Honokiol ameliorates angiotensin II‐induced cardiac hypertrophy by promoting dissociation of the Nur77–LKB1 complex and activating the AMPK pathway

Abstract Pathological cardiac hypertrophy is a key contributor to heart failure, and the molecular mechanisms underlying honokiol (HNK)‐mediated cardioprotection against this condition remain worth further exploring. This study aims to investigate the effect of HNK on angiotensin II (Ang II)‐induced myocardial hypertrophy and elucidate the underlying mechanisms. Sprague–Dawley rats were exposed to Ang II infusion, followed by HNK or vehicle treatment for 4 weeks. Our results showed that HNK treatment protected against Ang II‐induced myocardial hypertrophy, fibrosis and dysfunction in vivo and inhibited Ang II‐induced hypertrophy in neonatal rat ventricular myocytes in vitro. Mechanistically, HNK suppressed the Ang II‐induced Nur77 expression at the transcriptional level and promoted ubiquitination‐mediated degradation of Nur77, leading to dissociation of the Nur77–LKB1 complex. This facilitated the translocation of LKB1 into the cytoplasm and activated the LKB1‐AMPK pathway. Our findings suggest that HNK attenuates pathological remodelling and cardiac dysfunction induced by Ang II by promoting dissociation of the Nur77–LKB1 complex and subsequent activation of AMPK signalling. This study uncovers a novel role of HNK on the LKB1‐AMPK pathway to protect against cardiac hypertrophy.

is associated with the reactivation of the fetal gene program and is triggered by the collective action of various signalling proteins that transactivate pro-hypertrophic genes.
The mammalian target of rapamycin (mTOR) is a serine/threonine-protein kinase that plays a crucial role in coordinating nutrient availability and growth factor signalling during cell metabolism and growth.mTORC1 and mTORC2 are two different complexes of mTOR that regulate protein synthesis and degradation. 2mTORC1 activity increases during the progression of both physiological and pathological hypertrophy, which is stimulated by various metabolic, mechanical and biochemical signals.This increase in activity leads to a boost in ribosomal protein production (mRNA translation) by activating ribosomal protein S6 kinase β1 (S6K1) and suppressing eIF4E-binding protein 1 (4EBP1), thus enabling unrestricted cap-dependent translation via eIF4E. 3AMPactivated protein kinase (AMPK) is a crucial energy balance sensor in mammalian cells, 4,5 and liver kinase B1 (LKB1) acts as its key upstream kinase, mediating the phosphorylation of AMPK at Thr172. 6Under normal physiological conditions, LKB1 is primarily localized in the nucleus.However, under certain conditions, LKB1 is phosphorylated at Ser428 (pLKB1) and then exported to the cytoplasm, eventually inducing AMPK activation. 7LKB1-AMPK can suppress mTORC1, and pharmacological inhibition of mTORC1 can alleviate pressure overload-and Ang II-induced pathological myocardial hypertrophy and heart failure. 8,9r77 (NR4A1), also known as TR3, is a member of the nuclear receptor superfamily that functions as an orphan receptor. 10It is classified as an immediate-early response gene due to its ability to be induced by various stimuli, including growth factors, inflammatory factors and stress, in different cell types. 11Nur77 possesses multiple biological functions, including the modulation of cell proliferation, 12,13 differentiation, development, apoptosis, 14 metabolism and immunity. 15Recently, studies have reported that Nur77 modulates liver kinase B1 (LKB1) by binding and sequestering it in the nucleus, leading to the attenuation of AMP-activated protein kinase (AMPK) activation.However, the function of Nur77 in this regard can be inhibited by certain compounds, such as ethyl (2,3,4-trimethoxy-6-octanoylphenyl) acetate (TMPA), 16 which interact with Nur77 at specific sites, leading to the release and nuclear export of LKB1 to the cytoplasm. 16,17This event, in turn, mediates the phosphorylation of AMPK.The results of these studies have established both the transcriptional and post-transcriptional links between Nur77 and LKB1.Honokiol (HNK), a bioactive constituent obtained from Magnolia officinalis bark, has been frequently employed in traditional Chinese medicine.9][20][21][22][23] Notably, HNK has been shown to mitigate hepatic triglyceride and lipogenic protein levels and attenuate fat deposition in high-fat diet-fed mice through a significant increase in LKB1 and AMPK phosphorylation. 24In relation to cancer, HNK has been found to curb breast tumorigenesis in mouse models by engaging in LKB1-dependent pathways. 25Collectively, these findings suggest the crucial role of LKB1-AMPK signalling in mediating HNK's cytoprotective effects.However, the precise mechanism underlying the modulation of LKB1-AMPK signalling by HNK still remains elusive.
The present study investigated the effect of HNK on Ang IIinduced myocardial hypertrophy both in vivo and in vitro.Our findings reveal that HNK modulates the LKB1-AMPK-p70S6K signalling pathway, leading to the attenuation of myocardial hypertrophy.Further, we observed that LKB1-mediated AMPK phosphorylation in the cytoplasm was suppressed by the binding of Nur77, which sequestered LKB1 in the nucleus.Notably, HNK disrupted the Nur77-LKB1 interaction, thereby promoting LKB1's nuclear export and facilitating AMPK phosphorylation.These results provide novel insights into the mechanism by which HNK mediates its cytoprotective effects through the regulation of the LKB1-AMPK signalling pathway.

