Medicinal plants and natural products for treating overactive bladder.

BACKGROUND: Overactive bladder (OAB) presents a high prevalence of 16-18% worldwide. The pathophysiology of OAB is still poorly understood while effective therapy or countermeasure are very limited. On the other hand, medicinal plants and herbal remedies have been utilized for treating lower urinary tract symptoms (LUTS) in both Eastern and Western cultures since ancient times. In recent years, accumulating progress has also been made in OAB treatment research by using medicinal plants. METHODS: Relevant literature on the studies of medicinal plants and herbs used to treat OAB was reviewed. The medicinal plants were summarized and categorized into two groups, single-herb medications and herbal formulations. RESULTS: The present review has summarized current understanding of OAB's pathophysiology, its available treatments and new drug targets. Medicinal plants and natural products which have been used or have shown potential for OAB treatment were updated and comprehensively categorized. Studies on a wide variety of medicinal plants showed promising results, although only a few phytochemicals have been isolated and identified. Until now, none of these herbal compounds have been further developed into clinical therapeutics for OAB. CONCLUSIONS: This review provides the basis for discovering and designing new phytopharmaceutical candidates with effective and well-tolerated properties to treat OAB. Increasing evidences indicate new strategies with alternative herbal treatment for OAB have high efficacy and safety, showing great promise for their clinical use. Future studies in a rigorously designed controlled manner will be beneficial to further support the eligibility of herbal treatment as OAB therapeutics.

Evaluation of therapeutic effects of tetramethylpyrazine nitrone in Alzheimer's disease mouse model and proteomics analysis.

The pathophysiology of Alzheimer's disease (AD) is multifactorial with characteristic extracellular accumulation of amyloid-beta (Aß) and intraneuronal aggregation of hyperphosphorylated tau in the brain. Development of disease-modifying treatment for AD has been challenging. Recent studies suggest that deleterious alterations in neurovascular cells happens in parallel with Aß accumulation, inducing tau pathology and necroptosis. Therefore, therapies targeting cellular Aß and tau pathologies may provide a more effective strategy of disease intervention. Tetramethylpyrazine nitrone (TBN) is a nitrone derivative of tetramethylpyrazine, an active ingredient from Ligusticum wallichii Franchat (Chuanxiong). We previously showed that TBN is a potent scavenger of free radicals with multi-targeted neuroprotective effects in rat and monkey models of ischemic stroke. The present study aimed to investigate the anti-AD properties of TBN. We employed AD-related cellular model (N2a/APPswe) and transgenic mouse model (3×Tg-AD mouse) for mechanistic and behavioral studies. Our results showed that TBN markedly improved cognitive functions and reduced Aß and hyperphosphorylated tau levels in mouse model. Further investigation of the underlying mechanisms revealed that TBN promoted non-amyloidogenic processing pathway of amyloid precursor protein (APP) in N2a/APPswe in vitro. Moreover, TBN preserved synapses from dendritic spine loss and upregulated synaptic protein expressions in 3×Tg-AD mice. Proteomic analysis of 3×Tg-AD mouse hippocampal and cortical tissues showed that TBN induced neuroprotective effects through modulating mitophagy, MAPK and mTOR pathways. In particular, TBN significantly upregulated PINK1, a key protein for mitochondrial homeostasis, implicating PINK1 as a potential therapeutic target for AD. In summary, TBN improved cognitive functions in AD-related mouse model, inhibited Aß production and tau hyperphosphorylation, and rescued synaptic loss and neuronal damage. Multiple mechanisms underlie the anti-AD effects of TBN including the modulation of APP processing, mTOR signaling and PINK1-related mitophagy.

Emerging roles of astrocytes in blood-brain barrier disruption upon amyloid-beta insults in Alzheimer's disease.

