Garcinone E suppresses breast cancer growth and metastasis by modulating tumor-associated macrophages polarization via STAT6 signaling.

Cancer metastasis remains the most common cause of death in breast cancer patients. Tumor-associated macrophages (TAMs) are a novel therapeutic target for the treatment of metastatic breast cancer. Despite the good anti-cancer activity of garcinone E (GE), there are no reports on its therapeutic effects on breast cancer metastasis. The objective of this study was to examine the anti-cancer effects of GE on metastatic breast cancer. RAW 264.7 and THP-1 cells were polarized to M2 macrophages by IL-4/IL-13 in vitro. A 4T1 mouse breast cancer model and the tail vein breast cancer metastasis model were used to explore the effect of GE on breast cancer growth and metastasis in vivo. In vitro studies showed that GE dose-dependently suppressed IL-4 + IL-13-induced expression of CD206 in both RAW 264.7 cells and differentiated THP-1 macrophages. However, GE did not affect the LPS + IFN-γ-induced polarization to the M1-like macrophages in vitro. GE inhibited the expression of the M2 macrophage specific genes in RAW 264.7 cells, and simultaneously impaired M2 macrophage-induced breast cancer cell proliferation and migration, and angiogenesis. In animal studies, GE significantly suppressed tumor growth, angiogenesis, and lung metastasis in 4T1 tumor-bearing mice, without causing toxicity. In both tumor and lung tissues, the proportion of M2-like TAMs was significantly decreased while the proportion of M1-like TAMs was markedly increased by GE treatment. Mechanistically, GE inhibited phosphorylation of STAT6 in vitro and in vivo. Our results demonstrate for the first time that GE suppresses breast cancer growth and pulmonary metastasis by modulating M2-like macrophage polarization through the STAT6 signaling pathway.

Study on the mechanism of Tong-Qiao-Huo-Xue decoction regulating apoptosis via ASK1/MKK4/JNK pathway in MCAO/R rats.

BACKGROUND: Activation of blood stasis is a crucial aspect of stroke treatment, and the Tong-Qiao-Huo-Xue-Decoction (TQHXD) formula is commonly utilized for this purpose. However, the mechanism underlying the protective effects of TQHXD against cerebral ischemia-reperfusion (I/R) injury is unclear. PURPOSE: Identification of the TQHXD components responsible for its protective effects and determination of their mode of action against cerebral I/R injury. METHODS: Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were carried out to determine the active aspects of TQHXD. The active components and targets of TQHXD were looked up in the TCMSP and HERB databases; the Genecards, OMIM, TTD, and DrugBank databases were used to identify targets related to cerebral infarction; and the intersecting targets were obtained. The drug-ingredient-target-disease network and PPI network were subsequently built using Cytoscape 3.7.1 and STRING websites. Autodock VINA was used to perform molecular docking between the core target ASK1 and the active components of TQHXD detected by HPLC and GC. After successfully creating a rat model of middle cerebral artery occlusion (MCAO), the therapeutic effect of TQHXD was observed using triphenyltetrazolium and hematoxylin-eosin staining. We used Tunel-NeuN staining and transmission electron microscopy (TEM) to quantify hippocampal apoptosis. RT-qPCR and western blotting were used to detect protein and mRNA expression, respectively. RESULTS: HPLC and GC identified six active ingredients. Network pharmacology analyses were performed to test 66 intersection targets, including ASK1, MKK4, and JNK. Ferulic acid, HSYA, ligustilide, paeoniflorin, and muscone all displayed high binding affinity with ASK1 in molecular docking studies. The neuroprotective effects of TQHXD in I/R rats were demonstrated in the experimental models. In comparison with the model group, TQHXD decreased the apoptosis rate and reduced the protein levels of p-ASK1, caspase 3, p-MKK4, CytC, p-c-Jun, Bax/Bcl-2, and p-JNK, while considerably increasing the mRNA levels of Bcl-2 and decreasing those of Bax. CONCLUSION: By controlling the ASK1/MKK4/JNK pathway, TQHXD protects neurons from I/R damage and prevents apoptosis. Thus, TQHXD may be effective for the treatment of ischemic stroke. And the mechanism behind these therapeutic actions of TQHXD is supported by this research.

