scholarly journals Potential Simultaneous Inhibitors of Angiotensin-Converting Enzyme 2 and Transmembrane Protease, Serine 2

2020 ◽  
Vol 11 ◽  
Author(s):  
Ching-Yuan Wu ◽  
Yu-Shih Lin ◽  
Yao-Hsu Yang ◽  
Li-Hsin Shu ◽  
Yu-Ching Cheng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Hepg2 Cells ◽  
Kidney Tissue ◽  
Herbal Formula ◽  
Angiotensin Converting Enzyme 2 ◽  
Mrna And Protein Expression ◽  
Almost All ◽  
Lung And Kidney

Outbreak of coronavirus disease 2019 occurred in Wuhan and has rapidly spread to almost all parts of world. GB-1, the herbal formula from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, is used for the prophylaxis of SARS-CoV-2 in Taiwan. In this study, we investigated that the effect of GB-1 and the index compounds of GB-1 on the ACE2 and TMPRSS2 expression through in vitro and in vivo study. In our result, GB-1 can inhibit ACE2 and TMPRSS2 protein expression in HepG2 cells, 293T cells, and Caco-2 cells without cytotoxicity. For the mouse model, GB-1 treatment could decrease ACE2 and TMPRSS2 expression levels of the lung and kidney tissue without adverse effects, including nephrotoxicity and hepatotoxicity. In the compositions of GB-1, 0.5–1 mg/ml of Glycyrrhiza uralensis Fisch. ex DC. extract could not inhibit ACE2 mRNA and protein expression in HepG2 cells. In addition, theaflavin-3-gallate could inhibit protein expression of ACE2 and TMPRSS2 without significant cytotoxicity. Our results suggest that GB-1 and theaflavin-3-gallate could act as potential candidates for prophylaxis or treatment of SARS-CoV-2 infection through inhibiting protein expression of ACE2 and TMPRSS2 for the further study.

scholarly journals Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972097873
Author(s):  
Jing Li ◽  
Youming Zhu ◽  
Na Li ◽  
Tao Wu ◽  
Xianyu Zheng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dental Pulp ◽  
Tube Formation ◽  
Endothelial Differentiation ◽  
Cell Source ◽  
Lentiviral Infection ◽  
Mrna And Protein Expression ◽  
Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

scholarly journals Extracellular SPARC cooperates with TGF-β signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts

Rheumatology ◽  
2019 ◽  
Vol 59 (9) ◽  
pp. 2258-2263 ◽  
Author(s):  
Tiago Carvalheiro ◽  
Beatriz Malvar Fernández ◽  
Andrea Ottria ◽  
Barbara Giovannone ◽  
Wioleta Marut ◽  
...  
Keyword(s):  
Protein Expression ◽  
Therapeutic Target ◽  
Dermal Fibroblasts ◽  
Dependent Manner ◽  
Healthy Controls ◽  
Multiple Organs ◽  
Extracellular Matrix Components ◽  
Mrna And Protein Expression

Abstract Objectives SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. Methods Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-β1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. Results Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-β and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-β signalling. Conclusion These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

Comparison of the GDF-9 mRNA and Protein Expression Between In Vivo and In Vitro Cultured Mouse Ovaries∗

2000 ◽  
Vol 74 (3) ◽  
pp. S194-S195
Author(s):  
S.J Yoon ◽  
C.S Shin ◽  
K.-H Baek ◽  
J.J Ko ◽  
N.Y Yoon ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna And Protein Expression

RNAi Supress VEGF, TERT, and Bcl-xl in HEp-2 Cells

2008 ◽  
Vol 139 (2_suppl) ◽  
pp. P98-P98
Author(s):  
Ze-Zhang Tao ◽  
Wang Yan
Keyword(s):  
Protein Expression ◽  
Recombinant Plasmid ◽  
Antitumor Effect ◽  
Squamous Carcinoma ◽  
Therapeutic Modality ◽  
Multiple Targets ◽  
Rnai Technology ◽  
Mrna And Protein Expression

