Oroxin B Induces Apoptosis by Down-Regulating MicroRNA-221 Resulting in the Inactivation of the PTEN/PI3K/AKT Pathway in Liver Cancer

Nannan Li; Wenxiao Men; Yibo Zheng; Hechen Wang; Xiansheng Meng

doi:10.3390/molecules24234384

Mechanism of KLF4 Protection against Acute Liver Injury via Inhibition of Apelin Signaling

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/6140360 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Weitao Ji ◽

Hongyun Shi ◽

Hailin Shen ◽

Jing Kong ◽

Jiayi Song ◽

...

Keyword(s):

Liver Injury ◽

Signaling Pathway ◽

Acute Liver Injury ◽

Control Group ◽

Necrosis Factor Alpha ◽

Protein Levels ◽

Klf4 Expression ◽

Mrna And Protein Expression

Krüppel-like factor 4 (KLF4) is a key transcription factor that regulates genes involved in the proliferation or differentiation in different tissues. Apelin plays roles in cardiovascular functions, metabolic disease, and homeostatic disorder. However, the biological function of apelin in liver disease is still ongoing. In this study, we investigated the mechanism of KLF4-mediated protection against acute liver injury via the inhibition of the apelin signaling pathway. Mice were intraperitoneally injected with carbon tetrachloride (CCl4; 0.2 mL dissolved in 100 mL olive oil, 10 mL/kg) to establish an acute liver injury model. A KLF4 expression plasmid was injected through the tail vein 48 h before CCl4 treatment. In cultured LX-2 cells, pAd-KLF4 or siRNA KLF4 was overexpressed or knockdown, and the mRNA and protein levels of apelin were determined. The results showed that the apelin serum level in the CCl4-injected group was higher than that of control group, and the expression of apelin in the liver tissues was elevated while KLF4 expression was decreased in the CCl4-injected group compared to the KLF4-plasmid-injected group. HE staining revealed serious hepatocellular steatosis in the CCl4-injected mice, and KLF4 alleviated this steatosis in the mice injected with KLF4 plasmid. In vitro experiments showed that tumor necrosis factor-alpha (TNF-α) could downregulate the transcription and translation levels of apelin in LX-2 cells and also upregulate KLF4 mRNA and protein expression. RT-PCR and Western blotting showed that the overexpression of KLF4 markedly decreased basal apelin expression, but knockdown of KLF4 restored apelin expression in TNF-α-treated LX-2 cells. These in vivo and in vitro experiments suggest that KLF4 plays a key role in inhibiting hepatocellular steatosis in acute liver injury, and that its mechanism might be the inhibition of the apelin signaling pathway.

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TLR4 Signaling in MPP+-Induced Activation of BV-2 Cells

Neural Plasticity ◽

10.1155/2016/5076740 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 19

Author(s):

Peng Zhou ◽

Ruihui Weng ◽

Zhaoyu Chen ◽

Rui Wang ◽

Jing Zou ◽

...

Keyword(s):

Western Blot ◽

Mtt Assay ◽

Inflammatory Responses ◽

Cell Model ◽

Critical Role ◽

Control Group ◽

Immunofluorescence Technique ◽

Rt Pcr ◽

Sirna Interference

Aims.This work was conducted to establish anin vitroParkinson’s disease (PD) model by exposing BV-2 cells to 1-methyl-4-phenylpyridinium (MPP+) and exploring the roles of TLR2/TLR4/TLR9 in inflammatory responses to MPP+.Methods/Results.MTT assay showed that cell viability of BV-2 cells was 84.78 ± 0.86% and 81.18 ± 0.99% of the control after incubation with 0.1 mM MPP+for 12 hours and 24 hours, respectively. Viability was not significantly different from the control group. With immunofluorescence technique, we found that MPP+incubation at 0.1 mM for 12 hours was the best condition to activate BV-2 cells. In this condition, the levels of TNF-α, IL-1β, and iNOS protein were statistically increased compared to the control according to ELISA tests. Real time RT-PCR and western blot measurements showed thatTLR4was statistically increased after 0.1 mM MPP+incubation for 12 hours. Furthermore, after siRNA interference ofTLR4mRNA, NF-κB activation and the levels of TNF-α, IL-1β, and iNOS were all statistically decreased in this cell model.Conclusion.MPP+incubation at the concentration of 0.1 mM for 12 hours is the best condition to activate BV-2 cells for mimicking PD inflammation in BV-2 cells. TLR4 signalling plays a critical role in the activation of BV-2 cells and the induction of inflammation in this cell model.

