Isoliquiritigenin inhibits TGF-β1-induced fibrogenesis through activating autophagy via PI3K/AKT/mTOR pathway in MRC-5 cells

Abstract Isoliquiritigenin (ISL), a natural flavonoid derived from the root of liquorice, has been reported to possess anti-inflammatory and antioxidant activities. Previous studies have found that ISL plays a crucial role in anti-fibrosis of adipose tissue and renal tissue; however, its effect on pulmonary fibrogenesis has not been demonstrated. In this study, we aimed to explore the roles and the underlying mechanisms of ISL in TGF-β1-induced fibrogenesis using human lung fibroblast-derived MRC-5 cells. Cell proliferation and migration were determined by MTT and wound healing assay, respectively. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (COLIA1) and fibronectin (FN), microtubule-associated protein light chain 3 (LC3) and related signaling molecules were detected by quantitative real-time PCR, western blot and immunofluorescence assay, correspondingly. EGFP-LC3 transfection was used for autophagy analysis. The results showed that ISL inhibited the TGF-β1-induced proliferation and migration, and down-regulated the expressions of α-SMA, COLIA1 and FN. ISL treatment led to up-regulation of LC3 in TGF-β1-treated MRC-5 cells, accompanied by significant decrease in the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In addition, the inhibitory effects of ISL on TGF-β1-induced fibrogenic features in MRC-5 cells were enhanced by pretreatment with autophagy activator Rapmycin and PI3K/AKT inhibitor LY294002 and reversed by autophagy inhibitor 3-methyladenine and PI3K/AKT activator IGF-1. Taken together, our results demonstrated that ISL could attenuate the fibrogenesis of TGF-β1-treated MRC-5 cells by activating autophagy via suppressing the PI3K/AKT/mTOR pathway. Therefore, ISL holds a great potential to be developed as a novel therapeutic agent for the treatment of pulmonary fibrosis.

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MicroRNA-145 Involves in the Pathogenesis of Renal Vascular Lesions and May Become a Potential Therapeutic Target in Patients with Juvenile Lupus Nephritis

Kidney & Blood Pressure Research ◽

10.1159/000500923 ◽

2019 ◽

Vol 44 (4) ◽

pp. 643-655 ◽

Cited By ~ 2

Author(s):

Zhaomin Cai ◽

Wei Xiang ◽

Xiaojie Peng ◽

Yan Ding ◽

Wang Liao ◽

...

Keyword(s):

Smooth Muscle ◽

Lupus Nephritis ◽

Smooth Muscle Actin ◽

Vascular Lesions ◽

Alpha Smooth Muscle Actin ◽

Migration Ability ◽

Phenotypic Transformation ◽

And Migration ◽

Renal Vascular ◽

Proliferation And Migration

Aims: The current study was conducted with the central objective of investigating the expression of microRNA-145 (miR-145) in renal vascular lesions (RVLs) in juvenile lupus nephritis (JLN) and its possible mechanism. Methods: The clinical data of 49 JLN patients confirmed by renal biopsy were collected and followed by grouping according to the RVLs score after hematoxylin-eosin staining: mild, moderate, and severe groups. In situ hybridization was used to detect the expression of miR-145 in renal vessels which was then being compared among different RVLs groups. Up-LV-miR-145 and LV-miR-NC lentiviral vectors were constructed and transfected into human vascular smooth muscle cells (HVSMCs), respectively. After HVSMCs were treated with 10.0 µg/L platelet-derived growth factor (PDGF)-BB for 24 h, the proliferation, migration, and apoptosis of endothelial cells were detected by MTT, Transwell assay, and flow cytometry, respectively. Western blot was used to detect expression of alpha-smooth muscle actin (α-SM-actin) and osteopontin (OPN). Results: The expression of miR-145 in renal vascular cells was statistically significant. The higher the inner membrane ratio, the lesser the miR-145 expression. After treatment with PDGF-BB, expression of miR-145 in HVSMCs decreased, proliferation and migration ability enhanced, apoptosis decreased, α-SM-actin decreased, and OPN increased. The proliferation and migration ability of HVSMCs in the LV-miR-145 group suppressed, apoptosis enhanced, α-SM-actin increased, and OPN decreased. Conclusions: Our study revealed that miR-145 expression decreased with the increase of vascular damage. miR-145 can inhibit proliferation, migration, and differentiation phenotypic transformation of HVSMCs induced by PDGF-BB. miR-145 may be involved in the pathogenesis of RVLs and may be a new target for treatment of RVLs in lupus nephritis.