Problem To develop an RNA-interference (RNAi) approach involving the hitting of multiple targets by a recombinant plasmid and evaluate its antitumor effect on laryngeal squamous carcinoma in vitro and in vivo. Methods A plasmid containing 3 different short hairpin RNA (shRNA) segments termed pEGFP-shVEGF-shTERT-shBcl-xl was constructed. Plasmids containing single shRNA against each target (VEFG, TERT, BCL-xl alone) individually were also constructed as control. Cells were treated with these plasmids. The expression of targeted genes as well as apoptosis of tumor cells were evaluated after treatment with multiple shRNA vectors or control vectors. The mRNA and protein expression were determined by RT-PCR and western blotting. Cell viability was examined using the MTT assay. Apoptotic morphological alterations were observed by Hoechst staining and electron microscopy. The in vivo antitumor effect was characterized in a nude mice model of laryngeal squamous carcinoma. Results We demonstrated that a recombinant plasmid containing multiple shRNAs could effectively and simultaneously inhibit VEGF, TERT and Bcl-xl mRNA and protein expression in the HEp-2 cells; the plasmid containing the 3 different shRNAs exhibited a potent antitumor effect on LSCC both in vitro and in vivo, and could much more effectively induced cell apoptosis than each single shRNA. We also demonstrated that the simultaneous blockage of these 3 genes have a better inhibitory effect on human HEp-2 cells than the blockage of each single shRNA. Conclusion Our study demonstrates that the application of vector-based RNAi technology that involves hitting multiple targets will be a promising therapeutic modality in the gene therapy of human laryngeal cancers. Significance Our study provides experimental evidence for the clinical application of this technology in the future. Support This work was supported by the grants from the National Natural Science Foundation of China (No. 30471873, No.30672313 and No.30740012).

scholarly journals STING protects against cardiac dysfunction and remodelling by blocking autophagy

2021 ◽  
Author(s):  
Rui Xiong ◽  
Ning Li ◽  
Wei Wang ◽  
Bo Wang ◽  
Wenyang Jiang ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Protein Expression ◽  
Cardiac Fibroblasts ◽  
Cardiac Remodelling ◽  
Ang Ii ◽  
Mrna And Protein Expression

Abstract Background Heart failure, which is characterized by cardiac remodelling, is one of the most common chronic diseases in the aged. Stimulator of interferon genes (STING) acts as an indispensable molecule modulating immune response and inflammation in many diseases. However, the effects of STING on cardiomyopathy, especially cardiac remodelling are still largely unknown. This study was designed to investigate whether STING could affect cardiac remodelling and to explore the potential mechanisms. Methods In vivo, aortic binding (AB) surgery was performed to construct the mice model of cardiac remodelling. A DNA microinjection system was used to trigger STING overexpression in mice. The STING mRNA and protein expression levels in mice heart were measured, and the cardiac hypertrophy, fibrosis, inflammation and cardiac function were also evaluated. In vitro, cardiomyocytes stimulated by Ang II and cardiac fibroblasts stimulated by TGF-β to performed to further study effects of STING on cardiac hypertrophy and fibroblast. In terms of mechanisms, the level of autophagy was detected in mice challenged with AB. Rapamycin, a canonical autophagy inducer, intraperitoneal injected into mice to study possible potential pathway. Results In vivo, the STING mRNA and protein expression levels in mice heart challenged with AB for 6 weeks were significantly increased. STING overexpression significantly mitigated cardiac hypertrophy, fibrosis and inflammation, apart from improving cardiac function. In vitro, experiments further disclosed that STING overexpression in cardiomyocytes induced by Ang II significantly inhibited the level of cardiomyocyte cross-section area and the ANP mRNA. Meanwhile, TGF-β-induced the increase of α-SMA content and collagen synthesis in cardiac fibroblasts could be also blocked by STING overexpression. In terms of mechanisms, mice challenged with AB showed higher level of autophagy compared with the normal mice. However, STING overexpression could reverse the activation of autophagy triggered by AB. Rapamycin, a canonical autophagy inducer, offset the cardioprotective effects of STING in mice challenged with AB. Finally, further experiments unveiled that STING may inhibit autophagy by phosphorylating ULK1 on serine757. Conclusions STING may prevent cardiac remodelling induced by pressure overload by inhibiting autophagy, which could be a promising therapeutic target in heart failure.