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Dachengqi Decoction Attenuates Intestinal Vascular Endothelial Injury in Severe Acute Pancreatitis in Vitro and in Vivo

Cellular Physiology and Biochemistry ◽

10.1159/000486155 ◽

2017 ◽

Vol 44 (6) ◽

pp. 2395-2406 ◽

Cited By ~ 7

Author(s):

Li-yun Pan ◽

Ya-feng Chen ◽

Hong-chang Li ◽

Li-ming Bi ◽

Wen-jie Sun ◽

...

Keyword(s):

Acute Pancreatitis ◽

Serum Amylase ◽

Endothelial Damage ◽

Intestinal Injury ◽

Control Group ◽

Rt Pcr ◽

Intestinal Tissue ◽

Tnf Α

Background/Aims: Dachengqi decoction (DCQD) is a well-known traditional Chinese herbal drug with strong anti-inflammatory effects. Angiopoietin-1 (Ang-1) plays a vital role in maintaining the stability and integrity of the vascular wall and prevents vascular leakage due to inflammatory mediators. Our previous work found that DCQD protects against pancreatic injury in rats with severe acute pancreatitis (SAP). This study aims to investigate the effects of DCQD on intestinal endothelial damage in both damaged human umbilical vein endothelial cells (HUVECs) and SAP rats. Methods: HUVECs were randomly divided into four groups: control group, TNF-α group, TNF-α plus Ang-1 group (Ang-1 group), and TNF-α plus DCQD group (DCQD group). Cells were incubated for 6 h, 12 h, and 24 h, before collection. The treatment concentration of DCQD was decided based on a Cell Counting Kit-8 (CCK-8) assay. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TEER). Apoptosis was analyzed by flow cytometry. mRNA and protein expression of aquaporin 1 (AQP-1), matrix metalloproteinase 9 (MMP9), and junctional adhesion molecule-C (JAM-C) was evaluated by RT-PCR, immunocytofluorescence, and western blot. Forty male Sprague-Dawley rats were randomized into a control group, SAP group, SAP plus Ang-1 group (Ang-1 group), and SAP plus DCQD group (DCQD group). SAP was induced by intraperitoneal injection of cerulein and lipopolysaccharide (LPS), while the control group received 0.9% saline solution. Evans blue was injected through the penile vein and the rats were then sacrificed 12 h after modeling. Levels of serum amylase, TNF-α, IL-1β, IL-2, and IL-6 were determined by using ELISA. Intestinal tissue was analysed by histology, and capillary permeability in the tissues was evaluated by Evans blue extravasation assay. Protein and mRNA expression of AQP-1, MMP9, and JAM-C were assessed by immunohistofluorescence, western blot, and RT-PCR. Results: DCQD reduced the permeability of HUVEC induced by TNF-α in vitro. Furthermore, DCQD altered the mRNA and protein levels of JAM-C, MMP9, and AQP-1 in HUVECs after TNF-α induction. SAP intestinal injury induced by cerulein combined with lipopolysaccharides was concomitant with increased expression of JAM-C and MMP9, and reduced expression of AQP-1 in intestinal tissue. Pretreatment with DCQD attenuated SAP intestinal injury and lowered the levels of serum amylase, TNF–α, IL-1β, IL-2, and IL-6 effectively. Our study demonstrated that DCQD decreased the expression of JAM-C and MMP9 and increased the expression of AQP-1 both in vitro and in vivo. Conclusion: DCQD can reduce capillary endothelial damage in acute pancreatitis-associated intestinal injury and the mechanism may be associated with the regulation of endothelial barrier function-associated proteins AQP-1, MMP9, and JAM-C.

