Overcoming IMiD resistance in T-cell lymphomas through potent degradation of ZFP91 and IKZF1.

Immunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here, we show that 2 factors determine resistance to IMiDs among TCLs. First, limited CRBN expression reduces IMiD activity in TCLs but can be overcome by newer-generation degrader CC-92480. Using mass spectrometry, we show that CC-92480 selectively degrades IKZF1 and ZFP91 in TCL cells with greater potency than pomalidomide. As a result, CC-92480 is highly active against multiple TCL subtypes and showed greater efficacy than pomalidomide across 4 in vivo TCL models. Second, we demonstrate that ZFP91 functions as a bona fide transcription factor that coregulates cell survival with IKZF1 in IMiD-resistant TCLs. By activating keynote genes from WNT, NF-kB, and MAP kinase signaling, ZFP91 directly promotes resistance to IKZF1 loss. Moreover, lenalidomide-sensitive TCLs can acquire stable resistance via ZFP91 rewiring, which involves casein kinase 2-mediated c-Jun inactivation. Overall, these findings identify a critical transcription factor network within TCLs and provide clinical proof of concept for the novel therapy using next-generation degraders.

Interactive effects of information and trust on consumer choices of organic food: Evidence from China.

A large number of studies have explored the separate roles of information and trust in consumer choices of organic food, but little attention has been paid to exploring the interactive effects of information and trust. Here, for the first time to our knowledge, we explored the joint effects of information and consumers' trust in shaping consumer preferences for organic food. A hypothetical choice experiment was employed to elicit consumer preferences for organic food, and a between-subject design approach was used to explore the effects of information. Our results from a sample of 2382 Chinese consumers indicated that consumers are willing to pay extra price for organic rice compared to the conventional rice. Notably, individuals with a high level of trust exhibited a significantly higher willingness to pay for organic rice than those with lower trust levels. Furthermore, the introduction of information leads to a substantial 40% increase in consumers' willingness to pay for organic rice, with an even more significant 50% increase observed among high-trust consumers. These results highlight the augmenting role of trust in amplifying the effects of information. Consequently, effective strategies should encompass both the provision of information and the cultivation of trust concurrently to promote consumer choices of organic food.

Inhibition of long noncoding RNA Gm41724 alleviates pressure overload-induced cardiac fibrosis by regulating lamina-associated polypeptide 2α.

Cardiac fibrosis is a pathological process underlying myocardial remodeling and is characterized by excessive deposition of the myocardial extracellular matrix. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of various biological processes. In this study, we investigated the role of a novel lncRNA, Gm41724, in cardiac fibrosis induced by pressure overload. High-throughput whole transcriptome sequencing analysis was performed to detect differentially expressed lncRNAs in cardiac fibroblasts (CFs) with or without TGF-ß1 treatment. Differential expression analysis and gene set enrichment analysis identified Gm41724 as a potential molecule targeting fibrosis. Gm41724 positively regulated the activation of CFs induced by TGF-ß1 and pressure overload. Knocking down Gm41724 could inhibit the differentiation of CFs into myofibroblasts and alleviate cardiac fibrosis induced by pressure overload. Mechanistically, comprehensive identification of RNA-binding proteins by mass spectrometry (CHIRP-MS) and RNA immunoprecipitation (RIP) assay combined with other methods of molecular biological revealed the important role of Gm41724 binding to lamina-associated polypeptide 2α (lap2α) for the activation of CFs. Further mechanistic studies indicated that the regulator of G protein signaling 4 (Rgs4), as the downstream effector of Gm41724/lap2α, regulated CFs activation. Our results implicated the involvement of Gm41724 in cardiac fibrosis induced by pressure overload and it is expected to be a promising target for anti-fibrotic therapy.

Biomarker-driven strategy for MCL1 inhibition in T-cell lymphomas.

