STAR Protocols
·
2021
·
IF 0
Yunjing Wang,Qian Gong,Fan Huang,Linfang He,Yule Liu
Summary Wounding evokes transient increases in cytosolic calcium (Ca 2+ ) concentration. Visualizing real-time Ca 2+ flux provides new insights into Ca 2+ -signaling pathways. Here, we outline a protocol to detect insect feeding-induced Ca 2+ flux elevation in Nicotiana benthamiana leaves based on the GCaMP3 reporter system by Leica fluorescence stereo microscopes (LFSM). LFSM combines super-fast manual screening with high-end imaging capabilities. Through this protocol, we can clearly observe the calcium flow after aphid’s piercing-sucking. Additionally, we describe a protocol to quantify Ca 2+ level using LFSM. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) .
研究领域:
植物生物学
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分子生物学
·
昆虫学
✓ 已验证
DOI
PubMed
JOURNAL OF ETHNOPHARMACOLOGY
·
2024
·
IF 4.8
Junjie Li,Yixu Wang,Jiawei Li,Sihan Xu,Shou Wang,Wenjuan Liu,Li Fu,Min Jiang,Gang Bai
Ethnopharmacological relevance Lian Qiao (LQ), the dried fruit of Forsythia suspensa (Thunb.) Vahl, is a well-documented traditional Chinese medicine known for its detoxifying and heat-clearing properties. Clinically, compounds containing LQ are widely used to treat thrombotic diseases, indicating that it may have antithrombotic effects. However, its exact mechanism of action remains unknown. Aim of the study This study aimed to verify the antithrombotic effect of LQ and further explore the material basis and target mechanism of its antithrombotic effect using various biological methods. Materials and methods An epinephrine-collagen-thrombin-induced mouse model of acute pulmonary embolism (APE) was established to study the effects of LQ on thrombus development. A UPLC/Q/TOF-MS screening and identification system based on the inhibition of platelet aggregation and Ca 2+ antagonism was established to determine the pharmacodynamic components of LQ that inhibit platelet activation. The inhibitory effect of active ingredients on platelet activation, and the determination of the target of their inhibitory effect on platelet activation have been studied using chemical proteomics. Furthermore, based on the structure and function of the target protein, a multidisciplinary approach was adopted to analyze the molecular mechanism of active ingredient binding to target proteins and to evaluate the effects of active ingredients on the downstream signaling pathways of target proteins. Results LQ showed significant anticoagulant effects in APE model mice. Phillyrin and phillygenin were the antiplatelet-activating components of LQ. PLCβ3 was identified as a target for inhibiting platelet activation by phillyrin and its metabolites. The mechanism underlying the effect involves phillyrin and its metabolites inhibiting PLCβ3 activity by blocking the binding of PLCβ3 to Gαq through non-covalently targeting the ASN260 of PLCβ3, thus inhibiting the downstream Gαq-PLCβ3-Ca 2+ signaling pathway, effectively hindering platelet activation and therefore playing an anticoagulant role. Conclusion This study not only proposes and validates the antithrombotic effect of LQ for the first time but also finds that phillyrin and phillygenin are the main pharmacological substances through which LQ exerts antithrombotic activity and reveals a novel mechanism by which they exert antiplatelet activity by directly targeting and inhibiting PLCβ3 activity. These findings significantly contribute to our understanding of the therapeutic potential of phillyrin and provide important clues for the discovery and development of new antiplatelet drugs.
研究领域:
药理学
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传统中药
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分子生物学
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生物化学
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血栓研究
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CARBOHYDRATE POLYMERS
·
2020
·
IF 7.182
Zhao Zhao,Mei Ji,Qianqing Wang,Nannan He,Yue Li
Off-target drug delivery, together with multidrug resistance (MDR), are two keys obstacles that account for the disappointing outcome in clinical chemotherapy of cancer. To solve these dilemmas, Herein, we constructed cancer cell membrane (CCM) modified silica (CS) nanoparticles (CCM/CS) to co-deliver Ca 2+ channel siRNA with doxorubicin (DOX) to construct a platform (CCM/CS/R-D) for the efficient therapy of cervical cancer. It was demonstrated that the optimal CCM/CS/R-D was spherical nanoparticles with size at 122.39 ± 4.69 nm and the surface charge of -27.76 ± 3.12 mV. In addition, the CCM/CS/R-D showed acid responsive drug release while high stability under physiological conditions with negligible hemolysis. The CCM/CS/R-D showed CCM mediated cellular uptake and efficient endosomal escape as well as siRNA transfection potential (comparable to that of PEI 25 K) on MDR cervical cancer cells (HeLa/DOX). Most importantly, the MDR of cancer cells was conquered through modulation of T-type Ca 2+ (Cav) channels. It was observed that the Cav channel siRNA could negatively regulate the level of cytosolic Ca 2+ concentration which triggered G0/G1 phase cell cycle arrest and elevated intracellular drug retention in HeLa/DOX cells without significantly affect the expression of P-glycolprotein (P-gp). The in vitro and in vivo experiments revealed that CCM/CS/R-D exerted greatly enhanced tumor targetability and therapeutic effect on HeLa/DOX, which was superior than CS/R-D or mono delivery system (CCM/CS/R or CCM/CS/D).