| Materials and reagents
Sigma-Aldrich provided HNK, Ang II and DAPI, while WZ Biosciences (Shandong, China) supplied VigeneFection transfection reagent.Antibodies against LKB1, p-LKB1, p70S6K, p-p70S6K, AMPKα, p-AMPKα and LaminB1 were obtained from Cell Signalling Technology, and anti-β-actin antibody was obtained from Santa Cruz Biotechnology.Abcam supplied antibodies against Nur77.Thermo Fisher Scientific provided anti-rabbit and anti-mouse secondary antibodies conjugated to HRP.These materials were utilized in the present study.

| Animals
Animal experiments were carried out in accordance with the

| Animal model and experimental design
Cardiac remodelling was induced in the experimental animals by chronic infusion of Ang II (Sigma-Aldrich) at a dose of 520 ng•kg −1 •min −1 for a duration of 4 weeks, using ALZET® mini-osmotic pumps (model number: 2006, DURECT Corporation), according to the manufacturer's guidelines. 26The control group received subcutaneous infusion of phosphate-buffered saline (PBS).The rats were anaesthetised with a mixture of 2.0% isoflurane and oxygen (100%, airflow velocity: 1 L•min −1 ), and an incision was made in the skin on their backs to implant the capsule pump into the subcutaneous tissue.After suturing, the rats were returned to their hous-  27 and our pre-experimental results.Blood pressure was measured weekly using the Softron BP-2010A system (Softron Beijing Biotechnology Co. Ltd.) via the tail-cuff method.
The BP data were obtained by taking three measurements in the resting state, and the average value was calculated. 28All rats were subjected to environmental adaptation before the tail-cuff measurements.

| Routine echocardiogram and quantitative tissue velocity imaging (QTVI)
All rats were anaesthetised with 2% isoflurane through a facial mask.Transthoracic echocardiography using a 12S probe with The relative wall thickness (RWT) was also calculated using the equation RWT = 2 × LVPWD/LVIDD.
Mitral orifice flow spectra and tissue Doppler dynamic images of the mitral annulus in apical four-chamber cardiac view were obtained.The peak early diastolic velocity (PVE) was measured from the mitral orifice flow spectrum.To attain better temporal and velocity resolution, the Q-analyze software (GE Healthcare) was employed for measuring tissue motion velocities of the mitral annulus on dynamic DTI.Specifically, peak velocities of tissue motion spectra of the lateral (s) and interstitial (s') septal mitral annular walls during systole were measured, and their mean (s ¯) was calculated.
Similarly, peak velocities of tissue motion in the lateral (e) and interstitial (e') walls of the mitral annulus during early diastole were measured, and the mean (ⅇ ¯) was calculated.Finally, the ratio PVE/s ¯ was calculated.

| Analysis of heart tissue and blood serum
At the end of the fourth week, the animals were weighed.
Following euthanasia by carbon dioxide, blood samples were obtained, and the heart was quickly excised and prepared for histology, protein and RNA extraction.The ratios of heart weight (HW) to tibia length (TBL) and left ventricular weight (LVW) to TBL were determined.The rat hearts were fixed in 10% formalin, dehydrated, paraffin-embedded and cut into 5 mm sections.
For histological evaluation, sections were collected on gelatincoated slides and stained with haematoxylin and eosin (H&E, Sigma-Aldrich) and Picrosirius Red (PSR; Solarbio).Images of three hearts from each group were acquired using an Eclipse Ti microscope (Nikon) with a 20× objective.Only myocytes cut along the short axis with a visible nucleus were included in the analysis (a minimum of 200 cells per heart). 29The cell borders were manually delineated by a blinded operator.The two-dimensional cross-sectional areas were calculated using NIS-Elements software (Nikon).
For collagen analysis in the myocardial tissue, five fields of view were randomly chosen from each slide.For collagen volume fraction (CVF) analysis in the border area, six separate views (×400) were chosen, and the following equation was used to measure CVF: CVF = collagen area/total area.Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were monitored continuously to assess liver function.Blood glucose, urea nitrogen, lipid and creatinine levels were measured using enzymatic and urease colorimetric assays.Furthermore, the concentrations of creatine kinase (CK), CK-MB, lactic dehydrogenase and uric acid were determined by employing a colorimetric method provided by Roche.The serum levels of natriuretic peptide A, natriuretic peptide B, galectin-3 (Gal-3) and Ang II were analysed by employing an ELISA kit (Cloud-Clone Corp).