Blood-brain barrier disruption occurs in the early stages of Alzheimer's disease. Recent studies indicate a link between blood-brain barrier dysfunction and cognitive decline and might accelerate Alzheimer's disease progression. Astrocytes are the most abundant glial cells in the central nervous system with important roles in the structural and functional maintenance of the blood-brain barrier. For example, astrocytic coverage around endothelial cells with perivascular endfeet and secretion of homeostatic soluble factors are two major underlying mechanisms of astrocytic physiological functions. Astrocyte activation is often observed in Alzheimer's disease patients, with astrocytes expressing a high level of glial fibrillary acid protein detected around amyloid-beta plaque with the elevated phagocytic ability for amyloid-beta. Structural alterations in Alzheimer's disease astrocytes including swollen endfeet, somata shrinkage and possess loss contribute to disruption in vascular integrity at capillary and arterioles levels. In addition, Alzheimer's disease astrocytes are skewed into proinflammatory and oxidative profiles with increased secretions of vasoactive mediators inducing endothelial junction disruption and immune cell infiltration. In this review, we summarize the findings of existing literature on the relevance of astrocyte alteration in response to amyloid pathology in the context of blood-brain barrier dysfunction. First, we briefly describe the physiological roles of astrocytes in blood-brain barrier maintenance. Then, we review the clinical evidence of astrocyte pathology in Alzheimer's disease patients and the preclinical evidence in animal and cellular models. We further discuss the structural changes of blood-brain barrier that correlates with Alzheimer's disease astrocyte. Finally, we evaluate the roles of soluble factors secreted by Alzheimer's disease astrocytes, providing potential molecular mechanisms underlying blood-brain barrier modulation. We conclude with a perspective on investigating the therapeutic potential of targeting astrocytes for blood-brain barrier protection in Alzheimer's disease.

Canthin-6-one (CO) from Picrasma quassioides (D.Don) Benn. ameliorates lipopolysaccharide (LPS)-induced astrocyte activation and associated brain endothelial disruption.

BACKGROUND: Canthin-6-one (CO) is an active ingredient found in Picrasma quassioides (D.Don) Benn. (PQ) that displays various biological activities including anti-inflammatory properties. Several studies reported PQ displayed neuroprotective activities, but its effects on astrocytes have not yet been investigated. Astrocytes are crucial regulators of neuroinflammatory responses under pathological conditions in the central nervous system (CNS). Proinflammatory astrocytes can induce the blood-brain barrier (BBB) breakdown, which plays a key role in the progression of neurodegenerative disorder (ND). PURPOSE: This study aims to investigate the anti-neuroinflammatory effects of CO in LPS-induced astrocyte activation and its underlying mechanisms in protecting the blood-brain barrier (BBB) in vitro. METHODS: Mouse astrocytes (C8-D1A) were activated with lipopolysaccharide (LPS) with or without CO pretreatment. Effects of CO on astrocyte cell viability, secretions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and nitric oxide (NO) were determined. Intracellular transcriptions and translations of proinflammatory mediators, molecular signaling, [Ca2+] and the levels of reactive oxygen species (ROS) were evaluated by RT-PCR, western blotting, and flow cytometry, respectively. Astrocyte-conditioned medium (ACM) was further prepared for incubating endothelial monolayer (bEnd.3) grown on transwell. Endothelial disruptions were evaluated by transendothelial electrical resistance (TEER), FITC-dextran permeability and monocyte adhesion assays. Endothelial tight junctions (TJs) and molecular signaling pathways were evaluated by immunofluorescence staining and western blotting. RESULTS: CO attenuated LPS-induced expression of astrocytic proinflammatory mediators (TNF-α, IL-1ß, IL-6, NO) and inhibited deleterious molecular activities including inducible nitric oxide synthase (iNOS), p-NFκB and p-STAT3 in astrocytes. Incubation of ACM collected from CO-treated astrocytes significantly ameliorated endothelial disruptions, reduced expressions of endothelial cytokine receptors (IL-6R, gp130 (IL-6RB), TNFR and IL-1R), suppressed proinflammatory pathways, MAPKs (p-AKT, p-MEK, p-ERK, p-p38, p-JNK) and p-STAT3, restored endothelial stabilizing pathways (p-Rac 1) and upregulated beneficial endothelial nitric oxide synthase (eNOS). CONCLUSION: Our study demonstrates for the first time CO exhibited potent protective effects against astrocyte-mediated proinflammatory responses and associated endothelial barrier disruptions.

