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1.
Article in Chinese | WPRIM | ID: wpr-1025701

ABSTRACT

Objective To investigate the effect and mechanism of melatonin on the epithelial-mesenchymal transition(EMT)of human peritoneal mesothelial cells.Methods A human peritoneal mesoepithelial cell line(HMrSV5)was cultured.Decorin(DCN)overex-pression and knockout plasmids were constructed.The cells were divided into the normal,TGF-β1,TGF-β1+melatonin,TGF-β1+mela-tonin+siDCN,TGF-β1+melatonin+siNC,and TGF-β1+DCN groups.The cell proliferation and survival rates were determined using Cell Counting Kit-8(CCK-8).The protein and mRNA expression levels of DCN,TGF-β1,Smad2,and E-cadherin were detected by Western blotting and real-time polymerase chain reaction(PCR),respectively.Results The CCK-8 assay showed that the cell survival rates were higher in the TGF-β1+melatonin,TGF-β1+melatonin+siDCN,and TGF-β1+DCN groups than the TGF-β1 group(P<0.05).The cell sur-vival rate was higher for the TGF-β1+melatonin group than the TGF-β1+melatonin+siDCN group(P<0.05).Western blotting and real-time PCR showed DCN and that E-cadherin were significantly down-regulated in the TGF-β1 group compared with the control group(P<0.05).Compared with the TGF-β1 group,the TGF-β1+melatonin and TGF-β1+melatonin+siDCN groups showed up-regulated E-cadherin and DCN expression levels and down-regulated TGF-β1 and Smad2 expression levels(P<0.05).Compared with the TGF-β1+melato-nin+siDCN group,the TGF-β1+melatonin+siDCN group showed up-regulated E-cadherin and DCN expression levels and down-regulated TGF-β1 and Smad2 expression levels(P<0.05).Conclusion Melatonin can delay the EMT of human peritoneal mesoepithelial cells,and the DCNgene may be an important target to inhibit the EMT of these cells.

2.
Protein & Cell ; (12): 105-122, 2023.
Article in English | WPRIM | ID: wpr-971612

ABSTRACT

Glioblastoma multiforme (GBM), a highly malignant and heterogeneous brain tumor, contains various types of tumor and non-tumor cells. Whether GBM cells can trans-differentiate into non-neural cell types, including mural cells or endothelial cells (ECs), to support tumor growth and invasion remains controversial. Here we generated two genetic GBM models de novo in immunocompetent mouse brains, mimicking essential pathological and molecular features of human GBMs. Lineage-tracing and transplantation studies demonstrated that, although blood vessels in GBM brains underwent drastic remodeling, evidence of trans-differentiation of GBM cells into vascular cells was barely detected. Intriguingly, GBM cells could promiscuously express markers for mural cells during gliomagenesis. Furthermore, single-cell RNA sequencing showed that patterns of copy number variations (CNVs) of mural cells and ECs were distinct from those of GBM cells, indicating discrete origins of GBM cells and vascular components. Importantly, single-cell CNV analysis of human GBM specimens also suggested that GBM cells and vascular cells are likely separate lineages. Rather than expansion owing to trans-differentiation, vascular cell expanded by proliferation during tumorigenesis. Therefore, cross-lineage trans-differentiation of GBM cells is very unlikely to occur during gliomagenesis. Our findings advance understanding of cell lineage dynamics during gliomagenesis, and have implications for targeted treatment of GBMs.


Subject(s)
Mice , Animals , Humans , Glioblastoma/pathology , Endothelial Cells/pathology , DNA Copy Number Variations , Brain/metabolism , Brain Neoplasms/pathology
3.
Article in Chinese | WPRIM | ID: wpr-1022794

