The inhibition of FTO attenuates the antifibrotic effect of leonurine in rat cardiac fibroblasts.

BACKGROUND: Myocardial fibrosis (MF) is a common pathological condition in cardiovascular diseases that often causes severe cardiac dysfunction. MF is characterized by changes in cardiomyocytes, cardiac fibroblasts (CFs), levels of collagen (Col) -1, -3, and overdeposition of the extracellular matrix. Our previous research showed that leonurine (LE) effectively inhibits collagen synthesis and differentiation of CFs, but the mechanism is not fully elucidated. Recent evidence indicates that fat mass and obesity-associated proteins (FTO) regulates the occurrence and development of MF. This study aimed to explore the role of FTO in the antifibrotic effects of LE. METHODS: Neonatal rat CFs were isolated, and induced using angiotensin II (Ang II) to establish a cell model of MF. Cell viability, wound healing and transwell assays were used to detect cell activity and migration ability. The protein and mRNA levels of MF-related factors were measured following stimulation with Ang II and LE under normal conditions or after FTO knockdown. The RNA methylation level was measured by dot blot assay. RESULTS: The results showed that LE (20, 40 µM) was not toxic to normal CFs. LE reduced the proliferation, migration and collagen synthesis of Ang II-induced CFs. Further investigation showed that FTO was downregulated by Ang II stimulation, whereas LE reversed this effect. FTO knockdown facilitated the migration of CFs, upregulated the protein levels of Col-3, α-SMA and Col-1 in Ang II and LE-stimulated CFs, and enhanced the fluorescence intensity of α-SMA. Furthermore, LE reduced N6-methyladenosine (m6A) RNA methylation, which was partially blocked by FTO knockdown. FTO knockdown also reduced the expression levels of p53 protein in Ang II and LE-stimulated CFs. CONCLUSIONS: Our findings suggest that the inhibition of FTO may attenuate the antifibrotic effect of LE in CFs, suggesting that FTO may serve as a key protein for anti-MF of LE.

The p53/miR-29a-3p axis mediates the antifibrotic effect of leonurine on angiotensin II-stimulated rat cardiac fibroblasts.

Overactivation of cardiac fibroblasts (CFs) is one of the main causes of myocardial fibrosis (MF), and inhibition of CF activation is a crucial strategy for MF therapy. A previous study by our group demonstrated that leonurine (LE) effectively inhibits collagen synthesis and myofibroblast generation originated from CFs, and eventually mitigates the progression of MF (where miR-29a-3p is likely to be a vital mediator). However, the underlying mechanisms involved in this process remain unknown. Thus, the present study aimed to investigate the precise role of miR-29a-3p in LE-treated CFs, and to elucidate the pharmacological effects of LE on MF. Neonatal rat CFs were isolated and stimulated by angiotensin II (Ang II) to mimic the pathological process of MF in vitro. The results show that LE distinctly inhibits collagen synthesis, as well as the proliferation, differentiation and migration of CFs, all of which could be induced by Ang II. In addition, LE promotes apoptosis in CFs under Ang II stimulation. During this process, the down-regulated expressions of miR-29a-3p and p53 are partly restored by LE. Either knockdown of miR-29a-3p or inhibition of p53 by PFT-α (a p53 inhibitor) blocks the antifibrotic effect of LE. Notably, PFT-α suppresses miR-29a-3p levels in CFs under both normal and Ang II-treated conditions. Furthermore, ChIP analysis confirmed that p53 is bound to the promoter region of miR-29a-3p, and directly regulates its expression. Overall, our study demonstrates that LE upregulates p53 and miR-29a-3p expression, and subsequently inhibits CF overactivation, suggesting that the p53/miR-29a-3p axis may play a crucial role in mediating the antifibrotic effect of LE against MF.

Effects of Leonurine on oocyte maturation and parthenogenetic embryo development in sheep.