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Photodynamic Therapy Enhanced the Antitumor Effects of Berberine on HeLa Cells

Open Chemistry ◽

10.1515/chem-2019-0048 ◽

2019 ◽

Vol 17 (1) ◽

pp. 413-421 ◽

Cited By ~ 3

Author(s):

Han-Qing Liu ◽

Ya-Wen An ◽

A-Zhen Hu ◽

Ming-Hua Li ◽

Guang-Hui Cui

Keyword(s):

Photodynamic Therapy ◽

Western Blot ◽

Hela Cells ◽

Mammalian Target Of Rapamycin ◽

Control Group ◽

Antitumor Effects ◽

Underlying Mechanisms ◽

And Control

AbstractIn this study we investigated the antineoplastic effects of Berberine (BBR)-mediated photodynamic therapy (PDT) on HeLa cells and its related mechanisms. The CCK-8 assay and flow cytometry were used to evaluate the proliferation and apoptosis of cells respectively. In addition, changes in protein expression levels were assessed using western blot. BBR at dose of 10 mg/kg was injected intraperitoneally to mice with tumors and PDT treatments were performed 24 hours later. In vivo imaging systems were used to evaluate the fluorescence of BBR. In vitro, PDT significantly enhanced the effects of BBR on inducing cell apoptosis and inhibiting proliferation. The in vivo results showed that the fluorescence intensity in the PDT group was decreased compared with that in the BBR group. Tumor weights and tumor size in the PDT group were less than those in the control group; however, when BBR was applied without PDT, no significant differences were observed between the BBR and control group. The results of western blot showed that PDT enhanced the inhibitory effects of BBR on the mammalian target of rapamycin (mTOR) signaling pathway, that may partly explain the potential underlying mechanisms.

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Μελέτη του ρόλου των συστατικών του μεταβολικού μονοπατιού της HDL στην παθογένεια της οστεοπόρωσης και της οστεοαρθρίτιδας

10.12681/eadd/42084 ◽

2017 ◽

Author(s):

Ελένη Καλυβιώτη

Keyword(s):