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TGF-β1/SMOC2/AKT and ERK axis regulates proliferation, migration, and fibroblast to myofibroblast transformation in lung fibroblast, contributing with the asthma progression

Hereditas ◽

10.1186/s41065-021-00213-w ◽

2021 ◽

Vol 158 (1) ◽

Author(s):

Yuebin Wang ◽

Huike Yang ◽

Xian Su ◽

Anqiang Cao ◽

Feng Chen ◽

...

Keyword(s):

Calcium Binding ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β ◽

Chronic Respiratory Disease ◽

Lung Fibroblasts ◽

Promoting Effect ◽

And Migration ◽

Tgf Β1 ◽

Proliferation And Migration

Abstract Background Asthma is a common chronic respiratory disease that influences 300 million people all over the world. However, the pathogenesis of asthma has not been fully elucidated. It has been reported that transforming growth factor-β (TGF-β) can activate myofibroblasts. Moreover, the fibroblast to myofibroblast transformation (FMT) can be triggered by TGF-β, which is a major mediator of subepithelial fibrosis. Secreted modular calcium-binding protein 2 (SMOC2) is a member of cysteine (SPARC) family and is involved in the progression of multiple diseases. However, its role in asthma remains poorly understood. RT-qPCR evaluated the expression of SMOC2. Bromodeoxyuridine assay and wound-healing assay detected the proliferation and migration of lung fibroblasts, respectively. IF staining was performed to assess the expression of α-smooth muscle actin (α-SMA). Western blot analysis detected the levels of proteins. Flow cytometry was utilized for determination of the number of myofibroblasts. Results We found the expression of SMOC2 was upregulated by the treatment of TGF-β1 in lung fibroblasts. In addition, SMOC2 promoted the proliferation and migration of lung fibroblasts. More importantly, SMOC2 accelerated FMT of lung fibroblasts. Furthermore, SMOC2 was verified to control the activation of AKT and ERK. Rescue assays showed that the inhibition of AKT and ERK pathway reversed the promoting effect of SMOC2 overexpression on proliferation, migration and FMT in lung fibroblasts. Conclusions This work demonstrated that SMOC2 modulated TGF-β1-induced proliferation, migration and FMT in lung fibroblasts and may promote asthma, which potentially provided a novel therapeutic target for the management of asthma.

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Angiotensin-(1–7) decreases the expression of collagen I via TGF-β1/Smad2/3 and subsequently inhibits fibroblast–myofibroblast transition

Clinical Science ◽

10.1042/cs20160193 ◽

2016 ◽

Vol 130 (21) ◽

pp. 1983-1991 ◽

Cited By ~ 8

Author(s):

Jian Ping Zhou ◽

Wei Tang ◽

Yun Feng ◽

Ning Li ◽

Chen Juan Gu ◽

...

Keyword(s):

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Renin Angiotensin System ◽

Collagen Type I ◽

Transforming Growth Factor Β1 ◽

Mitogen Activated Protein Kinase ◽

Signalling Pathways ◽

Type I ◽

Human Lung Fibroblast ◽

Tgf Β1

Previous studies have shown that the RAS (renin–angiotensin system) might participate in airway remodelling in asthma. As a main component of the RAS, Ang-(1–7) [angiotensin-(1–7)] has been reported in few studies regarding its protective effect on asthma. However, the functional roles and relevant signalling pathways of Ang-(1–7) have not been well illustrated. In the present study, we analysed the effect of Ang-(1–7) on AngII (angiotensin II)-induced HLF (human lung fibroblast)–MF (myofibroblast) transition by detecting Col-I (collagen type I), TGF-β1 (transforming growth factor-β1) and α-SMA (α-smooth muscle actin) expression. We explored further the possible signalling pathways involved in HLF–MF transition. Our results showed that Ang-(1–7) could down-regulate the expression of Col-I, α-SMA and TGF-β1/Smad2/3 (all P<0.05). A significant decrease was found in phosphorylation of PI3K (phosphoinositide 3-kinase), Akt, p38-MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) signalling pathways during HLF–MF transition (all P<0.05). Our data suggests that Ang-(1–7) decreases the expression of Col-I via TGF-β1/Smad2/3 and subsequently inhibits HLF–MF transition.