ABSTRACT

Objective:To investigate the inhibitory effect of indocyanine green (ICG) on biological behavior and transdifferentiation of human lens epithelial cells (HLECs) and its mechanism.Methods:HLECs were divided into blank control group, 5% glucose solution (GS) group and 0.5% ICG group, 1.5% ICG group and 2.5% ICG group, which were treated with balanced salt solution, 5% GS and 0.5%, 1.5% and 2.5% ICG solutions for 3 minutes, respectively, and then were incubated in fresh medium for 24 hours.The apoptosis level of HLECs was detected by flow cytometry.The expression levels of apoptosis-related proteins, Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), caspase-3 and caspase-9 were detected by Western blot.Cell proliferation was detected via the cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay.The migration ability of HLECs was detected by cell scratch assay.Cell migration and invasion were determined by Transwell assays.The expression levels transdifferentiation-related proteins, α-smooth muscle actin (α-SMA), nerve calcium adhesion protein (N-cadherin), fibronectin (FN) and vimentin were assessed by Western blot.Results:The apoptosis rates of blank control group, 5% GS group, 0.5% ICG group, 1.5% ICG group and 2.5% ICG group were (4.35±0.60)%, (4.63±0.19)%, (8.17±0.69)%, (13.90±0.33)% and (23.08±1.12)%, with a statistically significant difference in the overall comparison ( F=412.74, P<0.05). The apoptosis rate was significantly higher in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The relative expressions of caspase-3, caspase-9 and Bax proteins were significantly higher in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the relative expression of Bcl-2 protein was lower in 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all at P<0.05). The rate of EdU-positive cells was significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG groups than in blank control group and 5% GS group (all at P<0.05). The survival rate of cells was significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The migration rates of scratch cells were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all P<0.05). The number of migrating cells and the number of invading cells were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The relative expressions of α-SMA, N-cadherin and FN were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the relative expression of vimentin was lower in 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all at P<0.05). Conclusions:ICG can promote HLECs apoptosis and inhibit HLECs proliferation, migration, invasion and transdifferentiation in a concentration-dependent manner.

4.
Chinese Journal of Rheumatology ; (12): 597-603, 2023.
Article in Chinese | WPRIM | ID: wpr-1027218

ABSTRACT

Objective:To investigate the IL-38 level in patients with primary biliary cholangitis (PBC), and to assess the modulatory function of IL-38 on the phenotype shift from regulatory T cells (Tregs) to Th17 cells in PBC.Methods:Forty-six PBC patients and 24 controls were included. Serum IL-38, IL-35 and IL-17 levels were measured by enzyme-linked immunosorbent assay. CD4 +CD25 +CD127 dim/-Tregs and CD4 +IL-17A +Th17 cell proportions in peripheral blood mononuclear cells (PBMC) were assessed by flow cytometry. Forkhead box P3 (FoxP3) and retinoid acid receptor related orphan receptor γt (RORγt) mRNA expressions were semi-quantified by real-time PCR. Purified Tregs were stimulated with recombinant IL-38, and were cocultured with autologous PBMC. Tregs function was assessed by measuring cellular proliferation and cytokines expression in the supernatants. Tregs were also polarized for Th17 culture, and recombinant IL-38 was added for stimulation. The influence of IL-38 on trans-differentiation of Tregs into Th17 phenotype was investigated by measuring CCR4/CCR6 expression, IL-17 secretion and RORγt mRNA expression. Student′s t test or Mann-Whitney U test were used for comparison between groups. Results:Serum IL-38 level was significantly lower in PBC group compared with control group [68.02(48.51, 96.74) pg/ml vs 89.6(58.0, 265.5)pg/ml, Z=3.25, P=0.037]. Proportion of Tregs [(6.9±1.3)% vs (11.3±3.5)%, t=7.64, P<0.001], FoxP3 mRNA expression [(1.0±0.3) vs (1.9± 0.7), t=7.74, P<0.001] and serum IL-35 level [25.87(16.08, 37.26)pg/ml vs 30.99(24.81, 52.29)pg/ml, Z=2.02, P=0.047] were notably lower in PBC group, while Th17 cell proportion [(5.5±1.4)% vs (3.8±1.0)%, t=5.43, P<0.001)], RORγt mRNA expression [(1.4±0.6) vs (1.0±0.4), t=2.72, P=0.008] and serum IL-17 level [202.0(121.6, 311.6)pg/ml vs 104.5(79.8, 155.5)pg/ml, Z=4.43, P<0.001] were remarkably higher in PBC group. Purified Tregs were co-cultured with autologous PBMC. Cellular proliferation was increased in PBC group compared with control group [(6.5±1.40)×10 5vs (5.3±1.1)×10 5, t=2.49, P=0.020]. IL-35 and IL-10 secretions in the supernatants were lower in PBC group ( P<0.05), but interferon- γ was higher in PBC group ( P=0.011). Tregs were also polarized for Th17 culture. CCR4 and CCR6 mean fluorescence intensity (MFI), RORγt mRNA and IL-17 secretion were higher in PBC group compared with control group ( P<0.05). IL-38 stimulation enhanced the inhibitory activity of Tregs from both PBC group and control group, which presented as reduced cellular proliferation as well as enhanced IL-35 and IL-10 secretion ( P<0.05). IL-38 stimulation only suppressed transdifferentiation of Tregs into Th17 phenotype in PBC group, which presented as down-regulation of CCR4 MFI, CCR6 MFI and IL-17 secretion ( P<0.05). However, IL-38 did not affect Tregs trans-differentiation into Th17 phenotype in control group. Conclusion:IL-38 played an immune-protective role and suppressed inflammatory response in the disease progression of PBC. IL-38 inhibited transdifferentiation of Tregs into Th17 phenotype in PBC patients.