scholarly journals Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett’s Esophageal Cell Line

2021 ◽  
Vol 22 (19) ◽  
pp. 10581
Author(s):  
Eszter Becskeházi ◽  
Marietta Margaréta Korsós ◽  
Eleonóra Gál ◽  
László Tiszlavicz ◽  
Zsófia Hoyk ◽  
...  
Keyword(s):  
Protein Expression ◽  
Compensatory Mechanism ◽  
Dependent Manner ◽  
Ion Transporters ◽  
Esophageal Diseases ◽  
Toxic Agent ◽  
Mrna And Protein Expression ◽  
Esophageal Epithelial Cells

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett’s esophagus, and this is presumably a compensatory mechanism against this toxic agent.

scholarly journals Eplerenone inhibits aldosterone-induced CRP generation in rat vascular smooth muscle cells by regulating the MR-ROS-ERK1/2 signal pathway

2017 ◽  
Vol 15 (3) ◽  
pp. 210-218 ◽  
Author(s):  
Xiaolu Zhang ◽  
Juntian Liu ◽  
Xiaoming Pang ◽  
Jingjing Zhao ◽  
Shouzhu Xu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Protein Expression ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Signal Pathway ◽  
Mrna And Protein Expression

Atherosclerosis is a chronic inflammatory disease in the vessel wall. As a representative inflammatory cytokine, C-reactive protein (CRP) participates in the formation and development of atherosclerosis. It is demonstrated that aldosterone induces CRP generation in vascular smooth muscle cells (VSMCs). This study explored the inhibitory effect of eplerenone on aldosterone-induced CRP expression in VSMCs and mechanism. In the in vitro experiments, rat VSMCs were cultured and aldosterone (10 nM) was used as a stimulant for CRP generation. VSMCs were pretreated with eplerenone for 1 h prior to the stimulation. Messenger RNA (mRNA) and protein expression were identified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot, respectively. The production of reactive oxygen species (ROS) was observed by a fluorescence microscope. In the in vivo experiment, a model of hyperaldosteronism was established by the subcutaneous administration of aldosterone to rats with the osmotic minipumps for 4 weeks. Serum aldosterone and CRP levels were determined with a radioimmunoassay and ELISA (enzyme-linked immunosorbent assay), respectively. The results showed that eplerenone inhibited aldosterone-induced mRNA and protein expression of CRP in VSMCs in vitro and in vivo, and decreased the circulating CRP level of hyperaldosteronism rats. Meanwhile, eplerenone reduced aldosterone-stimulated ROS generation and aldosterone-activated ERK1/2 phosphorylation in VSMCs. In summary, eplerenone inhibits aldosterone-induced CRP generation in VSMCs by regulating the MR-ROS-ERK1/2 signal pathway. These results provide new evidence for the potential anti-inflammatory effect of eplerenone.