Tong-Qiao-Huo-Xue decoction activates PI3K/Akt/mTOR pathway to reduce BMECs autophagy after cerebral ischemia/reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Tong-Qiao-Huo-Xue Decoction (TQHXD) is a traditional classic Chinese Medicinal Formula (CMF) used for clinical treatment of ischemic stroke. TQHXD leads to improvement in the symptoms of the acute period of cerebral infarction and recovery period after stroke. Our previous studies also showed that TQHXD produced a significant protective effect on the brain after cerebral ischemia-reperfusion (I/R) injury. It is reported that autophagy is closely related to ischemic brain injury; however, the functional contribution of TQHXD to brain microvascular endothelial cell (BMEC) autophagy and its underlying mechanism remains unclear. AIM OF THE STUDY: The purpose of this study was to investigate the effects and mechanism of TQHXD in inhibiting cerebral ischemia-induced endothelial autophagy. MATERIALS AND METHODS: The high-performance liquid chromatography (HPLC) fingerprint of the chemical constituents from TQHXD was established for the quality control, and the Longa method was used to evaluate the efficacy of TQHXD in rats with middle cerebral artery occlusion (MCAO). The expression of LC3 was determined by immunofluorescence double staining. To evaluate the protective effects of TQHXD-containing cerebrospinal fluid (CSF) on BMECs injured by oxygen-glucose deprivation and reperfusion, cell survival rate was determined using the CCK-8 assay and cell apoptosis was determined by fluorescein isothiocyanate (FITC)-Annexin V/PI. Autophagy was detected using transmission electron microscopy. RESULTS: The results showed that TQHXD-CSF significantly ameliorated oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury in BMECs. Confocal microscopy and Western blot results showed that TQHXD-CSF reduced autophagy-related protein expression and autophagosome number. The results of the western blotting indicated that TQHXD-CSF caused a marked increase in the phosphorylation of protein kinase B and phosphoinsotide-3 kinase (Akt/p-Akt and PI3K/p-PI3K, respectively) and their expression levels were down-regulated after treatment with pathway inhibitor, ZSTK474. Furthermore, in a MCAO model in rats, TQHXD markedly increased p-PI3K, p-Akt and p-mTOR, whereas the autophagy related proteins decreased. CONCLUSIONS: Taken together, these findings demonstrate that TQHXD protects against ischemic insult by inhibiting autophagy through the regulation of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway and that TQHXD may have therapeutic value for protecting BMECs from cerebral ischemia.

Ligustilide ameliorates hippocampal neuronal injury after cerebral ischemia reperfusion through activating PINK1/Parkin-dependent mitophagy.

BACKGROUND: Mitophagy plays a critical role in cerebral ischemia/reperfusion by timely removal of dysfunctional mitochondria. In mammals, PINK1/Parkin is the most classic pathway mediating mitophagy. And the activation of PINK1/Parkin mediated mitophagy exerts neuroprotective effects during cerebral ischemia reperfusion injury (CIRI). Ligustilide (LIG) is a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis (Oliv.) diels that exerts neuroprotective activity after cerebral ischemia reperfusion injury (CIRI). However, it still remains unclear whether LIG could attenuates cerebral ischemia reperfusion injury (CIRI) through regulating mitophagy mediated by PINK1/Parkin. PURPOSE: To explore the underlying mechanism of LIG on PINK1/Parkin mediated mitophagy in the hippocampus induced by ischemia reperfusion. METHODS: This research used the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model and oxygen-glucose deprivation and reperfusion (OGD/R) as in vitro model. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of LIG in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP) and activities of Na+-K+-ATPase were detected to reflect mitochondrial function. Moreover, transmission electron microscope (TEM) and fluorescence microscope were used to observe mitophagy and the western blot was performed to explore the changes in protein expression in PINK1/Parkin mediated mitophagy. Finally, exact mechanism between neuroprotection of LIG and mitophagy mediated by PINK1/Parkin was explored by cell transfection. RESULTS: The results show that LIG improved mitochondrial functions by mitophagy enhancement in vivo and vitro to alleviate CIRI. Whereas, mitophagy enhanced by LIG under CIRI is abolished by PINK1 deficiency and midivi-1, a mitochondrial division inhibitor which has been reported to have the function of mitophagy, which could further aggravate the ischemia-induced brain damage, mitochondrial dysfunction and neuronal injury. CONCLUSION: LIG could ameliorate the neuronal injury against ischemia stroke by promoting mitophagy via PINK1/Parkin. Targeting PINK1/Parkin mediated mitophagy with LIG treatment might be a promising therapeutic strategy for ischemia stroke.

Ligustilide attenuates ischemic stroke injury by promoting Drp1-mediated mitochondrial fission via activation of AMPK.