Leonurine (LEO), an alkaloid isolated from Leonurus spp., has anti-oxidant, anti-inflammatory and anti-apoptotic effects and can prevent damage caused by reactive oxygen species (ROS). These properties suggest that it can improve the maturation rate of oocytes and developmental ability of embryos, which are key parameters in animal breeding. In this study, the effects of LEO on in vitro maturation and early embryonic development in sheep oocytes were evaluated. Among various doses examined (0, 10, 20 and 40 µM), a dose of 20 µM was optimal with respect to the oocyte maturation rate. Compared with estimates in the control group, GSH levels and mitochondrial membrane potential of sheep oocytes treated with 20 µM LEO were significantly higher, and 40 µM LEO would affect oocyte maturation. Additionally, ROS levels were significantly lower, expression levels of the antioxidant genes CAT and SOD1 were significantly higher, and there was no significant difference in GPX3 expression. The Bax/Bcl-2 ratio and Caspase-3 expression were significantly reduced in the 20 µM LEO group. During early embryonic development in vitro, the cleavage rate and blastocyst rate were significantly higher in the 20 µM LEO treatment group compared to other groups. GSH levels and mitochondrial membrane potential were significantly higher, while ROS levels were significantly lower, and expression levels of the antioxidant genes CAT, GPX3 and SOD1 were significantly higher in eight-cell embryos treated with 20 µM LEO than in the control group. The Bax/Bcl-2 ratio and Caspase-3 levels were significantly decreased. In summary, LEO can reduce the effect of oxidative stress, improve the oocyte maturation rate and enhance embryonic development.

Leonurine Inhibits Hepatic Lipid Synthesis to Ameliorate NAFLD via the ADRA1a/AMPK/SCD1 Axis.

Leonurine is a natural product unique to the Lamiaceae plant Leonurus japonicus Houtt., and it has attracted attention due to its anti-oxidative stress, anti-apoptosis, anti-fibrosis, and metabolic regulation properties. Also, it plays an important role in the prevention and treatment of nonalcoholic fatty liver disease (NAFLD) through a variety of biological mechanisms, but its mechanism of action remains to be elucidated. Therefore, this study aims to preliminarily explore the mechanisms of action of leonurine in NAFLD. Mice were randomly divided into four groups: the normal control (NC) group, the Model (M) group, the leonurine treatment (LH) group, and the fenofibrate treatment (FB) group. The NAFLD model was induced by a high-fat high-sugar diet (HFHSD) for 12 weeks, and liver pathological changes and biochemical indices were observed after 12 weeks. Transcriptomic analysis results indicated that leonurine intervention reversed the high-fat high-sugar diet-induced changes in lipid metabolism-related genes such as stearoyl-CoA desaturase 1 (Scd1), Spermine Synthase (Sms), AP-1 Transcription Factor Subunit (Fos), Oxysterol Binding Protein Like 5 (Osbpl5), and FK506 binding protein 5 (Fkbp5) in liver tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results suggest that leonurine may exert its lipid-lowering effects through the AMP-activated protein kinase (AMPK) signaling pathway. Liver lipidomic analysis showed that leonurine could alter the abundance of lipid molecules related to fatty acyl (FAs) and glycerophospholipids (GPs) such as TxB3, carnitine C12-OH, carnitine C18:1-OH, and LPC (20:3/0:0). Molecular biology experiments and molecular docking techniques verified that leonurine might improve hepatic lipid metabolism through the alpha-1A adrenergic receptor (ADRA1a)/AMPK/SCD1 axis. In summary, the present study explored the mechanism by which leonurine ameliorated NAFLD by inhibiting hepatic lipid synthesis via the ADRA1a/AMPK/SCD1 axis.

Cell death regulation: A new way for natural products to treat osteoporosis.

Osteoporosis is a common metabolic bone disease that results from the imbalance of homeostasis within the bone. Intra-bone homeostasis is dependent on a precise dynamic balance between bone resorption by osteoclasts and bone formation by mesenchymal lineage osteoblasts, which comprises a series of complex and highly standardized steps. Programmed cell death (PCD) (e.g., apoptosis, autophagy, ferroptosis, pyroptosis, and necroptosis) is a cell death process that involves a cascade of gene expression events with tight structures. These events play a certain role in regulating bone metabolism by determining the fate of bone cells. Moreover, existing research has suggested that natural products derived from a wide variety of dietary components and medicinal plants modulate the PCDs based on different mechanisms, which show great potential for the prevention and treatment of osteoporosis, thus revealing the emergence of more acceptable complementary and alternative drugs with lower costs, fewer side effects and more long-term application. Accordingly, this review summarizes the common types of PCDs in the field of osteoporosis. Moreover, from the perspective of targeting PCDs, this review also discussed the roles of currently reported natural products in the treatment of osteoporosis and the involved mechanisms. Based on this, this review provides more insights into new molecular mechanisms of osteoporosis and provides a reference for developing more natural anti-osteoporosis drugs in the future.

[Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway].

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 µmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 µmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 µmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 µmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 µmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 µmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.

Leonurine inhibits the TXNIP/NLRP3 and NF-κB pathways via Nrf2 activation to alleviate carrageenan-induced pleurisy in mice.

Oxidative stress and inflammation play important roles in pleurisy. Leonurine (Leo) has been confirmed to exert antioxidative and antiinflammatory effects in many preclinical experiments, but these effects have not been studied in pleurisy. The aim of this study was to explore the therapeutic effect and mechanism of Leo in a carrageenan (CAR)-induced pleurisy model. In this study, we found that the increase of reactive oxygen species (ROS), myeloperoxidase (MPO), and malondialdehyde (MDA) and decrease of glutathione (GSH) induced by CAR could be reversed by the treatment of Leo. Leo effectively reduced the levels of proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and the percentages of mature macrophages and increased the levels of antiinflammatory cytokines (IL-10). Furthermore, Western blotting revealed that Leo significantly activated the Nrf2 pathway to restrain the thioredoxin-interacting protein/NOD-like receptor protein 3 (TXNIP/NLRP3) and nuclear factor kappa-B (NF-κB) pathways. However, the protective effect of Leo was significantly weakened in Nrf2-deficient mice. These results indicate that Leo confers potent protection against CAR-induced pleurisy by inhibiting the TXNIP/NLRP3 and NF-κB pathways dependent on Nrf2, which may serve as a promising agent for attenuating pleurisy.

Leonurine Preconditioning Attenuates Ischemic Acute Kidney Injury in Rats by Promoting Nrf2 Nuclear Translocation and Suppressing TLR4/NF-κB Pathway.

Despite the precise mechanisms for renal ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) are poorly understood, nuclear factor erythroid 2 related factor 2 (Nrf2) and Toll-like receptor 4 (TLR4) pathways were considered as the important targets. Leonurine (LEO) is a special alkaloid extracted from Chinese motherwort (Leonurus japonicus Houtt), which has an anti-inflammatory effect and reduces oxidative stress. We conducted the study to explore the efficacy of LEO against I/R-induced AKI in rats and further investigated the underlying mechanisms. Ischemic renal injury was induced by temporary vascular clamping for 45 min. We have measured the levels of inflammation-related biomarkers and antioxidative stress markers. Next, Western blot analysis and Real-time PCR were performed to analyze whether the Nrf2 and TLR4/nuclear factor-kappaB (NF-κB) pathways were involved in this process. We found that LEO pretreatment remarkably decreased serum creatinine and blood urea nitrogen (BUN) in I/R rats and attenuated acute tubular damage. In addition, LEO markedly increased the expression of antioxidant proteins and decreased the levels of inflammatory factors. Further study revealed that LEO promoted Nrf2 into the nucleus, promoted the expression of heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1), and suppressed the TLR4/NF-κB signal pathway in kidney tissues of ischemic AKI rats. The study reveals that LEO has a protective effect to prevent ischemic AKI through activation of Nrf2 nuclear translocation resisting oxidative stress injury and inhibition of the TLR4/NF-κB pathway mediated inflammatory gene expression.

Leonurine suppresses prostate cancer growth in vitro and in vivo by regulating miR-18a-5p/SLC40A1 axis.

Prostate cancer is a leading cause of cancer-associated death in males. Leonurine (Leo) is a pleiotropic anti-tumor agent isolated from traditional Chinese herb that was used in gynecologic treatments. However, its pharmacological effect against prostate cancer progression remains unclear. Here, we showed that Leo dose dependently inhibited prostate cancer cell proliferation, promoted cell apoptosis, and induced cell cycle arrest. Moreover, we noticed that miR-18a-5p was downregulated and the solute carrier family 40 member 1 (SLC40A1) is upregulated by Leo treatment. SLC40A1 knockdown by siRNA abrogated the inhibitory effect of Leo on prostate cancer progression. Notably, Leo also significantly inhibited prostate cancer progression in a subcutaneous xenograft tumor mouse model in vivo. This study further unveiled the mechanism by which Leo inhibited prostate cancer progression, which provides a promising potential for its future clinical application.

Leonurine Reduces Oxidative Stress and Provides Neuroprotection against Ischemic Injury via Modulating Oxidative and NO/NOS Pathway.

Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric oxide synthase (NOS) pathway. We firstly explored the effects of NO/NOS signaling on oxidative stress and apoptosis in in vivo and in vitro models of cerebral ischemia. Further, we evaluated the protective effects of Leo against oxygen and glucose deprivation (OGD)-induced oxidative stress and apoptosis in PC12 cells. We found that the rats showed anxiety-like behavior, and the morphology and number of neurons were changed in a model of photochemically induced cerebral ischemia. Both in vivo and in vitro results show that the activity of superoxide dismutase (SOD) and glutathione (GSH) contents were decreased after ischemia, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were increased, indicating that cerebral ischemia induced oxidative stress and neuronal damage. Moreover, the contents of NO, total NOS, constitutive NOS (cNOS) and inducible NOS (iNOS) were increased after ischemia in rat and PC12 cells. Treatment with L-nitroarginine methyl ester (L-NAME), a nonselective NOS inhibitor, could reverse the change in NO/NOS expression and abolish these detrimental effects of ischemia. Leo treatment decreased ROS and MDA levels and increased the activity of SOD and GSH contents in PC12 cells exposed to OGD. Furthermore, Leo reduced NO/NOS production and cell apoptosis, decreased Bax expression and increased Bcl-2 levels in OGD-treated PC12 cells. All the data suggest that Leo protected against oxidative stress and neuronal apoptosis in cerebral ischemia by inhibiting the NO/NOS system. Our findings indicate that Leo could be a potential agent for the intervention of ischemic stroke and highlighted the NO/NOS-mediated oxidative stress signaling.

[Effect and mechanism of leonurine on pressure overload-induced cardiac hypertrophy in rats].

To investigate the effects of leonurine(Leo) on abdominal aortic constriction(AAC)-induced cardiac hypertrophy in rats and its mechanism. A rat model of pressure overload-induced cardiac hypertrophy was established by AAC method. After 27-d intervention with high-dose(30 mg·kg~(-1)) and low-dose(15 mg·kg~(-1)) Leo or positive control drug losartan(5 mg·kg~(-1)), the cardiac function was evaluated by hemodynamic method, followed by the recording of left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVESP), as well as the maximum rate of increase and decrease in left ventricular pressure(±dp/dt_(max)). The degree of left ventricular hypertrophy was assessed based on heart weight index(HWI) and left ventricular mass index(LVWI). Myocardial tissue changes and the myocardial cell diameter(MD) were measured after hematoxylin-eosin(HE) staining. The contents of angiotensin Ⅱ(AngⅡ) and angiotensin Ⅱ type 1 receptor(AT1 R) in myocardial tissue were detected by ELISA. The level of Ca~(2+) in myocardial tissue was determined by colorimetry. The protein expression levels of phospholipase C(PLC), inositol triphosphate(IP3), AngⅡ, and AT1 R were assayed by Western blot. Real-time quantitative PCR(qRT-PCR) was employed to determine the mRNA expression levels of ß-myosin heavy chain(ß-MHC), atrial natriuretic factor(ANF), AngⅡ, and AT1 R. Compared with the model group, Leo decreased the LVSP, LVEDP, HWI, LVWI and MD values, but increased ±dp/dt_(max) of the left ventricle. Meanwhile, it improved the pathological morphology of myocardial tissue, reduced cardiac hypertrophy, edema, and inflammatory cell infiltration, decreased the protein expression levels of PLC, IP3, AngⅡ, AT1 R, as well as the mRNA expression levels of ß-MHC, ANF, AngⅡ, AT1 R, c-fos, and c-Myc in myocardial tissue. Leo inhibited AAC-induced cardiac hypertrophy possibly by influencing the RAS system.

Leonurine protects against dexamethasone-induced cytotoxicity in pancreatic ß-cells via PI3K/Akt signaling pathway.

Glucocorticoid excess induces pancreatic ß-cell apoptosis and insulin secretion impairment, which may lead to hyperglycemia and steroid diabetes. Leonurine is a natural alkaloid extracted from the Herba leonuri, which has been widely used in the treatment of obstetric and gynecological diseases. However, whether leonurine performs a protective role in pancreatic ß-cells remains unknown. In this study, we evaluated the effect of leonurine on dexamethasone -treated ß-cells. Our data showed that leonurine inhibited dexamethasone-induced INS-1 cell apoptosis and facilitated cell proliferation. Moreover, leonurine attenuated dexamethasone-impaired insulin secretion in mice islets. Leonurine ameliorated dexamethasone-induced dephosphorylation of Akt, Bad and GSK-3ß. Importantly, the protective role of leonurine on dexamethasone-induced cytotoxicity was blocked by LY294002 in INS-1 cells. Our findings revealed for the first time that leonurine could protect against dexamethasone-induced cytotoxicity in pancreatic ß-cells via PI3K/Akt signaling pathway, suggesting leonurine may be a promising therapeutic agent for steroid diabetes.