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Forsythiaside A Regulates Activation of Hepatic Stellate Cells by Inhibiting NOX4-Dependent ROS

Oxidative Medicine and Cellular Longevity ◽

10.1155/2022/9938392 ◽

2022 ◽

Vol 2022 ◽

pp. 1-17

Author(s):

Mengting Zhou ◽

Xingtao Zhao ◽

Li Liao ◽

Ying Deng ◽

Meichen Liu ◽

...

Keyword(s):

Liver Fibrosis ◽

Western Blot ◽

Hepatic Fibrosis ◽

Hepatic Stellate Cells ◽

Antioxidant Activities ◽

Stellate Cells ◽

Tnf Α ◽

And Migration ◽

Tgf Β1 ◽

Proliferation And Migration

Hepatic stellate cells (HSCs) activation is an important step in the process of hepatic fibrosis. NOX4 and reactive oxygen species expressed in HSCs play an important role in liver fibrosis. Forsythiaside A (FA), a phenylethanoid glycoside extracted and isolated from Forsythiae Fructus, has significant antioxidant activities. However, it is not clear whether FA can play a role in inhibiting the HSCs activation through regulating NOX4/ROS pathway. Therefore, our purpose is to explore the effect and mechanism of FA on HSCs activation to alleviate liver fibrosis. LX2 cells were activated by TGF-β1 in vitro. MTT assay and Wound Healing assay were used to investigate the effect of FA on TGF-β1-induced LX2 cell proliferation and migration. Elisa kit was used to measure the expression of MMP-1 and TIMP-1. Western blot and RT-qPCR were used to investigate the expression of fibrosis-related COLI, α-SMA, MMP-1 and TIMP-1, and inflammation-related TNF-α, IL-6 and IL-1β. The hydroxyproline content was characterized using a biochemical kit. The mechanism of FA to inhibit HSCs activation and apoptosis was detected by DCF-DA probe, RT-qPCR, western blot and flow cytometry. NOX4 siRNA was used to futher verify the effect of FA on NOX4/ROS pathway. The results showed that FA inhibited the proliferation and migration of LX2 cells and adjusted the expression of MMP-1, TIMP-1, COLI, α-SMA, TNF-α, IL-6 and IL-1β as well as promoted collagen metabolism to show potential in anti-hepatic fibrosis. Mechanically, FA down-regulated NOX4/ROS signaling pathway to improve oxidation imbalances, and subsequently inhibited PI3K/Akt pathway to suppress proliferation. FA also promoted the apoptosis of LX2 cells by Bax/Bcl2 pathway. Furthermore, the effects of FA on TGF-β1-induced increased ROS levels and α-SMA and COLI expression were weaken by silencing NOX4. In conclusion, FA had potential in anti-hepatic fibrosis at least in part by remolding of extracellular matrix and improving oxidation imbalances to inhibit the activation of HSCs and promote HSCs apoptosis.

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Metformin attenuates carotid neointimal hyperplasia by modulating the vascular smooth muscle cell phenotype transformation through upregulation of TET2, Nur77 and calponin

Archives of Biological Sciences ◽

10.2298/abs201103009l ◽

2021 ◽

Vol 73 (1) ◽

pp. 135-144

Author(s):

Hao Lin ◽

Shuo Cheng ◽

Zhichao Yuan ◽

Zhiqiang Yan ◽

Jifa Zhang

Keyword(s):

