Amlodipine accelerates bone healing in a stable closed femoral fracture model in mice

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.

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Differential Role of Rapamycin in Epidermis-Induced IL-15-IGF-1 Secretion via Activation of Akt/mTORC2

Cellular Physiology and Biochemistry ◽

10.1159/000479443 ◽

2017 ◽

Vol 42 (5) ◽

pp. 1755-1768 ◽

Cited By ~ 4

Author(s):

Yang Bai ◽

Rui Xu ◽

Xueyuan Zhang ◽

Xiaorong Zhang ◽

Xiaohong Hu ◽

...

Keyword(s):

Wound Healing ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Low Dose ◽

Underlying Mechanism ◽

High Dose ◽

Mtorc1 Pathway ◽

Hematoxylin And Eosin

Backgroud/Aims: The effects of rapamycin (RPM) on wound healing have been previously studied. However, reciprocal contradictory data have been reported, and the underlying mechanism remains unclear. This study aims to uncover differential role of RPM in regulation of wound healing and explore the possible mechanism. Methods: C57BL/6J mice and epidermal cells were treated with different doses of RPM. The wound re-epithelialization was observed by hematoxylin and eosin (HE) staining. The expression of IL-15 and IGF-1 were detected by immunohistochemistry and quantitative real-time PCR. Epidermal cell survival was determined by CCK-8 assays. Moreover, the mTORC1 and mTORC2 pathway were examined by western blot analysis. Results: This study showed that differential doses of RPM could lead to separate consequences in epidermis. Histological analyses showed that low-dose RPM promoted wound healing, and enhanced the expression of IL-15 and IGF-1. Furthermore, western blot analysis showed that the effect of low-dose RPM in epidermis were not through mTORC1 pathway. Instead, activation of the Akt/mTORC2 pathway was involved in low-dose RPM-induced IL-15 and IGF-1 production in epidermis, while high-dose RPM inhibited the expression of IL-15 and IGF-1 and the activity of mTORC1 and mTORC2 pathway. Conclusion: This study for the first time demonstrated that RPM-mediated wound healing was dose-dependent.

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Sennoside A induces GLP-1 secretion through activation of the ERK1/2 pathway in L-cells

10.21203/rs.2.22093/v1 ◽

2020 ◽

Author(s):

Li Ma ◽

Xinyu Cao ◽

Xiaotong Ye ◽

Jianping Ye ◽

Yongning Sun

Keyword(s):

Type 2 Diabetes ◽

Insulin Secretion ◽

Blood Glucose ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Oral Glucose ◽

High Dose ◽

L Cells

Abstract Background Glucagon-like peptide-1 (GLP-1) is secreted from the intestinal L-cells to stimulate insulin secretion in the control of blood glucose. Sennoside A (SA), derived from Rhubarb extract of traditional Chinese medicine, is often used to treat constipation and lose weight. Our previous study suggests that SA can increase the plasma GLP-1 level in a mouse model of type 2 diabetes. However, the mechanism of SA activity remains unknown. This issue was addressed in this study. Methods C57bl/6 mice were divided randomly into four groups at n = 12. Group one was used as a control group without drug treatment. The other three groups were treated with (SA) at three dosages: a low dose (15 mg/kg/day), medium dose (30mg/kg/day) or high dose (45 mg/kg/day). SA was delivered into the mice through drinking water. Bodyweight was monitored. After treatment, blood glucose was assayed by OGTT. Plasma GLP-1 and insulin were determined at 15 mins of oral glucose challenge. Colon tissues were collected for mRNA or western blot analysis. Immunofluorescence staining assays was used to evaluate the number of β-cells and L-cells. NCI-H716 cells were employed to investigate the mechanism of SA-induced GLP-1 secretion, and the cells were subjected to western blot analysis. In the study of extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, NCI-H716 cells were pretreated with ERK1/2 inhibitors (PD98059, 50 μM) for 30 min in the presence of SA (100 μM). Results In the current study, SA can reduce body weight during 5 weeks of weight monitoring and improve OGTT in C57BL/6 mice on the Chow diet. Furthermore, plasma GLP-1 was significantly elevated in the mouse treated by SA at the dosage of 45 mg/kg/day. The SA activity was supported by improving glucose-induced insulin secretion. Meanwhile, increased expression of EKR1/2 and prohormone convertase 1/3 (PC1/3) proteins was observed in the large intestine of SA-treated mice. The number of L-cells was not altered in each group. In the NCI-H716 cells, GLP-1 secretion was induced by SA with activation of the ERK1/2 pathway and elevation of PC1/3 protein. The SA effect was blocked by the ERK1/2 inhibitor. These data suggest that SA induced GLP-1 secretion in L-cells through activation of the ERK1/2 pathway in the mouse intestine. Conclusion Our study provides direct evidence that SA interacts with L cells for GLP-1 secretion. The data suggest that the SA effect is dependent on the ERK1/2 signaling pathway. Therefore, SA is a new drug candidate for the treatment of type 2 diabetes by induction of GLP-1 secretion.

