated with 10 μmol/L DCFH-DA for 30 min at 37 °C. DCFH-DA levels (Ex/Em: 504 nm/529 nm) were quantified using a fluorescence microscope (Leica, Biberach, Germany). In heart tissues, the levels of intracellular ROS were measured using DHE (Beyotime, Shanghai, China). The sections were treated with 1 μmol/L DHE for 30 min at 37 °C. The signals (Ex/Em: 300 nm/610 nm) were then analyzed using a fluorescence microscope (Nikon, Japan). 2.13. Cytokines immunoassay Following various treatments, the supernatant of H9c2 medium was harvested and the amount of IL-1β was measured by ELISA (ABclonal, Wuhan, China). Serum levels of IL-1β, IL-18, and IL-6 were measured simultaneously using a mouse cytokine cytometric bead array kit and LEGEND plex Data Analysis Software Suite (Biolegend, USA). 25 μL each of serum and assay buffer were mixed with the pre-mixed beads. After shaking incubation, 25 μL of detection antibody was added. Following a 1-h incubation, SA-PE (25 μL per well) was added. Samples were analyzed using a CytoFLEX S (BECKMAN, USA), with 1000 events recorded for each sample. 2.14. Cell transfection siRNA was utilized to downregulate CaMKⅡδ, while the pcDNA3.1(+) plasmid was employed to upregulate CaMKⅡδ in H9c2 cardiomyocytes. The specific siRNA sequences detailed in [79]Table S2. Initially, GP-transfect-Mate (GenePharma, Suzhou, China) was diluted in DMEM, followed by the dilution of siRNA/DNA in DMEM to create a combined siRNA/DNA mixture. Subsequently, the diluted siRNA/DNA was merged with the diluted GP-transfect-Mate at a 1:1 ratio. Finally, the siRNA/DNA lipid complex was introduced into the cells. The transfection efficiency of both siRNAs and plasmids was assessed using Western blot ([80]Fig. S2). 2.15. Molecular docking The receptor CaMKⅡδ was retrieved from the PDB (PDB ID: [81]2vn9), while the ligand sweroside was acquired from PubChem (PubChem CID: 161036). Protein dehydration and hydrogenation were carried out using the Discovery Studio Client software. Molecular docking was conducted utilizing Pyrx-0.8 and Autodock Vina [[82]27]. The optimal orientations of the ligand-receptor complex were determined based on the docking score. 2.16. Surface plasmon resonance (SPR) According to previous methods, all SPR experiments were performed with a Biacore T200 instrument (Cytiva, USA) [[83]28]. Recombinant human CaMKIIδ (CUSABIO, Wuhan, China) was immobilised on the CM5 sensor chip (Cytiva, USA) by using 1.0 × HBS-P+ (Cytiva, USA). The immobilization level was fixed at 14176 response units (RU), and gradient concentrations of sweroside were injected continuously to flow across the chip surface. Sweroside was dissolved in 5 % DMSO/buffer at different dose (20 mmol/L HEPES, 150 mmol/L NaCl, 0.005 % (v/v) tween 20, 5 mmol/L MgCl[2], and 2 mmol/L DTT, PH = 7.4) [[84]29]. Solvent correction was performed according to the standard scheme of the Biacore T200 instrument. In experiments to investigate the effects of Ca^2+ and ATP on sweroside-CaMKIIδ interactions, 1.5 mmol/L CaCl[2] and 1 mmol/L ATP were added supplementally to the SPR running buffer. 2.17. Cellular thermal shift assay H9c2, AC16 and NRCM cardiomyocytes were treated with either 100 μmol/L sweroside or PBS for 1 h. Cells were harvested with PBS containing protease and phosphatase inhibitors. The soluble proteins obtained was divided into eight portions and heated at varying temperatures (42, 46, 50, 54, 58, 62, 66, and 70 °C) on a PCR instrument (Bio-Rad, USA). The soluble fraction was analyzed using Western blot. 2.18. Western blot As described previously [[85]30,[86]31], cell- or tissue-extracted proteins were separated in 6 % or 12.5 % SDS-PAGE and blotted on PVDF membranes (Millipore, USA). After blocking with 5 % BSA, the membranes were incubated with primary antibodies and HRP-conjugated antibodies successively. Immunoreactive bands were detected