scholarly journals Nonmuscle myosin II shRNA inhibit migration and contraction in rat hepatic stellate cells through regulating AKT/mTOR/S6K/4EBP1 signaling pathway

2018 ◽  
Author(s):  
Zhenghong Li ◽  
Yun Feng ◽  
Ruling Zhang ◽  
Peiwen Wang ◽  
Lungen Lu ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Hepatic Stellate Cells ◽  
Real Time Pcr ◽  
Stellate Cell ◽  
Myosin Ii ◽  
Stellate Cells ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii

AbstractMigration and contraction of activated hepatic stellate cell (HSC) are essential factors for cirrhosis formation and development. It has been demonstrated that blebbistatin, a nonmuscle myosin II (NMMII) inhibitor, can inhibit the migration and contraction of HSC, whereas the main cell signaling pathway is still unknown. Mammalian target of rapamycin (mTOR) signaling pathway may be involved in many cells migration and contraction, whether NMMII and mTOR have any crosslinks draw our attention. In the currently study, we used LV-RNAi to specifically attenuate mTOR and NMMII in rat HSC. We aimed to examine the effect of mTOR LV-RNAi on the migration and contraction of HSC and explore the crosslink between mTOR cell signal and NMMII. Using real-time PCR and western blot, we found that mTOR and the downstream factors including S6K and 4EBP1 all up-regulated with the activation of HSC, mTOR and NMMII LV-RNAi was transfected into activated HSC using lipofectamine 2000. The levels of mRNA and proteins were also examined using real-time PCR and western blot respectively. The expression of mTOR can be down-regulated by NMMII LV-RNAi significantly, as well as the expression of S6K, 4EBP1, α-SMA and collagen I, but the level of AKT was up-regulated. Then we used Transwell system and collagen lattices to examine the NMMII and mTOR LV-RNAi efficiency on HSC migration and contraction, as we hypothesized, both of the LV-RNAi could inhibit HSC migration and contraction significantly. These results indicated that nonmuscle myosin II shRNA inhibit migration and contraction in rat hepatic stellate cells through the regulation of mTOR/S6K/4EBP1 signaling pathway

[331] EXPRESSION, LOCALISATION AND ROLE OF NONMUSCLE MYOSIN II ISOFORMS IN MOUSE HEPATIC STELLATE CELLS

2007 ◽  
Vol 46 ◽  
pp. S130 ◽  
Author(s):  
Z.A. Liu ◽  
H. Reynaert ◽  
E. Van Rossen ◽  
B. Schroyen ◽  
L. van Grunsven ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Myosin Ii ◽  
Stellate Cells ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii

scholarly journals Astragaloside positively regulated osteogenic differentiation of pre-osteoblast MC3T3-E1 through PI3K/Akt signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Wei Bing Jing ◽  
Hongjuan Ji ◽  
Rui Jiang ◽  
Jinlong Wang
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Akt Signaling ◽  
Calcium Deposition ◽  
Control Group ◽  
Akt Signaling Pathway ◽  
Western Blot Assay

Abstract Background Osteoporosis is a widespread chronic disease characterized by low bone density. There is currently no gold standard treatment for osteoporosis. The aim of this study was to explore the role and mechanism of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. Methods MC3T3-E1 cells were divided into control and different dose of Astragaloside (10, 20, 40, 50, and 60 μg/ml). Then, ALP and ARS staining were performed to identify the effects of Astragaloside for early and late osteogenic capacity of MC3T3-E1 cells, respectively. Real-time PCR and western blot were performed to assess the ALP, OCN, and OSX expression. PI3K/Akt signaling pathway molecules were then assessed by Western blot. Finally, PI3K inhibitor, LY294002, was implemented to assess the mechanism of Astragaloside in promoting osteogenic differentiation of MC3T3-E1 cells. Results Astragaloside significantly increased the cell viability than the control group. Moreover, Astragaloside enhanced the ALP activity and calcium deposition than the control groups. Compared with the control group, Astragaloside increased the ALP, OCN, and OSX expression in a dose-response manner. Western blot assay further confirmed the real-time PCR results. Astragaloside could significantly increase the p-PI3K and p-Akt expression than the control group. LY294002 partially reversed the promotion effects of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. LY294002 partially reversed the promotion effects of Astragaloside on ALP, OCN, and OSX of MC3T3-E1 cells. Conclusion The present study suggested that Astragaloside promoted osteogenic differentiation of MC3T3-E1 cells through regulating PI3K/Akt signaling pathway.

