Leonurine exerts its anti-atherosclerotic effects by promoting the expression of ATP-binding cassette transporter A1/G1 (ABCA1/G1) in a peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) signaling pathway-dependent manner. [1]
It enhances the mRNA and protein levels of PPARγ, LXRα, ABCA1, and ABCG1 in THP-1 macrophage-derived foam cells and in the aortic roots of apoE⁻/⁻ mice. [1]

Less lipid buildup, higher cellular cholesterol content (including total cholesterol (TC), free cholesterol (FC), and cholesteryl esters (CE), and apoA-I, or HDL-mediated cholesterol efflux, were the outcomes of leonurus alkaloids (0, 5, 10, 20, 40, and 80 µM) administration 24 hours later. In human THP-1 cells, lenurine can also dramatically and dose-dependently boost the mRNA and protein expression of ABCA1 and ABCG1, an action that is linked to PPARγ [1]. When palmitic acid (PA) is added as a free fatty acid (FFA) to HepG2 and HL-7702 cells for a whole day, leonurine hydrochloride (LH) protects the cells' survival. By triggering the AMPK/SREBP1 pathway, leonurineHCl (125, 250, and 500μM) enhances cellular lipid accumulation in HepG2 and HL-7702 cells [2]. In human chondrocytes, leonurine (5, 10, 20 µM) reduces the production of iNOS, COX-2, PGE2, NO, TNF-α, and IL-6 caused by IL-1β. It also inhibits the breakdown of extracellular matrix (ECM) in human OA chondrocytes. Furthermore, in a dose-dependent way, blocking IL-1β causes PI3K and Akt activation.

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In human THP-1 macrophage-derived foam cells, leonurine treatment (0, 5, 10, 20, 40, 80 µM for 24 h) resulted in a concentration-dependent decrease in lipid accumulation, as shown by Oil Red O staining. [1]
HPLC analysis revealed that leonurine significantly reduced cellular total cholesterol (TC), free cholesterol (FC), and cholesteryl ester (CE) levels in a concentration-dependent manner. For example, at 80 µM, TC was reduced from 788 ± 176 mg/g to 275 ± 25 mg/g, FC from 304 ± 19 mg/g to 112 ± 16 mg/g, and CE from 484 ± 24 mg/g to 163 ± 17 mg/g. The CE/TC ratio remained relatively constant (approx. 59-61%). [1]
Leonurine increased apoA-I- and HDL-mediated cholesterol efflux in a concentration-dependent manner, as measured by liquid scintillation counting. [1]
Western blot and RT-qPCR analyses demonstrated that leonurine (0-80 µM for 24 h) significantly increased the mRNA and protein expression of ABCA1, ABCG1, PPARγ, and LXRα in THP-1 macrophage-derived foam cells in a dose-dependent manner. [1]
Transfection of THP-1 macrophages with LXRα siRNA abolished the leonurine-induced up-regulation of ABCA1 and ABCG1 mRNA and protein levels and reversed the leonurine-induced increase in cholesterol efflux. Conversely, co-incubation with the LXRα agonist T0901317 enhanced the leonurine-induced increase in ABCA1, ABCG1, and LXRα expression. [1]
Transfection with PPARγ siRNA significantly decreased LXRα, ABCA1, and ABCG1 mRNA and protein levels and negated the stimulatory effects of leonurine on these proteins and on cholesterol efflux. [1]

In mouse serum, motherwort (10 mg/kg/d) significantly increased the expression of PPARγ, LXRα, ABCA1, and ABCG1, while lowering the levels of TG and TC [1]. By stimulating the AMPK/SREBP1 pathway, motherwort hydrochloride (50, 100, or 200 mg/kg) decreases liver peroxidized lipids, increases antioxidant levels, and improves intracellular lipid accumulation in mice [2]. In an orthopaedic mouse model of DMM, motherwort (20 mg/kg) ameliorates the development of osteoarthritis [3].