after incubation with enhanced chemiluminescence reagent (Med Chem Express, USA). Densitometric quantification was performed using Image J. 2.19. Quantitative real-time PCR (qRT-PCR) Cell or tissue extraction of total RNA and reverse transcribed were performed according to the kit instructions (Accurate Biology, China). qPCR kit (Accurate Biology, China), cDNA, primers were used and performed on a Step One Plus Systems (Thermo Fisher, USA). Primer sequence information is shown in [87]Table S3. qRT-PCR data was presented using 2^−ΔΔCT. 2.20. Statistical analysis Experiments were independently conducted at least 3 times, with specific N numbers and technical replicates provided in each figure. In vivo studies, each analysis used data collected per animal. Data were presented as the mean ± standard error of the mean. The statistical significance of differences between groups was determined using the Student's t-test or one way analysis of variance (ANOVA) multiple comparisons in GraphPad Prism 8.3. Normality and homogeneity of variance were conducted prior to t-test and ANOVA. Differences are significant at P < 0.05. 3. Results 3.1. Sweroside improves Ang II-induced cardiomyocyte hypertrophic phenotype and cardiac function in TAC-mice High levels of Ang II lead to cardiomyocyte hypertrophy, which has been considered a significant pathological process in heart failure [[88]32]. We demonstrated the cardio-protective properties of sweroside against cardiac hypertrophy [[89]13]. To verify this protective effect, we examined the effects of sweroside on rat cardiac tissue derived H9c2 cell line, human ventricular tissue derived AC16 cell line, and primary cardiomyocytes isolated from rat neonatal cardiac tissue. By examining the concentration gradient, we confirmed that sweroside treatment had no negative effect on cardiomyocyte survival ([90]Fig. 1B). Sweroside notably reduced ANP and BNP gene expression in Ang II-stimulated cardiomyocytes ([91]Fig. 1C, 1D, 1E). Phalloidin labeling of H9c2 cardiomyocytes revealed that the effect of Ang Ⅱ to increase cardiomyocyte cross-sectional area was counteracted by sweroside ([92]Fig. 1F). We carried out additional studies to evaluate the ability of sweroside to ameliorate TAC-induced mice. TAC results in heart failure in mice due to the progression of cardiac hypertrophy, myocardial fibrosis, and other factors [[93]33]. By utilizing echocardiography, we found that sweroside significantly enhanced cardiac function in mice, demonstrating a notable increase in EF and FS ([94]Fig. 1G). As shown in [95]Fig. S3, sweroside reduced the levels of LVIDs and LVVols and elevated the levels of LVAWs and LVPWs in TAC-induced mice. Additionally, we performed RNA sequencing analysis of mouse myocardial tissues after sweroside intervention, and obtained differential expressed genes in sweroside-intervened TAC mice ([96]Fig. S4). The outcomes of GO enrichment analysis can be seen in [97]Fig. 1H. In comparison to the TAC group, sweroside influenced the innate immune response, collagen-containing extracellular matrix, and cytokine receptor activity, among other factors. The results of KEGG enrichment analysis can be viewed in [98]Fig. 1I. Contrasted with the TAC group, sweroside impacted the calcium signaling pathway and cytokine-cytokine receptor interaction, among other pathways. The sequencing results suggested that sweroside enhances cardiac function in mice with heart failure primarily by regulating cardiac fibrosis and inflammatory mechanisms. 