| Detecting of capillary and arteriolar density
For capillary and arteriolar density analysis, six heart cross sections were taken from each group of experimental animals and stained for CD31 (28083-1-AP, Proteintech) and IB4 (L2895, Sigma-Aldrich), and the ratios of CD31 and IB4 positive areas to the total area were detected, respectively.

| TUNEL assay
For apoptosis analysis, 5-μm paraffin-embedded heart sections were subjected to TUNEL staining using the TUNEL assay kit (11684795910, Roche) following the manufacturer's protocol.Six to eight fields were randomly selected from the heart of rats, for each section, under an optical microscope.The percentage of apoptotic cells was calculated as TUNEL-positive nuclei number divided by the total number of nuclei identified by haematoxylin staining.

| Cell culture
Neonatal rat ventricular myocytes (NRVMs) were isolated and cultured according to previously described methods. 30,31In brief, the ventricles of newborn rats were minced, and the cells were isolated by digestion with type-II collagenase (150 U•mL −1 ; Gibco) and stirring at 37°C.The isolated cardiomyocytes were seeded on 0.1% gelatincoated culture dishes (Merck) at a density of 7.5 × 10 4 cells•cm −2 to obtain a confluent monolayer of spontaneously contracting cells after 24 h.Mitomycin C (10 μg•mL −1 ; Sigma-Aldrich) was used for 4 h to inhibit cardiac fibroblast proliferation after seeding.The culture medium consisted of Dulbecco's modified Eagle's medium with 25 mM glucose, 5% fetal bovine serum, GlutaMAX™ and HEPES (all from Gibco).Cardiomyocyte purity was determined by immunofluorescence using α-actinin antibody (Sigma-Aldrich), and NRVMs with a purity of over 90% were used.HEK293T cells from the American Type Culture Collection were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U•mL −1 penicillin and 100 μg•mL −1 streptomycin and maintained in a 5% CO2 atmosphere at 37°C.Empty adenovirus (Ad-empty) or adenoviruses carrying cDNA of rat Nur77 or dominant negative Nur77 were applied to freshly isolated and cultured cardiomyocytes at a multiplicity of infection (MOI) of 100 for 40 h prior to 30 min application of HNK.

| Subcellular fractionation
Nuclear and cytosolic fractions were harvested using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), according to the manufacturer's instructions.

| Western blot, co-immunoprecipitation and immunofluorescence assays
Western blot, co-immunoprecipitation and immunofluorescence assays were conducted according to established protocols. 32,33r western blot, cellular proteins ranging from 25 to 50 μg were separated via SDS-PAGE and transferred to PVDF membranes.
The membranes were blocked with 5% skim milk in TBST buffer (150 mM NaCl, 0.1% Tween-20 and 50 mM Tris-HCl, pH 7.4) and subsequently incubated overnight with primary antibodies, followed by detection with secondary antibodies.Detection of the final immunoreactive products was performed using an Invitrogen iBright™ FL1500 chemiluminescence system (ThermoFisher Scientific).Coimmunoprecipitation assay was conducted using the Dynabeads™ Co-Immunoprecipitation Kit (ThermoFisher Scientific), following the manufacturer's instructions.
The immunofluorescence analysis was conducted by culturing cells on glass slides for 24 h, and subjecting them to experimental treatment.The cells were then permeabilized with PBS containing 0.05% Triton™ X-100 and 0.1 M glycine on ice for 20 min and subsequently blocked with 1% BSA in PBS for 30 min at 18-25°C.Mouse anti-Nur77 antibody (1:100) and rabbit anti-LKB1 antibody (1:100) were added to the cells and incubated at 4°C for 12 h, followed by detection using an anti-mouse immunoglobulin G (IgG) conjugated with Alexa Fluor® Plus 594 (1:200) and anti-rabbit IgG conjugated with Alexa Fluor® Plus 488 (1:200, all from Thermo Fisher Scientific), respectively.Images were acquired with an LSM-510 confocal laser-scanning microscope (Carl Zeiss).