The Effect of Leonurine on Multiterritory Perforator Flap Survival in Rats.

BACKGROUND: Leonurine (Leo), a natural active compound of Leonurus cardiaca, has been shown to possess various biological activities. However, it is not known whether Leo promotes perforator flap survival. METHODS: In this study, a perforator flap was outlined in the rat dorsum. The rats that survived surgery were divided randomly to control and Leo groups (n = 36 per group). Flap viability, flap perfusion, and level of protein linked with oxidative stress, cell apoptosis, and angiogenesis were evaluated. RESULTS: Relative to control group, the Leo group showed significantly higher the flap survival percentage (70.5% versus 90.2%, P < 0.05) and blood perfusion (197.1 versus 286.3, P < 0.05). Leo also increased 1.8-fold mean vessel density and upregulated 2.1-fold vascular endothelial growth factor protein expression compared with the control group, both of which indicate increased angiogenesis. Moreover, it significantly inhibited apoptosis by lowering caspase-3 activity. Superoxide dismutase expression was remarkably elevated in Leo group compared with the control group (56.0 versus 43.2 U/mg/protein, P < 0.01), but malondialdehyde quantities were significantly lower in the Leo group compared with control group (41.9 versus 57.5 nmol/mg/protein, P < 0.05). CONCLUSIONS: Leo may serve as an effective drug for improving perforator flap survival in rats via antioxidant and antiapoptotic mechanisms and promotion of angiogenesis.

Leonurine suppresses neuroinflammation through promoting oligodendrocyte maturation.

Focal inflammation and remyelination failure are major hallmarks of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we found that leonurine, a bioactive alkaloid, alleviated EAE disease severity along with reduced central nervous system inflammation and myelin damage. During the pathogenesis of EAE, leonurine dramatically suppressed the recruitment of encephalitogenic T cells into the central nervous system, whereas did not impair periphery immune responses and microglia activation. Mechanistically, leonurine protected mice against demyelination along with enhanced remyelination through promoting the maturation of oligodendrocytes in both EAE and cuprizone-induced demyelination mouse models. Moreover, we identified that the expression of demethylase jumonji domain-containing protein D3 was significantly enhanced upon treatment of leonurine, which suppressed the trimethylation of histone H3 lysine-27 and enhanced oligodendrocyte maturation accordingly. Collectively, our study identified the therapeutic effect of leonurine on EAE model, which potentially represents a promising therapeutic strategy for multiple sclerosis, even other demyelination disorders.

Inhibition of PI3K/Akt/NF-κB signaling with leonurine for ameliorating the progression of osteoarthritis: In vitro and in vivo studies.

Osteoarthritis (OA) is characterized as the degeneration and destruction of articular cartilage. In recent decades, leonurine (LN), the main active component in medical and edible dual purpose plant Herba Leonuri, has been shown associated with potent anti-inflammatory effects in several diseases. In the current study, we examined the protective effects of LN in the inhibition of OA development as well as its underlying mechanism both in vitro and in vivo experiments. In vitro, interleukin-1 beta (IL-1ß) induced over-production of prostaglandin E2, nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6 and tumor necrosis factor alpha were all inhibited significantly by the pretreatment of LN at a dose-dependent manner (5, 10, and 20 µM). Moreover, the expression of thrombospondin motifs 5 (ADAMTS5) and metalloproteinase 13 (MMP13) was downregulated by LN. All these changes led to the IL-1ß induced degradation of extracellular matrix. Mechanistically, the LN suppressed IL-1ß induced activation of the PI3K/Akt/NF-κB signaling pathway cascades. Meanwhile, it was also demonstrated in our molecular docking studies that LN had strong binding abilities to PI3K. In addition, LN was observed exerting protective effects in a surgical induced model of OA. To sum up, this study indicated LN could be applied as a promising therapeutic agent in the treatment of OA.

Comparative pharmacokinetics study of leonurine and stachydrine in normal rats and rats with cold-stagnation and blood-stasis primary dysmenorrhoea after the administration of Leonurus japonicus houtt electuary.

