Study on the mechanism of Atractylodes macrocephala extract regulating mitochondrial endoplasmic reticulum stress through PI3K/Akt signaling pathway to reverse epithelial-mesenchymal transition in uterine fibroids.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Cholesterol metabolism affects endoplasmic reticulum (ER) stress and modulates epithelial-mesenchymal transition (EMT). Our previous study demonstrated that 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) attenuated EMT by blocking the transforming growth factor (TGF)-β/Smad signaling pathway and activating ER stress in MDA-MB-231 cells. To further assess the detailed mechanisms between cholesterol metabolism, ER stress, and EMT, LXR-623 (an agonist of LXRα) and simvastatin were used to increase and decrease cholesterol efflux and synthesis, respectively. Here, we found that high HP-β-CD concentrations could locally increase cholesterol levels in the ER by decreasing LXRα expression and increasing Hydroxymethylglutaryl-Coenzyme A reductase (HMGCR) expression in MDA-MB-231 and BT-549 cells, which triggered ER stress and inhibited EMT. Meanwhile, tunicamycin-induced ER stress blocked the TGF-β/Smad signaling pathway. However, low HP-β-CD concentrations can decrease the level of membrane cholesterol, enhance the TGF-β receptor I levels in lipid rafts, which helped to activate TGF-β/Smad signaling pathway, inhibit ER stress and elevate EMT. Based on our findings, the use of high HP-β-CD concentration can lead to cholesterol accumulation in the ER, thereby inducing ER stress, which directly suppresses TGF-β pathway-induced EMT. However, HP-β-CD is proposed to deplete membrane cholesterol at low concentrations and concurrently inhibit ER stress and induce EMT by promoting the TGF-β signaling pathways.

BackgroundUterine leiomyoma is one of the most common benign tumors in women. It dramatically decreases the quality of life in the affected women. However, there is a lack of effective treatment paradigms. Micro-RNAs are small noncoding RNA molecules that are extensively expressed in organisms, and they are interrelated with the occurrence and development of the tumor. miR-139-5p was found to be downregulated in various cancers, but its function and mechanism in uterine leiomyoma remain unknown. The aim of this study was to investigate the role of miR-139-5p and its target gene in uterine leiomyoma.MethodsBy using a bioinformatic assay, it was found that TPD52 was a potential target gene of miR-139-5p. Then, expressions of miR-139-5p and TPD52 in uterine leiomyoma and adjacent myometrium tissues were evaluated by quantitative real-time polymerase chain reaction and Western blot. Proliferation, apoptosis, and cell cycle of uterine leiomyoma cells transfected by miR-139-5p mimics or TPD52 siRNA were determined.ResultsIt was observed that the expression of miR-139-5p in uterine leiomyoma tissues was significantly lower (P<0.001) than that in the adjacent myometrium tissues. Overexpression of miR-139-5p inhibited the growth of uterine leiomyoma cells and induced apoptosis and G1 phase arrest. Dual-luciferase reporter assay and Western blot validated that TPD52 is the target gene of miR-139-5p. Furthermore, downregulation of TPD52 by siRNA in uterine leiomyoma cells inhibited cell proliferation and induced cell apoptosis and G1 phase arrest.ConclusionData suggested that miR-139-5p inhibited the proliferation of uterine leiomyoma cells and induced cell apoptosis and G1 phase arrest by targeting TPD52.