There is a pressing need for more effective therapies to treat patients with T-cell lymphomas (TCLs), including first-line approaches that increase the response rate to cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) chemotherapy. We characterized the mitochondrial apoptosis pathway in cell lines and patient-derived xenograft (PDX) models of TCL and assessed the in vitro efficacy of BH3 mimetics, including the BCL2 inhibitor venetoclax, the BCL2/BCL-xL inhibitor navitoclax, and the novel MCL1 inhibitor AZD5991. The abundance of antiapoptotic BCL2 family members based on immunoblotting or RNA transcript levels correlated poorly with the activity of BH3 mimetics. In contrast, the functional approach BH3 profiling reliably predicted sensitivity to BH3 mimetics in vitro and in vivo. We used BH3 profiling to select TCL PDX that were dependent on MCL1. Mice xenografted with these PDX and treated with AZD5991 had markedly improved survival. The combination of AZD5991 and CHOP achieved synergy based on survival improvement beyond a mathematical "sum of benefits" model. Thus, MCL1 inhibition is a promising strategy as both a single agent and in combination with chemotherapy for patients with TCL and functional dependence on MCL1.

Parametric Surface Modelling for Tea Leaf Point Cloud Based on Non-Uniform Rational Basis Spline Technique.

Plant leaf 3D architecture changes during growth and shows sensitive response to environmental stresses. In recent years, acquisition and segmentation methods of leaf point cloud developed rapidly, but 3D modelling leaf point clouds has not gained much attention. In this study, a parametric surface modelling method was proposed for accurately fitting tea leaf point cloud. Firstly, principal component analysis was utilized to adjust posture and position of the point cloud. Then, the point cloud was sliced into multiple sections, and some sections were selected to generate a point set to be fitted (PSF). Finally, the PSF was fitted into non-uniform rational B-spline (NURBS) surface. Two methods were developed to generate the ordered PSF and the unordered PSF, respectively. The PSF was firstly fitted as B-spline surface and then was transformed to NURBS form by minimizing fitting error, which was solved by particle swarm optimization (PSO). The fitting error was specified as weighted sum of the root-mean-square error (RMSE) and the maximum value (MV) of Euclidean distances between fitted surface and a subset of the point cloud. The results showed that the proposed modelling method could be used even if the point cloud is largely simplified (RMSE < 1 mm, MV < 2 mm, without performing PSO). Future studies will model wider range of leaves as well as incomplete point cloud.

[The potential role of long non-coding RNA Dnm3os in the activation of cardiac fibroblasts].

Long non-coding RNA (lncRNA) Dnm3os plays a critical role in peritendinous fibrosis and pulmonary fibrosis, but its role in the process of cardiac fibrosis is still unclear. Therefore, we carried out study by using the myocardial fibrotic tissues obtained by thoracic aortic constriction (TAC) in an early study of our group, and the in vitro cardiac fibroblast activation model induced by transforming growth factor-ß1 (TGF-ß1). Quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and collagen gel contraction test were used to identify the changes of activation phenotype and the expression of Dnm3os in cardiac fibroblasts. Small interfering RNA was used to silence Dnm3os to explore its role in the activation of cardiac fibroblasts. The results showed that the expression of Dnm3os was increased significantly in myocardial fibrotic tissues and in the activated cardiac fibroblasts. And the activation of cardiac fibroblasts could be alleviated by Dnm3os silencing. Furthermore, the TGF-ß1/Smad2/3 pathway was activated during the process of cardiac fibroblasts activation, while was inhibited after silencing Dnm3os. The results suggest that Dnm3os silencing may affect the process of cardiac fibroblast activation by inhibiting TGF-ß1/Smad2/3 signal pathway. Therefore, interfering with the expression of lncRNA Dnm3os may be a potential target for the treatment of cardiac fibrosis.

Matrix metalloprotein-triggered, cell penetrating peptide-modified star-shaped nanoparticles for tumor targeting and cancer therapy.