3.2. Sweroside inhibits Ang II-induced inflammatory response in cardiomyocytes Ang Ⅱ can induce cardiomyocyte hypertrophy while activating NF-κB/NLRP3 [[99]7,[100]8]. Thus, we detected that NF-κB and NLRP3 mRNA expression were significantly increased in cardiomyocytes induced by Ang II, and the results were consistent among H9c2, AC16, and NRCM ([101]Fig. 2A, 2B, 2C). Following sweroside treatment, NF-κB and NLRP3 expression was significantly decreased ([102]Fig. 2A, 2B, 2C). ROS, as an activator, increases significantly after Ang II stimulation and then activates NLRP3 [[103]34]. The fluorescence probe detection ([104]Fig. 2D) showed that the Ang II-treated group produced significantly more ROS compared with the control group. Furthermore, the concentration of ROS in the Ang II + Sweroside-treated group was significantly decreased. Sweroside reduced the production of ROS in H9c2 cells after Ang II-induced. Consistently, another oxidative stress marker, MDA, was also significantly reduced in Ang II-induced cardiomyocytes after sweroside treatment ([105]Fig. 2E). The proinflammatory cytokine expression of IL-6, IL-1β, and IL-18 were increased after the NF-κB/NLRP3 pathway of cardiomyocytes was induced [[106]8]. Treatment with sweroside significantly reduced IL-6, IL-1β, and IL-18 in cardiomyocytes ([107]Fig. 2F). It is important to note that cytokines function as secreted proteins with biological roles. The findings of IL-1β seen in the medium of cardiomyocytes aligned with those observed in the Western blot ([108]Fig. 2G). These results suggested that treatment with sweroside counteracted the inflammatory response induced by Ang Ⅱ in cardiomyocytes. Fig. 2. [109]Fig. 2 [110]Open in a new tab Sweroside suppresses cardiomyocyte inflammation. (A) qRT-PCR results of NF-κB and NLRP3 in H9c2 (n = 6); (B) qRT-PCR results of NF-κB and NLRP3 in AC16 (n = 6); (C) qRT-PCR results of NF-κB and NLRP3 in NRCM (n = 6); (D) ROS staining of H9c2 cells (n = 6). Scale bar = 500 μm; (E) Protein levels of MDA and representative immunoreactive bands in H9c2 (n = 6); (F) Protein levels of IL-6, IL-1β, and IL-18 in H9c2. Representative immune response band images (n = 6); (G) The levels of IL-1β in the medium of H9c2 (n = 6). The results are expressed as the mean ± SEM. Comparisons among multiple groups were made by ANOVA, followed by Tukey's post hoc analysis. *P < 0.05, **P < 0.01. 3.3. Sweroside inhibits Ang II-induced inflammatory response in cardiomyocytes associated with CaMKIIδ/NF-κB/NLRP3 Cardiomyocyte activation of the NF-κB/NLRP3 mechanism is linked to CaMKⅡδ [[111]6]. Upon Ang II stimulation, we observed upregulation of the CaMKIIδ gene in H9c2, AC16, and NRCM cardiomyocytes. After treatment with sweroside, the level of CaMKⅡδ in cardiomyocytes decreased ([112]Fig. 3A, 3B, 3C). Consistently, CaMKⅡδ changes in cardiomyocytes were also detected in Western blot ([113]Fig. 3D). The NLRP3 inflammasome activation, triggered by Ang II, leads to an increase in IL-1β and IL-18 [[114]3]. NF-κB, as a transcription factor, induces IL-6 expression [[115]7]. In order to investigate how sweroside inhibits inflammatory factors in cardiomyocytes, we examined its impact on NLRP3 inflammasome and NF-κB. The mRNA level of caspase-1 was significantly increased in cardiomyocytes exposed to Ang II for 24 h. Sweroside treatment was able to reduce the level of caspase-1 ([116]Fig. 3A, 3B, 3C). Correspondingly, the results of protein level assay in H9c2 cardiomyocytes showed that sweroside was able to inhibit the high level of NLRP3 and p20 induced by Ang II ([117]Fig. 3D). Therefore, sweroside significantly inhibited Ang Ⅱ-induced inflammasome activation. Immunofluorescence analysis revealed that Ang II substantially promoted NF-κB translocation into the nucleus, whereas sweroside inhibited this translocation ([118]Fig. 3E, 3F, 3G). Fig. 3. [119]Fig. 3 [120]Open in a new tab Sweroside inhibits cardiomyocyte inflammation by regulating CaMKIIδ/NF-κB/NLRP3. (A) qRT-PCR results of CaMKIIδ and caspase-1 in H9c2 (n = 6); (B) qRT-PCR results of CaMKIIδ and caspase-1 in AC16 (n = 6); (C) qRT-PCR results of CaMKIIδ and caspase-1 in NRCM (n = 6); (D) Protein levels of CaMKIIδ, NLRP3, and p20 in H9c2. Representative immune response band images (n = 6); (E) Representative images of NF-κB immunofluorescence detection in H9c2. Scale bar = 75 μm; (F) Mean fluorescence intensity of NF-κB in H9c2 calculated by Image J (n = 4); (G) Distribution of nucleus-plasmic location of NF-κB in H9c2 analyzed by Image J. The results are expressed as the mean ± SEM. Comparisons among multiple groups were made by ANOVA, followed by Tukey's post hoc analysis. *P < 0.05, **P < 0.01. 