Sodium tanshinone IIA sulfonate protects against hyperhomocysteine-induced vascular endothelial injury via activation of NNMT/SIRT1-mediated NRF2/HO-1 and AKT/MAPKs signaling in human umbilical vascular endothelial cells.

Homocysteine (Hcy) is one of the independent risk factors of cardiovascular disease. Sodium tanshinone IIA sulfonate (STS) is a hydrophilic derivate of tanshinone IIA which is the main active constitute of Chinese Materia Medica Salviae Miltiorrhizae Radix et Rhizoma, and exhibits multiple pharmacological activities. However, whether STS could prevent from Hcy-induced endothelial cell injury is unknown. We found that STS dramatically reversed Hcy-induced cell death concentration dependently in human umbilical vascular endothelial cells (HUVECs). STS ameliorated the endothelial cell cycle progression, proliferation and cell migratory function impaired by Hcy, which might be co-related to the inhibition of intracellular oxidative stress and mitochondrial dysfunction. STS also elevated the phosphorylation of AKT and MAPKs and protein expression of sirtuin1 (SIRT1), NRF2 and HO-1 which were suppressed by Hcy. The protective effect of STS against Hcy-induced endothelial cell toxicity was partially attenuated by PI3K, AKT, MEK, ERK, SIRT1, NRF2 and HO-1 inhibitors. Besides, knockdown of SIRT1 by its siRNA dramatically decreased the endothelial protective effect of STS accompanied with suppression of SIRT1, NRF2, HO-1 and phosphorylated AKT. The activation of AKT or NRF2 partially reversed SIRT1-knockdown impaired cyto-protective effect of STS against Hcy-induced cell injury. Furthermore, STS prevented from Hcy-induced intracellular nicotinamide N-methyltransferase (NNMT) reduction along with elevation of intracellular methylnicotinamide (MNA), and MNA enhanced STS protecting against Hcy induced endothelial death. Knockdown of NNMT reduced the protective effect of STS against Hcy induced endothelial cell injury. Collectively, STS presented potent endothelial protective effect against Hcy and the underlying molecular mechanisms were involved in the suppression of intracellular oxidative stress and mitochondria dysfunction by activation of AKT/MAPKs, SIRT1/NRF2/HO-1 and NNMT/MNA signaling pathways.

Tribulus terrestris L. extract ameliorates atherosclerosis by inhibition of vascular smooth muscle cell proliferation in ApoE-/- mice and A7r5 cells via suppression of Akt/MEK/ERK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis (AS) is one of major threatens of death worldwide, and vascular smooth muscle cell (VSMC) proliferation is an important characteristic in the progression of AS. Tribulus terrestris L. is a well-known Chinese Materia Medica for treating skin pruritus, vertigo and cardiovascular diseases in traditional Chinese medicine. However, its anti-AS activity and inhibition effect on VSMC proliferation are not fully elucidated. AIMS: We hypothesize that the furostanol saponins enriched extract (FSEE) of T. terrestris L. presents anti-AS effect by inhibition of VSMC proliferation. The molecular action mechanism underlying the anti-VSMC proliferation effect of FSEE is also investigated. MATERIALS AND METHODS: Apolipoprotein-E deficient (ApoE-/-) mice and rat thoracic smooth muscle cell A7r5 were employed as the in vivo and in vitro models respectively to evaluate the anti- AS and VSMC proliferation effects of FSEE. In ApoE-/- mice, the amounts of total cholesterol, triglyceride, low density lipoprotein and high density lipoprotein in serum were measured by commercially available kits. The size of atherosclerotic plaque was observed by hematoxylin & eosin staining. The protein expressions of α-smooth muscle actin (α-SMA) and osteopontin (OPN) in the plaque were examined by immunohistochemistry. In A7r5 cells, the cell viability and proliferation were tested by MTT and Real Time Cell Analysis assays. The cell migration was evaluated by wound healing assay. Propidium iodide staining followed by flow cytometry was used to analyze the cell cycle progression. The expression of intracellular total and phosphorylated proteins including protein kinase B (Akt) and mitogen-activated protein kinases (MAPKs), such as mitogen-activated extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), were detected by western blotting analysis. RESULTS: FSEE significantly reduced the area of atherosclerotic plaque in high-fat diet-fed ApoE-/- mice. And FSEE increased the protein expression level of α-SMA and decreased the level of OPN in atherosclerotic plaque, which revealed the inhibition of VSMC phenotype switching and proliferation. In A7r5 cells, FSEE suppressed fetal bovine serum (FBS) or oxidized low density lipoprotein (oxLDL)-triggered VSMC proliferation and migration in a concentration dependent manner. FSEE protected against the elevation of cell numbers in S phase induced by FBS or oxLDL and the reduction of cell numbers in G0/G1 phase induced by oxLDL. Moreover, the phosphorylation of Akt and MAPKs including MEK, ERK and JNK could be facilitated by FBS or oxLDL, while co-treatment of FSEE attenuated the phosphorylation of Akt induced by oxLDL as well as the phosphorylation of MEK and ERK induced by FBS. In addition, (25R)-terrestrinin B (JL-6), which was the main ingredient of FSEE, and its potential active pharmaceutical ingredients tigogenin (Tigo) and hecogenin (Heco) also significantly attenuated FBS or oxLDL-induced VSMC proliferation in A7r5 cells. CONCLUSION: FSEE presents potent anti- AS and VSMC proliferation activities and the underlying mechanism is likely to the suppression of Akt/MEK/ERK signaling. The active components of FSEE are JL-6 and its potential active pharmaceutical ingredients Tigo and Heco. So, FSEE and its active compounds may be potential therapeutic drug candidates for AS.