Objective To investigate the expression of aromatase cytochrome P450 (P450arom), the secretion of estradiol and the suppressive effect of aromatase inhibitor letrozole on the proliferation of uterine leiomyoma cells in vitro. Methods Uterine leiomyoma cells were cultured in vitro, after the action of letrozole with different concentration, MTT was used to examine the proliferative activity of uterine leiomyoma cells, RT-PCR and immunocytochemistry was used to determine the expression of the P450arom protein and mRNA, and the concentration of estradiol in media supernatant was measured by radioimmunity. Results Low dosage (0.06 μmol/L) of letrozole had no effect on the expression of P450arom protein and mRNA, and estradiol synthesis in uterine leiomyoma cell (P>0.05). However, high dosage(0,6-6 μmol/L) of letrozole could down-regulate the expression of P450arom, and to inhibit estradiol synthesis and the proliferation activity of uterine leiomyoma cells(P<0.05). Furthermore, the inhibition were shown to decline gradually along with the increase in the concentrations of letrozole. Conclusion The aromatase inhibitor letrozole could suppress the proliferation of uterine leiomyoma cells in the dosage-dependent manner by down-regulating the expression of the P450arom and decreasing the estradiol synthesis, suggesting that the drug may possibly be effective in the clinical treatment of uterine leiomyoma. Key words: Uterine neoplasms; Letrozole; Aromatase; Estradiol

Inositol-requiring protein 1 – X-box-binding protein 1 pathway promotes epithelial–mesenchymal transition via mediating snail expression in pulmonary fibrosis

Details Unfold: The Endoplasmic Reticulum Stress Response in Intestinal Inflammation and Cancer

The present research aimed to evaluate the expression of insulin-like growth factor-1 (IGF-1) in uterine leiomyoma, and explore its relationship with the occurrence and development of uterine leiomyoma and potential signal pathways. qRT-PCR and enzyme-linked immunosorbent assay were used for estimating the levels of IGF-1 in human uterine leiomyoma compared to myometrium. The expression of cell proliferation and PI3K/AKT/mTOR signaling pathway-related proteins in uterine leiomyoma cells was evaluated by western blot. Cell viability analysis was performed by CCK-8 assay. Lentivirus infection was used for IGF-1 overexpression and knockdown in uterine leiomyoma cells. The IGF-1 expression level was elevated in human uterine leiomyomas compared to myometrium. IGF-1 promoted the cell viability of human uterine leiomyoma cells. Overexpression of IGF-1 induced the expression of pro-proliferation markers including c-Myc, PCNA, and cyclin D1 in uterine leiomyoma cells. IGF-1 elevated the phosphorylation levels of PI3K, AKT, and mTOR, thus modifying PI3K/AKT/mTOR signaling in uterine leiomyoma cells. IGF-1 promotes proliferation of human uterine leiomyoma cells via PI3K/AKT/mTOR pathway.

Uterine leiomyomas are ovarian hormone-responsive tumors originating in the smooth muscle layer of the uterus called the myometrium. These benign tumors require surgery or other clinical intervention in ∼25% of all premenopausal women in the United States. Despite the high frequency and clinical impact of these tumors, the pathogenesis of uterine leiomyomas is not understood. The estrogen-responsive growth of uterine leiomyomas has been well established, however the role of G protein-coupled estrogen receptor (GPER) in leiomyoma cell regulation has not been studied. The membrane bound estrogen receptor GPER, formerly known as GPR30, is a 7-transmembrane G protein-coupled receptor that is activated by 17β-estradiol (E2). A role for GPER has been previously established in cancers that originate in E2-dependent tissues such as the breast and uterus. Other reports show that GPER is expressed in myometrial cells and activation of GPER modulates