Piceatannol Protects Brain Endothelial Cell Line (bEnd.3) against Lipopolysaccharide-Induced Inflammation and Oxidative Stress.

Dysfunction of the blood-brain barrier (BBB) is involved in the pathogenesis of many cerebral diseases. Oxidative stress and inflammation are contributing factors for BBB injury. Piceatannol, a natural ingredient found in various plants, such as grapes, white tea, and passion fruit, plays an important role in antioxidant and anti-inflammatory responses. In this study, we examined the protective effects of piceatannol on lipopolysaccharide (LPS) insult in mouse brain endothelial cell line (bEnd.3) cells and the underlying mechanisms. The results showed that piceatannol mitigated the upregulated expression of adhesion molecules (ICAM-1 and VCAM-1) and iNOS in LPS-treated bEnd.3 cells. Moreover, piceatannol prevented the generation of reactive oxygen species in bEnd.3 cells stimulated with LPS. Mechanism investigations suggested that piceatannol inhibited NF-κB and MAPK activation. Taken together, these observations suggest that piceatannol reduces inflammation and oxidative stress through inactivating the NF-κB and MAPK signaling pathways on cerebral endothelial cells in vitro.

Murine Beta-Amyloid (1-42) Oligomers Disrupt Endothelial Barrier Integrity and VEGFR Signaling via Activating Astrocytes to Release Deleterious Soluble Factors.

Transgenic mouse models of Alzheimer's disease (AD) overexpress mutations of the human amyloid protein precursor (APP) and presenilin-1 (PSEN1) genes, which are known causes of amyloid pathology in familial AD. However, animal models for studying AD in the context of aging and age-related co-morbidities, such as blood-brain barrier (BBB) disruptions, are lacking. More recently, aged and progeroid mouse models have been proposed as alternatives to study aging-related AD, but the toxicity of murine amyloid-beta protein (Aß) is not well defined. In this study, we aimed to study the potential toxicity of murine Aß on brain endothelial cells and astrocytes, which are important components of the BBB, using mouse brain endothelial cells (bEnd.3) and astrocytes (C8-D1A). Murine-soluble Aß (1-42) oligomers (sAßO42) (10 µM) induced negligible injuries in an endothelial monolayer but induced significant barrier disruptions in a bEnd.3 and C8-D1A co-culture. Similar results of endothelial perturbation were observed in a bEnd.3 monolayer treated with astrocyte-conditioned medium (ACM) generated by astrocytes exposed to sAßO42 (ACM-sAßO42), while additional exogenous sAßO42 did not cause further damage. Western blot analysis showed that ACM-sAßO42 altered the basal activities of vascular endothelial growth factor receptor 2 (VEGFR2), eNOS, and the signaling of the MEK/ERK and Akt pathways in bEnd.3. Our results showed that murine sAßO42 was moderately toxic to an endothelial and astrocyte co-culture. These damaging effects on the endothelial barrier were induced by deleterious soluble factors released from astrocytes, which disrupted endothelial VEGFR2 signaling and perturbed cell survival and barrier stabilization.

Anti-Hyperglycemic Effects of Refined Fractions from Cyclocarya paliurus Leaves on Streptozotocin-Induced Diabetic Mice.

