Abstract 308: Chronic Aspirin Treatment Possibly Poses a Risk to Ischemic Heart via Impairment of Cardiac Proteasome Functions

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Chunjiang Tan ◽  
Wenlie Chen ◽  
Yanbing Wu ◽  
Jiumao Lin ◽  
Ruhui Lin ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cell Viability ◽  
Cultured Cells ◽  
Ventricular Myocytes ◽  
Cell Model ◽  
Selective Inhibition ◽  
Ischemic Heart ◽  
Saline Control ◽  
Normal Male

Background: Impaired cardiac proteasome (CP) has been reported in ischemia and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, it’s unclear whether it affects CP functions. Objective: We investigated the influence of aspirin on CP in the rat model of myocardial infarction (MI). Methods and Results: MI, sham or normal male SD rats were injected intraperitoneally with high (300mg/kg), low (5mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated cell model of primary human ventricular myocytes incubated with different concentrations of aspirin. Myocardial hypertrophy, cardiac function, cell viability and the functions of 26S, 20S and 19S, including the β1, β2 and β5 subunits of 20S were determined. The activity of 26S, 20S and 19S declined by about 30%, and β5, β2 and β1 by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats ( P <0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, and β5 further decreased by 30% and 12%, and β1 by 40% and 30%, respectively, and their lost activity was correlated with compromised cardiac function in the MI rats. The dose-related and selective inhibition of β5 and β1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. However, the CP in the normal settings, or 19S and β2 in all the groups of animals or cultured cells were less affected by aspirin treatment. The impaired CP, dose-dependently enhanced by aspirin, led to the elevation of oxidative and ubiquitinated proteins in the MI rat hearts or in the hypoxia/reoxygenated cells. These aberrant proteins in turn constituted the ultimate causal factor for the cardiac dysfunction and the loss of cell viability. In vitro experiments confirmed that the purified CP from the MI rats or hypoxic cells, but was more susceptible to aspirin treatment, whereas the complexes in the normal settings appeared less affected by aspirin. Conclusions: Chronic aspirin treatment, via the dose-dependent and selective inhibition of CP, enhanced the ischemic CP dysfunction, which constitutes a potential risk factor for the ischemic heart.

Abstract 9: Temperature-responsive Cell Delivery Biopolymers for Cardiac Tissue Engineering

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Brisa Pena ◽  
Valentina Martinelli ◽  
Susanna Bosi ◽  
Carmen Sucharov ◽  
Mark Jeong ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Body Temperature ◽  
Cell Viability ◽  
Cultured Cells ◽  
Ventricular Myocytes ◽  
3D Culture ◽  
Polymeric Matrix ◽  
Neonatal Rat ◽  
Cell Delivery

Background: Advances in cell therapy and material science have made tissue engineering a promising strategy for heart regeneration. We developed an injectable biomimetic reverse thermal gel (RTG) that is liquid at room temperature but gel-like at body temperature, with the ultimate goal of being able to serve as a vehicle for cell-based delivery (liquid) to targeted tissue areas (gel-phase at 37°C). In this study we tested the suitability of this biomimetic RTG on cell viability. Methods and results: We tested different biomimetic RTG systems with and without the chemical incorporation of lysine. In vitro 3D culture experiments were performed with neonatal rat ventricular myocytes (NRVM) by mixing 3x104 cells with 50 μl of polymeric solution and allowing gel formation at 37°C. The cultured cells were incubated for 21 days. For controls we used NRVMs plated on 2D traditional gelatin coated dishes. We found that the 3D polymeric matrix induces rapid coordinated contraction with improved functionality when compared with standard 2D-cultured NRVM. By immunostaining for the morphology of the sarcomere (alpha-actinin) and DAPI, we also observed that the 3D polymeric matrix stimulates cells to spread and form 3D syncytia. Conclusion: These proof-of-concept results demonstrate long-term cell viability in this unique biomimetic system and therefore provide feasibility of a polymeric cell delivery system that permits reversible liquid-to-gel transition at body temperature. These results offer potential for a tissue engineering approach to cardiac regeneration.