| Statistical analysis
The data are presented as the mean ± SEM.Statistical analysis was conducted using one-way analysis of variance (anova) followed by Tukey's post-hoc test if the data were normally distributed.
Additionally, the post-hoc test was conducted only if the F-statistic was significant (p < 0.05).For non-normally distributed data, the Kruskal-Wallis H test was used, and individual mean comparisons were performed using the Mann-Whitney U test.The statistical analyses were conducted using SPSS version 16, and the significance level was set at p < 0.05.

| HNK eliorates Ang II-induced myocardial hypertrophic remodelling and diastolic dysfunction
In this study, the effect of HNK on myocardial dysfunction and remodelling induced by continuous Ang II infusion was investigated using a rat model.Our findings indicated that Ang II administration induced cardiac hypertrophy and dysfunction, as evidenced by elevated IVSD, LVPWD, RWT, HW, LVW values, as well as HW/TL and LVW/TL ratios (Figure 1A-C) and Figure S1A-D), along with decreased EDV, ESV, LVEF, LVFS, and LVDD, LVDS (Figure 1E-H), Figure S1E,F and Table 1).Moreover, s ¯ and ⅇ ¯ decreased (Figure 1I,J), while the PVE/s ¯ ratio increased in the Ang II-induced group (Figure 1K), as detected by QTVI technology.However, HNK treatment reversed these changes.In addition, Ang II infusion increased serum Ang II levels, as well as diastolic and systolic BP levels in rats, while HNK treatment did not alter serum Ang II levels or Ang II-induced BP (Tables 1 and 2) and Figure S1G,H).No changes in body weight were observed in any experimental groups.In summary, our results suggest that HNK can ameliorate myocardial remodelling and function in Ang II-infused rats, without altering BP.

| HNK improves lipid metabolism but increases serum uric acid
Our investigation of Ang II-infused rats revealed significant elevations in total cholesterol (TC), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) levels compared to the control group (Table 2) and Figure S2A-C).Following low-or high-dose HNK treatment for 4 weeks, we observed a marked reduction in TC, ANP, BNP and triglyceride (TG) levels in Ang II-infused rats (Table 2) and Figure S2A-D).Nevertheless, the administration of HNK increased serum uric acid levels in a dose-dependent manner (Table 2) and Figure S2E).We did not observe any signs of liver, skeletal muscle or kidney toxicity associated with HNK treatment (Table 2).

| HNK inhibits cardiomyocyte hypertrophy in vivo and in vitro
Galactin-3 (Gal-3) has been implicated in fibrogenesis, inflammation, and myocardial remodelling.Previous studies have shown that repression of Gal-3 can reverse left ventricular dysfunction induced by isoproterenol by attenuating myocardial inflammation and fibrogenesis. 34Three pro-hypertrophy markers, namely natriuretic peptide A, natriuretic peptide B and myosin heavy chain beta, are commonly used to evaluate myocardial hypertrophy. 35In this study, we examined whether HNK could protect against Ang II-induced myocardial hypertrophy in rats by performing H&E staining, SR staining, and Gal-3 testing assays in vivo.The results showed that the Ang II-infused rats exhibited thicker endocardium and larger cross-sectional area of myocardial cells compared to the control rats (Figure 2A-C).Additionally, the SR staining showed increased and scattered red-stained fibres in the myocardial tissue of the rats with 4 weeks of Ang II infusion (Figure 2D,E).Furthermore, the Gal-3 levels were significantly increased in the Ang II-infused group (Figure 2F).After 4 weeks of Ang II infusion, HNK was found to attenuate cardiac hypertrophy, fibrosis and serum Gal-3 levels in a dose-dependent manner (Figure 2F).Staining of CD31 and IB4 showed a bit increase in arteries and significant increase in capillaries with HNK + Ang II group (Figure 2H,I), Figure S3A,B.Meanwhile, a TUNEL assay was performed to detect cardiomyocyte apoptosis.
The results showed that cardiomyocyte apoptosis can be attenuated after treating with HNK (Figure 2, Figure S3C).Furthermore, in vitro experiments demonstrated that HNK inhibited ANP protein levels in a concentration-dependent manner, Ang II and HNK concentration as indicated (Figure 2G).