To identify the chemical components responsible for the anti-hyperglycemic effect of Cyclocarya paliurus (Batal.) Iljinsk (Juglandaceae) leaves, an ethanol extract (CPE) and a water extract (CPW) of C. paliurus leaves, as well as their total flavonoids (CPF), triterpenoids (CPT) and crude polysaccharides (CPP), were prepared and assessed on streptozotocin (STZ)-induced diabetic mice. After being orally administrated once a day for 24 days, CPF (300 mg/kg), CPP (180 mg/kg), or CPF+CPP (300 mg/kg CPF + 180 mg/kg CPP) treatment reversed STZ-induced body weight and muscle mass losses. The glucose tolerance tests and insulin tolerance tests suggested that CPF, CPP, and CPF+CPP showed anti-hyperglycemic effect in STZ-induced diabetic mice. Furthermore, CPF enhances glucose-stimulated insulin secretion in MIN6 cells and insulin-stimulated glucose uptake in C2C12 myotubes. CPF and CPP suppressed inflammatory cytokine levels in STZ-induced diabetic mice. Additionally, CPF and CPP improved STZ-induced diabetic nephropathy assessed by H&E staining, blood urea nitrogen content, and urine creatinine level. The molecular networking and Emperor analysis results indicated that CPF showed potential anti-hyperglycemic effects, and HPLC-MS/MS analysis indicated that CPF contains 3 phenolic acids and 9 flavonoids. In contrast, CPT (650 mg/kg) and CPC (300 mg/kg CPF + 180 mg/kg CPP + 650 mg/kg CPT) did not show anti-hyperglycemic effect. Taken together, polysaccharides and flavonoids are responsible for the anti-hyperglycemic effect of C. paliurus leaves, and the clinical application of C. paliurus need to be refined.

Clonazepam attenuates neurobehavioral abnormalities in offspring exposed to maternal immune activation by enhancing GABAergic neurotransmission.

Ample evidence indicates that maternal immune activation (MIA) during gestation is linked to an increased risk for neurodevelopmental and psychiatric disorders, such as autism spectrum disorder (ASD), anxiety and depression, in offspring. However, the underlying mechanism for such a link remains largely elusive. Here, we performed RNA sequencing (RNA-seq) to examine the transcriptional profiles changes in mice in response to MIA and identified that the expression of Scn1a gene, encoding the pore-forming α-subunit of the brain voltage-gated sodium channel type-1 (NaV1.1) primarily in fast-spiking inhibitory interneurons, was significantly decreased in the medial prefrontal cortex (mPFC) of juvenile offspring after MIA. Moreover, diminished excitatory drive onto interneurons causes reduction of spontaneous gamma-aminobutyric acid (GABA)ergic neurotransmission in the mPFC of MIA offspring, leading to hyperactivity in this brain region. Remarkably, treatment with low-dose benzodiazepines clonazepam, an agonist of GABAA receptors, completely prevented the behavioral abnormalities, including stereotypies, social deficits, anxiety- and depression-like behavior, via increasing inhibitory neurotransmission as well as decreasing neural activity in the mPFC of MIA offspring. Our results demonstrate that decreased expression of NaV1.1 in the mPFC leads to abnormalities in maternal inflammation-related behaviors and provides a potential therapeutic strategy for the abnormal behavioral phenotypes observed in the offspring exposed to MIA.

Crocetin and Its Glycoside Crocin, Two Bioactive Constituents From Crocus sativus L. (Saffron), Differentially Inhibit Angiogenesis by Inhibiting Endothelial Cytoskeleton Organization and Cell Migration Through VEGFR2/SRC/FAK and VEGFR2/MEK/ERK Signaling Pathways.

Crocetin and crocin are two important carotenoids isolated from saffron (Crocus sativus L.), which have been used as natural biomedicines with beneficial effects for improving the suboptimal health status associated with abnormal angiogenesis. However, the anti-angiogenic effects and underlying mechanisms of the effects of crocetin and crocin have not been investigated and compared. The anti-angiogenic effects of crocetin and crocin were tested on human umbilical vein endothelial cells (HUVECs) in vitro, and in zebrafish in vivo. In vivo, crocetin (20 µM) and crocin (50 and 100 µM) significantly inhibited subintestinal vein vessels formation, and a conversion process between them existed in zebrafish, resulting in a difference in their effective concentrations. In the HUVEC model, crocetin (10, 20 and 40 µM) and crocin (100, 200 and 400 µM) inhibited cell migration and tube formation, and inhibited the phosphorylation of VEGFR2 and its downstream pathway molecules. In silico analysis further showed that crocetin had a higher ability to bind with VEGFR2 than crocin. These results suggested that crocetin was more effective than crocin in inhibiting angiogenesis through regulation of the VEGF/VEGFR2 signaling pathway. These compounds, especially crocetin, are potential candidate natural biomedicines for the management of diseases associated with abnormal blood vessel growth, such as age-related macular degeneration.

