MMP-2; MMP-9; ERK1; ERK2; JNK
This study investigates the anti-inflammatory mechanisms of Astragaloside IV in a mouse model of cerebral ischemia and reperfusion. The compound modulates several key targets in the inflammatory pathway:

- Toll-like receptor 4 (TLR4) and its downstream signaling pathway (MyD88, TRIF, TRAF6). [1]
- Nuclear factor-kappa B (NF-κB), specifically inhibiting its phosphorylation (p-p65). [1]
- NLRP3 inflammasome, suppressing its activation. [1]
- Microglial activation (reducing Iba1 expression). [1]
- Reactive oxygen species (ROS) production. [1]
- Pro-inflammatory cytokines (IL-1β and TNF-α). [1]

Astragaloside IV (10, 20, 40 ng/mL) inhibits NSCLC cell growth, whereas low concentrations of astragaloside IV (1, 2.5, 5 ng/mL) has no obvious cytotoxicity on cell viability. Additionally, astragaloside IV combined therapy significantly raises the chemosensitivity of NSCLC cells to cisplatin. When cisplatin is present, astragaloside IV co-treatment significantly reduces the mRNA and protein levels of B7-H3[2]. Astragaloside IV reduces the activity of the matrix metalloproteases (MMP)-2 and -9, suppresses the activation of the mitogen activated protein kinase (MAPK) family members ERK1/2 and JNK, and inhibits the viability and invasive potential of MDA-MB-231 breast cancer cells[4].

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Astragaloside IV demonstrates significant effects in NSCLC cell lines, particularly in combination with cisplatin.

1. Cytotoxicity and Cell Viability: In A549, HCC827, and NCI-H1299 NSCLC cells, high doses of Astragaloside IV (10, 20, 40 ng/mL) significantly inhibited cell growth after 48 hours of treatment. Low concentrations (1, 2.5, 5 ng/mL) had no obvious cytotoxic effect on cell viability. Similarly, high concentrations of cisplatin (5, 10, 20, 40 μM) reduced cell viability, while low doses (0.5, 1, 2.5 μM) did not. [2]

2. Enhancement of Cisplatin Chemosensitivity: Combined treatment with a low, non-cytotoxic dose of Astragaloside IV (5 ng/mL) and a low dose of cisplatin (2.5 μM) significantly inhibited cell viability in A549, HCC827, and NCI-H1299 cells compared to treatment with cisplatin alone, as measured by CCK-8 assay. This indicates that Astragaloside IV sensitizes NSCLC cells to cisplatin. [2]

3. Induction of Apoptosis: Flow cytometry analysis with propidium iodide (PI) staining showed that treatment with 2.5 μM cisplatin alone did not induce obvious apoptosis. However, co-treatment with 2.5 μM cisplatin and 5 ng/mL Astragaloside IV significantly increased the apoptotic cell population to 39.57% in A549 cells, 36.77% in HCC827 cells, and 34.85% in NCI-H1299 cells. [2]

4. Inhibition of B7-H3 Expression: Real-time PCR and Western blot analyses revealed that co-treatment with Astragaloside IV and cisplatin significantly inhibited both the mRNA and protein levels of B7-H3 in A549 and HCC827 cells compared to treatment with cisplatin alone. [2]

5. Role of B7-H3 in Chemosensitization: Ectopic overexpression of B7-H3 via plasmid transfection in A549, HCC827, and NCI-H1299 cells significantly diminished the chemosensitizing effect of Astragaloside IV. In B7-H3-overexpressing cells, the combined treatment with Astragaloside IV and cisplatin no longer showed a significant reduction in cell viability compared to cisplatin alone, confirming that the effect of Astragaloside IV is mediated through inhibition of B7-H3. [2]

Astragaloside IV (10, 20 mg/kg, p.o.) exhibits a potent ability to prevent cognitive deficits brought on by transient cerebral ischemia and reperfusion. In comparison to the Model group, Astragaloside IV (10 mg/kg) and Astragaloside IV (20 mg/kg) can significantly lower these cytokine levels. It is possible that both MyD88-dependent and -independent pathways are crucial for Astragaloside IV's anti-inflammatory effects given that it significantly reduces the level of TLR4 and its downstream proteins. In addition to lowering the expression of Iba1 protein, astragaloside IV also inhibits the expression of NLRP3 and cleaved-caspase-1. In the mouse model, the high-dose astragaloside IV group significantly improved the 48-hour survival rate [60% (9/15) vs 13.3% (2/15), P < 0.05], significantly decreased serum ALT and AST levels (P < 0.01), significantly decreased liver histopathological indices and the degree of hepatocyte apoptosis (P < 0.01), significantly increased SOD activity (P < 0.01), and significantly decreased the content of MDA.