3.4. Sweroside inhibition of Ang II-induced NF-κB/NLRP3 in cardiomyocytes is dependent on CaMKIIδ In order to explore the possible connection between sweroside's regulation of NF-κB/NLRP3 and CaMKⅡδ, we utilized siRNA to knock down the level of CaMKⅡδ in cardiomyocytes. The findings revealed that sweroside suppressed the activation of NLRP3 inflammasome, and this impact was counteracted when CaMKⅡδ was silenced in H9c2 ([121]Fig. 4A). It is important to highlight the significant role of the NLRP3-ASC complex in promoting NLRP3 inflammasome activation [[122]35]. Furthermore, sweroside treatment led to a significant down-regulation of NLRP3 protein expression, while having minimal impact on ASC. Subsequently, we conducted immunofluorescence experiments to investigate inflammasome recruitment. The results demonstrated that Ang II induced NLRP3 inflammasome recruitment in cardiomyocytes. Notably, sweroside reduced the recruitment of NLRP3 inflammasome, but this effect was impeded by CaMKⅡδ silencing ([123]Fig. 4B). Additionally, the ability of sweroside to counteract the level of the NF-κB p65 nucleus-cytoplasm ratio was found to be hindered in CaMKⅡδ-silenced cells ([124]Fig. 4C). Therefore, the anti-inflammatory effect of sweroside in CaMKⅡδ-silenced cells was nullified. Specifically, the reduction in IL-6, IL-1β, and IL-18 was impeded in Ang Ⅱ-induced CaMKⅡδ-silenced cardiomyocytes ([125]Fig. 4D). We transfected cardiomyocytes with CaMKⅡδ-pcDNA. As a result, the enhanced reduction in IL-6, IL-1β, and IL-18 by sweroside was not observed in Ang II-induced CaMKⅡδ overexpressing cardiomyocytes ([126]Fig. 4E). Specifically, after Negative-pcDNA transfection of cardiomyocytes, the expression of IL-1β in sweroside treated groups were decreased by 24 % and 30 %, respectively, when compared with the Ang II group. In cardiomyocytes overexpressing CaMKIIδ, the expression of IL-1β in sweroside treated groups were decreased by 23 % and 40 %, respectively, when compared with the Ang II group ([127]Fig. 4E). Fig. 4. [128]Fig. 4 [129]Open in a new tab Effect of sweroside on regulation of NF-κB/NLRP3 when H9c2 are transfected with CaMKIIδ-siRNA/CaMKIIδ-pcDNA. (A) Levels of NLRP3, ASC, and p20 when H9c2 were transfected with CaMKIIδ-siRNA (n = 4); (B) Immunofluorescence labeling of NLRP3 and ASC in H9c2 cardiomyocytes transfected with CaMKIIδ-siRNA (n = 4). Scale bar = 75 μm; (C) Cytosolic nuclear-to-plasmic ratio levels of NF-κB p65 when CaMKIIδ-siRNA was introduced into H9c2 (n = 4); (D) Expression of IL-1β, IL-18, and IL-6 when H9c2 were transfected with CaMKIIδ-siRNA (n = 4); (E) Expression of IL-1β, IL-18, and IL-6 when H9c2 were transfected with CaMKIIδ-pcDNA (n = 4). The results are expressed as the mean ± SEM. Comparisons among multiple groups were made by ANOVA, followed by Tukey's post hoc analysis. ns, no significance, *P < 0.05, **P < 0.01. 