Tribulus terrestris L. Extract Protects against Lipopolysaccharide-Induced Inflammation in RAW 264.7 Macrophage and Zebrafish via Inhibition of Akt/MAPKs and NF-κB/iNOS-NO Signaling Pathways.

Inflammation response is a regulated cellular process and excessive inflammation has been recognized in numerous diseases, such as cardiovascular disease, neurodegenerative disease, inflammatory bowel disease, and cancer. Tribulus terrestris L. (TT), also known as Bai Jili in Chinese, has been applied in traditional Chinese medicine for thousands of years while its anti-inflammatory activity and underlying mechanism are not fully elucidated. Here, we hypothesize Tribulus terrestris L. extract (BJL) which presents anti-inflammatory effect, and the action mechanism was also investigated. We employed the transgenic zebrafish line Tg(MPO:GFP), which expresses green fluorescence protein (GFP) in neutrophils, and mice macrophage RAW 264.7 cells as the in vivo and in vitro model to evaluate the anti-inflammatory effect of BJL, respectively. The production of nitric oxide (NO) was measured by Griess reagent. The mRNA expression levels of inflammatory cytokines and inducible nitric oxide synthase (iNOS) were measured by real-time PCR, and the intracellular total or phosphorylated protein levels of NF-κB, Akt, and MAPKs including MEK, ERK, p38, and JNK were detected by western blot. We found that BJL significantly inhibited fin transection or lipopolysaccharide- (LPS-) induced neutrophil migration and aggregation in zebrafish in vivo. In mice macrophage RAW 264.7 cells, BJL ameliorated LPS-triggered excessive release of NO and transcription of inflammatory cytokine genes including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß). BJL also reduced the LPS-induced elevations of intracellular iNOS and nuclear factor kappa B (NF-κB) which mediate the cellular NO and inflammatory cytokine productions, respectively. Moreover, LPS dramatically increased the phosphorylation of Akt and MAPKs including MEK, ERK, p38, and JNK in RAW 264.7 cells, while cotreatment BJL with LPS suppressed their phosphorylation. Taken together, our data suggested that BJL presented potent anti-inflammatory effect and the underlying mechanism was closely related to the inhibition of Akt/MAPKs and NF-κB/iNOS-NO signaling pathways.

Pharmacological Activity, Pharmacokinetics, and Toxicity of Timosaponin AIII, a Natural Product Isolated From Anemarrhena asphodeloides Bunge: A Review.