| HNK alleviates cardiomyocyte hypertrophy via the LKB1/AMPK/p70S6K signalling pathway
In a previous study, we demonstrated that LKB1/AMPK/p70S6K signalling plays a pivotal role in the development of hypertensioninduced myocardial hypertrophy in rats. 36Through our in vivo experiments, we found that the myocardial levels of p-p70S6K and ANP were significantly higher in Ang II-infused rats than in the control group (Figure 3A-C), whereas the levels of LKB1, p-LKB1 and p-AMPK were markedly reduced in the Ang II-infused group (Figure 3D-F).Notably, HNK treatment dose-dependently reduced p-p70S6K expression (Figure 3A-C) and increased the expression of LKB1, p-LKB1 and p-AMPK (Figure 3D-F).However, the protein levels of p70S6K and AMPK were comparable among the four groups (Figure 3G,H), and HNK treatment alone did not alter the expression of these molecules in healthy control rats (Figure S4A-I).In and left ventricular posterior wall thickness (LVPWD) (n = 6-9 rats per group).(E-H) Echocardiography measurements of LV end-diastolic volume (EDV), LV end-systolic volume (ESV), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (n = 6-9 rats per group), at 4 weeks after Ang II infusion.(I) s , mean of peak velocity of the lateral wall and interval septum of the mitral annulus during systole.(J) ⅇ, mean of peak velocities in the lateral and interstitial walls of the mitral annulus during early diastole.(K) The PVE is the peak flow rate at the orifice of the mitral valve.ns indicates no statistical significance.*p < 0.05 and **p < 0.01, as assessed using one-way anova followed by Tukey's multiple comparison test.Data have been presented as mean ± S.E.M.

TA B L E 1
Physical and conventional echocardiographic parameters in SD rats and Ang II-infused rats treated with vehicle or HNK.

Note:
Values have been presented as mean ± SEM (n = 6-9 rats per group).
Abbreviations: e, mean of peak velocities in the lateral and interstitial walls of the mitral annulus during early diastole; s, mean of peak velocity of the lateral wall and the interval septum of the mitral annulus during systole; Ang

| HNK mediates the modulation of the LKB1/AMPK signalling pathway by Nur77
Numerous studies have highlighted the essential roles of Nur77 in animals, including myocardial hypertrophy, 29,37 fibrosi [39] and apoptosis. 38Consistent with previous findings, our in vivo experiment showed that infusion of Ang II for 4 weeks significantly increased Nur77 expression (Figure 5A).In vitro, we also observed that Nur77 expression was upregulated in NRVMs stimulated with Ang II for 1 h (Figure 5B).Moreover, upon exploring the time-effect relationship of Ang II intervention on Nur77 expression, we noted that Ang II

| HNK promotes dissociation of the Nur77-LKB1 complex to activate downstream signalling pathways
In the current study, we investigated the mechanism by which HNK counteracts Ang II-induced cardiomyocyte hypertrophy.In the nucleus, LKB1 binds to Nur77 to form a complex 16 ; TMPA promotes the dissociation of this complex, and thereby activates the downstream AMPK, to reduce lipid accumulation in HepG2 cells and mouse  primary hepatocytes. 39Our immunoprecipitation experiments confirmed that Nur77 can bind to LKB1 in the nucleus (Figure 6A).
Further experiments using immunofluorescence and nucleoplasmseparation experiments showed that HNK reduced the expression of Nur77, attenuated the binding of Nur77 to LKB1, and activated the AMPK signalling pathway in a concentration-dependent manner (Figure 6B,C,F) and Figure S8).In NRVMs, Ang II intervention increased the aggregation of Nur77 and LKB1 in the nucleus, while downregulating their expression in the cytoplasm, which led to more LKB1 sequestration in the nucleus and attenuated the downstream AMPK signalling cascade response.We have confirmed that HNK could block the formation of the Nur77-LKB1 complex and promote nuclear export of LKB1 to the cytoplasm and TMPA also have this effect, so we wanted to know if there was a synergistic effect between the two drugs.From the results, it is clear that there is no synergy between the two drugs (Figure 6C-E).Our findings suggest that HNK counteracts Ang II-induced cardiomyocyte hypertrophy by promoting the dissociation of the Nur77-LKB1 complex.

| HNK inhibits Nur77 transcription and promotes ubiquitous degradation of Nur77 protein
The present study investigated the effects of HNK on Nur77 protein levels and AMPK signalling.The observed results revealed that HNK treatment alone has no effect on Nur77 expression but only reverses Ang II effect on Nur77.Therefore, we next checked Nur77 transcription and degradation after an HNK alone treatment to confirm that HNK inhibits transcription of Nur77 and promotes its ubiquitous degradation.Real-time PCR experiments revealed that Ang II upregulated Nur77 mRNA levels while downregulating LKB1 mRNA levels in NRVMs.Notably, HNK administration significantly reversed the effects of Ang II on Nur77 and LKB1 mRNA levels (Figure 7A).