BACKGROUND: Specific targeting ability and good cell penetration are two critical requirements of tumor-targeted delivery systems. In the present work, we developed a novel matrix metalloprotein-triggered, cell-penetrating, peptide-modified, star-shaped nanoparticle (NP) based on a functionalized copolymer (MePEG-Peptide-Tri-CL), with the peptide composed of GPLGIAG (matrix metalloprotein-triggered peptide for targeted delivery) and r9 (cell-penetrating peptide for penetration improvement) to enhance its biological specificity and therapeutic effect. RESULTS: Based on the in vitro release study, a sustained release profile was achieved for curcumin (Cur) release from the Cur-P-NPs at pH 7.4. Furthermore, the release rate of Cur was accelerated in the enzymatic reaction. MTT assay results indicated that the biocompatibility of polymer NPs (P-NPs) was inversely related to the NP concentration, while the efficiency toward tumor cell inhibition was positively related to the Cur-P-NP concentration. In addition, Cur-P-NPs showed higher fluorescence intensity than Cur-NPs in tumor cells, indicating improved penetration of tumor cells. An in vivo biodistribution study further demonstrated that Cur-P-NPs exhibited stronger targeting to A549 xenografts than to normal tissue. Furthermore, the strongest tumor growth inhibition (76.95%) was observed in Cur-P-NP-treated A549 tumor xenograft nude mice, with slight pulmonary toxicity. CONCLUSION: All results demonstrated that Cur-P-NP is a promising drug delivery system that possesses specific enzyme responsiveness for use in anti-tumor therapy.

[The Observation of Liver and Kidney Injury and the Activation of Macrophages in the Overload Pressure Induced Cardiac Hypertrophy/Heart Failure Mouse Model].

OBJECTIVE: The purpose of this study is to investigate the injury of liver and kidney tissues in overload pressure induced cardiac hypertrophy/heart failure mice model and the changes of macrophage activation level. METHODS: 6-8 week-old C57BL/6 mice were subjected to transverse aortic constriction (TAC) surgery to establish the cardiac hypertrophy/heart failure mouse model induced by pressure overload, while the aortic was not ligated in the Sham group. At 4 weeks and 8 weeks after TAC, the mice of each group were subjected to echocardiography and blood collection. And mice were sacrificed to collect samples of the heart, liver, and kidney tissues. The contents of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and serum creatinine (Scr) in Sham group and two operation groups were determined. The histological changes of liver, heart and kidney tissues were observed by HE staining, and the expression of the marker of macrophage activation, F4/80 protein, was detected in the heart, liver and kidney tissue by immunohistochemical staining. RESULTS: Cardiac hypertrophy occurred at 4 weeks after TAC operation in C57BL/6 mice and developed into heart failure at 8 weeks after TAC. The echocardiography showed that, compared with the Sham group, the left ventricular end-diastolic posterior wall thickness (LVPWd) and the left ventricular internal diameter in diastole (LVIDd) were significantly increased, while the left ventricular ejection fraction (EF) and the left ventricular fractional shortening (FS) were significantly decreased ( P<0.05) in the 4-week-TAC group and 8-week-TAC group. The plasma content of ALT, AST, TBil and Scr in the 4-week-TAC group and 8-week-TAC group were significantly higher than those in the Sham group ( P<0.05). HE staining showed obvious liver pathological changes in TAC mice, such as vacuolation, mild hepatic sinusoid congestion and inflammatory infiltration in mice post 4 weeks after surgery, and such liver injury was worse in mice post 8 weeks after surgery. Besides, there was a slight damage in renal tissue shown by HE staining, such as slight glomerular injury and slight bleeding. F4/80 protein immunohistochemical staining results demonstrated that the activation of macrophages in the heart and liver in the 4-week-TAC group and 8-week-TAC group was significantly increased than that in the sham group ( P<0.05), but there was no significant difference in kidney tissues in groups. CONCLUSION: Macrophages are involved in the process of liver and kidney injury in cardiac hypertrophy/heart failure.

[Study on visfatin-induced inflammation and necroptosis via LOX-1 in human umbilical vein endothelial cells].