BACKGROUND: Ischemic stroke is a major global cause of death and permanent disability. Studies have suggested that mitochondria play a critical role in maintaining cellular energy homeostasis and inevitably involved in neuronal damage during cerebral ischemic. Ligustilide is the main active ingredient of Angelica sinensis and Ligusticum chuanxiongs with neuroprotective activity. PURPOSE: These study sought to exlopre the role of LIG in improving mitochondrial function and the relationship between LIG induced mitochondrial fission and mitophagy in ischemic stroke. METHODS: Cerebral I/R injury was established by the model of Oxygen-glucose deprivation/reperfusion (OGD/R) in HT22 cells and middle cerebral artery occlusion (MCAO) in rats. Mitochondrial functions of were detected by flow cytometry and immunofluorescence, and mitochondrial fission were detected by western blots. Furthermore, we studied the role of AMPK pathway in the neuroprotective effect of LIG. RESULTS: LIG treatment significantly increased the MMP and ATP production, decreased the reactive oxygen species (ROS) generation and Ca2+ overload, and further induced mitochondrial fission and mitophagy. Moreover, we found that blocking mitochondrial fission by mdivi-1 resulted in accumulation of damaged mitochondria mainly through selectively blocking mitophagy, thereby inhibiting viability of HT-22 cells after OGD/R. Also, Drp-1 inhibitor mdivi-1 increased the infarct volume and aggravated the neurological deficits after MCAO operation in vivo. Additionally, LIG triggered AMP-activated protein kinase (AMPK) pathway. AMPKα2 knockdown attenuated LIG-induced mitochondrial fission through inhibiting the expression of Drp1 and Fis1, and led to nerve cell apoptosis. CONCLUSION: Our study indicate that LIG attenuated the injury of ischemic stroke by improving mitochondrial function and highlight the critical role of LIG in the regulation of LIG-induced mitochondrial fission and mitophagy via an AMPK-dependent manner. These findings indicate that LIG protects nerve damage against ischemic stroke by inducing Drp1-mediated mitochondrial fission via activation of AMPK signaling pathway in vivo and in vitro.

Chemical composition and protective effect of cerebrospinal fluid of Dan-Deng-Tong-Nao capsules on brain microvascular endothelial cells injured by OGD/R.

ETHNOPHARMACOLOGICAL RELEVANCE: Dan-Deng-Tong-Nao Capsules (DDTNC) is a Chinese patent medicine and has been used in treating cerebral ischemic stroke (IS) for a long time in China, protection of brain microvascular endothelial cells (BMECs) is the main treatment strategy. But the holistic chemical information and potential bioactive components of DDTNC on protecting BMECs and its underlying mechanism is still unclear. AIM OF THE STUDY: To identify the active ingredients of DDTNC and to explore the protective effects of DDTNC on BMECs associated with Wnt/ß-catenin pathway. MATERIALS AND METHODS: The components of DDTNC and cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF) were detected by High performance liquid chromatography combined with Diode array detector (HPLC-DAD) and Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), respectively. The experiment rat model was established with middle cerebral artery occlusion (MCAO), the therapeutic effects of DDTNC were assessed by Longa assay and TTC staining. The cerebral micro vessel density was determined by immunofluorescence staining. The injured BMECs caused by oxygen-glucose deprivation and reperfusion (OGD/R) was used to evaluate the protective effect of cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF). The cell survival rate was detected by the method of CCK-8, the intracellular Ca2+ and reactive oxygen species (ROS) was estimated by Fluo-3. Moreover, the proteins of Bax, Bcl-2, Wnt, ß-catenin, GSK-3ß was determined by Western blotting. RESULTS: The RSD values of all methodological studies were less than 3.0%. A total of 20 compounds were detected under the optimized HPLC-DAD chromatographic condition. In the UPLC-Q-TOF-MS negative mode, peak 1 and peak 2 were detecteted in DDTNC-CSF and was identified as Danshensu and Puerarin, respectively. In the UPLC-Q-TOF-MS positive mode, peak 1 and peak 3 were detecteted in DDTNC-CSF and was identified as Danshensu and Scutellarin, respectively. DDTNC significantly decreased the Longa values and infarct volume and significantly increased the cerebral microvessel density of the MCAO rats. The accumulation of intracellular Ca2+ and ROS in BMECs injured by OGD/R decreased significantly in DDTNC-CSF group. The expression of Bcl-2, ß-catenin, wnt-1 was upregulated by DDTNC-CSF and the level of Bax and GSK3ß could be downregulated by DDTNC-CSF. CONCLUSION: The present study provided a scientific basis for revealing the mechanism of DDTNC in the treatment of IS and DDTNC is expected to be an effective drug for the treatment of IS.

Hydroxysafflor yellow A alleviates cerebral ischemia reperfusion injury by suppressing apoptosis via mitochondrial permeability transition pore.