Serum levels of miR-21-5p and miR-339-5p associate with occupational trichloroethylene hypersensitivity syndrome.

BACKGROUND: Trichloroethylene (TCE) hypersensitivity syndrome (THS) is a dose-independent and potentially life-threatening disease. In this study, we sought to identify THS-related miRNAs and evaluate its potential clinical value. METHODS: Serum samples of five patients and five matched TCE contacts were used for screening differential miRNAs. Another 34 patients and 34 matched TCE contacts were used for verifying significantly differential miRNAs with SYBR™ Green PCR and MGB PCR. The diagnostic model based on these miRNAs was established via the support vector machine (SVM) algorithm. Correlation between differential miRNAs and liver function was analyzed via the Spearman correlation test. RESULTS: A total of 69 miRNAs was found to be differentially expressed. MiR-21-5p and miR-339-5p were verified to have significant higher expressions in patients. The sensitivity, specificity and accuracy of disease model were 100, 75 and 86%, respectively. The two miRNAs showed significant correlations with liver function. CONCLUSION: These findings suggested that miRNAs profiles in serum of THS patients had changed significantly, and miR-21-5p and miR-339-5p were associated with THS.

Iron overload induced by IRP2 gene knockout aggravates symptoms of Parkinson's disease.

Parkinson's disease (PD) is accompanied by iron overload in the brain. However, whether iron accumulation is the cause or effect of PD is still unknown. Iron regulatory protein 2 (IRP2) plays a critical role in keeping iron homeostasis, and our previous data showed that the deletion of the IRP2 gene caused iron deposits in organs of mice. Therefore, we further investigated the role of iron overload induced by IRP2 gene deletion in the development of the MPTP-induced PD mouse model in vivo, and the underlying regulatory mechanisms in primary cultures of astrocytes in vitro. Data from neurobehavioral, immunohistochemistry, TUNEL and Elisa studies showed that MPTP treatment enhanced the symptoms of PD in vivo, increased cell apoptosis and decreased dopamine levels in IRP2-/- mice. In addition, the expression of L-ferritin and iron contents increased significantly in the substantia nigra (SN) of IRP2-/- mice. Moreover, MPTP treatment significantly increased the expression of DMT1 (-IRE) and decreased the expression of TfR1 in IRP2-/- mice. Further investigations with primary cultures of astrocytes from IRP2-/- mice showed that MPP+ increased the expression of L-ferritin and DMT1 (-IRE), and decreased the expression of TfR1. Our results demonstrated that IRP2 gene deletion induced iron accumulation in the SN, which exacerbated the neuronal apoptosis and Parkinsonism symptoms. At the same time, IRP2 gene deletion increased the iron contents in astrocytes around neurons, which further decreased their protection for neurons and increased the cell apoptosis, ultimately forming a vicious cycle that leads to the onset and progression of PD.

A Danshensu-Tetramethylpyrazine Conjugate DT-010 Overcomes Multidrug Resistance in Human Breast Cancer.