scholarly journals β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Guoying Zhang ◽  
Cheng Xue ◽  
Yiming Zeng
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Cell Model ◽  
Protective Efficacy ◽  
Rabbit Model ◽  
Akt Pathway ◽  
Loss Of Function ◽  
Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

scholarly journals PRC1 promotes GLI1-dependent osteopontin expression in association with the Wnt/β-catenin signaling pathway and aggravates liver fibrosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shenzong Rao ◽  
Jie Xiang ◽  
Jingsong Huang ◽  
Shangang Zhang ◽  
Min Zhang ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Cell Viability ◽  
Signaling Pathway ◽  
Cell Model ◽  
Stellate Cell ◽  
Underlying Mechanism ◽  
Histopathological Analysis ◽  
Osteopontin Expression

Abstract Background PRC1 (Protein regulator of cytokinesis 1) regulates microtubules organization and functions as a novel regulator in Wnt/β-catenin signaling pathway. Wnt/β-catenin is involved in development of liver fibrosis (LF). We aim to investigate effect and mechanism of PRC1 on liver fibrosis. Methods Carbon tetrachloride (CCl4)-induced mice LF model was established and in vitro cell model for LF was induced by mice primary hepatic stellate cell (HSC) under glucose treatment. The expression of PRC1 in mice and cell LF models was examined by qRT-PCR (quantitative real-time polymerase chain reaction), western blot and immunohistochemistry. MTT assay was used to detect cell viability, and western blot to determine the underlying mechanism. The effect of PRC1 on liver pathology was examined via measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and hydroxyproline, as well as histopathological analysis. Results PRC1 was up-regulated in CCl4-induced mice LF model and activated HSC. Knockdown of PRC1 inhibited cell viability and promoted cell apoptosis of activated HSC. PRC1 expression was regulated by Wnt3a signaling, and PRC1 could regulate downstream β-catenin activation. Moreover, PRC1 could activate glioma-associated oncogene homolog 1 (GLI1)-dependent osteopontin expression to participate in LF. Adenovirus-mediated knockdown of PRC1 in liver attenuated LF and reduced collagen deposition. Conclusions PRC1 aggravated LF through regulating Wnt/β-catenin mediated GLI1-dependent osteopontin expression, providing a new potential therapeutic target for LF treatment.

Ranolazine and TDM1 treatment: Cardioprotective effects in vitro and in vivo.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23102-e23102
Author(s):  
Nicola Maurea ◽  
Carmela Coppola ◽  
Giovanna Piscopo ◽  
Gennaro Riccio ◽  
Domenica Rea ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cell Viability ◽  
Cell Counting ◽  
In Vivo Studies ◽  
Breast Cancer Patients ◽  
Antibody Drug Conjugate ◽  
Fetal Cardiomyocytes ◽  
Covalently Linked

e23102 Background: Ado trastuzumab emtansine (TDM1) is a novel antibody–drug conjugate consisting of trastuzumab (TRAS) covalently linked to the highly potent microtubule inhibitory agent DM1 via a stable thioether linker. TDM1 is used in metastatic ErbB2 positive breast cancer patients. Although, the potential cardiotoxic effects of TDM1 have not yet been fully elucidated, they can include changes in Ca2+ regulation related to blockade of ErbB2, PI3K-Akt and MAPK pathways. Here, we aim to elucidate whether Ranolazine (R), administered after TDM1 treatment, blunts or not cardiotoxicity in vivo and in vitro. Methods: In vitro, human fetal cardiomyocytes (HFC) were treated with TDM1 for 3 days and then treated in the absence or presence of R for 3 days. Cell viability was assessed by cell counting and MTT assay. To evaluate cardiac function in vivo, C57/BL6 mice, 2-4 months old, were daily treated with TDM1 (44.4 mg/kg/day). At day 0 and after 7 days, fractional shortening (FS) and ejection fraction (EF) were measured, by M/B mode echocardiography, and radial and longitudinal strain (RS and LS) were evaluated using 2D speckle-stracking. These measurements were repeated after 5 days of R treatment (305 mg/Kg/day), started at the end of TDM1 treatment. Results: R reduces TDM1 toxicity in HFC, as evidenced by the higher percentage of viable cells treated with TDM1+ R with respect to the cells treated with TDM1 alone (p < 0.01). In in vivo studies: after 7 days with TDM1 administration, FS decreased to 53.6±0.9%, versus 61.0±0.8 % (sham), (p < 0.01), and EF decreased to 85.5±3.5 % versus 91.0±0.8% (sham), (p < 0.01). Moreover, RS decreased to 20.92±3.2 % versus 42.2±10.1% (sham) (p < 0.01), and LS decreased to -15.5±2.8 % versus -23.6±6.7% (sham), (p < 0.01).In mice treated with TDM1 and, successively treated with R for 5 days, the indices of cardiac function partially recovered: FS 58±2.4 % (p < 0.05), EF 88.8±1.7 %, (p < 0.05), RS (35.7±8.2 %, p > 0.05), whereas the alteration of LS persists even after treatment with R (-17.3±3.7 %, p > 0.05) Conclusions: Here we show that in vivo R post-treatment reduces cardiotoxic effects due to TDM1, as demonstrated by the recovery of FS, EF and RS values. As expected, R increases cell viability of HFC treated with TDM1.