Leonurus japonicus houtt, a well-known herb of traditional Chinese medicine, is widely used to treat gynaecological diseases. In this study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method for simultaneously quantifying leonurine and stachydrine, the two main bioactive components in Leonurus japonicus houtt, was developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile and separation by a Hewlett Packard XDB-C8 column (150 × 4.6 mm, id, 5 µm) equipped with a gradient elution system containing methanol-water and 0.1% formic acid at a flow-rate of 0.4 mL/min. Components were then detected by a mass spectrometer in positive electrospray ionization mode. This method showed good linearity, precision, accuracy, recovery, stability, and negligible matrix effects, which were within acceptable ranges. The method was successfully applied to compare the pharmacokinetics in normal rats and rats with cold-stagnation and blood-stasis primary dysmenorrhoea treated with Leonurus japonicus houtt electuary. The result showed significant differences (p < 0.05) in the pharmacokinetic parameters between the primary dysmenorrhoea and normal groups. This result implied that Leonurus japonicus houtt electuary remained longer and was absorbed slower in rats with primary dysmenorrhoea and exhibited higher bioavailability and peak concentration.

Inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate crystal-induced inflammation.

Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E synthase-1 (mPGES-1)-derived eicosanoids play an essential role in human inflammatory disorders. Here, we investigated whether inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate (MSU) crystal-induced inflammation. Virtual screening assay and in vitro enzyme inhibition assay showed that leonurine was a potential inhibitor of COX-2, mPGES-1 and 5-LOX. Compared with COX-2 inhibitor celecoxib, leonurine (30 mg/kg) significantly decreased ankle perimeter, gait score and neutrophil number in synovial fluid in MSU crystal-treated rats, accompanied with the decreased expression of COX-2, mPGES-1 and 5-LOX and production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the synovial fluid macrophages. In addition, leonurine decreased representative M1 marker (iNOS and CD86) expression, NLRP3 inflammasome activation and M1 cytokine (TNF-α and IL-1ß) production. In the in vitro cultured RAW264.7 and human monocyte-derived macrophages (MDMs), blockade of COX-2/mPGES-1 and 5-LOX by leonurine inhibited macrophage M1 polarization and NLRP3 inflammasome activation in response to MSU crystals, and thus down-regulated IL-1ß and TNF-α with STAT1 and NF-κB inactivation. Conversely, these effects were partially abolished by overexpression of COX-2, mPGES-1, 5-LOX or STAT1. Furthermore, leonurine prevented a positive feedback loop between COX-2/mPGES-1/5-LOX and IL-1ß/TNF-α in MSU crystal-induced inflammation. Together, simultaneous down-regulation of COX-2/mPGES-1 and 5-LOX by leonurine ameliorates MSU crystal-induced inflammation through decreasing IL-1ß and TNF-α production. Our study may provide novel multi-target agents toward the arachidonic acid (AA) network for gouty arthritis therapy.

Leonurine Prevents Atherosclerosis Via Promoting the Expression of ABCA1 and ABCG1 in a Pparγ/Lxrα Signaling Pathway-Dependent Manner.

BACKGROUND/AIMS: Previous studies have demonstrated that leonurine, a unique alkaloid compound of Herba leonuri, can exert anti-oxidative and anti-inflammatory effects on the development of atherosclerosis (AS). This study was designed to investigate the effects of leonurine on cholesterol efflux from THP-1 macrophage-derived foam cells and development of atherosclerotic lesions in apoE-/- mice, and further determine the potential mechanisms. METHODS: Human THP-1 cells were fully differentiated into foam cells by the pre-treatment with phorbol-12-myristate-13-acetate (PMA) and oxidized density lipoproteins (ox-LDL). After cells were incubated with various concentrations of leonurine, Oil Red O staining and high-performance liquid chromatography (HPLC) assays were utilized to detect cellular lipid accumulation and cholesterol content, respectively. Cellular cholesterol efflux was determined by liquid scintillation counting. The mRNA and protein levels of ATP-binding cassette transporter A1/G1 (ABCA1/G1), peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptor α (LXRα) in foam cells were assessed using real-time quantitative PCR (RT-qPCR) and western blot analyses, respectively. Plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) levels in apoE-/- mice were evaluated using enzymatic methods. The atherosclerotic lesion sizes and collagen contents in aortic roots were determined by Oil Red O and Masson's trichrome staining, respectively. RESULTS: Oil Red O staining and liquid scintillation counting assays showed that leonurine significantly inhibited lipid accumulation and promoted 3H-cholesterol efflux in human THP-1 macrophage-derived foam cells in a concentration-dependent manner. Besides, both the mRNA and protein levels of ABCA1/G1, PPARγ and LXRα were enhanced by leonurine, which were attenuated by LXRα siRNA or PPARγ siRNA transfection. Finally, leonurine improved plasma lipid profile, decreased atherosclerotic lesion sizes, increased collagen contents and amplified PPARγ, LXRα and ABCA1/G1 expressions in aortic roots of apoE-/- mice. CONCLUSIONS: Leonurine can promote cholesterol efflux and alleviate cellular lipid accumulation by magnifying the expression of ABCA1/G1 in a PPARγ/LXRα signaling pathway-dependent manner in human THP-1 macrophage-derived foam cells and abate atherogenesis in apoE-/- mice, which may offer a promising therapeutic intervention of leonurine in protecting against AS.