miR-184 Inhibits Inflammation Through Targeting Toll-Like Receptor 4 (TLR4)/NLR Family Pyrin Domain Containing 3 (NLRP3) Signaling Pathway in Neonatal Purulent Meningitis

2020 ◽  
Vol 10 (4) ◽  
pp. 569-575
Author(s):  
Jiang Wu ◽  
Shixiong Yang ◽  
Jin Shi ◽  
Yibing Shi
Keyword(s):  
Cell Proliferation ◽  
Cerebrospinal Fluid ◽  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Caspase 3 ◽  
Caspase 1 ◽  
Purulent Meningitis ◽  
Inflammation Group

Neonatal purulent meningitis (NPM) leads to higher mortality and neurological sequelae rates. miR184 involves in inflammation and tumor, but the role of miR-184 in NPM remains unclear. NPM patients and non-intracranial infected neonates were collected and miR-184 expression in cerebrospinal fluid was assessed by real-time PCR. The Neuro-2a cell line was cultured and divided into control group, inflammation group (treated with LPS), and miR-184 inhibitor group, which was transfected with miR-184 inhibitor on the basis of inflammation followed by analysis of miR-184 and TLR4 expression by Real time PCR, Caspase 3 activity, cell proliferation by MTT assay, secretion of IL-1β and IL-6 by ELISA, NLRP3 expression by real time PCR and western blot, and Caspase-1 p20 and NF- B level by western blot. miR-184 expression level was significantly increased in cerebrospinal fluid of NPM group (P < 0 05) and also elevated in inflammation group along with significantly inhibited cell proliferation was inhibited, increased Caspase 3 activity, IL-1β and IL-6 secretion, and decreased TLR4, NLRP3, Caspase-1 p20 and NFκ- B expression (P < 0 05). miR-184 inhibitor significantly down-regulated miR-184 expression in the inflammation group, promoted cell proliferation, decreased Caspase 3 activity, IL-1β and IL-6 secretion, and increased TLR4, NLRP3, Caspase1 p20 and NF- κB expression (P < 0 05). miR-184 expression is increased in neonatal purulent meningitis and it can inhibit inflammation by targeting TLR4/NLRP3 signaling pathway, leading to amelioration of the progression of neonatal purulent meningitis.

miR-184 Inhibits Hypertrophic Scar Through Regulating Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (AKT) Signaling Pathway

2020 ◽  
Vol 10 (1) ◽  
pp. 133-138
Author(s):  
Peng Zhao ◽  
Junxia Qin ◽  
Lili Liang ◽  
Xinzhong Zhang
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Hypertrophic Scar ◽  
Scar Tissue ◽  
Akt Signaling ◽  
Scar Formation ◽  
Akt Signaling Pathway