scholarly journals Repression of Matrix Metalloproteinases and Cytokine Secretion in Glioblastoma by Targeting K+ Channel: An in Vitro Study

2021 ◽  
Author(s):  
Farshid Saadat ◽  
◽  
Zohreh Zareighane ◽  
Farnaz Safavifar ◽  
Seyedeh Zohreh Jalali ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Cell Viability ◽  
Malignant Tumors ◽  
Cell Model ◽  
Gelatin Zymography ◽  
In Vitro Study ◽  
K Channel ◽  
Kv Channels ◽  
Vitro Model

Introduction: Glioblastoma is an aggressive malignancy of human brain with poorly understood pathogenesis. Voltage-gated potassium (Kv) channels and Matrix metalloproteinases (MMPs) are highly expressed in malignant tumors and involved in the progression and metastasis of glioblastoma. The purpose of this study was to determine whether a voltage-dependent potassium channel blocker could modulate astrocytes as a cell which involved in immunopathogenesis of glioblastoma. Methods: The cytotoxic effect of 4-aminopyridine (4-AP) at different doses in cell model of glioblastoma was measured by MTT assay. ELISA technique and gelatin zymography were used to assess cytokines levels and MMP-9 after 4-AP treatment, respectively. Results: Cytotoxicity analysis showed that cell viability reduced by increasing 4-AP level and cell growth reduced gradually by removing 4-AP from cell medium. 4-AP inhibits secretion of IL-6 and IL-1 (p<0.05). MMP9 activity significantly inhibits with increased 4-AP dose as compared to non-treated cells. Conclusion: Reduction of cell viability, IL-6 secretion and MMP-9 activity in an in vitro model of glioblastoma, might be assumed 4-AP as an agent for chemoprevention of cancer.

The Microenvironmental Stromal Cells Abrogate NF-κb Inhibitor Induced Apoptosis in Chronic Lymphocytic Leumkemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3208-3208
Author(s):  
Katharina Foerster ◽  
Carl Philipp Simon-Gabriel ◽  
Dorothee Bleckmann ◽  
Marco Benkisser-Petersen ◽  
Nicolas Thornton ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Viability ◽  
Cultured Cells ◽  
Cd40 Ligand ◽  
Binding Activity ◽  
Parp Cleavage ◽  
Dna Binding Activity ◽  
Induced Apoptosis