The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours' treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.

[Overexpression of miR-130a-3p attenuates cardiomyocyte hypertrophy].

This study aimed to explore the role of miR-130a-3p in cardiomyocyte hypertrophy and its underlying mechanisms. Pressure-overload induced myocardial hypertrophy mice model was constructed by thoracic aortic constriction (TAC). In vitro, norepinephrine (NE) was used to stimulate neonatal rat cardiomyocytes (NRCMs) and H9c2 rat cardiomyocytes to induce hypertrophic phenotypes. The expression of miR-130a-3p was detected in mice hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. The mimics and inhibitors of miR-130a-3p were transfected into H9c2 cells to observe the role of miR-130a-3p on the hypertrophic phenotype change of cardiomyocytes separately. Furthermore, whether miR-130a-3p regulated hypertrophic related signaling pathways was explored. The results showed that the expression of miR-130a-3p was significantly decreased in hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. After transfection of miR-130a-3p mimics, the expression of hypertrophic marker genes, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC), and the cell surface area were notably down-regulated compared with the control group (mimics N.C. + NE group). But after transfection of miR-130a-3p inhibitor, the expression of ANP, BNP and ß-MHC in H9c2 cells increased significantly, and the cell area increased further. By Western blot, it was found that the protein phosphorylation level of Akt and mTOR were down-regulated after over-expression of miR-130a-3p. These results suggest that miR-130a-3p mimics may alleviate the degree of cardiomyocyte hypertrophy, meanwhile its inhibitor can further aggravate cardiomyocyte hypertrophy. Over-expression of miR-130a-3p may attenuate cardiomyocytes hypertrophy by affecting the Akt pathway.

[The potential role of calnexin in the activation of cardiac fibroblasts].

Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca 2+ homeostasis, and Ca 2+ signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts in vivo, and the in vitro cardiac fibroblasts activation model was established by transforming growth factor ß1 (TGFß1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFß1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

Different effects of additional ganglion plexus ablation on catheter and surgical ablation for atrial fibrillation: a systemic review and meta-analysis.

The effect of ganglion plexus (GP) ablation in addition to pulmonary veins isolation (PVI) for atrial fibrillation (AF) remained controversial between the catheter and surgical-based studies. Eleven studies (five randomized controlled trials and six nonrandomized studies) of 1750 patients were included in a meta-analysis to elucidate the incremental benefit of additional GP ablation in patients undergoing catheter or surgical ablation. Risk ratios were calculated for freedom from AF or AT recurrence after a single procedure. Additional GP ablation was associated with a better rhythm outcome for patients undergone catheter ablation but did not seem to increase freedom from AF/AT for surgical patients. Both paroxysmal and non-paroxysmal AF showed a positive outcome comparing additional GP ablation with PVI alone.

In-situ evaluation of porosity in thermal barrier coatings based on the broadening of terahertz time-domain pulses: simulation and experimental investigations.

Porosity is one of the most important indicators for the characterization of the comprehensive performance of thermal barrier coatings (TBCs). Herein, we explored a fast, nondestructive porosity evaluation method based on the terahertz time-domain broadening effect. Different preparation process parameters were used to deposit the ceramic coatings, and the porosity ranged from 9.09% to 21.68%. Monte Carlo simulations were conducted to reveal the transitive relation between porosity and the terahertz time-domain broadening at different extinction coefficients and transmission distances. A transmission mode with an incidence angle of 0° was used to estimate the terahertz dielectric properties of ceramic coatings and the relative broadening ratio of terahertz pulses at different porosities. As a result, the Monte Carlo simulations showed that the time-domain broadening effect was enhanced when the extinction coefficient and transmission distances increased. As the porosity increased, the refractive index decreased and the extinction coefficient increased. The latter was more sensitive to minor porosity changes as demonstrated by linear fitting comparisons. Meanwhile, the relative broadening ratio increased when the porosity increased, and reserved the sensitivity of the extinction coefficient to porosity changes. The effect was more obvious on the relative broadening ratio which experienced multiple transmissions and reflections. Moreover, the relative broadening ratio could be obtained faster and in an easier manner compared to the dielectric parameters in both the transmission and reflection modes, based on single-step tests with the use of actual terahertz wave inspection. Finally, this study proposed a novel, convenient, online, nondestructive, and noncontact porosity evaluation method that could be potentially utilized to evaluate the integrity of TBCs in gas turbine blades.