研究领域:
癌症研究
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纳米医学
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药理学
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分子生物学
✓ 已验证
DOI
PubMed
高影响因子
ACS Nano
·
2019
·
IF 13.903
Jieni Fu,Xiangmei Liu,Lei Tan,Zhenduo Cui,Yufeng Zheng,Yanqin Liang,Zhaoyang Li,Shengli Zhu,Kelvin Wai Kwok Yeung,Xiaobo Feng,Xianbao Wang,Shuilin Wu
Using noninvasive stimulation of cells to control cell fate and improve bone regeneration by optical stimulation can achieve the aim of precisely orchestrating biological activities. In this study, we create a fast and repeatable photoelectric-responsive microenvironment around an implant using a bismuth sulfide/hydroxyapatite (BS/HAp) film. The unexpected increase of photocurrent on the BS/HAp film under near-infrared (NIR) light is mainly due to the depletion of holes through PO43– from HAp and interfacial charge transfer by HAp compared with BS. The electrons activate the Na+ channel of mesenchymal stem cells (MSCs) and change the cell adhesion in the intermediate environment. The behavior of MSCs is tuned by changing the photoelectronic microenvironment. RNA sequencing reveals that when photoelectrons transfer to the cell membrane, sodium ions flux and the membrane potential depolarizes to change the cell shape. Meanwhile, calcium ions fluxed and FDE1 was upregulated. Furthermore, the TCF/LEF in the cell nucleus began transcription to regulate the downstream genes involved in osteogenic differentiation, which is performed through the Wnt/Ca2+ signaling pathway. This research has created a biological therapeutic strategy, which can achieve in vitro remotely, precisely, and noninvasively controlling cell differentiation behaviors by tuning the in vivo photoelectric microenvironment using NIR light.
研究领域:
生物医学工程
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组织工程
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细胞生物学
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光遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FASEB JOURNAL
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2018
·Vol. 32
·期 6
·3398-3410
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IF 5.595
Yugai Jia,Yumeng Miao,Mengfan Yue,Mei Shu,Zhifeng Wei,Yue Dai
Tetrandrine, a bisbenzylisoquinoline alkaloid, was previously demonstrated to attenuate inflammation and cartilage destruction in the ankles of mice with collagen-induced arthritis (CIA). Here, we explored the underlying mechanism by which tetrandrine prevented arthritis-induced bone erosion by focusing on the differentiation and function of osteoclasts. We found that daily administration of tetrandrine (30 mg/kg) markedly reduced the bone damage and decreased the number of osteoclasts in CIA rats. In vitro , tetrandrine inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis at the early stage and reduced the expressions of osteoclast-related marker genes. In bone marrow-derived macrophages and RAW264.7 cells, tetrandrine inhibited RANKL-induced translocation of NF-κB-p65 and nuclear factor of activated T cell 1 (NFATc1) through suppressing spleen tyrosine kinase (Syk)-Bruton's tyrosine kinase-PLCγ2-Ca 2+ signaling. Of interest, tetrandrine did not affect the phosphorylation of immunoreceptor tyrosine-based activation motifs, the conventional upstream of Syk, but it inhibited the activity of Syk by enhancing its ubiquitination and degradation. The anti–osteoclastogenesis effect of tetrandrine nearly disappeared when it was used in combination with the Syk inhibitor piceatannol or in constitutively activated Syk-overexpressing cells. Taken together, tetrandrine attenuated CIA-induced bone destruction by inhibiting osteoclastogenesis through hindering the translocation of NF-κB-p65 and NFATc1 via reducing the activation of Syk.—Jia Y., Miao, Y., Yue, M., Shu, M., Wei, Z., Dai Y. Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase. FASEB J. 32, 3398–3410 (2018). www.fasebj.org
研究领域:
药理学
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免疫学
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风湿病学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）