Background: We previously demonstrated that a Danshensu-Tetramethylpyrazine conjugate DT-010 enhanced anticancer effect of doxorubicin (Dox) in Dox-sensitive human breast cancer cells, and protected against Dox-induced cardiotoxicity. This work was designed to see whether DT-010 overcomes Dox resistance in resistant human breast cancer cells. Methods: The effects of DT-010, Dox or their combination on cell viability of Dox-resistant human breast cancer MCF-7/ADR cells were conducted using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was examined by flow cytometry after Annexin V-FITC/PI co-staining. Dox accumulation in MCF-7/ADR cells was detected by flow cytometry and fluorescence microscopy. A fluorometric multidrug resistance (MDR) assay kit was used to evaluate the effect of DT-010 on MDR transporter activity. P-glycoprotein (P-gp) expression and activity were analyzed by Western blot and rhodamine 123 (Rh123) efflux assay, respectively. The effects of DT-010 on glycolysis and mitochondrial stress were detected using an Extracellular Flux Analyzer. A Succinate Dehydrogenase Activity Assay kit was used to measure mitochondrial complex II activity. Results: At non-cytotoxic concentrations, DT-010 in combination with Dox led to a significant growth inhibition of MCF-7/ADR cells, suggesting a synergy between DT-010 and Dox to reverse Dox resistance. DT-010 restored Dox-mediated apoptosis and p53 induction in MCF-7/ADR cells. DT-010 increased Dox accumulation in MCF-7/ADR cells via inhibiting P-gp activity, but without changing P-gp expression. Further studies showed that DT-010 significantly inhibited glycolysis and mitochondrial function of MCF-7/ADR cells. Mitochondrial complex II activity was inhibited by DT-010 or DT-010/Dox combination, but not by Dox. The DT-010-mediated suppression of metabolic process may render cells more vulnerable to Dox treatment and thus result in enhanced efficacy. Conclusions: The results indicate that DT-010 overcomes Dox resistance in human breast cancer cells through a dual action via simultaneously inhibiting P-gp-mediated drug efflux and influencing metabolic process.

Memantine Improves Cognitive Function and Alters Hippocampal and Cortical Proteome in Triple Transgenic Mouse Model of Alzheimer's Disease.

Memantine is a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist clinically approved for moderate-to-severe Alzheimer's disease (AD) to improve cognitive functions. There is no report about the proteomic alterations induced by memantine in AD mouse model yet. In this study, we investigated the protein profiles in the hippocampus and the cerebral cortex of AD-related transgenic mouse model (3×Tg-AD) treated with memantine. Mice (8-month) were treated with memantine (5 mg/kg/bid) for 4 months followed by behavioral and molecular evaluation. Using step-down passive avoidance (SDA) test, novel object recognition (NOR) test and Morris water maze (MWM) test, it was observed that memantine significantly improved learning and memory retention in 3xTg-AD mice. By using quantitative proteomic analysis, 3301 and 3140 proteins in the hippocampus and the cerebral cortex respectively were identified to be associated with AD abnormalities. In the hippocampus, memantine significantly altered the expression levels of 233 proteins, among which PCNT, ATAXIN2, TNIK, and NOL3 were up-regulated, and FLNA, MARK 2 and BRAF were down-regulated. In the cerebral cortex, memantine significantly altered the expression levels of 342 proteins, among which PCNT, PMPCB, CRK, and MBP were up-regulated, and DNM2, BRAF, TAGLN 2 and FRY1 were down-regulated. Further analysis with bioinformatics showed that memantine modulated biological pathways associated with cytoskeleton and ErbB signaling in the hippocampus, and modulated biological pathways associated with axon guidance, ribosome, cytoskeleton, calcium and MAPK signaling in the cerebral cortex. Our data indicate that memantine induces higher levels of proteomic alterations in the cerebral cortex than in the hippocampus, suggesting memantine affects various brain regions in different manners. Our study provides a novel view on the complexity of protein responses induced by memantine in the brain of AD.

G-protein coupled receptor 55 agonists increase insulin secretion through inositol trisphosphate-mediated calcium release in pancreatic ß-cells.

G-protein coupled receptor 55 (GPR55) is an orphan G-protein coupled receptor, which is activated by endocannabinoids and lipid transmitters. Recently, GPR55 was shown to play a role in glucose and energy homeostasis, and insulin secretion is essential to maintain glucose homeostasis in the body. In Type 2 Diabetes Mellitus (T2DM), chronic insulin resistance and a progressive decline in ß-cell function result in ß-cell dysfunction, this leads to defect in insulin secretion, which is the key process in the development and progression of T2DM. GPR55 agonists were shown to increase insulin secretion, however the underlying mechanisms were not fully understood. Therefore the aim of the present study was to examine the effects of potent GPR55 agonists, O-1602 and abnormal cannabidiol (Abn-CBD), on glucose-induced insulin secretion in a mouse pancreatic ß-cell line, MIN6, and the underlying mechanisms with a focus on intracellular calcium (Ca2+). Our results demonstrated that O-1602 and Abn-CBD increased glucose-induced insulin secretion in MIN6 cells, which was abolished by a PLC inhibitor, U73122. Glucose-induced Ca2+ transients were enhanced by O-1602 and Abn-CBD, and this was significantly reduced by U73122 and inositol trisphosphate (IP3) receptor inhibitors, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C, as well as by Y-27632, a Rho-associated protein kinase (ROCK) inhibitor. Interestingly, O-1602 and Abn-CBD could directly induce intracellular Ca2+ transients through IP3-mediated Ca2+ release. In conclusion, GPR55 agonists increased insulin secretion through calcium mobilisation from IP3-sensitive ER stores in ß-cells.