Hypertrophic scar (HS) is a process of tissue repair and healing, and excessive fibrosis of local tissue leads to scar formation. During HS formation, fibroblasts (Fb) proliferate, synthesize and secrete and promote HS development. miR-184 regulates skin formation and tissue development. However, miR-184’s role in HS remains unclear. miR-184 expression in HS patients and normal healthy (Control) tissues was measured by real-time PCR. pAKT expression was analyzed by Western blot. Fb cells from human HS were cultured and divided into 2 groups, siRNA NC group and miR-184 siRNA group followed by analysis of miR-184 expression by real time PCR, cell proliferation by MTT assay, secretion of inflammatory factors IL-1β and IL-6 by ELISA, as well as expression of pAKT and AKT by western blot. Compared with control group, miR-184 and pAKT expression was significantly increased in the HS group. Transfection of miR-184 siRNA into Fb significantly downregulated miR-184 expression, inhibited cell proliferation, promoted Caspase 3 activity, decreased IL-1β and IL-6 secretion, and reduced pAKT level (P < 0.05). miR-184 expression is increased in hypertrophic scar tissue. Down-regulation of miR-184 expression in proliferative scar tissue fibroblasts can down-regulate PI3K/AKT signaling pathway, inhibit inflammation, promote apoptosis, inhibit fibroblast proliferation, and regulate hypertrophic scar formation.

scholarly journals Liraglutide Exerts Antidiabetic Effect via PTP1B and PI3K/Akt2 Signaling Pathway in Skeletal Muscle of KKAy Mice

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Wenjun Ji ◽  
Xinlin Chen ◽  
Juan Lv ◽  
Meng Wang ◽  
Shuting Ren ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Glucose Transporter ◽  
Tyrosine Phosphatase ◽  
Gene Expressions ◽  
Protein Levels ◽  
Kkay Mice

Background. Liraglutide (a glucagon-like peptide 1 analog) was used for the treatment of type 2 diabetes (T2DM) which could produce glucose-dependent insulin secretion.Aim. The aim was to investigate whether liraglutide could improve myofibril and mitochondria injury in skeletal muscle and the mechanisms in diabetic KKAy mice.Method. We divided the male KKAy mice into 2 groups: liraglutide group (250 μg/kg/day liraglutide subcutaneous injection) and model group; meanwhile, the male C57BL/6J mice were considered as the control. After 6 weeks, the ultrastructure of skeletal muscle was observed by electron microscope. The gene expressions of protein tyrosine phosphatase 1B (PTP1B), phosphatidylinositol 3-kinase (PI3K), and glucose transporter type 4 (GLUT4) were determined by real-time PCR. The protein levels of the above molecules and phospho-Akt2 (p-Akt2) were measured by Western blot.Results. Liraglutide significantly ameliorated the injury of mitochondria by increasing the number (+441%) and the area (+113%) of mitochondria and mitochondrial area/100 µm2(+396%) in skeletal muscle of KKAy mice. The results of real-time PCR and Western blot showed that liraglutide downregulated PTP1B while it upregulated PI3K and GLUT4 (P<0.01). The protein level of p-Akt2/Akt2 was also increased (P<0.01).Conclusion. These results revealed that liraglutide could improve myofibril and mitochondria injury in skeletal muscle against T2DM via PTP1B and PI3K/Akt2 signaling pathway.

scholarly journals MiR-571 affects the development and progression of liver fibrosis by regulating the Notch3 pathway

2021 ◽  
Author(s):  
Shuo Cong ◽  
Yongmei Liu ◽  
Yi Li ◽  
Yu Chen ◽  
Rui Chen ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Signaling Pathway ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Rt Pcr ◽  
Promote Cell Proliferation ◽  
Notch 3 ◽  
And Migration ◽  
Set Up