| DISCUSS ION
Although several recent studies have demonstrated the beneficial effect of honokiol on cardiac hypertrophy, our study evaluates a new molecular mechanism by which honokiol mediates cardio-protection.The study uncovers a novel molecular mechanism downstream of honokiol action.Pathological myocardial hypertrophy is known to contribute to the pathogenesis of heart failure, a condition that has become increasingly prevalent and lethal worldwide. 40Recently, studies have highlighted the critical role of the LKB1/AMPK signalling pathway in the development of pathological myocardial hypertrophy. 41,42Our previous research has demonstrated that retinoid X receptor agonists can alleviate cardiomyopathy in streptozotocininduced type-1 diabetes by exerting LKB1-dependent anti-fibrotic effects. 43This, once again, reinforces the essential role of the LKB1-AMPK axis in the pathological remodelling of the heart.
In order to investigate the mechanism by which HNK ameliorates pathological myocardial remodelling and cardiac dysfunction, we assessed the impact of HNK intervention on the LKB1-AMPK pathway both in vitro and in vivo.Our findings revealed that in Ang II-induced hypertrophic hearts and NRVMs, the levels of p-LKB1 and p-AMPK were diminished, whereas those of p-p70S6K and ANP were significantly elevated.However, HNK significantly attenuated the effects of Ang II on p-LKB1, p-AMPK and p-p70S6K, resulting in reduced collagen deposition in hypertrophic hearts.These results demonstrate that the modulation of the LKB1-AMPK-p70S6K pathway plays a pivotal role in HNK-mediated mitigation of Ang II-induced pathological myocardial remodelling.
Nur77, among the three NR4A receptors, is highly expressed in the adult mouse heart. 44Numerous stressors, including isoproterenol 44 and transverse aortic constriction, 45 have been shown to rapidly increase Nur77 protein expression in the myocardium.
Consistent with these findings, our in vivo and ex vivo experiments F I G U R E 4 HNK antagonizes the regulatory effects of Ang II on the LKB1/AMPK/p70S6K pathway in NRVMs cultured in vitro.(A-G) Representative immunoblots and quantification of the major proteins of the LKB1/AMPK/p70S6K signalling pathway in NRVMs subjected to treatment with HNK (10 −9 to 10 −7 mol⋅mL −1 ) for 30 min, followed by activation with Ang II (10 −7 mol⋅mL −1 ) for 1 h, to induce hypertrophy (n = 3).(H-K) Representative immunoblots and quantification of the major proteins of the LKB1/AMPK/p70S6K signalling pathway in NRVMs subjected to treatment with HNK (10 −8 mol⋅mL −1 ) and Dorsomorphin (10 −5 mol⋅mL −1 ) for 30 min, followed by activation with Ang II (10 −7 mol⋅mL −1 ) for 1 h, to induce hypertrophy (n = 3).ns indicates no statistical significance.*p < 0.05 and **p < 0.01, as assessed using a oneway analysis of variance followed by Tukey's multiple comparison test.Data have been shown as mean ± S.E.M.
F I G U R E 5 HNK mediates modulation of the LKB1/AMPK signalling pathway by Nur77.(A) Nur77 protein levels in the myocardial tissues from CTL and cardiac hypertrophy rats were examined by means of immunoblotting, following which the results were quantified (n = 6 rats per group).(B) Representative immunoblots and quantification of Nur77 protein levels in NRVMs treated as described in Figure 4.
(C-H) NRVMs were treated with HNK for 30 min, following which they were transfected with Flag-His-tagged Nur77 plasmid using Lipofectamine™ 3000.HNK, honokiol; Ang II, angiotensin II; ns, no statistical significance.**p < 0.01, as assessed using one-way anova followed by Tukey's multiple comparison test.Data have been shown as mean ± S.E.M.
demonstrated that Ang II intervention significantly upregulated the protein levels of Nur77 in the heart.Furthermore, previous study revealed that Nur77 in cardiac fibroblasts promotes the transition of fibroblast-to-myofibroblast induced by isoproterenol and transforming growth factor (TGF)-β, while in myocytes, Nur77 inhibits the ability of myocytes to induce paracrine TGF-β-mediated transition of fibroblast-to-myofibroblast, thereby maintaining the balance of cardiac fibrotic response [39] .These results highlight the important role of Nur77 in modulating heart function and remodelling in various cardiac pathologies.
The Nur77-LKB1 interaction has been shown to play a role in various physiological and pathological processes.Previous studies have demonstrated that Nur77 specifically binds to LKB1 in the nucleus, which represses the nuclear export of LKB1 and its subsequent phosphorylation. 17 Recent studies have identified TMPA as a Nur77 antagonist that inhibits the binding of Nur77 to LKB1 and promotes the nuclear export of LKB1 to the cytoplasm and its subsequent phosphorylation, which stimulates downstream molecules associated with lipid metabolism and prevents fatty acid aggregation. 39In this study, we demonstrated that Nur77 interacts with LKB1 in the nucleus and that overexpression of Nur77 enhances its binding to LKB1.Our findings indicate that HNK blocks the formation of the Nur77-LKB1 complex, promotes the nuclear export of LKB1 to the cytoplasm and enhances its phosphorylation.This, in turn, promotes the phosphorylation of AMPKα and suppresses the downstream p70S6K.Our data suggest that promoting the dissociation of the Nur77-LKB1 complex in the nucleus may be a vital mechanism by which HNK ameliorates Ang II-induced myocardial hypertrophy.
As previously mentioned, the interaction between Nur77 and LKB1 in the nucleus of cardiomyocytes, as well as downstream AMPK and p70S6K activities, are directly influenced by the level