PEGylated self-assembled enzyme-responsive nanoparticles for effective targeted therapy against lung tumors.

BACKGROUND: Matrix-metalloproteinases, which are overexpressed in many types of cancer, can be applied to improve the bioavailability of chemotherapeutic drugs and guide therapeutic targeting. Thus, we aimed to develop enzyme-responsive nanoparticles based on a functionalized copolymer (mPEG-Peptide-PCL), which was sensitive to matrix metalloproteinase, as smart drug vesicles for enhanced biological specificity and reduced side effects. RESULTS: The rate of in vitro curcumin (Cur) release from Cur-P-NPs was not markedly accelerated in weakly acidic tumor microenvironment, indicating a stable intracellular concentration and a consistent therapeutic effect. Meanwhile, P-NPs and Cur-P-NPs displayed prominent biocompatibility, biostability, and inhibition efficiency in tumor cells. In addition, Cur-P-NPs showed higher fluorescence intensity than Cur-NPs in tumor cells, implying enhanced cell permeability and targeting ability. Moreover, the internalization and intracellular transport of Cur-P-NPs were mainly via macropinocytosis. Studies of pharmacodynamics and cellular uptake in vitro and biodistribution in vivo demonstrated that Cur-P-NPs had stronger target efficiency and therapeutic effect than Cur-DMSO and Cur-NPs in tumor tissue. CONCLUSION: Results indicate that Cur-P-NPs can be employed for active targeted drug delivery in cancer treatment and other biomedical applications.

Circulating hematopoietic stem and progenitor cells are myeloid-biased in cancer patients.

Cancer is associated with a profound perturbation in myelopoiesis that results in the accumulation of myeloid-derived suppressor cells (MDSCs) to promote disease progression. Recent studies in mice suggest that tumor-derived factors could regulate the differentiation of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and subsequently contribute to dysregulation of hematopoiesis. However, the nature and role of HPSCs in patients with cancer remain unknown. Here we show, in detailed studies of the peripheral blood from 133 untreated patients with seven different types of tumors, that the composition of circulating HSPCs was significantly altered in patients with solid tumors. The frequencies of circulating granulocyte-monocyte progenitors (GMPs) were increased four to seven fold in all types of tumors examined, and the circulating hematopoietic precursors exhibited myeloid bias with a skew toward granulocytic differentiation in patients with solid tumors. These myeloid precursors are selectively enriched in tumor tissues, and the high levels of circulating GMPs were positively correlated with disease progression. By using cord blood-derived CD34(+) cells, we developed an in vitro short-term culture model to effectively induce the rapid generation of MDSCs. We found that, among the factors produced by various tumors, GM-CSF, granulocyte colony-stimulating factor, and IL-6 could not only promote the myeloid-biased differentiation, but also induce the differentiation of myeloid precursors into functional MDSCs. These findings suggest that the altered circulating HSPCs may serve as an important link between dysregulated bone marrow hematopoiesis and accumulated MDSCs in patients with cancer.

Pd-Catalyzed C(sp3)-C(sp2) cross-coupling of Y(CH2SiMe3)3(THF)2 with vinyl bromides and triflates.