myometrial contraction suggesting an important regulatory role for this receptor in the myometrium. Based on these reports, we hypothesized that GPER regulates uterine leiomyoma cells and may represent a putative therapeutic target for this tumor type. The Eker rat model-derived uterine leiomyoma (ELT-3) cell line was used as a model system to study GPER in uterine leiomyoma cells. First, the expression of GPER in uterine leiomyoma cells was determined using RNA and protein samples from ELT-3 cells and real-time PCR and immunoblot analysis. Our findings indicate that GPER transcript and protein were expressed in ELT-3 cells. To determine if activation of GPER regulated uterine leiomyoma cell proliferation, ELT-3 cells were treated with G1, a GPER-specific ligand, or vehicle control and counted 5 days after treatment. Data from these experiments indicated that G1 treatment, and potentially GPER activation, reduced the number of cells over time compared to vehicle control. GPER activation is known to result in the accumulation of pERK and intracellular free Ca2+ and such accumulation is used as a biomarker for GPER activation. Upon G1 treatment, pERK and intracellular free Ca2+ were elevated compared to vehicle treatment in ELT-3 cells suggesting that GPER is functional in uterine leiomyoma cells and is activated by G1. In an effort to determine the mechanism of inhibition of uterine leiomyoma cell proliferation by G1 treatment, we hypothesized that treatment of estrogen-stimulated ELT-3 cells with G1 would inhibit the characteristic E2-induced proliferation of this cell type. Growth curve experiments were performed and data obtained in these experiments suggested that G1 treatment resulted in the inhibition of E2-induced ELT-3 cell proliferation. Taken together, these data suggest that GPER is an important regulator of uterine leiomyoma cell proliferation and is a putative target for novel molecular therapeutics for this tumor type. Citation Format: Maryann Castillo, Angelique M. Wimbley, Jacob J. Mayfield, Jenifer C. Lascano, Kevin D. Houston. Activation of G-protein coupled estrogen receptor (GPER) inhibits ELT-3 uterine leiomyoma cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2013-1305

Upon detecting endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) orchestrates adaptive cellular changes to reestablish homeostasis. If stress resolution fails, the UPR commits the cell to apoptotic death. Here we show that in hematopoietic cells, including multiple myeloma (MM), lymphoma, and leukemia cell lines, ER stress leads to caspase-mediated cleavage of the key UPR sensor IRE1 within its cytoplasmic linker region, generating a stable IRE1 fragment comprising the ER-lumenal domain and transmembrane segment (LDTM). This cleavage uncouples the stress-sensing and signaling domains of IRE1, attenuating its activation upon ER perturbation. Surprisingly, LDTM exerts negative feedback over apoptotic signaling by inhibiting recruitment of the key proapoptotic protein BAX to mitochondria. Furthermore, ectopic LDTM expression enhances xenograft growth of MM tumors in mice. These results uncover an unexpected mechanism of cross-regulation between the apoptotic caspase machinery and the UPR, which has biologically significant consequences for cell survival under ER stress.