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guidelines and regulations set forth by the US National Institutes of Health's 'Guide for the Care and Use of Laboratory Animals' and approved by the Animal Welfare and Policy Committee of Fujian Medical University (approval number: 2019-0085).The experiments were also compliant with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.Eight-week-old male Sprague-Dawley rats (180 ± 20 g) were procured from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences and kept in a specific pathogen-free environment.The animals were randomly allocated into experimental groups using the random number generator function of SPSS version 24.All rats were euthanized by carbon dioxide.Investigators who were blinded to the treatment groups performed the statistical analysis and evaluated the results of all rats.All animal experimentation was conducted at the Central Laboratory of the First Affiliated Hospital of Fujian Medical University.et al.
ing cages and provided individualized feed till the completion of the experiment.The animals were assigned randomly to one of the five experimental groups, including control (CTL), control group treated with HNK (2.5 mg•kg −1 •d −1 ; CTL + HNK), Ang II-treated group (520 ng•kg −1 •min −1 ; Ang II), Ang II plus low-dose HNK-treated group (2.5 mg•kg −1 •d −1 ; Ang II + HNK 2.5) and Ang II plus highdose HNK-treated group (5 mg•kg −1 •d −1 ; Ang II + HNK 5).HNK was dissolved in peanut oil and administered intraperitoneally twice daily.The high dose of HNK (5 mg•kg −1 •d −1 ) was determined based on the literature frequencies ranging from 4 to 12 MHz was then performed.The M-mode echocardiographic images of the parasternal left ventricle in the short-axis view were recorded.Pulsed-wave Doppler blood flow images of the apical four-chamber view at the mitral level and Doppler tissue images (DTI) of the lateral and septal mitral annulus were also captured.Heart rates were synchronously measured using electrocardiography.Image quantification and analysis were done using the EchoPAC 202 Image Analysis System by GE Healthcare.From the parasternal left ventricular long-axis view, the diastolic inner diameter of the aortic sinus and systolic anterior and posterior diameters of the left atrium were calculated.Using M-mode echocardiography in the short-axis view, the end-diastolic left ventricular (LV) posterior wall thickness (LVPWD), end-diastolic interventricular septum thickness (IVSD) as well as end-systolic and end-diastolic LV internal diameters (LVIDS and LVIDD, respectively) were measured at the LV papillary muscle level.The LV fractional shortening (LVFS) and LV ejection fraction (LVEF) were calculated using equations LVFS = [(LVIDD-LVIDS)/LVIDD] × 100 and LVEF = [(LV end-diastolic volume − LV end-systolic volume)/LV end-diastolic volume] × 100, respectively.