5.
Article in Chinese | WPRIM | ID: wpr-979469

ABSTRACT

Renal fibrosis, the final pathological outcome of end-stage chronic kidney diseases, is associated with inflammation, oxidative stress, epithelial-mesenchymal transdifferentiation (EMT), and extracellular matrix deposition. It belongs to the categories of edema, ischuria, anuria and vomiting, and consumptive disease in traditional Chinese medicine (TCM), with the key pathogenesis of Qi deficiency and blood stasis and the primary treatment principle of replenishing Qi and activating blood. Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma mainly contains astragalosides, polysaccharides, calycosin, salvianolic acid, and tanshinone, with the effect of tonifying Qi and activating blood. Studies have shown that this herb pair and its active components can delay the progress of renal fibrosis by regulating multiple signaling pathways. With consideration to the pathogenesis of Qi deficiency and blood stasis, this article reviews the research progress in the mitigation of renal fibrosis by Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma from the aspects of protecting glomerular filtration barrier, inhibiting EMT and mesangial cell proliferation, improving renal hemodynamics, and protecting renal function. Furthermore, the mechanisms were summarized. Specifically, Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma and its effective components can improve mitochondrial function and fatty acid metabolism, alleviate endoplasmic reticulum stress and autophagy disorders, and inhibit immune inflammation and oxidative stress by regulating nuclear factor E2-related factor 2 (Nrf2)/PTEN-induced kinase 1 (Pink1), Nrf2/antioxidant response element (ARE), tumor necrosis factor-α (TNF-α)/nuclear transcription factor-κB (NF-κB), miR-21/Smad7/transforming growth factor beta (TGF-β), Wnt/β-catenin, long non-coding RNA-taurine up-regulated gene 1 (lncRNA-TUG1)/tumor necrosis factor receptor-associated factor 5 (TRAF5), Ras-related C3 botulinum toxin substrate 1 (Rac1)/cell division cycle protein 42 (CDC42), Ras homolog (Rho)/Rho-associated coiled-coil containing protein kinase (ROCK), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), peroxisome proliferator-activated receptor α (PPARα)/peroxisome proliferator-activated receptor γ coactivator l alpha (PGC-1α), and p38 mitogen-activated protein kinase (p38 MAPK). This review aims to provide references for the relevant research, give play to the role of Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma, and provide guidance for the clinical treatment of renal fibrosis.

6.
International Eye Science ; (12): 1627-1633, 2023.
Article in Chinese | WPRIM | ID: wpr-987880

ABSTRACT

AIM: To scrutinize the role of the Wnt/β-catenin signaling pathway in the epithelial-mesenchymal transition(EMT)of lens epithelial cells under hypoxic conditions, and to further analyze the effect of Dickkopf-1(DKK-1)expression on EMT of lens epithelial cells.METHODS: Human lens epithelial cells(HLEB3 cells)were propagated in vitro and then separated into two groups: one exposed to standard oxygen levels, added DMEM culture solution containing 10% FBS(normoxic group)and another subjected to low oxygen levels(hypoxic group). The hypoxic condition was emulated by applying a concentration of 100 μmol/L cobalt chloride(CoCl2)for 6, 12, 24, and 48h. The utilization of immunofluorescence staining enabled the detection of Wnt3a and DKK-1 expressions, along with the expression and localization of β-catenin protein in these groups. The expression of DKK-1 mRNA was discerned by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS: Immunofluorescence assays indicated an escalating trend in the Wnt3a and DKK-1 protein expression, which corresponded with the increasing duration of hypoxia. Likewise, an intensified nuclear accumulation of β-catenin protein was observed to be directly proportional to the length of hypoxia treatment. The qRT-PCR demonstrated that the difference in DKK-1 mRNA expression between the normoxic group and the group exposed to hypoxia for 6h was not statistically significant(P&#x003E;0.05), whereas the DKK-1 mRNA expression of the 12, 24, and 48h hypoxia groups were significantly increased(P&#x003C;0.001).CONCLUSION: Hypoxia can activate Wnt/β-catenin pathway in lens epithelial cells and induce the expression of DKK-1, thus regulating the Wnt/β-catenin pathway and affecting the EMT process.