Western Blot ◽

High Density Lipoprotein ◽

Real Time ◽

Density Lipoprotein ◽

High Density ◽

Rt Pcr ◽

Knock Out

Εισαγωγή: Πρόσφατα δεδομένα υποδεικνύουν ότι διαταραχές στον λιπιδικό μεταβολισμό επηρεάζουν τη λειτουργία των κυττάρων του οστού με αποτέλεσμα την ανάπτυξη εκφυλιστικών και μεταβολικών νόσων, όπως η οστεοπόρωση (ΟΠ). Η ΟΠ αποτελεί μια κοινή μεταβολική διαταραχή, η οποία χαρακτηρίζεται από μειωμένη οστική μάζα και σταδιακή επιδείνωση της μικροαρχιτεκτονικής δομής του οστίτη ιστού. Επακολούθως, οι ασθενείς που πάσχουν από τη νόσο διατρέχουν αυξημένο κίνδυνο καταγμάτων. Τα τελευταία χρόνια αρκετές μελέτες ανέδειξαν την ύπαρξη μιας ισχυρής σύνδεσης μεταξύ του οστικού και λιπιδικού μεταβολισμού. Επιπλέον, τόσο η ομάδα μας όσο και άλλοι ερευνητές έχουν δείξει ότι διαταραχές στο μεταβολισμό της υψηλής πυκνότητας λιποπρωτεΐνης (High Density Lipoprotein-HDL) ενέχονται στην εμφάνιση μεταβολικών νοσημάτων, όπως είναι η Οστεοαρθρίτιδα (ΟΑ). Γνωρίζοντας τον πολύ σημαντικό ρόλο της HDL στον μεταβολισμό των λιπιδίων στο πλάσμα και στους ιστούς και οδηγούμενοι από τα ευρήματα της δικής μας ομάδας αλλά και άλλων ερευνητών, στην παρούσα διδακτορική διατριβή, μελετήσαμε το ρόλο της απολιποπρωτεΐνης Α1 (APOA1), βασικού συστατικού του μονοπατιού βιοσύνθεσης της HDL, στη ρύθμιση της λειτουργίας των κυττάρων του οστού και στην παθογένεια της οστεοπόρωσης, σε πειραματικά μοντέλα ποντικών.Μεθοδολογία: Για τον λόγο αυτό, χρησιμοποιήσαμε μοντέλα ποντικών με έλλειψη του γονιδίου της ApoA1 (ApoΑ1-/-) και αγρίου τύπου (C57BL/6) (ομάδα ελέγχου). Δύο και επτά ημέρες προ της ευθανασίας πραγματοποιήθηκε ενδοπεριτοναϊκή ένεση καλσεΐνης. Πριν την ευθανασία λήφθηκε πλάσμα αίματος και ακολούθως τα ζώα θυσιάστηκαν. Από τα πειραματόζωα απομονώθηκαν χειρουργικά το μηριαίο οστό και οι οσφυϊκοί σπόνδυλοι, τα οποία χρησιμοποιήθηκαν για ιστολογικές, ιστομορφομετρικές, εμβιομηχανικές και in vitro αναλύσεις. Με τη χρήση microCT scanner εκτιμήθηκε η ποιότητα και η ποσότητα του σπογγώδους και του φλοιώδους οστού (στατική ιστομορφομετρία). Με τη χρώση TRAP και τη χρώση καλσεΐνης (δυναμική ιστομορφομετρία) ελέγχθηκε η οστική αποδόμηση και ο ρυθμός σύνθεσης νεοσχηματιζόμενου οστού, αντίστοιχα. Με τη χρήση φασματοσκοπίας Raman, αξιολογήθηκε η κατάσταση του δικτύου κολλαγόνου των μηριαίων οστών, ενώ αναλύθηκαν και οι εμβιομηχανικές ιδιότητες του οστού (αντοχή σε θραύση, ελαστικότητα κλπ) με τη χρήση του 3-point bending, στις δύο ομάδες πειραματόζωων. Επιπλέον, μεσεγχυματικά κύτταρα (MSC) του μυελού των οστών απομονώθηκαν από το μηριαίο οστό των πειραματόζωων και καλλιεργήθηκαν με σκοπό την εκτίμηση της έκφρασης, τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο mRNA, μορίων που εμπλέκονται στην οστεοβλαστική (RUNX2, OSX, COL1A1) και στην οστεοκλαστική λειτουργία (OPG, RANKL, OPG/RANKL, RANK, TRAP, cathepsin Κ) καθώς και στην λιπογένεση (PPARγ, CEBPA). Επίσης, μελετήθηκε η έκφραση των γονιδίων που εμπλέκονται στο μεταβολικό μονοπάτι της χοληστερόλης (ApoA2, Ggps, Hmgcr, Hdlbp, Fdps1, Cpt1a, Scarb1), αλλά και των γονιδίων που σχετίζονται με τη διατήρηση και την κινητοποίηση των μεσεγχυματικών κυττάρων (Ptprc, Sca-1, Cxcl12, Cxcr4, Anxa2). Για την εκτίμηση των επιπέδων έκφρασης των πρωτεϊνών, χρησιμοποιήσαμε τις μοριακές τεχνικές Western Blot και κυτταρομετρία ροής ενώ για την αξιολόγηση των επιπέδων έκφρασης του mRNA χρησιμοποιήσαμε τη real time RT-PCR. Αποτελέσματα: Η ανάλυση των αποτελεσμάτων έδειξε στατιστικά σημαντική μείωση της οστικής μάζας στα ΑpoΑ1-/- συγκριτικά με τα ποντίκια αγρίου τύπου. Η έκφρασης της ACTH ήταν όμοια και στις δυο ομάδες ζώων, ενώ τα επίπεδα mRNA του Scarb1 ήταν σημαντικά αυξημένα στα ΑpoΑ1-/- ποντίκια. Τα αποτελέσματα της στατικής και δυναμικής ιστομορφομετρίας έδειξαν ότι η μειωμένη οστική μάζα στα ΑpoΑ1-/- ποντίκια αντικατοπτρίζει τη μειωμένη οστική σύνθεση. Η ανάλυση της βιοχημικής σύνθεσης και των εμβιομηχανικών ιδιοτήτων των μηριαίων οστών, έδειξε ότι αυτές οι παράμετροι ήταν διαταραγμένες στα ΑpoΑ1-/- συγκριτικά με τα αγρίου τύπου ποντίκια. Επιπρόσθετα, η επαγωγή διαφοροποίησης των MSC, κάτω από τις ίδιες συνθήκες, από τα ΑpoΑ1-/- ποντίκια φαίνεται να εμφανίζει μειωμένη παραγωγή οστεοβλαστών και αυξημένη παραγωγή λιποβλαστών σε αντίθεση με τα MSC από αγρίου τύπου ποντίκια. Αυτό φανερώνει την ύπαρξη μιας μετατόπισης στην ισορροπία της διαδικασίας διαφοροποίησης των MSC, η οποία ευνοεί την λιπογένεση. Η παρατήρηση αυτή έρχεται σε συμφωνία με την αρχική ιστολογική παρατήρηση της ύπαρξης αυξημένων λιποκυττάρων στο μυελό των οστών των ΑpoΑ1-/- ποντικιών σε σχέση με τα ποντίκια ελέγχου. Αντίθετα με τα ευρήματα για τη δυσλειτουργία των οστεοβλαστών, η οστεοκλαστική διαφοροποίηση in vitro και η μέτρηση της οστεοκλαστικής επιφάνειας in vivo εμφανίζονται ανεπηρέαστες. Επιπρόσθετα, σε ολικά κύτταρα του μυελού των οστών, σε συμφωνία με τα αυξημένα λιποκύτταρα και τα δεσμευμένα προς λιποβλάστες αρχέγονα MSC, τα επίπεδα έκφρασης του PPARγ είναι στατιστικώς σημαντικά αυξημένα στα ΑpoΑ1-/- ποντίκια. Ακόμα, στο μυελό των οστών στα apoΑ1-/- ποντίκια η έκφραση της κυτταροκίνης CXCL12 είναι μειωμένη, ενώ του CXCR4 αυξημένη, στοιχεία που συμφωνούν με μειωμένη σηματοδότηση στο μονοπάτι που εμπλέκεται στη διατήρηση των MSC (homing) και την οστεοβλαστική διαφοροποίηση. Επιπλέον, στα ApoΑ1-/- ποντίκια παρατηρείται σημαντική μείωση στους παράγοντες που σχετίζονται με τη διαφοροποίηση και λειτουργία των οστεοβλαστών, όπως είναι ο RUNX2, ο OSX και ο COL1A1. Ενώ, επίσης τα knock out ποντίκια εμφανίζουν αυξημένη έκφραση του παράγοντα CEPBa, υποδηλώνοντας την ύπαρξη πολύπλοκων αλλαγών στην ανάπτυξη και διαφοροποίηση που απαιτούν περαιτέρω διερεύνηση.Συμπεράσματα: Η έλλειψη της APOA1 οδηγεί σε ελάττωση της οστικής μάζας, κυρίως λόγω μειωμένης λειτουργίας των οστεοβλαστών. Επιπλέον, η έλλειψη της APOA1 οδηγεί σε αύξηση της λιπογένεσης και μείωση της οστεοβλαστογένεσης, η οποία με τη σειρά της οδηγεί σε ελαττωματική σύνθεση οστού, ενώ παράλληλα η δράση των οστεοκλαστών παραμένει ανεπηρέαστη. Τα αποτελέσματα μας, για πρώτη φορά, αναδεικνύουν ότι η APOA1 ρυθμίζει τη λειτουργία των οστεοβλαστών και των λιποβλαστών και αυξάνει την πιθανότητα ότι η APOA1 μπορεί να δράσει ως πιθανός θεραπευτικός στόχος για τη θεραπεία της οστεοπόρωσης και άλλων μεταβολικών νοσημάτων.