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Astragaloside IV demonstrates significant neuroprotective and anti-inflammatory effects in a mouse model of transient cerebral ischemia and reperfusion injury.

1. Improvement of Cognitive Impairment (Morris Water Maze): In mice subjected to bilateral common carotid artery occlusion (BCCAO), oral administration of Astragaloside IV (10 and 20 mg/kg, once daily for 7 days pre-surgery and 7 days post-surgery) significantly attenuated memory deficits. This was evidenced by decreased escape latency during training trials (20 mg/kg group showed significant improvement from day 2 onward; 10 mg/kg group on day 5) and improved performance in the probe trial, with increased time spent in the target quadrant (10 mg/kg, p<0.05; 20 mg/kg, p<0.01) and increased frequency of platform crossings (both doses, p<0.01) compared to the model group. [1]

2. Reduction of Pro-inflammatory Cytokines: Astragaloside IV treatment (10 and 20 mg/kg) significantly reduced the elevated levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the hippocampus of BCCAO mice, as measured by ELISA. [1]

3. Modulation of Oxidative Stress Markers: Astragaloside IV treatment significantly reversed the changes in oxidative stress markers induced by ischemia-reperfusion. It increased the activity of superoxide dismutase (SOD) and decreased the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in the hippocampus. [1]

4. Inhibition of TLR4 Signaling Pathway: Western blot analysis of hippocampal tissue showed that Astragaloside IV significantly down-regulated the protein expression of TLR4 and its downstream adaptors, including MyD88, TRIF, and TRAF6. It also inhibited the phosphorylation of NF-κB p65 subunit. [1]

5. Suppression of NLRP3 Inflammasome Activation: Astragaloside IV treatment significantly reduced the protein expression of NLRP3 and cleaved caspase-1 in the hippocampus, as determined by Western blot and immunohistochemistry, indicating suppression of the NLRP3 inflammasome. [1]

6. Inhibition of Microglial Activation: Astragaloside IV significantly decreased the expression of Iba1, a marker of activated microglia, in the hippocampus of BCCAO mice, as shown by Western blot. [1]

In a nutshell, MDA-MB-231 cells treated as advised or tumor tissues are harvested and lysed in Mg2+ lysis buffer containing 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.5 M NaCl, and protease inhibitor cocktail. Equal amounts of lysates are incubated with PAK-PBD beads at 4°C for one hour. Centrifugation is used to pellet PAK-PBD beads, which are then washed in a solution of 25 mM Tris (pH 7.5), 30 mM MgCl2, and 40 mM NaCl. Western blotting is used to find active Rac1.

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The CCK-8 assay is used to determine cell viability. In a nutshell, 4×104 (cells/well) of cultured NSCLC cells are seeded into 96-well plates. Then, for an additional 2 hours at 37°C in the dark, a 10 µL/well CCK8 solution is added. The wavelength of 490 nm is used to calculate absorbance.

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Several cell-based assays were used to evaluate the effects of Astragaloside IV.

1. Cell Lines and Culture: Human NSCLC cell lines A549, HCC827, and NCI-H1299 were used. Cells were cultured in DMEM supplemented with 10% FBS at 37°C in a 5% CO₂ incubator. [2]

2. Cell Viability Assay (CCK-8): Cells were seeded into 96-well plates at a density of 4 × 10⁴ cells/well. After treatment with Astragaloside IV and/or cisplatin for 48 hours, 10 μL/well of CCK-8 solution was added and incubated for 2 hours at 37°C in the dark. Absorbance was measured at 490 nm to determine cell viability. [2]

3. Apoptosis Assay (Flow Cytometry): After treatment, cells were washed with PBS, trypsinized, and harvested by centrifugation. Cells were resuspended in binding buffer at a concentration of 9 × 10⁵ cells/mL. Propidium iodide (PI) was added, and the mixture was incubated for 5 minutes in the dark at 4°C. Apoptosis rate was assessed by flow cytometry, with 10,000 events recorded per analysis. [2]

4. RNA Extraction and Real-time PCR: Total RNA was isolated from cells using TRIzol reagent. Reverse transcription was performed using a commercial kit. Real-time PCR was carried out using SYBR Green Premix on an ABI 7500 system to quantify B7-H3 mRNA expression, with GAPDH as an endogenous control. [2]

5. Western Blot: Cells were lysed with RIPA buffer, and protein concentration was quantified using a BCA assay. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with anti-B7-H3 primary antibody overnight at 4°C, followed by HRP-conjugated secondary antibody. Protein bands were detected by chemiluminescence. [2]

6. Plasmid Transfection: The cDNA fragment encoding B7-H3 was cloned into the pcDNA3.1(+) vector. Cells were transfected with the B7-H3-expressing plasmid or empty vector using Lipofectamine 2000 according to the manufacturer's protocol. Transfection medium was replaced with complete medium after 12 hours. [2]