7.
Article in Chinese | WPRIM | ID: wpr-989126

ABSTRACT

Neonatal brain injury refers to the pathological damage of brain tissue caused by various factors in the perinatal period.The most common disorders are hypoxic-ischemic encephalopathy, periventricular-intraventricular hemorrhage, and periventricular leukomalacia.After a severe brain injury, the repair and reconstruction of the central nervous system (CNS) is crucial in restoring CNS architecture and function.The studies have shown that neural stem cells (NSC) have the potential for multidirectional differentiation and the ability to maintain self-renewal.Endogenous neural stem cells (eNSC) can proliferate, migrate to the lesion sites and finally differentiate into astrocytes, oligodendrocytes, and neurons, to provide new options for the treatment of neural regeneration.This paper aims to review the recent progress of eNSC in treating neonatal brain injury.

8.
Chinese Pharmacological Bulletin ; (12): 1014-1019, 2023.
Article in Chinese | WPRIM | ID: wpr-1013775

ABSTRACT

Fibrosis is a repair response initiated by tissues and organs after injury, and is a self-protection mechanism of the body. It has been found that endothelium-to-interstitial transdifferentiation (EndMT) is involved in the physiological and pathological processes of various organ fibrosis, which has become a focus of the research on fibrotic diseases. In recent years, the study has found that EndMT plays an important role in many pathological processes in cardiovascular system, lungs, kidneys, liver, pancreas fibrosis, and so on. This article summarizes EndMT regulatory mechanism and its role in each organ fibrosis, as well as the related treatment progress of EndMT targets, so as to provide new targets for prevention and control of organ fibrosis.

9.
Zhongnan Daxue xuebao. Yixue ban ; (12): 526-537, 2023.
Article in English | WPRIM | ID: wpr-982319

ABSTRACT

OBJECTIVES@#Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms.@*METHODS@#Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA.@*RESULTS@#Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited.@*CONCLUSIONS@#MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.


Subject(s)
Animals , Rats , Adrenal Medulla , Cell Transdifferentiation , Chromaffin Cells , Dexamethasone , Epinephrine/pharmacology , Mammals , Nerve Growth Factor , Neurons , Peripherins , Protein Kinases , Tyrosine 3-Monooxygenase
10.
Article in Chinese | WPRIM | ID: wpr-931702

ABSTRACT

Objective:To investigate the effects of tamoxifen on high glucose-induced epithelial-to-mesenchymal transition of rat peritoneal mesothelial cells and the underlying mechanism.Methods:The peritoneal mesothelial cells of normal male SD rats were selected between January 2015 and June 2016 and then cultured and divided into blank control, high-glucose stimulation and drug intervention groups. High-glucose stimulation group: primary cultured rat peritoneal mesothelial cells (RPMCs) were treated with 60 mmol/L high-concentration glucose to induce epithelial-to-mesenchymal transition. Drug intervention group: (1) RPMCs were treated with 60 mmol/L high-concentration glucose and different concentrations (0.5 μmol/L, 2 μmol/L) of tamoxifen. After 72 hours of stimulation, protein was extracted. (2) RPMCs were treated with 60 mmol/L high-concentration glucose and 2 μmol/L tamoxifen with or without 2 μmol/L ER-α antagonist for 1 hour to extract protein and for 6 hours to extract RNA. (3) RPMCs were treated with high-concentration glucose and 2 μmol/L tamoxifen with or without 1 μmol/L 1 μM proteasome inhibitor for 1 hour to extract protein. Western blot analysis was performed to analyze change in E-cadherin, α-SMA, Smad2, p-Smad2, Smad3, p-Smad3 and Smad4 protein. Real-time fluorescence quantitative PCR was performed to detect the change in mRNA expression of Smad2, Smad3, connective tissue growth factor and plasminogen activator inhibitor 1.Results:Tamoxifen attenuated epithelial-to-mesenchymal transition on RPMCs induced by high-level glucose, showing increased expression of epithelial cell marker E-cadherin and decreased expression of α-SMA in a concentration-dependent manner ( tE-cadherin = 2.31, tα-SMA =-2.53, both P < 0.05).TGF-β1/R-Smad signal pathway was activated by high-concentration glucose. Phosphorylation of Smad2/3 and mRNA expression of CTGF and PAI-1 were increased. Tamoxifen remarkably reduced protein and mRNA level of above mentioned protein and related target genes ( tp-Smad2 = -3.38, tCTGF = -3.81, P < 0.05), which could be blocked by ER-α antagonist. Finally, proteasome inhibitor could weaken the inhibitory effects of tamoxifen on p-Smad2/3 and increase p-Smad2/3 protein level ( tp-Smad2 = 3.94, P < 0.05). Conclusion:Tamoxifen activates ER-α on RPMCs, weakens the activation of TGF-β1/R-Smad signal pathway through decreasing p-Smad2 protein level, and effectively inhibits the progression of high-concentration glucose-induced epithelial-to-mesenchymal transition possibly through degrading p-Smad2 protein through proteasome. The role of tamoxifen in epithelial-to-mesenchymal transition may provide a possible guide for research, prevention and treatment of peritoneal fibrosis.

