Herein, for the first time, we show that NOT directly inhibits cardiomyocyte hypertrophy by reducing the activity of the GSK3 signal pathway. The MI sudden onset and its exceedingly limited therapeutic window make accurate drug administration challenging. Time-insensitive strategy is urgently needed to develop against ischemic heart injury[1]. We tried multiple dosing times to explore the action of NOT. Interestingly, NOT has a cardioprotective effect on HF development and progression as well as targets numerous HF pathogeneses, such as cardiac remodeling, hypertrophy, fibrosis, as well as inflammatory mechanisms, when administered either early (0.5 h p-MI) or delayed (12 h p-MI) or therapeutically (post-HF). Our data supported NOT with a broad therapeutic window after acute MI, and the longest effective time for the first dose was 12 h after MI in mice. The duration of administration was suggested at 28 days, although it works at 14 days with a slightly weaker effect. In vitro, NOT presented an anti-hypertrophy property in cultured HL1 cells subjected to AngⅡ-stimulation. It can regulate the activity of GSK3β (inhibitory phosphorylation), which is a known combing target of NOT and a negative regulator of HF. Pharmacological inhibition of GSK3β abolished the anti-hypertrophy action NOT. The findings confirm that the beneficial action of NOT against myocardial hypertrophy is mediated by GSK3β. Herein, regarding toxicity, no significant changes were found in the NOT-treated mice body or organ weight, except for the injured heart, indicating that there was no significant toxicity resulting from NOT treatment.
NOT, a furanocoumarin-type is an active compound extracted from N. incisum; the latter had an extensive spectrum impact on biomedical pharmacology[21]. NOT exhibited anti-arthritis, anti-cancer, and anti-dementia activities, respectively, by inhibiting the JAK-STAT pathway and reducing inflammatory reaction; by suppressing proliferation and promoting apoptosis in acute human leukemia HL-60 and HEPG-2 cell lines; and by suppressing BACE1 and GSK3β activity, then decreasing Aβ and phosphorylated tau in APP/PS1 AD mice[18–23]. Focus on heart disease; there is only one paper about PAH[23]. In MCT-induced PAH rats, NOT improved mortality rate and right ventricular function, parallelled with decreased right ventricular hypertrophy and fibrosis, eased pulmonary vascular remodeling and attenuated inflammation of the lung. In vitro, the human pulmonary arterial smooth muscle cells (HPASMCs) proliferation and migration were inhibited by NOT. To our knowledge, pulmonary hypertension leads to right ventricle overload and eventually progresses to right ventricular failure. In that study, MCT induced PAH model did not affect left ventricular ejection fraction (LVEF). Comparably, LVEF showed no significant effect after NOT administration. HF can be triggered by several risk factors, and HF developed from MI still accounts for the largest proportion[27]. Therefore, our study first focuses on AMI-induced HF. Using LAD-ligation for four weeks, chronic HF was induced, and it has similar features to human disorders in hemodynamic changes, vascular remodeling, and HF [3]. Similarly to an earlier study, we found the beneficial NOT effect on HF development and progression by alleviating heart dysfunction and inhibiting cardiac remodeling, hypertrophy, fibrosis, and inflammation. Our study enriched efficacy data of pharmacology. NOT (100 mg/kg, not 10 and 30 mg/kg) exhibited cardioprotection, not only by early (0.5 h-pMI) and delayed pretreatment (12 h-pMI) but also by treatment after HF. The dose-effect and time-effect relationship were exhibited. In addition, GSK3β is recognized as a key target of NOT and is involved in the anti-hypertrophy action, supported by intervention studies. It should be noted that, in the same as in intragastric administration, our effective dose of NOT is higher than that used in previous studies. In murine RA mice, the therapeutic schedule of NOT is 20 mg/kg for 22 days. In APP/PS1 mice, NOT works at 40 mg/kg, not 20 mg/kg, for two months. In PAH rats, a dosage of 20 mg/kg for 21 days was administered. Our cardioprotective dose of NOT is 100 mg/kg daily for 28 days in HF mice, with 30 mg/kg tending to affect heart function. Moreover, the beneficial effect is already observed after NOT-treated 14 days, in a time-dependent manner, which is slightly weaker than that after 28 days. Our broad dose setting to cover the range of effective doses may partially explain the difference, which weakens the ability to explore the minimum effective dose.