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Overcoming Drug Resistance In Acute Lymphoblastic Leukemia by Inhibition of CBP/γ-Catenin

Blood ◽

10.1182/blood.v116.21.3264.3264 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3264-3264

Author(s):

Enzi Jiang ◽

Eugene Park ◽

Cu Nguyen ◽

James Yoon ◽

Yao-Te Hsieh ◽

...

Keyword(s):

Colon Cancer ◽

Western Blot ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Saline Control ◽

Control Group ◽

Apoptosis Protein

Abstract Abstract 3264 Survivin, an inhibitor of apoptosis protein (IAP) family, has been associated with poor prognosis in cancer including leukemia. Survivin can be downregulated in colon cancer cells by inhibition of the β-catenin/Creb-binding protein (CBP) interaction using ICG-001, a small molecule specific inhibitor of the β-catenin/CBP interaction. We have shown previously that combined ICG-001 and chemotherapy can downregulate Survivin and sensitize ALL cells to chemotherapy in vitro and in a pilot study in vivo. In this study, we determine the CBP interaction with ICG-001 in primary ALL cells and preclinically evaluate ICG-001 in vitro and in vivo as an adjuvant against primary ALL and. For this purpose, primary ALL cells were co-cultured with OP9 cells and treated for 4 days with ICG-001 (10mM, 20mM) or DMSO as vehicle control. Mean viability (trypan blue exclusion) of cells treated with ICG-001 was significantly lower (ICG-001 10mM: 75.12% ± 3.15%; 20mM: 41.18%± 7.88%) compared to cells treated with DMSO (84.99% ± 0.42%) (% cell viability relative to initial control) (p=0.03). Real time RT-PCR showed ICG-001 dose-dependent downregulation of Survivin in ALL compared to control (ICG10mM vs. control: p=0.0037 and 20mM vs. control: p=0.0031). Immunoblotting demonstrated reduction of Survivin after ICG-001 treatment. Primary ALL cells incubated with a combination of VDL (Vincristine, Dexamethasone and L-Asparaginase) and ICG-001 showed decreased viability (28.7%± 4.9%) versus VDL only (79.3%± 13.6%) (p=0.014) determined by MTT assay. To elucidate if ICG-001 interacts with β-catenin/CBP as shown previously in colon cancer, we analyzed ten primary pre-B ALL cells and found significantly greater γ-catenin and Survivin expression versus normal pre-B-Cells. β-catenin was absent or in some cases expressed only weakly. Expression of v-catenin and b-catenin in ALL xenograft cells were detected by Western blot. One primary ALL was selected and incubated with γ-catenin and β-catenin siRNA for 48hrs, followed by 6hrs incubation with Wnt3a. Wnt3a induced both of γ-catenin and β-catenin expression. Survivin was reduced by γ-catenin siRNA but not β-catenin siRNA treatment. Addition of Wnt3a partially recovered the decrease of Survivin. In addition, Survivin was knocked down in primary ALL using shRNA and non-silencing shRNA control or ICG-001 (2uM) and DMSO control. Western blot analysis showed that survivin shRNA or ICG-001 treatment lead to downregulation of Survivin and γ-catenin. Using a ChIP assay we could demonstrate occupancy of TCF4 and CBP association at the Survivin promoter, which was not altered by ICG-001 in primary ALL. Moreover, ICG-001 treatment of primary ALL cells prevents CBP but not p300 occupancy. For further preclinical in vivo evaluation of ICG-001, one Philadelphia chromosome positive ALLs (Ph+) and two Ph− primary ALL were injected into sublethally irradiated NOD/SCID IL2Rγ−/-mice and treated with ICG-001 (50mg or 100mg/kg/day per subcutaneous miniosmotic pump) with or without chemotherapy including VDL for Ph− ALL (per intraperitoneal injections) or Nilotinib for Ph+ ALL (per os). For analysis we pooled the survival of all three primary leukemias. The saline control group (n=10) (MST= 55.5.days) and the ICG-001 only groups (n=3) (MST=61 days) died rapidly. The group treated with chemotherapy (n=13) had a median survival time (MST) of 85 days. In marked contrast, the group treated with the combined chemotherapy+ICG-001 (n=15) lived significantly longer (MST=100) (p<0.05). Taken together, our data shows that Survivin transcription can be mediated by γ-catenin in primary ALL and that targeting CBP/γ-catenin by using ICG-001 ALL can sensitize ALL cells to chemotherapy in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

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Intermittent Hypoxia Composite Abnormal Glucose Metabolism-Mediated Atherosclerosis In Vitro and In Vivo: The Role of SREBP-1

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/4862760 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Linqin Ma ◽