11.
Chinese Journal of Trauma ; (12): 947-954, 2022.
Article in Chinese | WPRIM | ID: wpr-956527

ABSTRACT

Spinal cord injury can be divided into primary and secondary injury. As an important process of spinal cord injury, secondary injury can be classified into acute phase, subacute phase and chronic phase according to the time and progression of the injury. Oxidative stress reaction, inflammatory reaction, tissue edema, scar formation and other pathological changes appear subsequently during the process. In the central nervous system, the astrocyte is one of the most widely distributed cell that has different shapes at different stages and plays a complex role such as anti-inflammatory or pro-inflammatory action and neuroprotective or anti-neurorestorative effect after spinal cord injury. The astrocyte has been a research focus in the field of spinal cord injury. The authors review the role and research progress of astrocyte in oxidative stress response, excitotoxicity, inflammatory response, tissue edema, scar formation, axonal regeneration and cell transformation in spinal cord injury based on the pathological changes of secondary injury, in order to provide new ideas to the related research of spinal cord injury.

12.
Article in Chinese | WPRIM | ID: wpr-1006634

ABSTRACT

【Objective】 In recent years, granulocyte colony stimulating growth factor (G-CSF) has been proved to be expressed in the posterior capsule of the lens of posterior capsular opacification (PCO), but its specific role remains unclear. The purpose of this study was to explore whether G-CSF plays a role in PCO. 【Methods】 First, human lens epithelial cells (HLEC-B3) were treated with different concentration of recombinant G-CSF protein and screened for effective appropriate concentration. Then, Western blotting was used to detect the effects of extracellular matrix (ECM) synthesis and epithelial mesenchymal transdifferentiation (EMT) marker genes after G-CSF treatment on HLEC-B3 cells. Finally, the effects of G-CSF treatment on the migration and invasion of HLEC-B3 cells were detected by scratch experiment and Transwell test. 【Results】 G-CSF at 80 μg/L could promote the proliferation of HLEC-B3 cells. After G-CSF was treated for 48 h, the expressions of EMT and ECM synthesis marker genes in HLEC-B3 cells were significantly upregulated with time. G-CSF could significantly promote HLEC-B3 cell invasion after 48 h induction. Similarly, G-CSF could also significantly induce cell migration compared to the CTRL group. 【Conclusion】 G-CSF can promote the proliferation, migration and invasion of HLEC-B3 cells, as well as EMT and ECM synthesis, which might be involved in the occurrence of PCO. Inhibiting the expression of G-CSF may be a new strategy for PCO prevention.

13.
Chinese Journal of Nephrology ; (12): 911-917, 2021.
Article in Chinese | WPRIM | ID: wpr-911912

ABSTRACT

Objective:To investigate the role and mechanism of (histone deacetylase 6, HDAC6) in the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells and the activation of renal interstitial fibroblasts.Methods:Human renal tubular epithelial cells (HK-2) and rat renal interstitial fibroblast (NRK-49F) were cultured in vitro, and divided into 4 groups: control group, Tubastatin A (TA) group (treated with 10 μmol/L HDAC6 inhibitor TA for 36 h), transforming growth factor-β1 (TGF-β1) group (10 ng/ml TGF-β1 for 36 h), and TGF-β1+TA group (treated with 10 ng/ml TGF-β1 and 10 μmol/L TA for 36 h). The expression levels of fibronectin, α-smooth muscle actin (α-SMA), collagen I, E-cadherin, HDAC6, acetyl histone H3, histone H3, acetyl α-tubulin, α-tubulin, TGF-β receptor (TGF-βR) 1, p-Smad3, Smad3, connective tissue growth factor (CTGF), epidermal growth factor receptor (EGFR) and p-EGFR in HK-2 and NRK-49F cell samples were detected by Western blotting, and quantitative analysis was performed according to gray level. Results:(1) In HK-2 cells stimulated by TGF-β1, TA decreased the expression of fibronectin, α-SMA, collagen I, and increased the expression of epithelial cell marker E-cadherin. Meanwhile, TA decreased the expression of HDAC6 and increased the expression levels of acetyl histone H3 and acetyl α-tubulin (all P<0.05). (2) Compared with the TGF-β1 group, the expressions of TGF-βR1, p-Smad3, CTGF and p-EGFR in TGF-β1+TA group were decreased (all P<0.05), while the total protein levels of Smad3 and EGFR were not significantly different (both P>0.05). (3) In NRK-49F cells stimulated by TGF-β1, TA decreased the expressions of fibronectin, α-SMA, collagen I, TGF-βR1 and p-Smad3 (all P<0.05). Conclusions:Blockade of HDAC6 by TA may inhibit the EMT of renal tubular epithelial cells and the activation of renal interstitial fibroblasts via regulating multiple signaling pathways including TGF-β/Smad3, CTGF and EGFR.