Metformin exerts anti-tumor effects via Sonic hedgehog signaling pathway by targeting AMPK in HepG2 cells

2022 ◽  
Author(s):  
Ang Hu ◽  
Zeming Hu ◽  
Jianming Ye ◽  
Yuwen Liu ◽  
Zhonghong Lai ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Sonic Hedgehog ◽  
Hepg2 Cells ◽  
Biological Behavior ◽  
Migration And Invasion ◽  
Shh Pathway ◽  
Mrna And Protein Expression ◽  
Proliferation And Metastasis

Metformin, a traditional first-line pharmacologic treatment for type 2 diabetes, has recently been shown to impart anti-cancer effects on hepatocellular carcinoma (HCC). However, the molecular mechanism of metformin on its antitumor activity is still not completely clear. The Sonic hedgehog (Shh) signaling pathway is closely associated with the initiation and progression of HCC. Therefore, the aim of the current study was to investigate the effects of metformin on the biological behavior of HCC and the underlying functional mechanism of metformin on the Shh pathway. The HCC cellular was induced in HepG2 cells by recombinant human Shh (rhShh). The effects of metformin on proliferation and metastasis were evaluated by proliferation, wound healing and invasion assays in vitro. The mRNA and protein expression levels of proteins related to the Shh pathway were measured by western blotting, quantitative PCR and immunofluorescence staining. Metformin inhibited rhShh-induced proliferation and metastasis. Furthermore, metformin decreased mRNA and protein expression of components of the Shh pathway including Shh, Ptch, Smo and Gli-1. Silencing of AMPK in the presence of metformin revealed that metformin could exert its inhibitory effect via AMPK. Our findings demonstrate that metformin can suppress the migration and invasion of HepG2 cells via AMPK-mediated inhibition of the Shh pathway.

scholarly journals STING Protects Against Cardiac Dysfunction and Remodelling by Blocking Autophagy

2021 ◽  
Author(s):  
Rui Xiong ◽  
Ning Li ◽  
Bohao Liu ◽  
Ruyuan He ◽  
Wenyang Jiang ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Protein Expression ◽  
Cardiac Fibroblasts ◽  
Cardiac Remodelling ◽  
Ang Ii ◽  
Mrna And Protein Expression

Abstract Background: Heart failure, which is characterized by cardiac remodelling, is one of the most common chronic diseases in the aged. Stimulator of interferon genes (STING) acts as an indispensable molecule modulating immune response and inflammation in many diseases. However, the effects of STING on cardiomyopathy, especially cardiac remodelling are still largely unknown. This study was designed to investigate whether STING could affect cardiac remodelling and to explore the potential mechanisms. Methods: In vivo, aortic binding (AB) surgery was performed to construct the mice model of cardiac remodelling. A DNA microinjection system was used to trigger STING overexpression in mice. The STING mRNA and protein expression levels in mice heart were measured, and the cardiac hypertrophy, fibrosis, inflammation and cardiac function were also evaluated. In vitro, cardiomyocytes stimulated by Ang II and cardiac fibroblasts stimulated by TGF-β to performed to further study effects of STING on cardiac hypertrophy and fibroblast. In terms of mechanisms, the level of autophagy was detected in mice challenged with AB. Rapamycin, a canonical autophagy inducer, intraperitoneal injected into mice to study possible potential pathway.Results: In vivo, the STING mRNA and protein expression levels in mice heart challenged with AB for 6 weeks were significantly increased. STING overexpression significantly mitigated cardiac hypertrophy, fibrosis and inflammation, apart from improving cardiac function. In vitro, experiments further disclosed that STING overexpression in cardiomyocytes induced by Ang II significantly inhibited the level of cardiomyocyte cross-section area and the ANP mRNA. Meanwhile, TGF-β-induced the increase of α-SMA content and collagen synthesis in cardiac fibroblasts could be also blocked by STING overexpression. In terms of mechanisms, mice challenged with AB showed higher level of autophagy compared with the normal mice. However, STING overexpression could reverse the activation of autophagy triggered by AB. Rapamycin, a canonical autophagy inducer, offset the cardioprotective effects of STING in mice challenged with AB. Finally, further experiments unveiled that STING may inhibit autophagy by phosphorylating ULK1 on serine757.Conclusion: STING may prevent cardiac remodelling induced by pressure overload by inhibiting autophagy, which could be a promising therapeutic target in heart failure.

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