Pd-Catalyzed C(sp3)-C(sp2) cross-coupling of Y(CH2SiMe3)3(THF)2 with vinyl bromides and triflates has been developed for efficient synthesis of various allyltrimethylsilanes. The cross-coupling reaction was conducted at room temperature with low catalyst loading of either Pd(PPh3)4 or Pd(PPh3)2Cl2, and exhibited high efficiency and a broad substrate scope. In combination with the cross-coupling by the Lewis-acid catalyzed Hosomi-Sakurai reaction, a novel three-component one-pot cascade reaction was then accomplished to deliver homoallylic alcohols and ethers with high regioselectivity and diastereoselectivity. The three-component reaction defined the yttrium complex as a novel one-carbon synthon, which could either trigger bifunctionalization of alkenes or link two electrophiles and would find applications in organic synthesis.

[Study on Potential Role of Calreticulin in Pressure Overload Induced Cardiac Hypertrophy].

This study aims to investigate the role of calreticulin in(CRT)pressure overload induced cardiac hypertrophy.In our study,cardiac hypertrophy was induced by left ventricular pressure overload in male SD rats subjected to transverse aortic constriction(TAC)operation.Expression of gene and protein of calreticulin,markers of cardiac hypertrophy and endoplasmic reticulum stress(ERS)were measured with real-time qPCR and Western blot respectively.Meanwhile,atorvastatin(a known ERS inhibitor)and calreticulin-specific small interference ribonucleic acid(siRNA)were used to inhibit the expression of ERS and calreticulin respectively.The experimental data demonstrated that the gene and protein levels of calreticulin,hypertrophic and ERS markers were increased significantly in the heart tissues of TAC rat models after 4weeks.Moreover,atorvastatin administration improved the cardiac function and reduced the expression of calreticulin and ERS markers in TAC rats.In addition,cultured primary neonatal rat cardiomyocytes(NCMs)were treated with norepinephrine(NE),angiotensionⅡ(AngⅡ)or isoprenaline(ISO)to induce hypertrophic phenotype and ERS.The expression of hypertrophic markers was reduced in NCMs transfected with calreticulin-siRNA.The results suggested that calreticulin might be a promising target for the treatment of cardiac hypertrophy.

[Effect of Biaxial Tensile Strain on Expression of Osteogenic Specificity Markers of Rat Bone Marrow-derived Mesenchymal Stem Cells in Vitro].

The purpose of this study was to investigate the effect of biaxial tensile strain on the osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSCs)in vitro.The rBMSCs were isolated from tibia and femur of 4weeks-old Sprague-Dawley(SD)rats.The rBMSCs were cultured in DMEM-LG complete culture medium and grew to subconfluence in the cell culture device for loading tensile strain.The biaxial tensile strain was applied to the rBMSCs for periods of 2,4and 6hours every day,respectively,lasting 3days.The amplitude of biaxial tensile strain applied to the rBMSCs were 1%,2% and 5%respectively,at a frequency of 1Hz.Unstrained rBMSCs were used as blank control(control group).The rBMSCs cultured with DMEM-LG complete culture medium containing100nmol/Lß-Estradiol(E2)were used as positive control.The mRNA expression of alkaline phosphatase(ALP),collagen typeⅠ(ColⅠ),Runt-related transcription factor 2(Runx2)and osteocalcin(OCN)was examined with real-time quantitative PCR and the protein expression of ALP,ColⅠ,Runx2 and OCN was detected with Western blot method.The results showed as follws:(1)The mRNA and protein expression of the ALP,ColⅠ,Runx2,OCN were significantly higher in rBMSCs of the E2 group than those in the control group(P<0.05).(2)The mRNA and protein expression level of the ALP,Runx2 were higher markedly in the 1% tensile strain groups than those in the control group(P<0.05),but lower than those in the E2group(P<0.05).(3)The mRNA and protein expression level of the ALP,ColⅠ,Runx2,OCN were significantly higher in the 2% tensile strain groups than those in the control group(P<0.05),and the mRNA and protein expression level of ColⅠ and Runx2 in the group applied with2% amplitude of tensile strain for 4h/d was significantly higher than those in E2group(P<0.05).(4)The mRNA and protein expression level of the ALP,ColⅠ,Runx2 were significantly higher in the groups applied with 5% amplitude of tensile strain for 2h/d or for 4h/d than those in the control group(P<0.05).In our study,E2 and mechanical stimulation played an important role in the regulation of differentiation of rBMSCs into osteoblasts,and the manner applied with the 2%amplitude of tensile strain for 4h/d,lasting 3days was an optimal stimulus for up-regulating the mRNA and protein expression of ALP,ColⅠ,Runx2,OCN of rBMSCs.