Anemarrhena asphodeloides Bunge is a famous Chinese Materia Medica and has been used in traditional Chinese medicine for more than two thousand years. Steroidal saponins are important active components isolated from A. asphodeloides Bunge. Among which, the accumulation of numerous experimental studies involved in Timosaponin AIII (Timo AIII) draws our attention in the recent decades. In this review, we searched all the scientific literatures using the key word "timosaponin AIII" in the PubMed database update to March 2020. We comprehensively summarized the pharmacological activity, pharmacokinetics, and toxicity of Timo AIII. We found that Timo AIII presents multiple-pharmacological activities, such as anti-cancer, anti-neuronal disorders, anti-inflammation, anti-coagulant, and so on. And the anti-cancer effect of Timo AIII in various cancers, especially hepatocellular cancer and breast cancer, is supposed as its most potential activity. The anti-inflammatory activity of Timo AIII is also beneficial to many diseases. Moreover, VEGFR, X-linked inhibitor of apoptosis protein (XIAP), B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1), thromboxane (Tx) A2 receptor, mTOR, NF-κB, COX-2, MMPs, acetylcholinesterase (AChE), and so on are identified as the crucial pharmacological targets of Timo AIII. Furthermore, the hepatotoxicity of Timo AIII was most concerned, and the pharmacokinetics and toxicity of Timo AIII need further studies in diverse animal models. In conclusion, Timo AIII is potent as a compound or leading compound for further drug development while still needs in-depth studies.

Antiangiogenesis effect of timosaponin AIII on HUVECs in vitro and zebrafish embryos in vivo.

Timosaponin AIII (Timo AIII) is a natural steroidal saponin isolated from the traditional Chinese herb Anemarrhena asphodeloides Bge with proved effectiveness in the treatment of numerous cancers. However, whether Timo AIII suppresses tumor angiogenesis remains unclear. In the present study, we investigated the antiangiogenesis effects of Timo AIII and the underlying mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish embryos in vivo. We showed that treatment with Timo AIII (0.5-2 µM) partially disrupted the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) growth in transgenic zebrafish Tg(fli-1a: EGFP)y1. Timo AIII (0.5-4 µM) dose-dependently inhibited VEGF-induced proliferation, migration, invasion, and tube formation of HUVECs, but these inhibitory effects were not due to its cytotoxicity. We further demonstrated that Timo AIII treatment significantly suppressed the expression of VEGF receptor (VEGFR) and the phosphorylation of Akt, MEK1/2, and ERK1/2 in HUVECs. Timo AIII treatment also significantly inhibited VEGF-triggered phosphorylation of VEGFR2, Akt, and ERK1/2 in HUVECs. Moreover, we conducted RNA-Seq and analyzed the transcriptome changes in both HUVECs and zebrafish embryos following Timo AIII treatment. The coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc., which was consistent with the antiangiogenesis effects of Timo AIII in HUVECs and zebrafish embryos. We conclude that the antiangiogenesis effect of Timo AIII is mediated through VEGF/PI3K/Akt/MAPK signaling cascade; Timo AIII potentially exerts antiangiogenesis effect in cancer treatment.

Pro-angiogenesis effect and transcriptome profile of Shuxinyin formula in zebrafish.

BACKGROUND: Angiogenesis plays a critical role in ischemia disease like coronary heart disease. Shunxinyin formula has been developed for treating coronary heart disease according to the principle of traditional Chinese medicine while its underlying mechanism is not fully elucidated. PURPOSE: Here, we hypothesize Shuxinyin formula could promote angiogenesis and microcirculation, and the underlying mechanism is also investigated. METHODS: We established the chemical profile of Shuxinyin (SXY) extract utilizing a UHPLC-Q/Exactive analysis system and evaluated its pro-angiogenesis effect in zebrafish model. The underlying mechanisms were investigated by combination of pharmacological experiments with transcriptome analysis in zebrafish. Zebrafish treated with VEGF was served as the positive control in present study. RESULTS: We found SXY significantly enhanced the sub-intestinal vessel plexus (SIVs) growth in zebrafish. Co-treatment and post-treatment SXY attenuated VEGF receptor tyrosine kinase inhibitor II (VRI)-induced deficiency of intersegmental vessels (ISVs) in a concentration dependent manner. Post-treatment VEGF, which is a well-known angiogenesis driver, also partially ameliorated VRI-induced ISVs deficiency. In addition, SXY inhibited the down-regulation of VEGF receptors, including kdr, flt1 and kdrl, induced by VRI in zebrafish. The pro-angiogenesis effect of SXY on VRI-induced ISVs deficiency was suppressed by PI3K and JNK inhibitors, and Akt inhibitor abolished the pro-angiogenesis effect of SXY. The transcriptome profile of SXY preventing from VRI-induced vascular growth deficiency revealed that the underlying mechanisms were also co-related to cell junction, apoptosis and autophagy. CONCLUSION: We could conclude that SXY presented pro-angiogenesis effect and the action mechanisms were involved in VEGF/PI3K/Akt/MAPK signaling pathways, cell junction, apoptosis and autophagy.