To explicit the targets of NOT is very important to evaluate the feasibility of patent medicine and also pave the way for developing more specific agents. Previous studies have shown that NOT, as a small natural molecule, can inhibit BACE1, GSK3β, and JAK2[19–22]. After recognizing NOT as BACE1 and GSK3β dual inhibitor, researchers designate a series of triple inhibitors to lead compounds on the basis of NOT furocoumarin scaffold, aimed to suppress the three AD classical targets (AChE/BACE1/GSK3β), which is expected to obtain ideal anti-cognitive impairment activity[27]. In the RA model and in zebrafish, direct NOT binding to JAK2 and JAK3 is necessary for its anti-inflammatory effects, with 931/arginine, 980/asparagine, and 981/leucine of JAK2 are essential binding sites[22]. In our study, NOT regulates the activation of GSK3β, in agreement with previous studies. Intervention with GSK3β inhibitors abolished NOT hypertrophy in the myocardium, suggesting that it is a critical target of NOT in cardioprotection. According to the literature, NOT showed a strong inhibitory effect against GSK3β (IC50: 1 µM)[21]. Their binding mode was predicted by virtual screening in silico, and NOT bound in kinase GSK3β hinge region and establishes two firmly hydrogen-bonding interactions with Asp133 and Tyr134 backbones. BCAE1 and JAK2 target functions were not studied in our study, which cannot be ruled out and require further studies.
GSK-3β is consistently active in resting cells, and it is crucial to maintain normal cardiac homeostasis that governs cardiac development, proliferation, hypertrophy, and fibrosis [28, 29]; it is found to be suppressed in hypertrophy and HF and serves as a negative regulator. Activated GSK-3β caused heart injury. GSK-3β pharmacological inhibition or knockdown is beneficial in several cardiovascular pathologies[7–16]. GSK-3β intervention results in increased cardiomyocyte proliferation in cultured cardiomyocytes through overexpression growth regulators (GATA4, cyclin D1, and c-Myc) and canonical Wnt signaling[30, 31]. It also promotes angiogenesis, prevents adverse cardiac remodeling by inhibition of Akt-GSK-3β-mTOR pathways, and triggers GSK-3β-β-catenin[14, 32, 33], downregulates mPTP opening, and induces antiapoptotic (decreased Bax/Bcl-2 ratio and the apoptotic cascade)[9–13]. GSK-3β phosphorylates a wide range of substrates, voltage-dependent anionic channel 1 (VDAC1) and E2-related factor (Nrf2) are also reported as downstream substrates [15, 16]. Nevertheless, other studies show contradicting results. GSK-3β inhibition conferred physiological hypertrophy induction[7]. In GSK-3β KO mice subjected to transverse aortic constriction surgery, no significant differences in hypertrophy were observed compared to WT mice[17]. We cannot explain this inconsistency in data, yet, we supposed that it might be because of protein phosphorylation at a different site that defines the protein activity. GSK-3β residues have the ability to modulate their substrate binding potential[28, 29]. GSK-3β phosphorylation at tyrosine 216 elevates GSK-3β enzymatic activity. In contrast, GSK-3β phosphorylation at serine 9 inactivates GSK-3β by blocking its active site [29]. These phosphorylation and dephosphorylation switches of GSK-3β can affect several molecular pathways. The GSK-3β phosphorylation inhibition at serine 9 was well demonstrated.
Divergent GSK-3β inhibitor cardioprotective effect, including SB216763, copper nanoparticles (NPs), and resveratrol, was reported[7, 8]. However, in human heart clinical trials, there were no GSK3β inhibitors; only limited inhibitors were utilized for conducting distinct phases of clinical trials for the diseased conditions, including neurodegenerative disorders, myotonic dystrophy, and several cancers. In the interest of fighting myocardial injury with cutting-edge therapeutic approaches, researchers should consider developing GSK3β inhibitors. Now we recognize NOT as a potential agent. A diverse-acting substrate of GSK3β may confer NOT to extensively participate in different pathogenesis of HF. Notably, NOT is a natural compound, which means it is safer than synthetic chemicals and more affordable to manufacture. Moreover, It can be easily extracted from N. incisum.
Our research attained crucial novel insights, but it also had some drawbacks. First, JAK2 and JAK3 are possible target pathways of NOT, but we did not identify the specific target molecule. Additional research is required to explore their role. Second, our effective dosage in vivo is greater than that in previous research. The dose setting of NOT in mice is broad, which weakens the capability to explore minimum effective dose. Although the action of NOT has been confirmed, the minimum effective dose is still obscure. A more refined dose setting is needed to clear NOT pharmacological activity.