Abstract Introduction: In recent years, the emergence of kinase inhibitors has drastically altered treatment strategies and improved outcomes in CLL patients, but lack of cure and resistance to therapy still remain serious issues. The transcription factor NF-κB influences several cellular functions such as proliferation, apoptosis and inflammation and is known as a key factor contributing to CLL development and progression. NF-κB is constitutively active in CLL and the NF-κB subunit RELA has been proposed as a prognostic marker in CLL with high RELA DNA-binding activity being predictive of short time to first treatment and overall survival. Therefore, NF-κB has gained attention as a promising therapeutic target. NF-kB inhibition induces apoptosis in CLL cells in vitro. However, whether this effect pertains in vivoremains unclear. Since the microenvironment is crucial for CLL cell viability circumventing apoptosis, we tested whether NF-κB inhibition modulates CLL viability in the presence of the microenvironment. Methods: The specific NF-κB inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ) was used alone (2-5 µg/ml) or combined with fludarabine (10 µM), rhBAFF (50 ng/ml), rhAPRIL (500 ng/ml), rhSDF-1a (100 ng/ml) or CD40 ligand (1 µg/ml) on primary CLL cells cultured alone (monoculture) or on bone marrow stromal cells (BMSC) (co-culture with a ratio of 20 CLL cells per stromal cell) for 48-144 h. Viability and apoptosis were measured by flow cytometry using AnnexinV/PI stainings. Protein expression was analyzed by western blot using standard protocols. NF-κB DNA-binding activity after DHMEQ treatment (5 µg/ml) for 6 h was measured by ELISA for all subunits using 1 µg of protein lysate for the NF-κB1 subunit and 10 µg protein lysate for the subunits RELA, NF-κB2, RELB and c-REL. RELA gene knockdown was performed by siRNA transfection (2 µM targeting and non-targeting siRNA). Results: NF-κB inhibition using DHMEQ led to apoptosis in monocultured CLL cells (viability 74% vs. 24%, n=17, p<0.0001) but surprisingly had no effect on cell viability of cells co-cultured with BMSC (viability 96% vs. 95%, p=0.9995). In monoculture, apoptosis induction was accompanied by downregulation of the NF-κB target protein TRAF1 (untreated vs. treated: expression reduced by 34 %, p=0,0044), upregulation of the proapoptotic protein BAX (expression increased by 3175 %, p=0,0268), and increased PARP cleavage (100% vs. 8393% expression, p=0,0078). Conversely, in co-culture, downregulation of TRAF1 by 52 % (p=0,0054) was observed without concomitant BAX upregulation or PARP cleavage matching the non-appearance of apoptosis induction in those cells. While co-culturing untreated CLL cells on BMSC led to tendentially increased expression levels of the non-canonical NF-κB subunits NF-κB2 (untreated monocultured cells vs. untreated co-cultured cells: 100% vs. 142%, p=0,8438) and RELB (100% vs. 128%, p=0,7422), NF-κB DNA-binding activities of all NF-κB subunits were equally suppressed by DHMEQ treatment in mono- and co-cultured cells (e.g. treated monocultured vs. treated co-cultured cells: 1,6% vs. 4,9%, p<0,9999 for NF-κB1). Gene knockdown of the NF-κB subunit RELA by siRNA transfection solely induced apoptosis in monocultured CLL cells as well. Adding soluble BAFF to monocultured treated CLL cells attenuated DHMEQ efficiency (viability 1,3% vs. 16%, p=0,0258, n=9), while adding APRIL, CD40 ligand and SDF-1a had little influence on the response to treatment. Finally, the combined use of DHMEQ with fludarabine in co-cultured CLL cells led to a higher rate of apoptosis than DHMEQ (viability 57% vs. 37%, p=0.0202) or fludarabine alone (viability 50% vs. 37%, p=0.1828). Conclusion: NF-κB inhibition in primary CLL cells shows great discrepancy between in vitro and in vivo scenarios. While DHMEQ treatment leads to apoptosis in mono-cultured cells by BAX upregulation and increased PARP cleavage, CLL cell viability is not affected in the presence of microenvironment, suggesting that the NF-κB pathway can be bypassed in vivo. Soluble ligands, especially BAFF, appear to be involved in mediating this protective effect. However, the combination of NF-κB inhibition with standard chemotherapy might represent a promising approach and warrants further clinical assessment. Disclosures No relevant conflicts of interest to declare.

scholarly journals KCNQ1OT1 promotes the proliferation and migration of psoriatic keratinocytes by regulating miR-183-3p/GAB1

2021 ◽  
Vol 49 (5) ◽  
pp. 125-130
Author(s):  
Ting Liu ◽  
Xi Duan ◽  
Jia He ◽  
Chuan Yang
Keyword(s):  
Cell Viability ◽  
Cell Model ◽  
Growth Factor Receptor ◽  
Keratinocyte Proliferation ◽  
Tnf Α ◽  
Keratinocyte Cell Line Hacat ◽  
Gated Channel ◽  
And Migration ◽  
Proliferation And Migration