14.
Article in Chinese | WPRIM | ID: wpr-906515

ABSTRACT

Objective:To observe the effect of modified Shengjiangsan on renal fibrosis in rats with membranous nephropathy (MN) and to explore the mechanism of its complications of renal fibrosis. Method:Rats were injected with cationized bovine serum albumin(C-BSA)in the tail vein to establish a rat model of membranous nephropathy. The normal group,model group,modified Shengjiangsan group (27.3 g·kg<sup>-1</sup>)and benazepril group(10 mg·kg<sup>-1</sup>)were established in this study. Each group was given corresponding dosage of the drug once a day for 4 weeks of continuous intervention. After the administration,we observed the pathological changes of rat kidneys by the technology of Masson staining, silverhexylamine iodate (PASM) staining, transmission electron microscopy (TEM), immunofluorescence technology (IF) was used to detect immunoglobulin(Ig)G deposition in rat kidneys. The levels of interleukin-1<italic>β</italic> (IL-1<italic>β</italic>), interleukin-6 (IL-6), tumor necrosis factor-<italic>α</italic> (TNF-<italic>α</italic>) in rat serum were detected by enzyme-linked immunosorbent assay (ELISA) method. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and immunohistochemistry (IHC) were used to detect the mRNA and protein expression levels of monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), nuclear factor kappa B (NF-<italic>κ</italic>B), Toll-like receptor 4 (TLR4), plasminogen activator inhibitor 1 (PAI-1), transforming growth factor-<italic>β</italic><sub>1</sub> (TGF-<italic>β</italic><sub>1</sub>), <italic>α</italic>-smooth muscle actin (<italic>α</italic>-SMΑ) and type Ⅳ Collagen (Collagen Ⅳ) in rat kidney tissues. Result:Compared with normal group, the kidney tissue of the model group was obviously fibrotic, the serum levels of IL-1<italic>β</italic>, IL-6, and TNF-<italic>α</italic> were significantly increased(<italic>P</italic><0.05), and the expressions of MCP-1, ICAM-1, NF-<italic>κ</italic>B, TLR4, PAI-1, TGF-<italic>β</italic><sub>1</sub>, <italic>α</italic>-SMA and Collagen Ⅳ mRNA and protein in kidney tissue were significantly increased(<italic>P</italic><0.05). Compared with model group, modified Shengjiangsan and benazepril significantly improved renal fibrosis in rats, reduced the levels of IL-1<italic>β</italic>, IL-6, and TNF-<italic>α</italic> in the serum of MN rats(<italic>P</italic><0.05), down-regulated MCP-1, ICAM-1, NF-<italic>κ</italic>B, TLR4, PAI-1, TGF-<italic>β</italic><sub>1</sub>, <italic>α</italic>-SMA and Collagen Ⅳ mRNA and protein expression in kidney tissue(<italic>P</italic><0.05). Conclusion:Modified Shengjiangsan can reduce the release and expression of inflammatory factors by down-regulating the TLR4/NF-<italic>κ</italic>B signaling pathway, inhibit renal fibrosis, and reduce renal damage in MN rats.