Leonurine attenuates fibroblast-like synoviocyte-mediated synovial inflammation and joint destruction in rheumatoid arthritis.

Objective: To explore the role of leonurine in the regulation of synovial inflammation and joint destruction inRA. Methods: Fibroblast-like synoviocytes were isolated from synovial tissue from RA patients. Pro-inflammatory cytokine and MMP expression was evaluated using real-time PCR and a cytometric bead array. Cell migration and invasion in vitro were measured using the Boyden chamber method and the scratch assay, respectively. Protein expression was measured by western blotting. Nuclear factor kappa B (NF-κB) nuclear translocation was detected by immunofluorescence. The in vivo effect of leonurine was evaluated in mice with CIA. Results: Leonurine treatment significantly decreased the production of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNFα) and MMPs (MMP-1 and MMP-3) and suppressed the migration and invasion of RA fibroblast-like synoviocytes. The molecular analysis revealed that leonurine impaired TNFα-induced NF-κB signalling by inhibiting the phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) and subsequently preventing the nuclear translocation of the NF-κB p65 subunit. Leonurine also inhibited the p38 and Jun N-terminal kinase mitogen-activated protein kinases signalling pathways without affecting ERK signalling. Intraperitoneal injection of leonurine reduced synovial inflammation, joint destruction and the serum IL-1ß, IL-6 and TNFα levels in mice with CIA. Conclusion: Our findings show that leonurine reduces synovial inflammation and joint destruction in RA through the NF-κB and mitogen-activated protein kinases pathways. Leonurine has potential as a therapeutic agent for RA.

Leonurine Exerts Antidepressant-Like Effects in the Chronic Mild Stress-Induced Depression Model in Mice by Inhibiting Neuroinflammation.

Background: Leonurine is an active alkaloid that is extracted from Traditional Chinese Medicine Herba leonuri. Emerging evidence indicates that leonurine produces neuroprotective effects in ischemic stroke, Parkinson's disease, and Alzheimer's disease. However, the effect of leonurine in neuropsychiatric disorders, especially in major depression, remains unknown. Methods: We used the chronic mild stress mouse model to explore the antidepressant effects of leonurine and the potential mechanisms. Behavioral tests including sucrose preference test, forced swimming test, and tail suspension test were taken to evaluate depression symptoms. Moreover, the contents of monoamine neurotransmitters in hippocampus and prefrontal cortex were measured by high-performance liquid chromatography. Neuronal morphology was detected by transmission electron microscopy. Results: Administration of leonurine (60 mg/kg) for 4 weeks significantly alleviated depression-like behaviors of chronic mild stress mice, including increased sucrose preference and reduced immobility time in forced swimming test and tail suspension test. We further found that leonurine (60 mg/kg) effectively restored the levels of 5-hydroxytryptamine, noradrenaline, and dopamine in the hippocampus and prefrontal cortex of chronic mild stress mice, accompanied by amelioration of hippocampal neuronal damage. Furthermore, leonurine (60 mg/kg) significantly inhibited the production of proinflammatory cytokine interleukin-1ß, interleukin-6 and TNF-α, and suppressed the nuclear factor kappa B signaling pathway. Conclusions: These findings demonstrate that leonurine exerts antidepressant-like effects, which may be mediated, at least in part, by improving monoamine neurotransmitters and inhibiting neuroinflammation. Our study provides insight into the potential of leonurine in depression therapy.