Qiangxinyin formula protects against isoproterenol-induced cardiac hypertrophy.

Heart failure is a life-threatening cardiovascular disease and characterized by cardiac hypertrophy, inflammation and fibrosis. The traditional Chinese medicine formula Qiangxinyin (QXY) is effective for the treatment of heart failure while the underlying mechanism is not clear. This study aims to identify the active ingredients of QXY and explore its mechanisms protecting against cardiac hypertrophy. We found that QXY significantly protected against isoproterenol (ISO)-induced cardiac hypertrophy and dysfunction in zebrafish. Eight compounds, including benzoylmesaconine (BMA), atractylenolide I (ATL I), icariin (ICA), quercitrin (QUE), psoralen (PRN), kaempferol (KMP), ferulic acid (FA) and protocatechuic acid (PCA) were identified from QXY. PRN, KMP and icaritin (ICT), an active pharmaceutical ingredient of ICA, prevented ISO-induced cardiac hypertrophy and dysfunction in zebrafish. In H9c2 cardiomyocyte treated with ISO, QXY significantly blocked the calcium influx, reduced intracellular lipid peroxidative product MDA, stimulated ATP production and increased mitochondrial membrane potential. QXY also inhibited ISO-induced cardiomyocyte hypertrophy and cytoskeleton reorganization. Mechanistically, QXY enhanced the phosphorylation of Smad family member 2 (SMAD2) and myosin phosphatase target subunit-1 (MYPT1), and suppressed the phosphorylation of myosin light chain (MLC). In conclusion, PRN, KMP and ICA are the main active ingredients of QXY that protect against ISO-induced cardiac hypertrophy and dysfunction largely via the blockage of calcium influx and inhibition of mitochondrial dysfunction as well as cytoskeleton reorganization.

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.

Oyster peptide prevents the occurrence of exercise-hypogonadal male condition by improving the function of pituitary gonadal axis in male rats.

This study evaluates the protective role of oyster peptide (OP) on the occurrence of Exercise-Hypogonadal Male Condition. Male rats were given heavy-load swimming training and / or OP was supplemented for 6 consecutive weeks. After heavy-load training, sperm count, sperm viability and sperm motility in epididymis, testosterone in serum and testis, glutathione peroxidase (GSH-px) and androgen receptor (AR) in testis and mating times were remarkably decreased, malondialdehyde (MDA), capture latency and mating latency were significantly increased, mRNA expression of steroidogenic acute regulatory (StAR) and P450 cholesterol side-chain cleavage enzyme (P450scc) were obviously down-regulated, but serum follicle-stimulating hormone (FSH) and luteinising hormone (LH) were not statistically changed. Conversely, when OP was supplemented at heavy-load training, sperm count, sperm viability and sperm motility in epididymis, serum FSH, LH, testosterone, GSH-px, superoxide dismutase (SOD), testosterone, AR in testis and mating times were dramatically increased, while testicular MDA, capture latency and mating latency were significantly decreased, and mRNA expression of StAR, StARD7, P450scc and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were significantly up-regulated. In conclusion, heavy-load training causes testicular spermatogenic and steroidogenic disorders by enhancing the generation of reactive oxygen species (ROS), which can be protected by the co-administration of OP by enhancing the function of pituitary gonad axis and lowering ROS generation.

[Clinical Analysis of 66 Patients with Essential Thrombocytopenia].