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In apoE⁻/⁻ mice, intragastric administration of leonurine at 10 mg/kg/day for 8 weeks significantly decreased atherosclerotic lesion sizes in the aortic roots compared to the control group, as assessed by Oil Red O staining. [1]
Masson's trichrome staining showed that leonurine treatment increased collagen content within the atherosclerotic lesions of the aortic roots. [1]
Western blot analysis of aortic root homogenates revealed that leonurine treatment significantly increased the protein expression of PPARγ, LXRα, ABCA1, and ABCG1 compared to the control group. [1]
Leonurine treatment significantly improved the plasma lipid profile: it decreased total cholesterol (TC) from 17.59 ± 2.12 mmol/L to 13.78 ± 1.85 mmol/L, triglycerides (TG) from 1.88 ± 0.45 mmol/L to 1.25 ± 0.23 mmol/L, and low-density lipoprotein cholesterol (LDL-C) from 10.54 ± 1.83 mmol/L to 8.26 ± 1.35 mmol/L. It also increased high-density lipoprotein cholesterol (HDL-C) from 0.95 ± 0.25 mmol/L to 1.42 ± 0.43 mmol/L. [1]

Cell Culture and Treatment: Human THP-1 cells were cultured in RPMI-1640 medium and pre-treated with phorbol-12-myristate-13-acetate (PMA, 160 nmol/L) for 24 hours to induce differentiation into macrophages. The medium was then replaced with serum-free medium containing oxidized low-density lipoprotein (ox-LDL, 50 µg/mL) for an additional 48 hours to fully differentiate the cells into foam cells. These foam cells were then treated with various concentrations of leonurine (0, 5, 10, 20, 40, 80 µM) for 24 hours for subsequent experiments. [1]
Oil Red O Staining: To assess cellular lipid accumulation, THP-1 macrophage-derived foam cells were fixed in 4% paraformaldehyde, rinsed with 60% isopropanol, and stained with 0.3% Oil Red O solution for 10 minutes. The cells were then counterstained with Gill III hematoxylin. Images of stained cells were captured using a microscope. [1]
Cholesterol Efflux Assay: Differentiated and radiolabeled THP-1 foam cells were incubated with 5 µCi/mL ³H-cholesterol. After treatment with leonurine for 24 hours, the cells were washed and then incubated with apoA-I (10 µg/mL) or HDL (50 µg/mL) as lipid acceptors for 6 hours. The ³H-cholesterol levels in the medium and cells were measured by liquid scintillation counting. The percentage of cholesterol efflux was calculated as [medium counts / (medium counts + cell counts)] × 100%. [1]
High-Performance Liquid Chromatography (HPLC) Assay: Cellular total cholesterol (TC) and free cholesterol (FC) were determined by HPLC. Cell lysates were sonicated, and lipids were extracted using isopropanol:hexane. The extracted samples were then reacted with cholesterol oxidase (for FC) or cholesterol oxidase and cholesterol esterase (for TC). The reaction products were analyzed on a C-18 HPLC column with an isopropanol: n-heptane: acetonitrile mobile phase at a flow rate of 1 mL/min. The absorbance was measured at 216 nm. [1]
RNA Extraction and RT-qPCR: Total RNA was extracted from cells using TRIzol reagent, and cDNA was synthesized. The mRNA expression levels of ABCA1, ABCG1, PPARγ, and LXRα were quantified by real-time PCR using SYBR Green detection and specific primers. β-actin was used as an internal control. The relative expression was calculated using the ΔΔCt method. [1]
Western Blot Analysis: Cells or aortic tissues were lysed, and protein concentrations were determined. Equal amounts of protein (20 µg per lane) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking, membranes were incubated with primary antibodies against ABCA1, ABCG1, PPARγ, LXRα, and β-actin, followed by a peroxidase-conjugated secondary antibody. Protein bands were visualized using a chemiluminescence detection system and quantified. [1]
Small Interfering RNA (siRNA) Transfection: THP-1 macrophages were transfected with siRNA specific for LXRα or PPARγ using a lipofectamine-based reagent according to the manufacturer's instructions. Transfection efficiency was evaluated by western blot analysis. [1]