To the Editor: Diabetic kidney disease (DKD) is one of the major causes of end-stage renal failure. Progressive mesangial expansion in the glomerulus is widely recognized. More and more evidence shows that tubulointerstitial fibrosis is also regarded as a prominent feature of DKD, in which tubular epithelial-mesenchymal transition (EMT) may play an important role.[1] During EMT, tubular epithelial cells lose their characteristic and produce high levels of myofibroblast makers such as α-smooth actin (α-SMA) which is the signature protein of EMT.[2] However, renal fibrogenesis is a complicated process in which the contribution of DKD is unknown. The endoplasmic reticulum (ER) is a key player in maintenance of protein homeostasis. Disturbances such as ischemia, oxidative stress and hypoxia can cause accumulation of misfolded proteins in the ER lumen, which induce endoplasmic reticulum stress (ERS).[3] ERS activates the highly conserved unfolded protein response which is initiated by three kinds of ER transmembrane signal protein pathways. PERK is one of them, which is combined with GRP78 to make it inactive in the physiological state. However, in the case of stress, PERK is dissociated with GRP78 and activated by its own phosphorylation, which serves as the central regulator of translational control during ERS. In the meantime, PERK has protein-kinase activity to phosphorylate the α-submit of eIF2α. Several mechanisms including hyperglycemia can cause ERS. On the other hand, ERS greatly affects the secretion and action of insulin, which leads to elevated blood glucose level.[4] Mice lacking PERK exhibit several defects including small size, bone abnormalities and type 1 diabetes.[5] Furthermore, there is evidence that ERS can lead to diabetic vascular complications.[6] ER stress has been demonstrated to contribute to the onset and progression of diabetic retinopathy by induction of multiple inflammatory signaling pathways.[7] Liu et al[8] has proved that ER stress contributes to premature senescence of renal tubular epithelial cells in patients with diabetes. But it is not very clear how ERS participates in the development of DKD. The purpose of the present study was to get a comprehensive understanding of how ERS participates in tubulointerstitial fibrosis in DKD and the mechanisms involved. The Ethics Committee of Renmin Hospital of Wuhan University confirmed that the ethics approval of this study was not needed. Anti-PERK, phosphor-PERK, GRP78, eIF2α antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phosphor-eIF2α and α-SMA antibody were obtained from Abcam Biotechnology (Cambridge, UK). Thapsigargin was purchased from Sigma (St. Louis, MO, USA). GSK2606414 was purchased from Selleck Chemicals (Houston, TX, USA). Rat renal proximal tubule epithelial cells (NRK-52E) were presented from the laboratory of nephrology department, Renmin Hospital of Wuhan University (obtained from American Type Culture Collection). NRK-52E cells were cultured (at 37°C, in a 5% CO2 atmosphere) in Dulbecco Modified Eagle's Medium (DMEM) containing glucose (5.6 mmol/L), 10% fetal bovine serum, 100 mg/mL streptomycin, 100 U/mL penicillin. Cells were seeded at 1 × 105 cells/well in 6-well dishes and were quiescent with serum-free medium for 24 h before use in all experiments. After finishing experiments, the media was removed and the cells were washed twice with ice-cold PBS. Cells were lysed with RIPA lysates (Beyotime Biotechnology, Shanghai, China), containing 1 mmol/L PMSF and quantified using bicinchoninic acid protein assay kit (Beyotime Biotechnology, Shanghai, China). Cell homogenates were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were then blocked for 1 h at room temperature with 5% skim milk powder in TBST. The blots were incubated with one of the following primary polyclonal antibodies: α-SMA, GRP78, PERK, phosphor-PERK, eIF2α, or phosphor-eIF2α. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G was used as the secondary antibody. After the chemiluminescence reaction, bands were detected by exposing the blots to X-ray film. The same membrane was reused to detect β-actin by incubating it with monoclonal anti-β-actin antibody. For a quantitative analysis, the bands were detected and evaluated densitometrically with Bandscan software (Glyko Inc., Novato, CA, USA), and normalized to corresponding density of β-actin. All data were expressed as mean ± standard deviation (SD). Each experiment was repeated three times independently. Data were compared among groups using one-way analysis of variance (ANOVA), while between-group comparisons of means were analyzed by Least Significant Difference (LSD) t test. All statistical analyses were performed by the SPSS Statistical Software version 21.0 (SPSS Inc., Chicago, USA). A value of P < 0.05 was considered statistically significant. As shown in Figure 1A, NRK-52E cells were cultured in mannitol (5.6 mmol/L D-glucose + 19.4 mmol/L D-mannitol; Mann group), or medium containing different level of glucose (NC group: 5.6 mmol/L; H1 group: 15 mmol/L; H2 group: 25 mmol/L; H3 group: 50 mmol/L) for 48 h, or medium containing 25 mmol/L glucose for 24 h (H/24 h group) or 48 h (H/48 h group). High glucose increased the protein of α-SMA in NRK-52E cells in a dose-dependent (NC: 0.21 ± 0.01 vs. H1: 0.40 ± 0.02, P < 0.01; NC: 0.21 ± 0.01 vs. H2: 0.45 ± 0.04, P < 0.01; NC: 0.21 ± 0.01 vs. H3: 0.53 ± 0.03, P < 0.01; F = 94.13, P < 0.01) and time-dependent manner (NC/24 h: 0.15 ± 0.01 vs. H/24 h: 0.25 ± 0.01, P < 0.01; NC/48 h: 0.15 ± 0 vs. H/48 h: 0.38 ± 0.02, P < 0.01; F = 199.67, P < 0.01). The addition of mannitol had no significant effect on α-SMA (NC: 0.21 ± 0.01 vs. Mann: 0.23 ± 0.02, P = 0.36).Figure 1: High concentration of glucose up-regulated α-SMA expression and triggered ERS in NRK-52E cells. Cells were cultured in medium with different level glucose (NC group: 5.6 mmol/L; H1 group: 15 mmol/L; H2 group: 25 mmol/L; H3 group: 50 mmol/L), or medium containing 25 mmol/L glucose in different time for 24 h (H/24 h group) or 48 h (H/48 h group). The results showed that high glucose induced the overexpression of α-SMA protein (A). The expression of GRP78 protein and phosphorylation of PERK and eIF2α were increased in high glucose medium, while the express of PERK and eIF2α protein did not change (B and C). * P < 0.05 vs. NC group, † P < 0.05 vs. H1 group and H/24 h group. α-SMA:α-smooth actin; ERS: Endoplasmic reticulum stress; eIF2α: Eukaryotic translation-initiation factor 2α; GRP78: 78kd-glucose-reglulated protein; Mann: Mannitol; NC: Normal control; PERK: Protein kinase R-like ER kinase.And as shown in Figure 1B and 1C, compared with the levels of NC group, the expression of GRP78 protein (NC: 0.90 ± 0.09 vs. H1: 1.27 ± 0.09, P = 0.001; NC: 0.90 ± 0.09 vs. H2: 1.42 ± 0.07, P < 0.01; NC: 0.90 ± 0.09 vs. H3: 1.14 ± 0.06, P = 0.03; F = 25.54, P < 0.01) and phosphorylation of PERK (NC: 0.43 ± 0.06 vs. H1: 0.65 ± 0.02, P < 0.01; NC: 0.43 ± 0.06 vs. H2: 0.77 ± 0.07, P < 0.01; NC: 0.43 ± 0.06 vs. H3: 0.40 ± 0.01, P = 0.43; F = 37.45, P < 0.01), eIF2α (NC: 0.37 ± 0.06 vs. H1: 0.47 ± 0.04, P = 0.018; NC: 0.37 ± 0.06 vs. H2: 0.63 ± 0.05, P < 0.01; NC: 0.37 ± 0.06 vs. H3: 0.56 ± 0.03, P < 0.01; F = 22.07, P < 0.01) were increased with the concentration of glucose. The peak expression of these proteins appeared after cells being cultured 48 h in the 25 mmol/L glucose. However, the protein of PERK (NC: 0.71 ± 0.06 vs. H1: 0.72 ± 0.03, P = 0.86; NC: 0.71 ± 0.06 vs. H2: 0.72 ± 0.07, P = 0.89; NC: 0.71 ± 0.06 vs. H3: 0.67 ± 0.03, P = 0.37; F = 1.64, P = 0.24) and eIF2α (NC: 0.27 ± 0.01 vs. H1: 0.29 ± 0.02, P = 0.20; NC: 0.27 ± 0.01 vs. H2: 0.27 ± 0.02, P = 0.58; NC: 0.27 ± 0.01 vs. H3: 0.27 ± 0.01, P = 0.87; F = 1.09, P = 0.41) did not change. Meanwhile, mannitol (osmotic control) had no effect on all these proteins. These results suggested that high glucose-induced ERS-related protein overproduction was not due to high osmotic stress. To examine whether ERS induced overexpression of α-SMA, NRK-52E cells were treated with the thapsigargin (Thaps), which was the inducing agent of ER-stress. Cells were incubated for 24 h or 48 h in Thaps concentration of 0.1 and 0.2 μmol/L respectively (Thaps 0.1/24 h group, Thaps 0.2/24 h group, Thaps 0.1/48 h group, Thaps 0.2/48 h group). The