[Research on Potential Role of Receptor-interacting Protein Kinase 1 in Phenotype Switching of Vascular Smooth Muscle Cells].

Vascular smooth muscle cells(VSMCs)phenotype switching plays an essential role in the pathogenesis of various vascular diseases.The present study aims to investigate the role of receptor-interacting protein kinases 1(RIPK1)in VSMCs phenotypic switching induced by AngiotensinⅡ(AngⅡ).Expression of mRNA and protein of RIPK1,markers of VSMCs phenotypic switching and secretion,phosphorylation of the P65 subunit of NF-κB were measured by real-time PCR and Western blot.Meanwhile,EdU incorporation assay and wound scratch assay were performed to determine the cell proliferation and migration respectively.At the same time,Necrostatin-1(Nec-1,an known RIPK1inhibitor)and RIPK1-specific small interference RNA(siRNA)were used to inhibit the expression of RIPK1.The experimental data demonstrated that the mRNA and protein levels of RIPK1 and P65phosphorylation were increased significantly in the process of VSMC phenotypic switching induced by Ang II.Moreover,the expression of RIPK1 and P65phosphorylation were significantly down-regulated in VSMCs pretreated with Nec-1or transfected with RIPK1-siRNA.Furthermore,the proliferation,secretion and migration of VSMCs were also markedly suppressed after inhibition of RIPK1 by Nec-1or its specific siRNA.The results suggested that RIPK1 might be involved in VSMC phenotypic switching induced by Ang II,which was possibly via up-regulating the NF-κB signaling pathway.

Nogo-B: A potential indicator for hepatic cirrhosis and regulator in hepatic stellate cell activation.

AIM: To evaluate plasma Nogo-B levels in liver cirrhotic patients and declare a novel molecular basis by which Nogo-B modulates hepatic stellate cell (HSC) activation. METHODS: Plasma Nogo-B levels from liver cirrhotic patients were detected by enzyme-linked immunosorbent assay. Rat primary HSC were culture activated or stimulated with transforming growth factor (TGF)-ß. Activated HSC were transfected for 48 h with Nogo-B shRNA to inhibit Nogo-B expression. Gene expressions of Nogo-B, α-smooth muscle actin (SMA), collagen type I, TGF-ß, endoplasmic reticulum (ER) stress key molecules, including C/EBP homologous protein (CHOP), glucose-regulated protein 78 (GRP78), activating transcription factor (ATF)4, ATF6, X-box binding protein 1 (Xbp-1) and calnexin, and the marker of autophagy beclin 1, were detected by quantitative reverse transcription polymerase chain reaction. The protein expressions of Nogo-B, α-SMA, collagen type I, CHOP, GRP78 and the marker of autophagy LC3B were evaluated by western blot. RESULTS: Liver cirrhotic patients showed a much higher level of plasma Nogo-B compared with the healthy controls. Nogo-B expression and ER stress could be induced during the process of cultured HSC activation. TGF-ß treatment increased Nogo-B expression time- and dose-dependently. Knockdown of Nogo-B in HSC reduced the activation of HSC. After Nogo-B gene knockdown, there was a decline of expression of ER stress markers and autophagic markers. Agonist or antagonist of ER stress could regulate autophagy level. CONCLUSION: Circulating Nogo-B may be an effective indicator for liver cirrhosis. Nogo-B inhibition could diminish HSC activation, in which alleviating ER stress may be one of the mechanisms, suggesting a potential approach to interference Nogo-B in liver fibrosis.