Autophagy-related proteins (Beclin 1, LC3B, p62). [1]

Notoginsenoside Fc (20 μM; for 24 hours) dramatically increases the expression of Beclin 1 and LC3B while decreasing p62 expression in RAOECs [1].

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Under high-glucose (HG, 30 mM D-glucose) conditions in rat aortic endothelial cells (RAOECs), Notoginsenoside Fc (20 µM, 24 h) prevented HG-induced autophagy reduction. Western blot and RT-qPCR analyses showed that HG decreased protein and mRNA expression of LC3B and Beclin 1 and increased p62 levels. Fc treatment markedly upregulated LC3B and Beclin 1 and downregulated p62 compared to the HG group. [1]
Using mRFP-eGFP-LC3B plasmid transfection and confocal microscopy, HG treatment decreased both yellow puncta (autophagosomes) and red puncta (autolysosomes). Pretreatment with Notoginsenoside Fc (20 µM) followed by HG increased yellow puncta with no obvious difference in red puncta compared to HG alone. [1]
Transmission electron microscopy showed small vacuoles in HG-treated RAOECs; the number of autophagosomes increased when cells were pretreated with Fc (20 µM). [1]
Cell cycle progression analysis showed that HG caused more RAOECs arrested in G1 phase; Notoginsenoside Fc (20 µM) promoted proliferation by reversing this change. This effect was decreased by pretreatment with the autophagy inhibitor 3-methyladenine (3-MA, 5 mM, 2 h). Western blot analysis of proliferating cell nuclear antigen (PCNA) showed lower expression in HG-treated cells; Fc increased PCNA expression, which was reduced by 3-MA. [1]
Wound healing assay showed that HG significantly reduced RAOEC migration; Notoginsenoside Fc (20 µM) increased migration under HG conditions. Migration was decreased following addition of 3-MA (5 mM, 2 h). Cell proliferation and migration did not differ significantly among NG, NG+Fc, and NG+Fc+3-MA groups. [1]

After carotid artery damage in diabetic Sprague-Dawley rats (200±20 g), notoginsenoside Fc (3.5 mg/kg/day) can prevent excessive neointima development and speed up re-endothelialization [1].

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In diabetic Sprague-Dawley rats (induced by streptozotocin, 60 mg/kg intraperitoneal injection), following carotid artery wire injury, Notoginsenoside Fc (3.5 mg/kg/d by gavage) accelerated reendothelialization. Evans blue staining at 14 and 28 days post-injury showed reendothelialization rates: DM group had 47.8±9.2% (day 14) and 64.4±4.8% (day 28); DM+Fc group had 75.9±8.7% (day 14) and 85±9.3% (day 28). Control (non-diabetic injured) rats had 80.5±5.8% (day 14) and 90.2±3.5% (day 28). [1]
Notoginsenoside Fc retarded excessive neointimal formation. Hematoxylin and eosin staining at 14 days after injury showed that diabetes significantly increased neointimal hyperplasia. The neointima area and neointima-to-media area ratio were significantly increased in DM group vs. control; these were attenuated by Fc treatment (DM+Fc group). Media area did not differ significantly among groups. Neointimal hyperplasia area was inversely associated with reendothelialization rate. [1]
Immunohistochemistry at 14 days after injury showed that Beclin 1 protein was found predominantly in the intima. The number of Beclin 1-positive cells was decreased in DM group vs. control; Notoginsenoside Fc treatment markedly improved Beclin 1 expression in the injured artery. [1]

Cell culture: RAOECs were cultured in DMEM with 10% fetal bovine serum at 37°C with 5% CO2. Passages 4-6 were used. Cells were divided into: normal glucose (NG, 5.6 mM D-glucose); NG+Fc (20 µM Fc for 24 h); high-glucose (HG, 30 mM D-glucose for 24 h); HG+Fc (30 mM D-glucose + 20 µM Fc for 24 h). For autophagy inhibition, cells were pretreated with 5 mM 3-methyladenine (3-MA) for 2 h. [1]
Cell proliferation assay: RAOECs were seeded onto 96-well plates (1×10^4 cells/well). After reaching 70-80% confluence, cells were treated with corresponding drugs for 24 h. Cell cycle analyses were performed using a Cell Cycle Phase Determination Kit. Each experiment was performed independently at least three times. [1]
Wound healing assay: RAOECs were inoculated into 6-well plates (5×10^5 cells/well) and grown for 24 h to 70-80% confluence. After 12 h of serum withdrawal, three scratches were made using a P-20 pipette. Cells were incubated for 24 h with indicated treatments, and images were taken to measure gap changes. [1]
Monitoring autophagy flux using mRFP-eGFP-LC3B plasmid: RAOECs were seeded onto glass bottom dishes. At 80% confluence, 4 µg of the plasmid was dissolved in Opti-MEM reduced serum medium and mixed with liposomes using Lipofectamine 2000. The mixture was added to cells and replaced with serum medium after 4-6 h. After 48 h, cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and stained with DAPI for 15 min. Samples were observed with a confocal microscope. At least eight cells per experiment were randomly selected to count red and yellow puncta. [1]
Transmission electron microscopy: RAOECs were fixed with glutaraldehyde at 4°C. After ethanol dehydration and embedding in epoxy resin, ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and observed with an electron microscope. [1]
RT-qPCR: Total RNA was extracted with TRIzol reagent. RNA concentration was measured, then reverse transcribed to cDNA using a PrimeScript RT Reagent Kit. PCR was performed with SYBR Premix Ex Taq using primers for LC3B, Beclin 1, p62, and GAPDH (internal control). Relative mRNA expression was normalized to GAPDH and presented as 2^(-ΔΔCt) values. [1]
Western blot analysis: Carotid arteries or cells were lysed with cell lysis buffer supplemented with 0.5 mM phenylmethanesulfonylfluoride. After centrifugation (12,000 rpm, 15 min, 4°C), protein concentrations were determined by Bicinchoninic Acid Protein Assay. Equal protein masses were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk in TBST for 2 h at room temperature, then probed with primary antibodies (1:500-1:1000 dilution) overnight at 4°C, followed by HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were detected with enhanced chemiluminescence. Antibodies against LC3B, p62, Beclin 1, PCNA, and β-actin were used. [1]