levels of α-SMA protein were all upregulated (NC: 0.35 ± 0.03 vs. Thaps 0.1/24 h: 0.80 ± 0.08, P < 0.01; NC: 0.35 ± 0.03 vs. Thaps 0.2/24 h: 0.63 ± 0.07, P < 0.01; NC: 0.35 ± 0.03 vs. Thaps 0.1/48 h: 0.72 ± 0.08, P < 0.01; NC: 0.35 ± 0.03 vs. Thaps 0.2/48 h: 0.52 ± 0.04, P < 0.01; F = 27.66, P < 0.01). The production of α-SMA was the highest when Thaps concentration was 0.1 μmol/L and the incubation time was 24 h (0.83 ± 0.08). To determine the mechanism of Thaps-induced α-SMA, we used GSK2606414 in subsequent intervene experiment. Cells were pre-treated with GSK2606414 (30 min), then cultured with Thaps. The overexpression of α-SMA was blocked greatly (0.91 ± 0.04 vs. 0.60 ± 0.03, P < 0.01), indicating that the PERK-e IF2α pathway was partly essential for ER-stress induced α-SMA activation [Figure 2A].Figure 2: Role of PERK- eIF2α pathway on α-SMA overexpression induced by Thaps and high glucose. Cells were incubated for 24 h or 48 h in different concentration of Thaps (Thaps 0.1/24 h group; Thaps 0.2/24 h group; Thaps 0.2/24 h group; Thaps 0.2/48 h group). The production of α-SMA was the highest when Thaps concentration was 0.1 mol/L and the incubation time was 24 h. Cells were pre-treated by GSK2606414 followed by cultured with Thaps (Thaps group) or high glucose (H group), the overexpression of α-SMA induced by Thaps was blocked greatly. * P < 0.05 vs. NC group, † P < 0.05 vs. Thaps 0.1/24 h group, ‡ P < 0.05 vs. Thaps group (A). GSK2606414 treatment inhibited high glucose-induced phosphorylation of eIF2α and α-SMA. * P < 0.05 vs. NC group, † P < 0.05 vs. H group (B). α-SMA:α-smooth actin; eIF2α: Eukaryotic translation-initiation factor 2α; PERK: Protein kinase R-like ER kinase; Thaps: Thapsigargin.Cells were pre-treated with different concentration of GSK2606414 (10 nmol/L, 100 nmol/L) for 30 min, then cultured with high glucose for 24 h. We observed that GSK2606414 (100 nmol/L) treatment inhibited high glucose-induced phosphorylation of eIF2α (0.55 ± 0.09 vs. 0.92 ± 0.13, P = 0.001) and α-SMA (0.56 ± 0.04 vs. 0.85 ± 0.08, P = 0.001) expression. The data indicated that high glucose activated α-SMA expression through PERK-e IF2α pathway [Figure 2B]. DKD is currently the leading cause of irreversible ESRD (end-stage renal disease) requiring dialysis. Rather than a solely glomerular disease, tubulointerstitial injury is also a major characteristic of DKD and an important predictor of renal dysfunction. The severity of tubulointerstitial fibrosis correlates with progressive renal damage. Various studies have demonstrated that EMT is a direct contributor to the kidney myofibroblast accumulation in the development of renal fibrosis, including DKD. In this process of EMT, tubular cells lost their cell markers and expressed a high level of mesenchymal markers, such as α-SMA. The present study showed that α-SMA as an index of fibrosis was increased in NRK-52E cells exposed to high glucose, which was consistent with previous research.[9] ER is a vast membranous network that is recognized as a protein-folding factory. A number of pathophysiological conditions are associated with ER stress, including diabetes and Alzheimer's disease. GRP78 is an important molecular indicator of ERS. The most immediate response to ER stress is the homodimerization and trans-phosphorylation of PERK, which is a Ser/Thr protein kinase and its activation results in the phosphorylate of eIF2α. Lakshmana et al[6] reported that the phosphorylation of myocardial PERK and eIF2α was shown to be significantly increased in the Spontaneous Diabetic Torill (SDT) rats, when compared with the SD rats. Loss of PERK in humans leads to Wallcot-Rallison syndrome (WRS), a disorder involving neonatal insulin-dependent diabetes resulting from destruction of pancreatic islet beta cells.