BACKGROUND: Mitochondria are key cellular organelles that are essential for cell fate decisions. Hydroxysafflor yellow A (HSYA) has displayed an impressively essential role in protection of cerebral ischemia/reperfusion (I/R). However, the mitochondrial effect of HSYA on Brain Microvascular Endothelial Cells (BMECs) under I/R remains to be largely unclear. PURPOSE: To evaluate the protective effects of HSYA-mediated mitochondrial permeability transition pore (mPTP) on cerebral I/R injury and its mechanism. METHODS: Cerebral I/R injury was established by the model of Middle cerebral artery occlusion (MCAO) in rats. Furthermore, to further clarify the relevant mechanism of HSYA's effects on mPTP, inhibition of extracellular regulated protein kinases (ERK) with U0126 and transfect with Cyclophilin D (CypD) SiRNA to reversely verified whether the protective effects of HSYA were exerted by regulating the Mitogen-activated protein kinase kinase (MEK)/ERK/CypD pathway. RESULTS: HSYA treatment significantly increased BMECs viability, decreased the generation of ROS, opening of mPTP and translocation of cytochrome c after OGD/R. In addition to inhibited CypD, HSYA potentiated MEK and increased phosphorylation of ERK expression in BMECs, inhibited apoptosis mediated by mitochondrial. Notably, HSYA also significantly ameliorated neurological deficits and decreased the infarct volume in rats. CONCLUSION: HSYA reduced the CytC export from mitochondrial by inhibited the open of mPTP via MEK/ERK/CypD pathway, contributing to the protection of I/R. Thus, our study not only revealed novel mechanisms of HSYA for its anti-I/R function, but also provided a template for the design of novel mPTP inhibitor for the treatment of various mPTP-related diseases.

Vitamin D deficiency promotes large rupture-prone abdominal aortic aneurysms and cholecalciferol supplementation limits progression of aneurysms in a mouse model.

Vitamin D deficiency has been associated with human abdominal aortic aneurysm (AAA); however, its role in AAA pathogenesis is unclear. The aim of the present study was to investigate the effect of vitamin D deficiency on AAA development and examine if administering cholecalciferol (CCF) could limit growth of established AAA within the angiotensin-II (AngII) infused apolipoprotein E-deficient mouse model. Mice were rendered vitamin D deficiency through dietary restriction and during AngII infusion developed larger AAAs as assessed by ultrasound and ex vivo morphometry that ruptured more commonly (48% vs. 19%; P=0.028) than controls. Vitamin D deficiency was associated with increased aortic expression of osteopontin and matrix metalloproteinase-2 and -9 than controls. CCF administration to mice with established aortic aneurysms limited AAA growth as assessed by ultrasound (P<0.001) and ex vivo morphometry (P=0.036) and reduced rupture rate (8% vs. 46%; P=0.031). This effect was associated with up-regulation of circulating and aortic sclerostin. Incubation of human aortic smooth muscle cells with 1,25-dihyroxyvitamin D3 (the active metabolite of vitamin D) for 48 h induced up-regulation of sclerostin (P<0.001) and changed the expression of a range of other genes important in extracellular matrix remodeling. The present study suggests that vitamin D deficiency promotes development of large rupture-prone aortic aneurysms in an experimental model. CCF administration limited both growth and rupture of established aneurysms. These effects of vitamin D appeared to be mediated via changes in genes involved in extracellular matrix remodeling, particularly sclerostin.

Pro-angiogenic effects of Ilexsaponin A1 on human umbilical vein endothelial cells in vitro and zebrafish in vivo.

BACKGROUND: Ilexsaponin A1 is the major bioactive ingredient of Ilex pubescens Hook. et Arn. This plant has been conventionally used in Traditional Chinese Medicine for the treatment of cardiovascular diseases including stroke, coronary arterial disease, and peripheral vascular diseases. PURPOSE: To investigate the pro-angiogenic effect of Ilexsaponin A1 and its mechanism of action. STUDY DESIGN: Human umbilical vein endothelial cells (HUVECs) and transgenic zebrafish Tg(fli1:EGFP) were employed as an in vitro and in vivo model respectively. METHODS: Pro-angiogenic effects of Ilexsaponin A1 were examined by assessing endothelial cell proliferation, migration, invasion and tube formation. The mechanism of pro-angiogenic effects was investigated by measuring the expression level of various signalling proteins. Furthermore, vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor II (VRI)-induced vascular insufficient transgenic zebrafish model was used to confirm the results of the HUVECs results in vivo. RESULTS: Ilexsaponin A1 significantly promoted cell proliferation, migration, invasion and tube formation in HUVECs, and rescued blood vessel loss in VRI-induced vascular insufficient zebrafish. Ilexsaponin A1 upregulated p-Akt, p-mTOR, p-Src, p-FAK, p-MEK, and p-Erk1/2 in HUVECs. CONCLUSION: This study showed that Ilexsaponin A1 exhibits pro-angiogenic activity in HUVECs and VRI-induced vascular insufficient zebrafish, probably by activating Akt/mTOR, MAPK/ERK and Src- and FAK-dependent signalling pathways. The findings suggest that Ilexsaponin A1 and probably I. pubescens, a major source of Ilexsaponin A1, could be developed as a potential therapeutic agent for preventing or treating cardiovascular diseases and/or other diseases related to vascular insufficiency.