15.
Article in Chinese | WPRIM | ID: wpr-801520

ABSTRACT

Objective@#Previous studies have found abnormal proliferation and transdifferentiation of alveolar epithelial cells(AECs)in hyperoxic lung injury of neonatal rats.The purpose of this study was to clarify the expression of zonula occludens 1(ZO-1) and ZO-1 related nucleic acid binding protein(ZONAB)in AECs in hyperoxic lung injury model, in order to investigate its effect on the proliferation and transdifferentiation of AECs in the injured lung tissue.@*Methods@#Full-term neonatal Wistar rats were randomly divided into two groups within 12 h after birth, model group(inhaled oxygen concentration 85%)and control group(inhaled air). Lung specimens were collected at 7, 14 and 21 days after exposure.The expression of ZONAB in typeⅡalveolar epithelial cells(AECⅡ)was observed by double immunofluorescence staining.At the same time, AEC Ⅱ was isolated from lung tissues of animal models at these time points, and the expression levels of ZO-1, ZONAB protein and mRNA in lung tissues and AECⅡof the two groups were detected by Western blot and Real-Time PCR.In addition, AEC Ⅱ was isolated from lung tissue of normal newborn rats and then divided into model group(85% oxygen concentration)and control group(21% oxygen concentration). After 48 hours of culture in vitro, the expression levels of ZO-1, ZONAB protein and mRNA were detected, and the expression level and location of ZONAB were observed by immunofluorescence staining.@*Results@#Double immunofluorescence staining showed that the expression of ZONAB in AECⅡin model group was significantly lower than that in control group.The protein and mRNA expression levels of ZO-1 and ZONAB in AECⅡisolated from lung tissue of model group were both significantly lower than those from control group, starting from 7 d after hyperoxia exposure.AECⅡisolated from lung tissue of normal newborn rats, were then incubated for 48 hours under hyperoxia or normoxia in vitro, the protein and mRNA expression levels of ZO-1 and ZONAB significantly decreased in model group compared with those in control group.The results of immunofluorescence staining showed that the expression of ZONAB was higher in AECⅡof the control group, and ZONAB was mostly located in the junction and nucleus of cells, while the expression of ZONAB in the model group significantly decreased than that in the control group, and the expression sites were clustered in the cytoplasm, with little expression in the junction and nucleus.@*Conclusion@#ZO-1, as a tight junction-related protein, is down-regulated in hyperoxic lung injury model.In addition to destroying pulmonary epithelial barrier to mediate pulmonary edema, it also participated in the regulation of proliferation and differentiation of AECs by regulating transcription factor ZONAB, suggesting that this may be another pathway leading to hyperoxic lung injury.

16.
Article in Chinese | WPRIM | ID: wpr-802340

ABSTRACT

Objective:To investigate the renal protective effect of Tangshenping capsule (Tangshenping) on diabetic nephropathy (DN) KKAy mice and its effect on Wnt/β-catenin signaling pathway. Method:Sixty female Sprague-Dawley KKAy mice aged 10 weeks old were induced with KKAy rat feed for 10 weeks. The DN animal model was successfully determined with blood glucose (>16.7 mmol·L-1) and 24 hour urine protein (>0.4 mg). The model mice were randomly divided into a model group, an irbesartan group, and low, medium and high-dose Tangshenping group, with 10 female C57BL/6J mice as a control group. The treatment groups were given the corresponding drugs by gavage. The normal group and the model group were given an equal volume of deionized water by gavage. The intragastric dose was 0.01 mL·g-1 body weight coefficient once a day. The general conditions of the mice were observed, the body mass was weighed every 4 weeks, and 24 h urine protein was quantified. At the 26th week, the blood was collected from eyeballs, and the mice were put to death. The quality of the kidneys, serum blood urea nitrogen (BUN), serum creatinine (SCr), triglyceride (TG), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) content were measured. In situ hybridization and immunohistochemistry were used to detect the expressions of Wnt4, glycogen synthase kinase 3β(GSK3β) and β-catenin in kidney tissues. Result:Compared with model group, body mass, kidney mass/body mass, and 24 h urine protein were significantly lower in high-dose Tangshenping group (PPPβ and β-catenin were decreased (PConclusion:Tangshenping may inhibit the activation of Wnt/β-catenin signaling pathway, reverse the transdifferentiation of renal tubular epithelial cells in DN KKAy mice, delay the progression of renal interstitial fibrosis, and then exert renal protection.