[5] These researches indicate that PERK-eIF2α pathway is closely related to diabetes. In the present study, we demonstrated that high glucose increased the levels of GRP78 protein expression and PERK, eIF2α phosphorylation. Otherwise, mannitol had no effect on all these proteins, which suggests these effects were not induced by hyperosmosis. Moderate ER stress can maintain cell homeostasis. When the stress is excessive or prolonged, it can initiate pathologic reactions. Recently, ERS has been revealed to be closely linked to inflammatory and stress signal networks, including the action of JNK-AP1, NF-κB pathways. Furthermore, the relationship between ERS and renal damage is also gradually concerned. It has been proved that ERS plays a vital role in the development of renal fibrosis. And inhibition of ERS can improve renal fibrosis progression.[10] Fang et al[11] reported ER stress seemed to play an important role in albuminuria-induced kidney epithelial cells injury. In order to clarify the relationship between ERS and EMT, we used ERS-inducer thapsigargin to incubate NRK-52E cells. The results showed that thapsigargin increased the level of α-SMA protein. Subsequently, we used a potent selective PERK inhibitor–GSK2606414 to pre-treat cells. After co-cultured with GSK2606414, the increased level of α-SMA induced by thapsigargin was blocked significantly. These results manifested that thapsigargin stimulated overexpression of α-SMA by inducing ERS, which is partially through the PERK-eIF2α pathway. However, it has remained unclear how hyperglycemia induces EMT mediated by ER stress. In cancer cells, EMT can drive tumor metastasis, and disruption of PERK-eIF2α axis compromises the malignant phenotype of EMT cancer cells. Because of that, PERK pathway inhibitors warrant further exploration as potential cancer therapies.[12] Qi et al[13] proved that 4-PBA exerted a marked renoprotective effect through suppressing the expression of p-PERK in STZ-induced diabetic rats. Furthermore, as is well-known, P58IPK is an important component whose intracellular effect on ERS was achieved by inhibiting PERK activation. The ultimate result of a lack of P58IPK is deficiency of insulin, which mimics β-cell failure in type 1 and late-stage type 2 diabetes.[4] Yang et al[7] revealed the protecting role of P58IPK against ER stress-mediated diabetic retinopathy. In the present study, NRK-52E cells were cultured for 48 h with high glucose after they were pre-treated with GSK2606414. We observed that GSK2606414 inhibited high glucose-induced P-eIF2α and α-SMA expression. Based on these researches, we hypothesized that high glucose induced α-SMA overexpression in NRK-52E cells partly through the PERK-eIF2α pathway in the process of ERS. Our results provided some evidences to support this theory. Firstly, it had been proved that high concentration glucose triggered ERS in NRK-52E cells. Secondly, our results indicated that ERS and high glucose both induced renal proximal tubular cells to undergo EMT. Lastly, the study proved that GSK2606414 treatment reduced significantly overexpression of P-eIF2α and α-SMA protein stimulated by high glucose. In conclusion, our findings confirmed that high glucose stimulated α-SMA overexpression and ER stress in NRK-52E cells. In addition, this study also showed that high glucose-induced EMT was at least partially through the PERK-eIF2α pathway. As is well-known, EMT contributes to the development of tubulointertial injury. Collectively, the finding suggests inhibiting ER stress is a potential novel therapy of alleviating DKD. This experiment was only done in cells. In the future, we expect that animal experiments will be conducted to further explore the corresponding mechanism. Funding This work was supported by grants from the Natural Science Foundation of Hubei Province (No. 2017CFB779) and the Fundamental Research Funds for the Central Universities (No. 2042017kf0133). Conflicts of interest None.