BCCAO is used to prepare transient cerebral ischemia and reperfusion because it is regarded as the best model for studying the injury-mediated inflammatory response to transient cerebral ischemia and reperfusion. The Sham, Model, Astragaloside IV (10 mg/kg), and Astragaloside IV (20 mg/kg) treatment groups are randomly assigned to mice. The IV astragaloside treatment groups begin 7 days prior to surgery and end on the day of sacrifice by intragastric administration. 2 hours before ischemia on the day of surgery, astragaloside IV is administered. Distilled water is used to treat the groups that operated as a sham and the model. The bilateral common carotid arteries are exposed and delicately separated with a small ventral neck incision after the mice have been given an intraperitoneal injection of chloral hydrate (350 mg/kg) to induce anesthesia. With a few minor modifications from the previous description, the common carotid arteries are then twice (20 min each) occluded with ligated surgical silk. Between each of the two occlusion periods (ischemia 20 min, reperfusion 10 min, ischemia 20 min), there is a 10 min reperfusion period. The same surgical procedure is performed on mice who underwent a sham operation without the surgical silk ligation. Heaters are used to keep the mouse's body temperature at 370±5°C throughout the procedure and until the animal wakes up from the anesthesia.

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1. **Animals:** Male ICR mice (20 ± 2 g) were used. They were housed under controlled conditions (25 ± 1°C, 60-65% humidity, 12h light/dark cycle) with free access to food and water. [1]

2. **Drug Administration:** Astragaloside IV (purity 98%) was dissolved in distilled water and administered orally (intragastrically) at doses of 10 mg/kg and 20 mg/kg. Treatment was given once daily, starting 7 days before the surgical induction of ischemia and continuing for 7 days after surgery until the day of sacrifice. On the day of surgery, the drug was administered 2 hours prior to inducing ischemia. The Sham and Model groups received distilled water only. [1]

3. **Induction of Cerebral Ischemia and Reperfusion:** Transient cerebral ischemia and reperfusion was induced by bilateral common carotid artery occlusion (BCCAO). Mice were anesthetized with chloral hydrate (350 mg/kg, i.p.). The bilateral common carotid arteries were exposed and occluded twice for 20 minutes each, with a 10-minute reperfusion interval between occlusions (20 min ischemia - 10 min reperfusion - 20 min ischemia). Sham-operated mice underwent the same surgical procedure without ligation. Body temperature was maintained at 37 ± 0.5°C during and after surgery until recovery. [1]

4. **Morris Water Maze (MWM) Test:** Spatial learning and memory were assessed starting on the 8th day post-surgery. The test was conducted over 5 days. For 4 consecutive training days, mice were given two trials per day to find a hidden platform (escape latency recorded, max 90s). On the 5th day (probe trial), the platform was removed, and the time spent in the target quadrant and frequency of crossing the former platform location were recorded over 90s. [1]

5. **Tissue Collection and Biochemical Assays:** After behavioral tests, mice were sacrificed, and hippocampi were dissected. Tissue homogenates were prepared for various assays: ELISA for IL-1β, TNF-α, and ROS; colorimetric assays for SOD activity and MDA content; and Western blot for protein expression analysis. [1]

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1. Animals: Male ICR mice (20 ± 2 g) were used. They were housed under controlled conditions (25 ± 1°C, 60-65% humidity, 12h light/dark cycle) with free access to food and water. [1]

2. Drug Administration: Astragaloside IV (purity 98%) was dissolved in distilled water and administered orally (intragastrically) at doses of 10 mg/kg and 20 mg/kg. Treatment was given once daily, starting 7 days before the surgical induction of ischemia and continuing for 7 days after surgery until the day of sacrifice. On the day of surgery, the drug was administered 2 hours prior to inducing ischemia. The Sham and Model groups received distilled water only. [1]

3. Induction of Cerebral Ischemia and Reperfusion: Transient cerebral ischemia and reperfusion was induced by bilateral common carotid artery occlusion (BCCAO). Mice were anesthetized with chloral hydrate (350 mg/kg, i.p.). The bilateral common carotid arteries were exposed and occluded twice for 20 minutes each, with a 10-minute reperfusion interval between occlusions (20 min ischemia - 10 min reperfusion - 20 min ischemia). Sham-operated mice underwent the same surgical procedure without ligation. Body temperature was maintained at 37 ± 0.5°C during and after surgery until recovery. [1]

4. Morris Water Maze (MWM) Test: Spatial learning and memory were assessed starting on the 8th day post-surgery. The test was conducted over 5 days. For 4 consecutive training days, mice were given two trials per day to find a hidden platform (escape latency recorded, max 90s). On the 5th day (probe trial), the platform was removed, and the time spent in the target quadrant and frequency of crossing the former platform location were recorded over 90s. [1]