Novel protective effect of O-1602 and abnormal cannabidiol, GPR55 agonists, on ER stress-induced apoptosis in pancreatic ß-cells.

Insulin resistance and ß-cell dysfunction are the main defects in Type 2 Diabetes Mellitus (T2DM), and ß-cell dysfunction and apoptosis is the critical determinant in the progression of T2DM. G-protein coupled receptor 55 (GPR55) is an orphan G-protein coupled receptor, which is activated by endocannabinoids and lipid transmitters. Recently, GPR55 was shown to regulate glucose and energy homeostasis, however its role in ß-cell apoptosis was not studied. Therefore, in this study, we investigated the novel effect of GPR55 agonists, O-1602 and abnormal cannabidiol (Abn-CBD), on endoplasmic reticulum (ER) stress-induced apoptosis in mouse pancreatic ß-cell lines, MIN6 and Beta-TC-6, and its underlying mechanisms. Our results showed that O-1602 and Abn-CBD reduced ER stress-induced apoptosis in MIN6 and Beta-TC-6 cells. This was through the phosphorylation of 3'-5'-cyclic adenosine monophosphate response element-binding protein (CREB) in ß-cells, hence activating CREB downstream anti-apoptotic genes, Bcl-2 and Bcl-xL. Moreover, O-1602 and Abn-CBD directly activated kinases, CaMKIV, Erk1/2 and PKA, to induce CREB phosphorylation. Therefore, our results indicated that GPR55 agonists protected from ß-cell apoptosis through CREB activation, thus up-regulating anti-apoptotic genes. In conclusion, our study provided a novel protective effect of GPR55 agonists on ER stress-induced apoptosis in ß-cells and its underlying mechanisms mediating this protection, therefore we suggested that GPR55 might be a therapeutic target for T2DM.

Neuroprotective Effects of BHDPC, a Novel Neuroprotectant, on Experimental Stroke by Modulating Microglia Polarization.

This study mainly investigated the therapeutic effects of BHDPC on ischemic stroke and its underlying mechanisms. In vivo, the transient middle cerebral artery occlusion (MCAO) was used to induce ischemic model. In vitro, oxygen and glucose deprivation/reperfusion (OGD/R)-induced ischemic stroke in BV-2 microglia and primary neurons, and bEnd.3 mouse cerebral microvascular endothelial cells (ECs) were also used. First, we found that BHDPC exerts considerable neuroprotection against MCAO-induced ischemic injury to mice via alleviating neurological deficits and brain infarcts, inhibiting neuronal cell loss and apoptosis, and attenuating blood-brain barrier disruption and tight junction protein changes. Next, we observed that BHDPC significantly reduced microglial M1 activation but enhanced M2 polarization in MCAO-induced ischemic brain. Further experiments in vitro indicated that BHDPC suppressed microglial activation but promoted M2 microglial polarization in OGD/R-induced BV-2 microglia. In addition, conditioned medium (CM) experiments showed that CM from BHDPC-treated BV-2 microglia provided protections against OGD/R-induced ischemic damage in primary neurons and bEnd.3 ECs. Moreover, we found that BHDPC actions on microglial inflammation were associated with the inactivation of NF-κB signaling. Interestingly, we also found that BHDPC enhanced phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The pharmacological inhibition or gene knockdown of PKA/CREB signaling diminished BHDPC-promoted microglial M2 polarization. In summary, BHDPC conferred neuroprotection against ischemic injury in experimental stroke models. Modulating microglial activation and polarization contributes to BHDPC-mediated neuroprotective actions, which in part were mediated by nuclear factor kappa B and PKA/CREB signaling pathway.