The present study aimed to determine whether isorhamnetin (Isor), a natural antioxidant polyphenol, has antifibrotic effects in a murine model of bleomycin-induced pulmonary fibrosis. A C57 mouse model of pulmonary fibrosis was established by intraperitoneal injection of a single dose of bleomycin (3.5 U/kg), and then Isor (10 and 30 mg/kg) was administered intragastrically. The level of fibrosis was assessed by hematoxylin and eosin and Sirius red staining. α-smooth muscle actin and type I collagen levels in lung tissues were determined by western blotting and immunohistochemistry (IHC). Epithelial-mesenchymal transition (EMT), endoplasmic reticulum stress (ERS) and related signaling pathways were examined by western blotting and IHC. In vitro, human bronchial epithelial cells (HBECs) and A549 cells were treated with transforming growth factor (TGF)β1 with or without Isor, and collagen deposition and the expression levels of EMT- and ERS-related genes or proteins were analyzed by reverse transcription-quantitative polymerase chain reaction, western blotting, and immunofluorescence. The results demonstrated that Isor inhibited bleomycin-induced collagen deposition, reduced type I collagen and α-SMA expression, and alleviated EMT and ERS in vivo. Furthermore, incubation of HBECs and A549 cells with TGFβ1 activated EMT and ERS, and this effect was reversed by Isor. In conclusion, Isor treatment attenuated bleomycin-induced EMT and pulmonary fibrosis and suppressed bleomycin-induced ERS and the activation of PERK signaling.

To study the effects of ginsenoside Rb1 and the molecular mechanisms on proliferation and apoptosis of uterine fibroid cells, Rb1 + pc DNA3.1, Rb1 + pc DNA3.1-HMGB1, si-NC or si-HMGB1 was transfected into uterine fibroid cells by liposome method; the inhibitory rate and proliferation of human uterine fibroid cells were detected by MTT assay; apoptosis of uterine fibroid cells was detected by flow cytometry assay; HMGB1 protein expression in uterine fibroid cells was detected by Western blot assay. Compared with untreated uterine fibroid cells, the inhibitory and apoptosis rate of uterine fibroid cells treated with Rb1 were significantly up-regulated, while the expression level of HMGB1 was significantly down-regulated (p < .05). HMGB1 knockdown inhibited proliferation and promoted apoptosis of uterine fibroid cells. HMGB1 overexpression reversed the inhibitory effect on proliferation and the promotion effect on apoptosis of Rb1 in uterine fibroid cells. Ginsenoside Rb1 could inhibit uterine fibroid cells proliferation and promote apoptosis. This mechanism might be directly related to the downregulation of HMGB1, providing a basis for the treatment of uterine fibroids with ginsenoside Rb1.

The epithelial-mesenchymal transition (EMT) and endoplasmic reticulum (ER) stress induced by urinary protein, particularly albumin, play an important role in tubulointerstitial injury. However, signaling pathways regulating both albumin-induced EMT and ER stress are not precisely known. We postulated that reactive oxygen species (ROS), c-Src kinase, and mammalian target of rapamysin (mTOR) would act as upstream signaling molecules. We further examined the effect of imatinib mesylate on these processes. All experiments were performed using HK-2 cells, a human proximal tubular cell line. Protein and mRNA expression were measured by Western blot analysis and real-time PCR, respectively. Exposure of tubular cells to albumin (5 mg/ml) for up to 5 days induced EMT in a time-dependent manner, as shown by conversion to the spindle-like morphology, loss of E-cadherin protein, and upregulation of α-smooth muscle actin mRNA and protein. Albumin also induced ER stress as evidenced by phosphorylation of eukaryotic translation initiation factor-2α and increased expression of GRP78 mRNA and protein. Albumin induced ROS, c-Src kinase, and mTOR as well. Antioxidants, c-Src kinase inhibitor (PP2), and mTOR inhibitor (rapamycin) suppressed the albumin-induced EMT and ER stress. Antioxidants and PP2 inhibited the albumin-induced c-Src kinase and mTOR, respectively. Imatinib suppressed the albumin-induced EMT and ER stress via inhibition of ROS and c-Src kinase. Imatinib also inhibited the albumin-induced mRNA expression of MCP-1, VCAM-1, transforming growth factor (TGF)-β1, and collagen I (α1). In conclusion, the ROS-c-Src kinase-mTOR pathway played a central role in the signaling pathway that linked albumin to EMT and ER stress. Imatinib might be beneficial in attenuating the albumin-induced tubular injury.