Endothelial-Dependent and Independent Vascular Relaxation Effect of Tetrahydropalmatine on Rat Aorta.

Tetrahydropalmatine (THP) is an active natural alkaloid isolated from Corydalis yanhusuo W.T. Wang which has been widely used for treating pain and cardiovascular disease in traditional Chinese medicine. Previous studies suggested THP have various pharmacological effects in neural and cardio tissue while the vascular reactivity of THP was not fully established. The present study found that THP relaxed rat aorta which contracted by phenylephrine (Phe), KCl, and U46619. The vascular relaxation effect of THP was partially attenuated by PI3K inhibitor wortmannin, Akt inhibitor IV, endothelial nitric oxide synthetase (eNOS) inhibitor L-NAME, guanylate cyclase inhibitors and the mechanical removal of endothelium. Also, the eNOS substrate L-arginine reversed the inhibition effect of L-NAME on THP-induced vascular relaxation. THP also induced intracellular NO production in human umbilical vein endothelial cells. However, Pre-incubation with ß-adrenergic receptor blocker propranolol, angiotensin II receptor 1 (AT1) inhibitor losartan, angiotensin II receptor 2 (AT1) inhibitor PD123319 or angiotensin converting enzyme inhibitor enalapril enhanced the vascular relaxation effect of THP. THP did not affect the angiotensin II induced vascular contraction. Cyclooxygenase-2 (COX2) inhibitor indomethacin did not affect the vascular relaxation effect of THP. Furthermore, pre-treatment THP attenuated KCl and Phe induced rat aorta contraction in standard Krebs solution. In Ca2+ free Krebs solution, THP inhibited the Ca2+ induced vascular contraction under KCl or Phe stress and reduced KCl stressed Ca2+ influx in rat vascular smooth muscle cells. THP also inhibited intracellular Ca2+ release induced vascular contraction by blocking Ryr or IP3 receptors. In addition, the voltage-dependent K+ channel (Kv) blocker 4-aminopyridine, ATP-sensitive K+ channel (KATP) blocker glibenclamide and inward rectifying K+ channel blocker BaCl2 attenuated THP induced vascular relaxation regardless of the Ca2+-activated K+ channel (KCa) blocker tetraethylammonium. Thus, we could conclude that THP relaxed rat aorta in an endothelium-dependent and independent manner. The underlying mechanism of THP relaxing rat aorta involved PI3K/Akt/eNOS/NO/cGMP signaling path-way, Ca2+ channels and K+ channels rather than COX2, ß-adrenergic receptor and renin-angiotensin system (RAS). These findings indicated that THP might be a potent treatment of diseases with vascular dysfunction like hypertension.

Potent anti-angiogenic activity of B19--a mono-carbonyl analogue of curcumin.

AIM: The compound B19 (C21H22O5) is a newly synthesized, mono-carbonyl analog of curcumin that has exhibited potential antitumor effects. This present study was performed to identify the anti-angiogenic activity of this compound. METHODS AND RESULTS: B19 inhibited migration and tube formation of human umbilical vein endothelial cells, and arrested microvessel outgrowth from rat aortic rings. In addition, B19 suppressed the neovascularization of chicken chorioallantoic membrane. Mechanistic studies revealed that B19 suppressed the downstream protein kinase activation of vascular endothelial growth factor (VEGF) by decreasing phosphorylated forms of serine/threonine kinase Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase, with or without stimulating vascular endothelial growth factor (VEGF). CONCLUSIONS: B19 exerted anti-angiogenic activity in vitro and ex vivo, which suggests that it merits further investigation as a promising anticancer angiogenesis compound.