Smooth Muscle ◽

Carotid Artery ◽

Vascular Smooth Muscle ◽

Smooth Muscle Actin ◽

Neointimal Hyperplasia ◽

Nuclear Antigen ◽

Cell Phenotype ◽

Alpha Smooth Muscle Actin ◽

And Migration ◽

Proliferation And Migration

Metformin is a drug used to treat type 2 diabetes based on its effectiveness as well as cardiovascular safety. Metformin has been shown to modulate proliferation and migration of vascular smooth muscle cells (VSMCs), but the underlying mechanisms of the effect of metformin on VSMC function remains unclear. We found that metformin inhibits VSMC proliferation and migration and upregulates the expression of nuclear receptor subfamily 4 group A member 1 (Nur77), ten-eleven translocation 2 (TET2), and calponin in vitro. In the carotid artery balloon injury model of rats, metformin effectively prevented neointimal hyperplasia in the carotid artery, including neointimal thickness, increased neointimal area, and the neointimal area/medial area ratio. It also reduced the number of proliferating cell nuclear antigen (PCNA)+ cells and increased the expression of Nur77, calponin and alpha-smooth muscle actin (?-SMA). These results show that metformin attenuates neointimal hyperplasia in balloon-injured carotid arteries via increased expression of TET2, Nur77 and calponin, and reduced expression of matrix metallopeptidase 9 (MMP-9).

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Platelet-Derived Exosomes Affect the Proliferation and Migration of Human Umbilical Vein Endothelial Cells Via miR-126

Current Vascular Pharmacology ◽

10.2174/1570161116666180313142139 ◽

2019 ◽

Vol 17 (4) ◽

pp. 379-387 ◽

Cited By ~ 11

Author(s):

Yan Sun ◽

Xiao-li Liu ◽

Dai Zhang ◽

Fang Liu ◽

Yu-jing Cheng ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Real Time ◽

Umbilical Vein ◽

Angiogenic Factors ◽

Healthy Donors ◽

And Migration ◽

Umbilical Vein Endothelial Cells ◽

Tgf Β1 ◽

Proliferation And Migration

Background:Intraplaque angiogenesis, the process of generating new blood vessels mediated by endothelial cells, contributes to plaque growth, intraplaque hemorrhage, and thromboembolic events. Platelet-derived Exosomes (PLT-EXOs) affect angiogenesis in multiple ways. The ability of miR-126, one of the best-characterized miRNAs that regulates angiogenesis, carried by PLT-EXOs to influence angiogenesis via the regulation of the proliferation and migration of endothelial cells is unknown. In this study, we aimed to investigate the effects of PLT-EXOs on angiogenesis by Human Umbilical Vein Endothelial Cells (HUVECs).Methods:We evaluated the levels of miR-126 and angiogenic factors in PLT-EXOs from Acute Coronary Syndrome (ACS) patients and healthy donors by real-time Polymerase Chain Reaction (PCR) and western blotting. We incubated HUVECs with PLT-EXOs and measured cell proliferation and migration with the Cell Counting Kit-8 assay and scratch assay, respectively. We also investigated the expression of miR-126 and angiogenic factors in HUVECs after exposure to PLT-EXOs by western blotting and real-time PCR.Results:PLT-EXOs from ACS patients contained higher levels of miR-126 and angiogenic factors, including Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), and Transforming Growth Factor Beta 1 (TGF-β1), than those from healthy donors (p<0.05). Moreover, the levels of exosomal miR-126 and angiogenic factors were increased after stimulation with thrombin (p<0.01). HUVEC proliferation and migration were promoted by treatment with activated PLT-EXOs (p<0.01); they were accompanied by the over-expression of miR-126 and angiogenic factors, including VEGF, bFGF, and TGF-β1 (p<0.01).Conclusion:Activated PLT-EXOs promoted the proliferation and migration of HUVECs, and the overexpression of miR-126 and angiogenic factors, thereby elucidating potential new therapeutic targets for intraplaque angiogenesis.

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The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms22041861 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1861

Author(s):

Jemima Seidenberg ◽

Mara Stellato ◽

Amela Hukara ◽

Burkhard Ludewig ◽

Karin Klingel ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Cardiac Fibrosis ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Beclin 1 ◽

Type I ◽

Alpha Smooth Muscle Actin

Background: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. Methods and Results: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-β. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-β-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. Conclusion: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-β-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

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mTOR inhibitors potentially reduce TGF-β2-induced fibrogenic changes in trabecular meshwork cells

Scientific Reports ◽

10.1038/s41598-021-93580-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nozomi Igarashi ◽

Megumi Honjo ◽

Makoto Aihara

Keyword(s):

Trabecular Meshwork ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Mtor Inhibitors ◽

In Vivo Model ◽

Type I ◽

Fibrotic Response ◽

Alpha Smooth Muscle Actin

AbstractWe examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.

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