17.
China Occupational Medicine ; (6): 551-558, 2019.
Article in Chinese | WPRIM | ID: wpr-881825

ABSTRACT

OBJECTIVE: To investigate the differentially expressed microRNAs(miRNAs) in human embryonic lung fibroblast MRC-5 cells stimulated by transforming growth factor-β1(TGF-β1) using microarray chip, and screen for key genes and signaling pathways of fibroblast trans-differentiation. METHODS: The miRNA expression gene chip dataset GSE43992 on TGF-β1 stimulated MRC-5 cells were downloaded from high-throughput Gene Expression Omnibus(GEO) database of National Center for Biotechnology Information of the United States. The R language Limma package was used to screen the differentially expressed miRNAs. Corresponding target genes were predicted by miRWalk database performed by Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analysis. The protein-protein interaction(PPI) network was constructed by the search tool for the Retrieval of Interacting Genes database. RESULTS: A total of five differentially expressed miRNAs were identified, including four up-regulated miRNAs and one down-regulated miRNA; and 42 corresponding differentially expressed target genes were predicted. GO analysis indicated that the target genes were significantly enriched in collagen catabolic process, extracellular matrix organization, membrane organization, collagen fibril organization, and cellular response to amino acid stimulus. The results of KEGG pathway analysis showed that the signaling pathways corresponding to miRNAs and target genes were mainly concentrated in 18 signaling pathways, that were mainly related to the age-ethnic signaling pathways and protein digestion and absorption miRNAs in tumors and diabetic complications. The core genes transfected into the myofibroblasts by the three fibroblasts screened by the PPI network were threonine kinase 1, estrogen receptor 1 and β-catenin. CONCLUSION: Five differentially expressed miRNAs, 42 target genes, 18 signaling pathways, and 3 core genes related to TGF-β1-induced MRC-5 cell trans-differentiation were screened. It can provide new reference for the treatment and research of many diseases including pneumoconiosis and pulmonary fibrosis.

18.
Asian j. androl ; Asian j. androl;(6): 233-240, 2019.
Article in Chinese | WPRIM | ID: wpr-842555

ABSTRACT

Prostate cancer (PCa) is the most common cause of malignancy in males and the third leading cause of cancer mortality in the United States. The standard care for primary PCa with local invasive disease mainly is surgery and radiation. For patients with distant metastases, androgen deprivation therapy (ADT) is a gold standard. Regardless of a favorable outcome of ADT, patients inevitably relapse to an end-stage castration-resistant prostate cancer (CRPC) leading to mortality. Therefore, revealing the mechanism and identifying cellular components driving aggressive PCa is critical for prognosis and therapeutic intervention. Cancer stem cell (CSC) phenotypes characterized as poor differentiation, cancer initiation with self-renewal capabilities, and therapeutic resistance are proposed to contribute to the onset of CRPC. In this review, we discuss the role of CSC in CRPC with the evidence of CSC phenotypes and the possible underlying mechanisms.

19.
Asian j. androl ; Asian j. androl;(6): 241-248, 2019.
Article in Chinese | WPRIM | ID: wpr-842556

ABSTRACT

Therapy resistance is a significant challenge for prostate cancer treatment in clinic. Although targeted therapies such as androgen deprivation and androgen receptor (AR) inhibition are effective initially, tumor cells eventually evade these strategies through multiple mechanisms. Lineage reprogramming in response to hormone therapy represents a key mechanism that is increasingly observed. The studies in this area have revealed specific combinations of alterations present in adenocarcinomas that provide cells with the ability to transdifferentiate and perpetuate AR-independent tumor growth after androgen-based therapies. Interestingly, several master regulators have been identified that drive plasticity, some of which also play key roles during development and differentiation of the cell lineages in the normal prostate. Thus, further study of each AR-independent tumor type and understanding underlying mechanisms are warranted to develop combinational therapies that combat lineage plasticity in prostate cancer.

20.
Asian j. androl ; Asian j. androl;(6): 241-248, 2019.
Article in English | WPRIM | ID: wpr-1009622

ABSTRACT

Therapy resistance is a significant challenge for prostate cancer treatment in clinic. Although targeted therapies such as androgen deprivation and androgen receptor (AR) inhibition are effective initially, tumor cells eventually evade these strategies through multiple mechanisms. Lineage reprogramming in response to hormone therapy represents a key mechanism that is increasingly observed. The studies in this area have revealed specific combinations of alterations present in adenocarcinomas that provide cells with the ability to transdifferentiate and perpetuate AR-independent tumor growth after androgen-based therapies. Interestingly, several master regulators have been identified that drive plasticity, some of which also play key roles during development and differentiation of the cell lineages in the normal prostate. Thus, further study of each AR-independent tumor type and understanding underlying mechanisms are warranted to develop combinational therapies that combat lineage plasticity in prostate cancer.


Subject(s)
Humans , Male , Androgen Antagonists/therapeutic use , Androgen Receptor Antagonists/therapeutic use , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Receptors, Androgen/drug effects
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