5. Tissue Collection and Biochemical Assays: After behavioral tests, mice were sacrificed, and hippocampi were dissected. Tissue homogenates were prepared for various assays: ELISA for IL-1β, TNF-α, and ROS; colorimetric assays for SOD activity and MDA content; and Western blot for protein expression analysis. [1]

[1]. Astragaloside IV attenuates cognitive impairments induced by transient cerebral ischemia and reperfusion in mice via anti-inflammatory mechanisms. Neurosci Lett. 2016 Dec 20. pii: S0304-3940(16)30994-

[2]. Astragaloside IV Enhances Cisplatin Chemosensitivity in Non-Small Cell Lung Cancer Cells Through Inhibition of B7-H3. Cell Physiol Biochem. 2016;40(5):1221-1229. Epub 2016 Dec 14.

[3]. [Protective effect of astragaloside IV against acute liver failure in experimental mice]. Zhonghua Gan Zang Bing Za Zhi. 2016 Oct 20;24(10):772-777.

[4]. Astragaloside IV inhibits breast cancer cell invasion by suppressing Vav3 mediated Rac1/MAPK signaling. Int Immunopharmacol. 2016 Dec 5;42:195-20.

Astragaloside IV is a pentacyclic triterpenoid compound belonging to the cycloastragalol group, with β-D-xylose and β-D-glucose residues linked at the O-3 and O-6 positions, respectively. It was isolated from Astragalus membranaceus var. mongholicus. Astragaloside IV possesses various activities, including as an EC 4.2.1.1 (carbonic anhydrase) inhibitor, anti-inflammatory agent, neuroprotective agent, antioxidant, pro-angiogenic agent, and plant metabolite. It is a triterpenoid saponin and a pentacyclic triterpenoid compound, functionally related to cycloastragalol. Astragaloside IV has also been reported to exist in Astragalus hoantchy, Astragalus lepsensis, and other organisms with relevant data.

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Astragaloside IV (AS-IV) is a small molecular (C₄₁H₆₈O₁₄, molecular weight = 784) saponin and the main active component isolated from the traditional Chinese medicinal herb Astragalus membranaceus. It has been widely used in China for treating various diseases, including cardiovascular, hepatic, and renal disorders. This study provides evidence for its neuroprotective effects against cognitive impairments induced by transient cerebral ischemia and reperfusion in mice. The mechanism of action is attributed to its anti-inflammatory properties, specifically through the inhibition of the TLR4 signaling pathway (including both MyD88-dependent and -independent branches) and the suppression of NLRP3 inflammasome overactivation. These effects lead to reduced microglial activation, decreased production of pro-inflammatory cytokines (IL-1β, TNF-α), and attenuation of oxidative stress, ultimately ameliorating memory deficits. [1]

C41H68O14

784.9702

784.46

C, 62.73; H, 8.73; O, 28.54

84687-43-4

84687-43-4

13943297

White to off-white solid powder

1.4±0.1 g/cm3

895.7±65.0 °C at 760 mmHg

295-296ºC

495.5±34.3 °C

0.0±0.6 mmHg at 25°C

1.621

1.96

9

14

7

55

1460

21

C[C@@]12C[C@H](O)[C@H]([C@]3(CC[C@@H](C(O)(C)C)O3)C)[C@@]1(C)CC[C@@]13C[C@]41CC[C@H](O[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O)C(C)(C)[C@@H]4[C@H](C[C@@H]23)O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1

QMNWISYXSJWHRY-YLNUDOOFSA-N

InChI=1S/C41H68O14/c1-35(2)24(54-33-29(48)26(45)20(44)17-51-33)9-11-41-18-40(41)13-12-37(5)31(39(7)10-8-25(55-39)36(3,4)50)19(43)15-38(37,6)23(40)14-21(32(35)41)52-34-30(49)28(47)27(46)22(16-42)53-34/h19-34,42-50H,8-18H2,1-7H3/t19-,20+,21-,22+,23-,24-,25-,26-,27+,28-,29+,30+,31-,32-,33-,34+,37+,38-,39+,40-,41+/m0/s1

(2R,3R,4S,5S,6R)-2-[[(1S,3R,6S,8R,9S,11S,12S,14S,15R,16R)-14-hydroxy-15-[(2R,5S)-5-(2-hydroxypropan-2-yl)-2-methyloxolan-2-yl]-7,7,12,16-tetramethyl-6-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-9-pentacyclo[9.7.0.01,3.03,8.012,16]octadecanyl]oxy]-6-(hydroxymethyl)oxane-3,4,5-triol

Astragaloside IV

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (~127.4 mM)

Solubility in Formulation 1: 2.5 mg/mL (3.18 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (3.18 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 (3.18 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.)