OBJECTIVE: To investigate the clinical characteristics of essential thrombocytopenia (ET) patients with positive mutations including JAK2, CALR, MPL, or negative mutations. METHODS: A total of 66 newly diagnosed ET cases from January 2016 to December 2018 in Department of Hematology, Huaian No.1 People's Hospital affiliated to Nanjing Medical University were analyzed. Statistical analysis data included the patient's sex, age, symptoms, thrombosis and embolism events, spleen omegaly, platelet count (Plt), leukocyte (WBC) count, hemoglobin (Hb), fibrinogen (FIB), thrombus elastic diagram (TEG), serum potassium, blood glucose (GLU), lactate dehydrogenase (LDH), JAK2, CALR and MPL mutations, treatment options, and efficacy. RESULTS: All the patients were not MPL-positive, and divided in three groups: JAK2 mutation (46 cases, 69.7%), CALR mutation (9 cases, 13.6%) and gene negative mutation (11 cases, 16.7%) group. The average age of patients in the JAK2 mutation group was 63.2 years old, and significantly higher than that in the CALR mutation group (51.8 year) and gene negative group (50.2 year) (P＜0.05). Compared with the JAK2 mutation group and gene negative group, the CALR mutation group had lower WBC count (6.3×109/L vs 13.79×109/L) (P=0.003) (6.3×109/L vs 9.70×109/L) (P=0.009). Also the Hb level of patients in CALR mutation group was lower than the JAK2 mutation group (121.22 g/L vs 136.2 g/L) (P=0.036). However, there was higher tumor burden in the CALR mutation group, compared with the gene negative mutation group (300.11 U/L vs 227.4 U/L) (P=0. 033). There was no significant difference among the three groups, such as the Plt counts, serum potassium level, GLU level and FIB level (P＞0.05). In addition, thrombus and embolism appeared in 30.3% (20/66) cases. 18.2% (12/66) cases were complicated with hyperkalemia, which significantly correlated with Plt counts (r=0.518). TEG was performed in 34 patients, of which 41.2% (14/34) had abnormal TEG and 55.9% (19/34) were accompanied by Plt count ＞ 1 000 ×109/L, but there was no significant correlation between them (r=0.134). After routine clinical treatment, all the 66 cases achieved partial or complete hematological remission, but the disease usually repeated. Until now 4.5% (3/66) cases had been converted to myelofibrosis (MF) all with JAK2 mutation, but without advancing to acute myeloid leukemia. CONCLUSION: ET patients with JAK2 mutation have higher incidence, moreover were in older age. However, the patients with CALR mutations display lower WBC count and Hb level, but higher tumor burden. In short, the multiple gene mutations of ET showed different clinical features closely relates with the prognosis, thus providing guidance for the clinical diagnosis and treatment.

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.

Transient acid treatment cannot induce neonatal somatic cells to become pluripotent stem cells.

Currently, there are genetic- and chemical-based methods for producing pluripotent stem cells from somatic cells, but all of them are extremely inefficient.  However, a simple and efficient technique has recently been reported by Obokata et al (2014a, b) that creates pluripotent stem cells through acid-based treatment of somatic cells.  These cells were named stimulus-triggered acquisition of pluripotency (STAP) stem cells. This would be a major game changer in regenerative medicine if the results could be independently replicated. Hence, we isolated CD45 (+) splenocytes from five-day-old Oct4-GFP mice and treated the cells with acidified (pH 5.7) Hank's Balanced Salt Solution (HBSS) for 25 min, using the methods described by Obokata et al 2014c. However, we found that this method did not induce the splenocytes to express the stem cell marker Oct4-GFP when observed under a confocal microscope three to six days after acid treatment. qPCR analysis also confirmed that acid treatment did not induce the splenocytes to express the stemness markers Oct4, Sox2 and Nanog.  In addition, we obtained similar results from acid-treated Oct4-GFP lung fibroblasts. In summary, we have not been able to produce STAP stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment reported by Obokata et al (2014a, b, c).

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.

Fennel-like nanoaggregates based on polysaccharide derivatives and their application in drug delivery.

Biodegradable polymeric nanoparticles, which hold hierarchical morphologies, are of importance for controlled drug delivery. In this work, nanoparticles with fennel-like morphology were prepared from graft copolymers of hydroxyethylcellulose and poly(p-dioxane). The effect of microstructure parameters on the morphological transition of the nano-aggregates was studied and the nanoparticles were investigated as a carrier of hydrophobic drug. The morphology and drug release property of the nanoparticles were found to be related with the degree of substitution and molecular weight of graft chain of the copolymer.