Background: Differentially expressed lncRNAs have been reported to be involved in keratinocyte proliferation and migration, and participate in the development of psoriasis. Potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) was implicated in the pathogenesis of various diseases, including cancer, sepsis, diabetic cardiomyopathy, and atherosclerosis. The influence of KCNQ1OT1 on proliferation and migration of psoriatic keratinocytes was unfolded in this study. Methods: Human keratinocyte cell line (HaCaT) was incubated with TNF-α to establish in vitro cell model of psoriasis. Cell viability and migration were assessed by MTT and wound healing, respectively. Target miRNA of KCNQ1OT1 was identified by luciferase activity and RNA immunoprecipitation (RIP) assays. Results: KCNQ1OT1 was up-regulated in TNF-α-induced HaCaT, and knockdown of KCNQ1OT1 reduced cell viability and suppressed migration of TNF-α-induced HaCaT. KCNQ1OT1 bind to miR-183-3p and negatively regulated expression of miR-183-3p. Over-expression of GAB1 (growth factor receptor binding 2-associated binding protein 1) counteracted with the suppressive effects of KCNQ1OT1 silence on cell viability and migration of TNF-α-induced HaCaT. Conclusion: Silence of KCNQ1OT1 suppressed proliferation and migration of TNF-α-induced HaCaT through regulation of miR-183-3p/GAB1, providing potential strategy for psoriasis.

scholarly journals Safflower Yellow a Attenuates Osteoarthritis via Regulating Inflammation and Cholesterol Metabolism

2020 ◽  
Author(s):  
Shaohua Ju ◽  
Lirong Tan ◽  
Panwang Liu ◽  
Xiaohong Li ◽  
Youli Tan ◽  
...  
Keyword(s):  
Cell Viability ◽  
Signaling Pathways ◽  
Complex Disease ◽  
Cholesterol Metabolism ◽  
Cell Model ◽  
Carthamus Tinctorius ◽  
Mtor Signaling ◽  
Processing Enzymes ◽  
Metabolism Disorder

Abstract BackgroundOsteoarthritis (OA) is known to be associated with inflammation and cholesterol metabolism disorder. As a chronic and complex disease, uncovering its molecular mechanism and finding effective therapy with low side effects are urgent. Hydroxysafflor yellow A (HSYA) is extracted from Carthamus tinctorius L, which has extensive pharmacological effects. MethodsIn this study, Interleukin 1β (IL-1β) was used to establish the OA model in vitro, and the impacts of HSYA on the OA cell model were analyzed. We used CCK-8 to measure the cell viability and Flow Cytometry to test the apoptosis. ELISA was performed to calculate the release of inflammatory cytokines. And WB was carried out to measure the expression of collagen and cholesterol relevant proteins. We also measured the protein levels in NF-κB and PI3K/Akt/mTOR signaling pathways. ResultsThe results showed that HSYA promoted cell viability and inhibited apoptosis. And it up-regulated the expression levels of collagen II (Col-II) and Sry related HMG box-9 (SOX9) while down-regulated the expression of matrix metalloproteinase-13 (MMP13). The IL-1β induced high levels of IL-6 and TNF-α were inhibited by HSYA. Also, HSYA regulated the expression of cholesterol relevant proteins. Compared with the model group, the levels of APT-binding cassette transporter 1 (ABCA1) and cholesterol transport gene (APOA-1) were significantly elevated. However, the levels of cholesterol-processing enzymes cholesterol 25-hydroxylase (CH25H) and 25-hydroxy-cholesterol 7-alpha- hydroxylase (CYP7B1) were inhibited. Besides, HSYA inhibited the protein expression in NF-κB and PI3K/Akt/mTOR signaling pathways. ConclusionsHSYA was proved to regulate inflammatory response and cholesterol metabolism in vitro.

Cellular Alterations Induced by Candida albicans RC Nosodes: an in vitro Study

2021 ◽  
Vol 11 (40) ◽  
pp. 209-210
Author(s):  
Fortune Homsani ◽  
Gleyce Moreno ◽  
Camila Siqueira ◽  
Juliana Grechi ◽  
André Luis Santos ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Viability ◽  
Cell Model ◽  
Etiologic Agent ◽  
Yeast Cells ◽  
In Vitro Assays ◽  
Mitochondrial Activity ◽  
No Production ◽  
Cellular Alterations