3.5. Identification of CaMKⅡδ as a direct sweroside binding protein To further validate the potential for direct interaction between sweroside and CaMKIIδ, we performed molecular docking, cellular thermal shift assay and SPR. The outcomes of molecular docking indicated that sweroside had an affinity of −7.1 kcal/mol for CaMKIIδ. The 3D image results showed that sweroside is able to incorporate into the docking pocket. They have docking activity between sweroside and CaMKIIδ. The enlarged 2D image showed that sweroside forms a favourable interaction with CaMKIIδ. Sweroside formed hydrogen bonds with ASP157, LYS43 of CaMKIIδ ([130]Fig. 5A). The interaction of ligands with target proteins can be shown through the cellular thermal shift assay, which identifies the enhanced resistance of target proteins to heat-induced precipitation by using immunoblotting. As anticipated, sweroside incubation led to the stabilization of CaMKIIδ in H9c2, AC16, and NRCM cardiomyocytes ([131]Fig. 5B). Given that CaMKIIδ is a member of the protein kinase family and that Ca^2+ and ATP play crucial roles in its activation and biological function [[132]36]. Our SPR experiments aimed to explore key regulatory parameters that may influence this interaction. We examined the affinity of sweroside-CaMKIIδ in the absence or presence of Ca^2+ and ATP. In the same way, the findings from SPR experiments indicated that sweroside directly interacts with CaMKIIδ in a manner dependent on the concentration (KD = 6.492 μmol/L). Following the addition of Ca^2+ and ATP to the buffer system, the interaction continued to be concentration-dependent, yet the strength of binding decreased by a factor of 4.8 (KD = 1.348 μmol/L) ([133]Fig. 5C). This indicated that the presence of Ca^2+ and ATP did not impede the interaction between sweroside and CaMKIIδ, but rather competitively influenced the strength of the interaction. Fig. 5. [134]Fig. 5 [135]Open in a new tab Identification of sweroside binding directly to CaMKIIδ. (A) Molecular docking predicts affinity for the binding of sweroside-CaMKIIδ; (B) Cellular thermal shift assay to detect thermal stability of CaMKIIδ after sweroside (100 μmol/L) treatment in H9c2, AC16, and NRCM cells; (C) SPR analysis of the binding affinity of sweroside to CaMKIIδ. The results are expressed as the mean ± SEM. 3.6. Sweroside inhibits myocardial inflammation in TAC/Ang II-mice by regulating CaMKIIδ/NF-κB/NLRP3 Our next objective was to determine whether sweroside inhibited TAC/Ang Ⅱ induced cardiac inflammation in mice. We used a murine model in which TAC was performed and a subcutaneous osmotic minipump for continuous infusion of Ang Ⅱ murine model. Furthermore, we induced high expression of CaMKⅡδ in the myocardium of mice through tail vein injection of AAV-CaMKⅡδ ([136]Fig. 6A, S5A). Fig. 6. [137]Fig. 6 [138]Open in a new tab Effect of sweroside on CaMKIIδ/NF-κB/NLRP3 in the heart of TAC-mice. (A) Time course of experiments. After mice were adapted for 7 days and underwent TAC surgery, AAV-CaMKIIδ was injected to induce high expression of CaMKIIδ in the myocardium of mice. 7 days later, mice were euthanized for heart and serum collection; (B) Cytometric bead array detection of sweroside on circulating IL-1β, IL-6, and IL-18 in TAC mice (n = 3); (C) Western blot detection of sweroside on IL-1β, IL-6, and IL-18 in the myocardium of TAC mice (n = 3); (D) DHE probe labelling of ROS levels in myocardial tissue of TAC-mice (n = 3). Scale bar = 60 μm; (E) Effects of sweroside on the nucleus-to-plasma ratio of NF-κB p65 in the myocardium of TAC mice (n = 3); (F) IP assay showed the levels of NLRP3-ASC, NLRP3-pro-caspase-1 in myocardial tissues of TAC mice (n = 3); (G) Immunofluorescence labelling of TAC mouse myocardial tissue for NLRP3 and α-SMA. Myocardial cell membranes were labeled using WGA. Scale bar = 75 μm. The results are expressed as the mean ± SEM. Comparisons among multiple groups were made by ANOVA, followed by Tukey's post hoc analysis. ns, no significance, *P < 0.05, **P < 0.01. It was previously shown that in mice with TAC-induced or sustained delivery of Ang II for 7 days, cardiomyocytes would specifically display high levels of CaMKⅡδ/NF-κB/NLRP3 [[139]7]. The TAC/Ang Ⅱ-mice displayed increased circulating IL-6, IL-1β, and IL-18, which were normalized by sweroside. The inhibitory effect of sweroside on TAC/Ang II-induced