Abstract Exploring the expression of miR-571 in patients with liver fibrosis and its role in the progression of liver fibrosis. A total of 74 patients with chronic hepatitis and cirrhosis accompanied by liver fibrosis in our institution from September to December 2018 were collected for study, and the expression of miR-571 in patients with different progressions of liver fibrosis was determined by RT-PCR and Western blot analysis. Set up Notch3 up group and Notch3 down regulated group, RT-PCR and Western blot were used to determine the effect of Notch signaling on the expression of fibrogenic α-SMA, collagen I. CCK-8, cell scratch assays, Transwell assays, flow cytometry were used to determine the effect of miR-571 on LX-2 proliferation, migration, apoptosis in human stem stellate cells, and RT-PCR, Western blot assays were performed to determine the effect of miR-571 on the Notch3 signaling pathway and the expression of profibrogenic factors. miR-571 is up-regulated in patients with liver fibrosis and is associated with the progression of liver fibrosis. Notch3 signaling pathway can promote the expression of fibroblast in human hepatic stellate cells; miR-571 can inhibit the apoptosis of human hepatic stellate cells, promote cell proliferation and migration; up regulation of miR-571 can promote the expression of Notch3 and Jagged 1; up regulation of miR-571 can also promote the expression of fibroblast. miR-571 can promote the activation of human stem stellate cells and the expression of fibroblasts through Notch 3 signaling pathway.

scholarly journals The Drosophila melanogaster Rab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function

2020 ◽  
Vol 31 (21) ◽  
pp. 2379-2397
Author(s):  
Amy Platenkamp ◽  
Elizabeth Detmar ◽  
Liz Sepulveda ◽  
Anna Ritz ◽  
Stephen L. Rogers ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Signaling Pathway ◽  
Cross Talk ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii ◽  
And Function

The Rab GAP RN-tre regulates the activity, coalescence, and function of nonmuscle myosin II in Drosophila melanogaster cells through cross-talk with the Rho1 signaling pathway. This regulation is partially independent of RN-tre’s GAP activity.

scholarly journals MiR-571 affects the development and progression of liver fibrosis by regulating the Notch3 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuo Cong ◽  
Yongmei Liu ◽  
Yi Li ◽  
Yu Chen ◽  
Rui Chen ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Signaling Pathway ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Rt Pcr ◽  
Related Factors ◽  
Promote Cell Proliferation ◽  
And Migration ◽  
Set Up

AbstractExploring the expression of miR-571 in patients with liver fibrosis and its role in the progression of liver fibrosis. A total of 74 patients with liver fibrosis in our institution from September to December 2018 were collected for study, and the expression of miR-571, Notch3 and Jagged1 in patients with different progressions of liver fibrosis was determined by RT-PCR and Western blot analysis. Set up Notch3 up group and Notch3 down regulated group, RT-PCR and Western blot were used to determine the effect of Notch signaling on the expression of fibrogenic factors. CCK-8, cell scratch assays, Transwell assays, flow cytometry were used to determine the effect of miR-571 on LX-2 proliferation, migration, apoptosis in human stem stellate cells, and RT-PCR, Western blot assays were performed to determine the effect of miR-571 on the Notch3 signaling pathway and the expression of profibrogenic factors. miR-571, Notch3 and Jagged1 are up-regulated in patients with liver fibrosis and is associated with the progression of liver fibrosis. Notch3 signaling pathway can promote the expression of fibroblast in human hepatic stellate cells; miR-571 can inhibit the apoptosis of human hepatic stellate cells, promote cell proliferation and migration; up regulation of miR-571 can promote the expression of Notch3 and Jagged1, and up-regulation of miR-571 also promoted the expression of related fibroblasts. MiR-571 can promote the activation of human stem cell stellate cells and the expression of fibroblast related factors through Notch3 signaling pathway.

scholarly journals Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255656
Author(s):  
Naoki Dohi ◽  
Momoka Yamaguchi ◽  
Reina Hase ◽  
Ryosuke Suzuki ◽  
Yumeto Wakabayashi ◽  
...  
Keyword(s):  
Real Time ◽  
Hepatic Stellate Cells ◽  
Kinase Inhibitor ◽  
Myosin Ii ◽  
Fluorescent Microscopy ◽  
Rho Kinase ◽  
Stellate Cells ◽  
Channel Blockers ◽  
Endothelin 1 ◽  
Activated State

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ETA receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca2+-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca2+ channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd3+ and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca2+-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ETA receptors, as well as via mechanisms involving Ca2+-calmodulin and Rho kinase.

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