Jingchun Zhang ◽

Yu Qiao ◽

Xinli Sun ◽

Ting Mao ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucose Metabolism ◽

Intermittent Hypoxia ◽

Control Group ◽

Abnormal Glucose Metabolism ◽

Mrna And Protein Expression ◽

Oil Red O Staining ◽

Oil Red O

Objective. The aim of this study was to establish a 3T3-L1 adipocyte model and ApoE−/− mouse model of intermittent hypoxia (IH) composite abnormal glucose metabolism (AGM) in vitro and in vivo and explore their synergistic damage effect leading to atherosclerosis (AS) and the influence of SREBP-1 signaling molecule-related mechanisms. Methods. Mature 3T3-L1 adipocytes were cultured with complete culture medium containing DEX 1×106 mol/L for 96 h to establish an AGM-3T3-L1 adipocyte model. Then, AGM-3T3-L1 adipocytes were treated with IH for 0 cycles, 2 cycles, 4 cycles, 8 cycles, 16 cycles, and 32 cycles and sustained hypoxia (SH). ApoE−/− mice were treated with high-fat diet and injection of STZ solution to establish an AGM-ApoE−/− mouse model. A total of 16 AGM-ApoE−/− mice were randomly and averagely divided into the normoxic control group (NC) and model group (CIH). AGM-ApoE−/− mice of the CIH group were treated with IH, which meant that the oxygen concentration fell to 10±0.5% in the first 90 seconds of one cycle and then increased to 21±0.5% in the later 90 seconds, continuous for eight hours per day (09 : 00-17 : 00) with a total of eight weeks. Eight C57BL/6J mice were used as the blank control group (Con) which was fed with conventional diet. qPCR and Western blotting were used to detect the expression level of SREBP-1c, FAS, and IRS-1. Oil Red O staining was used to compare the plaque of the aorta among each mouse group. Results. As a result, within 32 cycles of IH, mRNA and protein expression levels of SREBP-1c and FAS in AGM-3T3-L1 adipocytes were elevated with the increase in IH cycles; the mRNA expression of IRS-1 was decreased after IH 32 cycles and lower than that of the SH group. For the study in vivo, Oil Red O staining showed a more obvious AS aortic plaque in the CIH group. After CIH treatment of 4 w and 8 w, fasting blood glucose (FBG) of the NC group and CIH group was higher than that of the Con group, and the insulin level of the CIH group was higher than that of the Con group after IH treatment of 8 w. The expressions of the IRS-1 mRNA level in the aorta, skeletal muscle, and liver of the CIH group were lower than those in the Con group. The mRNA and protein expression of SREBP-1c and its downstream molecule FAS in the aorta, skeletal muscle, and liver significantly enhanced in the CIH group in contrast with those in the Con group. Conclusion. The CIH composite AGM could promote the progress of AS, which might be related to the modulation of the expression of SREBP-1-related molecular pathways.

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Human amniotic stem cells-derived exosmal miR-181a-5p and miR-199a inhibit melanogenesis and promote melanosome degradation in skin hyperpigmentation, respectively

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02570-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiao-Yu Wang ◽

Xiao-Hui Guan ◽

Zhen-Ping Yu ◽

Jie Wu ◽

Qi-Ming Huang ◽

...

Keyword(s):

Stem Cells ◽

Western Blot ◽

Melanin Synthesis ◽

Skin Substitutes ◽

Rt Pcr ◽

B16f10 Cells ◽

Skin Hyperpigmentation ◽

Underlying Mechanisms

Abstract Background Hyperpigmentation of skin is caused by an imbalance between the melanosome/melanin synthesis in melanocytes and the melanosome/melanin degradation in keratinocytes. Although studies showed that stem cells play a role in hypopigmentation, the underlying mechanisms are far not elucidated. Human amniotic stem cells (hASCs) including human amniotic mesenchymal stem cells (hAMSCs) and human amniotic epithelial stem cells (hAESCs) were considered to be a promising cell source for stem cells-based therapy of many diseases clinically due to their pluripotent potential, no tumorigenesis and immunogenicity, no ethical issues, and potent paracrine effects. Here, we reported that both hASCs and their conditional medium (CM) had a potent anti-hyperpigmentation in skin in vivo and in vitro. Methods hAESCs and hAMSCs were identified by RT-PCR, flow cytometric analysis and immunofluorescence. Effects of hASCs and hASC-CM on pigmentation were evaluated in B16F10 cells stimulated with α-melanocyte-stimulating hormone (α-MSH), and mouse ears or human skin substitutes treated with ultraviolet radiation B (UVB). Expressions of the key proteins related with melanogenesis and autophagic flux were detected by western blot in B16F10 cells for further exploring the effects and the underlying mechanisms of hAESC-CM and hAMSC-CM on melanogenesis and melanosome degradation. The hAMSCs exosomes-derived miRNAs were determined by sequencing. RT-PCR, western blot, melanin content analysis and luciferase activity assay were used to determine the hypopigmentation of miR-181a-5p and miR-199a. Results In our study, we observed that both hASCs and their CM significantly alleviated the α-MSH in B16F10 cells or UVB-induced hyperpigmentation in mouse ears or human skin substitutes by suppressing melanin synthesis and promoting melanosome degradation in vivo and in vitro. Furthermore, we demonstrated that miR-181a-5p and miR-199a derived from hASCs exosomes remarkably inhibited melanogenesis by suppressing MITF (microphthalmia-associated transcription factor) which is a master regulator for governing melanogenesis and promoting melanosome degradation through activating autophagy, respectively. Conclusions Our studies provided strong evidence that the conditional medium and exosomes derived from hAMSCs inhibit skin hyperpigmentation by suppressing melanogenesis and promoting melanosome degradation, indicating that the hASCs exosomes or their released microRNAs might be as reagents for cell-free therapy in hyperpigmented disorders clinically.