Animal model: Male Sprague-Dawley rats (200±20 g) were randomly separated into four groups: sham, control (injured non-diabetic), DM, and DM+Fc (n=12 each). Diabetes was induced by intraperitoneal injection of 60 mg/kg streptozotocin (STZ) after 12 h fasting. Fasting blood glucose was measured on days 3 and 7 from tail vein; fasting blood glucose >16.7 mmol/L on both days indicated successful diabetic model. [1]
Carotid artery injury: After successful modeling, animals were fasted for 12 h before wire injury. A 2-French balloon catheter was inserted through the left external carotid artery into the common carotid artery and insufflated three times with 2 atm of pressure. After injury, the external carotid artery was ligated and blood flow resumed. The DM+Fc group began drug treatment with gavage of 3.5 mg/kg/d Notoginsenoside Fc until sacrifice. The other three groups received same dose of saline. [1]
Evans blue staining: At 14 and 28 days after injury, reendothelialization was assessed by injection of 0.5% Evans blue dye into the left thigh femoral vein after anesthesia. The tissue was cut longitudinally, washed with PBS, fixed with 4% paraformaldehyde, and photographed. Deendothelialized areas (stained blue) were measured using ImageJ software. Blood glucose of rats was measured at days 0 (before injury), 14, and 28. [1]
HE staining: At 28 days after injury, rat arteries were harvested, fixed in 4% paraformaldehyde for 24-48 h, embedded in paraffin. Sections (3-5 mm thick) were dewaxed, stained, examined microscopically, and photographed. Neointimal and media areas were computed: neointimal area = internal elastic lamina area - lumen area; media area = external elastic lamina area - internal elastic lamina area. [1]
Immunohistochemistry: At 14 days after injury, arteries were harvested, embedded in OCT compound, snap-frozen in liquid nitrogen, stored at -80°C. Sections (7 µm thick) at 500 mm intervals of the injured carotid artery (4 mm) were cut and stained with HE for immunohistochemistry. Samples were immunostained with rabbit anti-Beclin 1 antibody, then with HRP-conjugated anti-rabbit IgG polymer, and colored with 3,3-diaminobenzidine. [1]

[1]. Notoginsenoside Fc Accelerates Reendothelialization following Vascular Injury in Diabetic Rats by Promoting Endothelial Cell Autophagy. J Diabetes Res. 2019 Sep 3;2019:9696521.

Reports have indicated that ginsenoside Fc exists in Japanese ginseng and Panax notoginseng, and relevant data is available for reference.

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Notoginsenoside Fc (molecular formula C58H98O26, molecular weight 1211.4 Da, purity ≥98%) is a novel saponin isolated from Panax notoginseng leaves. It is a protopanaxadiol (PPD)-type saponin. This study is the first to report that Fc promotes endothelial cell proliferation and migration under high-glucose conditions in vitro and accelerates reendothelialization in diabetic rats in vivo by promoting autophagy. The findings suggest that Fc may exert therapeutic benefits for early endothelial injury and restenosis following intervention in diabetes-associated vascular diseases (e.g., peripheral vascular disease). [1]

C58H98O26

1211.3831

1210.634

88122-52-5

75412556

Off-white to light yellow solid

1.47±0.1 g/cm3

1.631

6.6

16

26

17

84

2210

33

C[C@@]12CC[C@H]([C@](C)(CC/C=C(\C)/C)O[C@H]3[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]4OC[C@@H](O)[C@H](O)[C@H]4O)O3)[C@H]1[C@H](O)C[C@@H]1[C@]3(CC[C@H](O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O[C@@H]4OC[C@@H](O)[C@H](O)[C@H]4O)C(C)(C)[C@@H]3CC[C@@]21C)C

XBGLCVZQMWKHFC-NMQALWILSA-N

InChI=1S/C58H98O26/c1-24(2)10-9-14-58(8,84-51-46(74)41(69)40(68)31(80-51)23-77-49-44(72)36(64)27(62)21-75-49)25-11-16-57(7)35(25)26(61)18-33-55(5)15-13-34(54(3,4)32(55)12-17-56(33,57)6)81-52-47(42(70)38(66)29(19-59)78-52)83-53-48(43(71)39(67)30(20-60)79-53)82-50-45(73)37(65)28(63)22-76-50/h10,25-53,59-74H,9,11-23H2,1-8H3/t25-,26+,27+,28+,29+,30+,31+,32-,33+,34-,35-,36-,37-,38+,39+,40+,41-,42-,43-,44+,45+,46+,47+,48+,49-,50-,51-,52-,53-,55-,56+,57+,58-/m0/s1

(2S,3R,4S,5S,6R)-2-[(2S)-2-[(3S,5R,8R,9R,10R,12R,13R,14R,17S)-3-[(2R,3R,4S,5S,6R)-3-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxyoxan-2-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-12-hydroxy-4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl]-6-methylhept-5-en-2-yl]oxy-6-[[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxymethyl]oxane-3,4,5-triol

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~100 mg/mL (~82.55 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.06 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.06 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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