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IFN-CSP Inhibiting Hepatitis B Virus in HepG2.2.15 Cells Involves JAK-STAT Signal Pathway

BioMed Research International ◽

10.1155/2015/959684 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Xuemei Lu ◽

Jie Wang ◽

Xiaobao Jin ◽

Yanting Huang ◽

Wenting Zeng ◽

...

Keyword(s):

Hepatitis B ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Molecular Mechanisms ◽

Core Protein ◽

Hbv Dna ◽

Enzyme Linked Immunosorbent Assay ◽

High Dose ◽

Oligoadenylate Synthetase

Frequent and high-dose administration of interferon to patients with viral hepatitis results in various side effects. In our previous study, a novel liver-targeting interferon (IFN-CSP) combiningPlasmodiumregion I peptide with IFNα2b was successfully designed and expressed in theEscherichia coliexpression systems. This targeting would target the IFNα2b specifically to the liver, thus reducing the adverse events. In the present study, we further investigated the anti-HBV effects and molecular mechanisms of recombinant IFN-CSP in HepG2.2.15 cell line. Hepatitis B surface antigen (HBsAg) and HBe antigen (HBeAg) in the culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). HBV-DNA was measured by real-time quantitative PCR. HBV core protein was assayed by immunofluorescent and western blot analysis. The expressions of signal transducers and transactivator 1 (STAT1), STAT2, IFN regulatory factor 9 (IRF-9), and 2′-5′-oligoadenylate synthetase 1 (OAS1) were investigated by the reverse transcription PCR and western blot analysis. Results indicate IFN-CSP efficiently inhibited HBsAg and HBeAg secretion, HBV-DNA replication, and HBV core protein expression in HepG2.2.15 cells. The anti-HBV mechanisms involve activation of JAK-STAT signaling and increase of the anti-HBV protein OAS expression. IFN-CSP could be a good substitute for IFNα2b for anti-HBV treatment.

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Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

Journal of Neurosurgery ◽

10.3171/2010.7.gks10972 ◽

2010 ◽

Vol 113 (Special_Supplement) ◽

pp. 228-235 ◽

Cited By ~ 4

Author(s):

Qiang Jia ◽

Yanhe Li ◽

Desheng Xu ◽

Zhenjiang Li ◽

Zhiyuan Zhang ◽

...

Keyword(s):

Western Blot ◽

Gamma Knife ◽

Blot Analysis ◽

Western Blot Analysis ◽

Glioma Cells ◽

C6 Glioma ◽

C6 Glioma Cells ◽

C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

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Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200731174216 ◽

2020 ◽

Vol 20 (23) ◽

pp. 2070-2079

Author(s):

Srimadhavi Ravi ◽

Sugata Barui ◽

Sivapriya Kirubakaran ◽

Parul Duhan ◽

Kaushik Bhowmik

Keyword(s):

Western Blot ◽

Cancer Cells ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cancer Cell Lines ◽

Atm Kinase ◽

Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

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The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200227092623 ◽

2020 ◽

Vol 20 (9) ◽

pp. 1147-1156

Author(s):

Hanrui Li ◽

GeTao Du ◽

Lu Yang ◽

Liaojun Pang ◽

Yonghua Zhan

Keyword(s):

Hepatocellular Carcinoma ◽

Western Blot ◽

Blot Analysis ◽

Tumor Size ◽

Hepg2 Cells ◽

Western Blot Analysis ◽

Bioluminescence Imaging ◽

Antitumor Effects ◽

In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

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Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽

10.3390/nu11112794 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2794 ◽

Cited By ~ 8

Author(s):

Cao ◽

Chen ◽

Ren ◽

Zhang ◽

Tan ◽

...

Keyword(s):

Western Blot ◽

Signaling Pathway ◽

Inflammatory Cytokines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Inflammatory Responses ◽

Raw264.7 Macrophages ◽

Tnf Α ◽

Autophagy Inhibition ◽

Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

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Protocol for determining protein cysteine thiol redox status using western blot analysis

STAR Protocols ◽

10.1016/j.xpro.2021.100566 ◽

2021 ◽

Vol 2 (2) ◽

pp. 100566

Author(s):

Bikram Datt Pant ◽

Sunhee Oh ◽

Kirankumar S. Mysore

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Redox Status ◽

Thiol Redox

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Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

Human & Experimental Toxicology ◽

10.1177/09603271211006168 ◽

2021 ◽

pp. 096032712110061

Author(s):

D Cao ◽

L Chu ◽

Z Xu ◽

J Gong ◽

R Deng ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Migration ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Cell Migration And Invasion ◽

Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

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OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

Disease Markers ◽

10.1155/2013/917898 ◽

2013 ◽

Vol 34 (4) ◽

pp. 257-267 ◽

Cited By ~ 15

Author(s):

Alessandro Bressan ◽

Francesca Bozzo ◽

Carlo Alberto Maggi ◽

Monica Binaschi

Keyword(s):

Ovarian Cancer ◽

Monoclonal Antibodies ◽

Western Blot ◽

Tandem Repeat ◽

Blot Analysis ◽

Western Blot Analysis ◽

Human Cancer ◽

Ovarian Cancer Cells ◽

Repeat Region ◽

Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

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