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WISP2/CCN5 gene knockdown in vitro and in vivo exhibits proliferation promotion of breast cancer through targeting Skp2 and p27Kip1

10.1101/2020.01.29.924688 ◽

2020 ◽

Author(s):

Yan Lv ◽

Chang Zhang ◽

Xiao Jiang Li ◽

Shan Gao ◽

Xu Zheng ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Gene Knockdown ◽

Control Group ◽

Rt Pcr ◽

Protein Levels ◽

Mcf 7

AbstractBackgroundEmerging evidence has demonstrated that WISP2/CCN5 is critically involved in tumorigenesis. However, the function of WISP2/CCN5 in breast cancer carcinogenesis is largely unclear.Methodswe aim to explore the effects and potential mechanisms of WISP2/CCN5 on proliferation of breast cancer cells and carcinogenesis of breast cancer xenograft. Lentivirus vector with WISP2/CCN5shRNA was transfected into MCF-7, and breast cancer cells and xenograft were conducted. Effect of WISP2/CCN5 on growth and carcinogenesis of breast cancer cells and xenografts was evaluated by MTT assay and tumor volume. The relationship between WISP2/CCN5, Skp2 and p27Kip1 was detected in vitro and in vivo by RT-PCR at mRNA level and Western blotting at protein level.ResultsThe result of MTT assay indicated that MCF-7 cell growth viability in WISP2/CCN5 gene knockdown group was significantly higher than negative vector group(P<0.05) or control group (P<0.05). It suggested that knockdown of WISP2/CCN5 gene by shRNA lentivirus plasmid promoted proliferation of MCF-7 cells. The growth curves of breast cancer xenograft showed that xenografts in WISP2/CCN5 knockdown group grew more quickly than negative vector group(P< 0.05) or control group (P< 0.05). Subsequently, the results of RT-PCR and Western blotting revealed that WISP2/CCN5 gene knockdown led to increased Skp2 and decreased p27Kip1 at mRNA and protein levels. WISP2/CCN5 exerts its inhibition on proliferation of MCF-7 cell line and suppressive functions on growth of breast carcinoma via regulation of Skp2 and p27Kip1at mRNA and protein levels. However, WISP2/CCN5 gene knockdown resulted in loss of inhibition effect on MCF-7 and breast cancer.ConclusionsOur findings suggest that WISP2/CCN5 could be a useful therapeutic strategy for the treatment of breast cancer through targeting Skp2 and p27Kip1.

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Expression and Significance of protein 4.1R in rat models with diaphragmatic weakness

10.21203/rs.2.17943/v1 ◽

2019 ◽

Author(s):

Yanhong Li ◽

Yongqiang Li ◽

Xinying Ji ◽

Huimin Li ◽

Dongdong Wu ◽

...

Keyword(s):

Western Blot ◽

Control Group ◽

Cell Models ◽

Rat Models ◽

L6 Cells ◽

Protein 4.1R ◽

Diaphragmatic Weakness ◽

Hematoxylin Eosin

Abstract Abstract Background To explore the significance of protein 4.1R expression in the diaphragmatic weakness animal and cell models and its preliminary mechanism.Methods Rats were intraperitoneally injected with Lipopolysaccharide (LPS) to construct diaphragmatic weakness models. Histopathology of diaphragmatic tissues was detected by Hematoxylin eosin(HE) staining. L6 cells were induced by LPS to establish the myasthenic cell models and transfected with 4.1R-siRNA to konckdown 4.1R. Immunohistochemistry was performed to detect the expression of acetylcholine receptor(AchR) and 4.1R; The expression of desmin and myosin in tissues and cells were detected by western blot.Results LPS could induce the diaphragmatic weakness of rats. The expression of AchR in diaphragmatic weakness tissues was lower, while that of desmin was higher than that in the control group. 4.1R was up-regulated in the diaphragmatic weakness models, and related to the severity. After knockdown of 4.1R in LPS induced L6 cells, the expression of AchR was up-regulated significantly. But there was not difference of contractile proteins.Conclusions Protein 4.1R was upregulated in diaphragmatic weakness model in vivo and in vitro and might be involved into the occurrence of myasthenia gravis by negatively regulating the expression of AchR.

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