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et al. addition, we observed higher levels of p-p70S6K and lower levels of LKB1, p-LKB1 and p-AMPK in NRVMs after 1 h of Ang II treatment compared to those in the control group (Figure 4A).HNK treatment concentration-dependently reduced p-p70S6K levels (Figure 4A,B) and increased LKB1, p-LKB1 and p-AMPK expression in NRVMs (Figure 4C-E), while the levels of p70S6K and AMPK proteins remained unchanged (Figure 4F,G).Meanwhile, to demonstrate the important role played by AMPK signalling in cardiomyocyte hypertrophy, we performed AMPK inhibition experiments in NRVMs, which showed a significant increase in ANP expression and significant hypertrophy of cardiomyocytes in the presence of Dorsomorphin, an AMPK inhibitor (Figure 4H-K).Nur77 knockdown was performed in order to proove that the effects on cardiomyocyte hypertrophy observed upon Ang II and HNK administration are really dependent on Nur77.The results indicated that the AMPK signalling pathway was activated after Nur77 knockdown (Figure S5A-G).Overall, our findings suggest that the LKB1/AMPK/p70S6K signalling pathway is implicated in the development of Ang II-induced myocardial hypertrophy, and that HNK can attenuate cardiomyocyte hypertrophy by modulating this pathway.F I G U R E 1 HNK ameliorates Ang II-induced myocardial hypertrophic remodelling and diastolic dysfunction.(A) Representative M-model echocardiograms and Doppler recordings at 4 weeks after Ang II infusion.(B-D) Quantitative analysis of IVSD, relative wall thickness (RWT) induced a time-dependent increase in Nur77 protein level, with the peak at 1 h of intervention (FigureS6A,B).Overexpression of Nur77 in 293T cells inhibited the LKB1/AMPK signalling, but treatment with HNK reversed this result (FigureS7A-G).Meanwhile, overexpression of Nur77 in NRVMs by adenoviral transfection showed that overexpression of Nur77 significantly inhibited the LKB1/AMPK signalling pathway, and that this inhibition could be reversed by HNK intervention (Figure5C-H).Therefore, Nur77 is an upstream molecule of the LKB1/AMPK signalling pathway that may play a crucial role in modulating the LKB1/AMPK pathway by HNK.

F I G U R E 2
HNK inhibits cardiomyocyte hypertrophy in vivo and in vitro.(A) Representative LV sections stained with haematoxylin and eosin (H&E).Scale bar: 2000 μm.(B-E) Representative LV sections stained with H&E and Sirius red (SR) to evaluate cardiomyocyte hypertrophy (B) and fibrosis (D), respectively.Scale bar: 50 or 200 μm.(C) Quantification of the cardiomyocyte cross-sectional area in the H&E-stained sections.(E) Quantification of collagen in the SR-stained cardiomyocytes.(F) Levels of galectine-3 (Gal-3) at the end of the study, as measured using ELISA assay.ns indicates no statistical significance.(G) Representative immunoblots and quantification of ANP protein upon treatment of NRVMs with Ang II (10 −7 mol⋅L −1 ) for 24 h (n = 3).(H,I) Representative confocal IF images of CD31 and IB4 obtained from capillaries and arteries in LV and the quantification of the positive area of ORI.(J) Representative LV sections stained with TUNEL.DAPI staining denotes nuclei; myocardial apoptosis index measured as the positive cell divided by total cell.Data have been shown as mean ± S.E.M. n = 6-9 rats per group; **p < 0.01, as assessed using one-way anova followed by Tukey's multiple comparison test.CTL, control; AII, angiotensin II; HNK, honokiol.F I G U R E 3 HNK alleviates cardiac hypertrophy via the LKB1/AMPK/p70S6K signalling pathway in AII-infused rats.(A) Major proteins of the LKB1/AMPK/p70S6K signalling pathway were examined in myocardial tissues from CTL and cardiac hypertrophy rats, by means of immunoblotting, the results for which are quantified in (B-G) (n = 6 rats per group).ns indicates no statistical significance.Data have been shown as mean ± S.E.M. *p < 0.05 and **p < 0.01, as assessed using one-way anova followed by Tukey's multiple comparison test.CTL, control; AII, angiotensin II; HNK, honokiol.

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Conversely, repression of Nur77 binding with LKB1 may have a positive effect on hepatic steatosis.HNK promotes dissociation of the Nur77-LKB1 complex to activate downstream signalling pathways.(A) Coimmunoprecipitation results for Nur77 and LKB1.Flag-His-tagged Nur77 plasmids were transfected to HEK293T cells incubated with or without HNK (10 −8 mol L −1 ).The precipitate was analysed using a western blot, with anti-Nur77 and anti-LKB1 antibodies.(B) Subcellular co-localization of Nur77 and LKB1.Immunofluorescence in the treated NRVMs was examined using confocal fluorescence microscopy.Scale bar: 10 μm (n = 3).(C-F) NRVMs were pre-treated with HNK (10 −9 -10 −8 mol⋅mL −1 ) and TMPA(10 −5 mol L −1 ) for 30 min and then stimulated with Ang II (10 −7 mol⋅mL −1 ) for 1 h, following which the expression levels of Nur77, total and phosphorylated LKB1 and AMPK were analysed in the cytosolic and nuclear proteins.Representative blots and quantification data are shown.HNK, honokiol; Ang II, angiotensin II.ns indicates no statistical significance.*p < 0.05 and **p < 0.01, as assessed using one-way anova followed by Tukey's multiple comparison test.Data have been presented as mean ± S.E.M. et al.