Animal Model and Treatment:** Eight-week-old male apoE⁻/⁻ mice were fed a chow diet for 2 weeks. They were then randomly divided into control and treatment groups (n=15 per group). Mice in the leonurine group received leonurine (10 mg/kg/day) by intragastric administration every day for 8 weeks. The control group received an equal volume of phosphate-buffered saline (PBS). [1]
**Sample Collection and Tissue Preparation:** At 16 weeks of age, mice were euthanized, and blood and tissue samples were collected. Hearts and proximal aortas were dissected, fixed in formalin, and embedded in OCT medium. Serial sections (8 µm thick) of the aortic sinus were cut for histological analysis. [1]
**Assessment of Atherosclerotic Lesions and Collagen Content:** Aortic sinus sections were stained with Oil Red O to identify atherosclerotic lesion areas and with Masson’s trichrome (MT) to identify collagen content. Lesion sizes and collagen content were quantified using image analysis software. [1]
**Lipid Analyses:** Plasma levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using enzymatic methods with commercial test kits. [1]

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Animal Model and Treatment: Eight-week-old male apoE⁻/⁻ mice were fed a chow diet for 2 weeks. They were then randomly divided into control and treatment groups (n=15 per group). Mice in the leonurine group received leonurine (10 mg/kg/day) by intragastric administration every day for 8 weeks. The control group received an equal volume of phosphate-buffered saline (PBS). [1]
Sample Collection and Tissue Preparation: At 16 weeks of age, mice were euthanized, and blood and tissue samples were collected. Hearts and proximal aortas were dissected, fixed in formalin, and embedded in OCT medium. Serial sections (8 µm thick) of the aortic sinus were cut for histological analysis. [1]
Assessment of Atherosclerotic Lesions and Collagen Content: Aortic sinus sections were stained with Oil Red O to identify atherosclerotic lesion areas and with Masson’s trichrome (MT) to identify collagen content. Lesion sizes and collagen content were quantified using image analysis software. [1]
Lipid Analyses: Plasma levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using enzymatic methods with commercial test kits. [1]

MTT assays demonstrated that LH at concentrations up to 1000 µM did not exhibit obvious cytotoxic effects on HepG2 and HL7702 cells after 24 h of treatment. [2]

[1]. Leonurine Prevents Atherosclerosis Via Promoting the Expression of ABCA1 and ABCG1 in a Pparγ/Lxrα Signaling Pathway-Dependent Manner. Cell Physiol Biochem. 2017;43(4):1703-1717.

[2]. Novel hepatoprotective role of Leonurine hydrochloride against experimental non-alcoholic steatohepatitis mediated via AMPK/SREBP1 signaling pathway. Biomed Pharmacother. 2018 Dec 7;110:571-581.

[3]. Inhibition of PI3K/Akt/NF-κB signaling with leonurine for ameliorating the progression of osteoarthritis: In vitro and in vivo studies. J Cell Physiol. 2018 Nov 11.

Leonurine is a trihydroxybenzoic acid. It has been reported that Leonurine is found in Siberian motherwort and lion's ear grass, and relevant data are available for reference.

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Leonurine (SCM-198) is a unique alkaloid compound extracted from Herba leonuri (also known as "Yi-Mu-Cao"), a traditional Chinese herbal medicine. While previous studies have demonstrated its anti-oxidative, anti-inflammatory, and anti-apoptotic properties in cardiovascular diseases, this study is the first to report that leonurine promotes cholesterol efflux and prevents atherosclerosis by upregulating ABCA1 and ABCG1 expression via the PPARγ/LXRα signaling pathway. The authors propose that this mechanism represents a novel therapeutic intervention for atherosclerosis. [1]

C14H21N3O5

311.33

311.148

24697-74-3

Leonurine hydrochloride;24735-18-0

161464

White to off-white solid powder

1.3±0.1 g/cm3

496.7±55.0 °C at 760 mmHg

191-193ºC

254.2±31.5 °C

0.0±1.3 mmHg at 25°C

1.554

0.22

3

6

9

22

360

0

WNGSUWLDMZFYNZ-UHFFFAOYSA-N

InChI=1S/C14H21N3O5/c1-20-10-7-9(8-11(21-2)12(10)18)13(19)22-6-4-3-5-17-14(15)16/h7-8,18H,3-6H2,1-2H3,(H4,15,16,17)

4-(diaminomethylideneamino)butyl 4-hydroxy-3,5-dimethoxybenzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~33.33 mg/mL (~107.06 mM)
H2O : ~1 mg/mL (~3.21 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)