Introduction: Candidiasis is an opportunist infection, caused by yeast of the genus Candida, which emerges as one of the main causes of systemic infections in hospitalized patients. Candida albicans is the most common causing agent of these infections. According to the Brazilian Homeopathic Pharmacopeia[1], nosodes are medicines compounded from chemically undefined biological products. Living nosodes are prepared using the etiologic agent of an illness in its infective form, were first developed by Brazilian physician Roberto Costa (RC). Roberto Costa’s research indicated that living nosodes present a higher capability to stimulate the host’s immunological system [2]. Aim: This study aims to evaluate cellular alterations induced in C. albicans yeasts and RAW 264-7 macrophages by Candida albicans RC. Methodology: To prepare Candida albicans RC, one part of C. albicans infective yeast suspension (108 cell/ml) was diluted in 9 parts of sterile distilled water and submitted to 100 mechanical succussions. This process was successively repeated to the potencies of 12x and 30x1. Water 30x was prepared by the same technique, as control. The cell viability of C. albicans previously treated with nosodes in both potencies and respective controls was evaluated using the samples at the concentration of 10% (V/V), in a volume of 1ml, distributed in 1-3 days. The viability of the yeast cells was analyzed by MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolic) (5mg/ml) assay [3] and by Propidium Iodide (PI) incorporation methods. Additionally, using macrophages RAW 264-7 as a cell model, Nitric Oxide (NO) production and cell viability were also evaluated. For this, the following protocol of cell treatment was employed: on each experimental day, RAW 264-7 cells were treated 4 times (4 stimuli) with RC nosode 30x at the concentration of 10% (V/V). Results: The nosodes (12x and 30x) did not present cytotoxic effects on macrophage cells (n=1), or on C. albicans yeasts (n=2), as detected by MTT and PI methods. Moreover, no statistically significant differences on NO production were detected among the experimental groups (n=6). Conclusion: Preliminary results of in vitro assays indicate that nosodes (12x and 30x) do not alter mitochondrial activity or cell viability of C. albicans. Similarly, treatment by RC nosodes does not seem to alter NO release and mitochondrial activity of RAW macrophages. New experiments are being performed to confirm these preliminary data.

scholarly journals Huang-Pu-Tong-Qiao Formula Ameliorates Tau Phosphorylation by Inhibiting the CaM-CaMKIV Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Shu Ye ◽  
Biao Cai ◽  
Peng Zhou ◽  
Guoquan Wang ◽  
Huawu Gao ◽  
...  
Keyword(s):  
Cell Viability ◽  
Learning And Memory ◽  
Neuronal Damage ◽  
Cell Model ◽  
Neuroprotective Effect ◽  
Tau Phosphorylation ◽  
Morris Water Maze Test ◽  
Mrna Levels

Alzheimer’s disease (AD) is a complex neurodegenerative disease. It is a chronic, lethal disease in which brain function is severely impaired and neuronal damage is irreversible. Huang-Pu-Tong-Qiao (HPTQ), a formula from traditional Chinese medicine, has been used in the clinical treatment of AD for many years, with remarkable effects. However, the neuroprotective mechanisms of HPTQ in AD have not yet been investigated. In the present study, we used AD models in vivo and in vitro, to investigate both the neuroprotective effect of HPTQ water extracts (HPTQ-W) and the potential mechanisms of this action. For the in vivo study, after HPTQ intervention, the Morris water maze test was used to examine learning and memory in rats. Transmission electron microscopy and immunofluorescence methods were then used to investigate neuronal damage. For the in vitro experiments, rat primary hippocampal neurons were cultured and cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Additionally, mRNA levels of CaM, CaMKK, CaMKIV, and tau were examined using qRT-PCR, and protein expression of CaM, CaMKK, p-CaMKIV, and p-tau were examined using western blot. In vivo, we revealed that HPTQ significantly improved learning and memory deficits and attenuated neuronal damage in the AD rat model. Furthermore, in vitro results showed that HPTQ significantly increased cell viability in the AD cell model. We also demonstrated that HPTQ significantly decreased the mRNA levels of CaM, CaMKK, CaMKIV, and tau and significantly decreased the protein expressions of CaM, CaMKK, p-CaMKIV, and p-tau. In conclusion, our results indicated that HPTQ improved cognition and ameliorated neuronal damage in AD models and implicated a reduction in tau phosphorylation caused by inhibition of the CaM-CaMKIV pathway as a possible mechanism.

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