Angiogenesis in Ischemic Stroke and Angiogenic Effects of Chinese Herbal Medicine.

Stroke is one of the major causes of death and adult disability worldwide. The underlying pathophysiology of stroke is highly complicated, consisting of impairments of multiple signalling pathways, and numerous pathological processes such as acidosis, glutamate excitotoxicity, calcium overload, cerebral inflammation and reactive oxygen species (ROS) generation. The current treatment for ischemic stroke is limited to thromolytics such as recombinant tissue plasminogen activator (tPA). tPA has a very narrow therapeutic window, making it suitable to only a minority of stroke patients. Hence, there is great urgency to develop new therapies that can protect brain tissue from ischemic damage. Recent studies have shown that new vessel formation after stroke not only replenishes blood flow to the ischemic area of the brain, but also promotes neurogenesis and improves neurological functions in both animal models and patients. Therefore, drugs that can promote angiogenesis after ischemic stroke can provide therapeutic benefits in stroke management. In this regard, Chinese herbal medicine (CHM) has a long history in treating stroke and the associated diseases. A number of studies have demonstrated the pro-angiogenic effects of various Chinese herbs and herbal formulations in both in vitro and in vivo settings. In this article, we present a comprehensive review of the current knowledge on angiogenesis in the context of ischemic stroke and discuss the potential use of CHM in stroke management through modulation of angiogenesis.

Zebrafish models of cardiovascular diseases and their applications in herbal medicine research.

The zebrafish (Danio rerio) has recently become a powerful animal model for cardiovascular research and drug discovery due to its ease of maintenance, genetic manipulability and ability for high-throughput screening. Recent advances in imaging techniques and generation of transgenic zebrafish have greatly facilitated in vivo analysis of cellular events of cardiovascular development and pathogenesis. More importantly, recent studies have demonstrated the functional similarity of drug metabolism systems between zebrafish and humans, highlighting the clinical relevance of employing zebrafish in identifying lead compounds in Chinese herbal medicine with potential beneficial cardiovascular effects. This paper seeks to summarise the scope of zebrafish models employed in cardiovascular studies and the application of these research models in Chinese herbal medicine to date.

Evaluation of anti-oxidant capacity of root of Scutellaria baicalensis Georgi, in comparison with roots of Polygonum multiflorum Thunb and Panax ginseng CA Meyer.

In Chinese communities, regular consumption of Chinese-medicated diets (CMD) (usually in the form of soup) is a traditional practice to promote health and prevent disease development. The overall improvement of health conditions is believed to be correlated with the anti-oxidant potentials of these herbs. Huangqin, roots of Scutellaria baicalensis Georgi (Lamiaceae), is one of the herbs commonly used in CMD. In this study, the anti-oxidant capacities of Huangqin extracts (water, ethanol and ether extracts) were evaluated and compared to commonly used CMD herbs, Heshouwu, roots of Polygonum multiflorum Thunb (Polygonaceae) and Renshen (or Ginseng), roots of Panax ginseng CA Meyer (Araliaceae). The anti-oxidant capacities were measured by using both cell-free assay [ferric reducing/anti-oxidant power (FRAP)] and biological methods [2,2'-azobis-(2-amidinopropane) (AAPH)-induced haemolysis assay and H(2)O(2)-induced cell damage on H9C2 cells]. Additionally, the total phenolic content was measured using Folin-Ciocalteu methods. Water extract of Huangqin has the highest anti-oxidant activities compared to the ethanol and ether extracts. A positive relationship between the anti-oxidant effects and total phenolic contents of extracts was demonstrated. This shows that Huangqin could be an effective dietary anti-oxidant that can be consumed regularly as a functional food for the prevention of oxidant/free radical-related diseases.

Formononetin, an isoflavone, relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways.

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 microM) and methylene blue (10 microM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS(Ser1177) protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 microM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 microM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 microM, an estrogen receptor (ER alpha/ER beta) antagonist) or mifepristone (10 microM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K(+) (K(ATP)) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK(Ca) and K(ATP) channels.