elevation of IL-6, IL-1β, and IL-18 was not enhanced after high CaMKIIδ expression in mouse myocardium ([140]Fig. 6B, S5B). Consistent with the in vitro results, the effects of sweroside on IL-6, IL-1β, and IL-18 in myocardial tissues of TAC/Ang Ⅱ-mice also exhibited the same pattern ([141]Fig. 6C, S6A). Myocardial tissue ROS and MDA assay showed that high levels of CaMKIIδ in TAC-mice hearts enhance ROS and MDA levels and reduce the ability of sweroside to inhibit ROS and MDA ([142]Fig. 6D, S7). As expected, sweroside decreased the nucleus-to-cytoplasm ratio levels of NF-κB p65 in cardiac tissue and could not be further blocked under conditions of high expression of CaMKⅡδ ([143]Fig. 6E, S8A). Sweroside reduced the interaction of NLRP3 with pro-caspase-1 or ASC in cardiac tissues, whereas the inhibitory effect was not enhanced in TAC/Ang II-induced myocardial tissues overexpressing CaMKIIδ ([144]Fig. 6F, S8B). Immunofluorescence showed that high levels of NLRP3 in the hearts of TAC-mice enhanced the levels of α-SMA. However, because of the inhibition of CaMKIIδ/NLRP3 by sweroside, the level of α-SMA in the myocardium was correspondingly reduced ([145]Fig. 6G). These results indicated that sweroside inhibits the activation of CaMKⅡδ/NF-κB/NLRP3, prevents TAC/Ang Ⅱ-induced inflammation, and reduces the activation of cardiac fibroblasts. 3.7. Sweroside improves TAC-induced cardiac remodeling in mice Our last objective was to determine the effect of sweroside on cardiac remodeling. To verify this, we performed TAC in mice and used captopril as a positive control. In this experimental model, we initiated sweroside treatment for 8 weeks following TAC induction ([146]Fig. 7A). Our results showed that sweroside reduced heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) in TAC-mice ([147]Fig. 7B). The administration of sweroside led to a decrease in the mRNA levels of ANP and BNP in the heart tissue ([148]Fig. 7C). Histological examination using HE staining indicated that sweroside treatment alleviated hypertrophic changes and reduced ventricular remodeling area in TAC-mice ([149]Fig. 7D). To visualize tissue collagen, Masson and Sirius red staining were used, while WGA staining was applied to illustrate cell morphology. Additionally, cTnT was utilized as a marker of cardiomyocyte damage [[150]37]. Histologically, TAC-mice exhibited collagen deposition, increased cardiomyocyte cross-sectional area and elevated cTnT levels. However, sweroside treatment normalized cardiomyocyte hypertrophy and fibrosis, which were in the same pattern as the effects of captopril ([151]Fig. 7D, 7E, 7F, 7G). Specifically, sweroside led to decreased levels of α-SMA and collagen I in the myocardial tissue of TAC-mice ([152]Fig. 7G). These findings collectively underscore the protective effect of sweroside in myocardial remodeling. Fig. 7. [153]Fig. 7 [154]Open in a new tab Effect of sweroside on cardiac remodeling in TAC mice. (A) A time course of experiments. When mice were adapted for 7 days and underwent TAC, they were intervened with sweroside or captopril for 8 weeks. Mice were euthanized for heart and serum collection; (B) Effect of sweroside on HW/BW and HW/TL in TAC-mice; (C) Gene expression of ANP and BNP in heart of mice (n = 6); (D) HE, Masson, Sirius red, and WGA staining of myocardial tissue in TAC mice. (HE: Scale bar = 2000 μm/100 μm; Masson, Sirius red, and WGA: Scale bar = 100 μm); (E) Analysis of collagen deposition in myocardial tissue by Image J (n = 6); (F) Analysis of cell size in myocardial tissue by Image J (n = 6); (G) Expression of collagen Ⅰ, α-SMA, cTnT in mouse myocardial tissues by Western blot (n = 6). The results are expressed as the mean ± SEM. Comparisons among multiple groups were made by ANOVA, followed by Tukey's post hoc analysis. *P < 0.05, **P < 0.01. (For interpretation of the references