Schisantherin A Attenuates Neuroinflammation in Activated Microglia: Role of Nrf2 Activation Through ERK Phosphorylation.

BACKGROUND/AIMS: In the present study, we investigated whether schisantherin A (StA) had anti-inflammatory effects under neuroinflammatory conditions. METHODS: The effects of StA and its underlying mechanisms were examined in lipopolysaccharide (LPS)-activated BV-2 microglial cells by ELISA, qPCR, EMSA, Western blot, and IHC. RESULTS: Firstly, we found that StA inhibited the inflammatory response in LPS-activated BV-2 microglia. Secondly, we found that StA suppressed LPS-induced activation of NF-κB via interfering with degradation of IκB and phosphorylation of IκB, IKK, PI3K/Akt, JNK, and p38 MAPK. Thirdly, StA conferred indirect antioxidative effects via quenching ROS and promoted expression of antioxidant enzymes, including HO-1 and NQO-1, via stimulating activation of Nrf2 pathways. Finally, we demonstrated that anti-neuroinflammatory actions of StA were dependent on ERK phosphorylation-mediated Nrf2 activation. CONCLUSION: StA induced ERK phosphorylation-mediated Nrf2 activation, which contributed to its anti-inflammation and anti-oxidation. The anti-neuroinflammatory and anti-oxidative effects of StA may show preventive therapeutic potential for various neuroinflammatory disorders.

Discovery of a Novel ERp57 Inhibitor as Antiplatelet Agent from Danshen (Salvia miltiorrhiza).

Danshen (Salvia miltiorrhiza) is a well-known herb in Traditional Chinese Medicine (TCM) for treating cardiovascular diseases, but the underlying mechanism remains to be fully elucidated. Here, we showed that Danshen and its active ingredient rosmarinic acid exhibited antiplatelet effects through the inhibition of ERp57, a member of protein disulfide isomerase (PDI) with potential roles in platelet aggregation. Danshen extract (DSE) exhibited potent inhibitory effects on the platelet aggregation induced by arachidonic acid- (AA-) induced platelet aggregation and the enzymatic activity of ERp57. Rosmarinic acid was identified by virtual screening and molecular docking as one of the hit compounds for ERp57. In line with this, rosmarinic acid displayed significant inhibitory effect on ERp57 activity and inhibited AA-induced platelet aggregation. Taken together, we demonstrated for the first time that DSE and rosmarinic acid displayed inhibitory effects on the catalytic activity of ERp57, providing evidence of the regulatory role of ERp57 underlying the antiplatelet effects of Danshen.

Tanshinone I prevents atorvastatin-induced cerebral hemorrhage in zebrafish and stabilizes endothelial cell-cell adhesion by inhibiting VE-cadherin internalization and actin-myosin contractility.

Defects in vascular integrity in cerebrovasculature lead to serious pathologies including hemorrhagic stroke. The stability of cell adhesion junctions and actin-myosin contractile machinery are two major determinants for the integrity of endothelial monolayer. Here we have evaluated the protective effects of tanshinone I (Tan I), a lipophilic compound presents in Salvia miltiorrhiza, against atorvastatin-induced cerebral hemorrhage in zebrafish in vivo, and further dissected the molecular mechanisms in HUVECs. We demonstrated that Tan I protected endothelial integrity by stabilizing cell-cell adhesion junctions via the inhibition of Src-mediated VE-cadherin internalization and subsequent junction-linked actin cytoskeleton depolymerization. In addition, Tan I inhibited ROCK-associated endothelial contractile machinery by dephosphorylating cofilin and MYPT1. These findings identified Tan I as an endothelial stabilizing agent and suggested Tan I as a potential treatment for vascular leakage in hemorrhagic stroke.