Efficacy of Sanqi (Radix Notoginseng) in treating cerebral hemorrhage in rats with traumatic brain injury.

OBJECTIVE: To evaluate the protective efficacy of Sanqi (Radix Notoginseng) on cerebral hemorrhage in a rat model of traumatic brain injury (TBI) by investigating plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA), nuclear factor-κB (NF-κB, p-p65), nitric oxide (NO), endothelin (ET), cluster differentiation (CD61CD62), and coagulation. METHODS: The free-fall method was used to create a rat model of TBI. Forty-eight rats were randomly divided into six groups: the blank group, sham group, model group, low-dose Sanqi (Radix Notoginseng) group, middle-dose Sanqi (Radix Notoginseng) group, and high-dose Sanqi (Radix Notoginseng) group. At 24 h after the model was created, we investigated brain MRI, brain tissue morphology using HE staining, flow cytometry, and immunohistochemical changes. RESULTS: Cerebral hemorrhage was aggravated in TBI rats (observed in brain specimens, brain MRI, and brain tissue HE). Cerebral immunohistochemistry results demonstrated that the expression of t-PA, PAI-1 and p-p65 increased significantly in TBI rats, while t-PA/PAI-1 had a significant decrease. In addition, CD61CD62, D2D, and ET were significantly increased in TBI rats, and PT and APTT were significantly prolonged; in contrast, NO was significantly decreased. Sanqi (Radix Notoginseng) decreased cerebral hemorrhage in TBI rats (observed in brain MRI and brain tissue HE), and increased t-PA/PAI-1, CD61CD62 significantly. It also significantly decreased the expression of t-PA, PAI-1, and p-p65 in brain immunohistochemistry and significantly decreased PT, APTT, D2D, and ET. However, there were no differences in NO between the model group and the Sanqi (Radix Notoginseng) group. CONCLUSION: Sanqi (Radix Notoginseng) can decrease the expression of p-p65, increase t-PA/PAI-1, and stem traumatic intracranial hemorrhage in a TBI rat model.

Molecular Mechanisms at the Basis of Pharmaceutical Grade Triticum vulgare Extract Efficacy in Prompting Keratinocytes Healing.

BACKGROUND: It has been shown that many plant- or microbial-derived oligos and polysaccharides may prompt tissue repair. Among the different extracts that have been studied, the aqueous one of Triticum vulgare (TVE) that was obtained from a whole germinated plant has been proven to have different biological properties that are useful in the process of wound healing. Nevertheless, with the long tradition of its use in pharmaceutical cream and ointments, especially in Italy, a new protocol was recently proposed (and patented) to improve the extraction process. METHODS: In a simplified in vitro model, human keratinocyte monolayers were scratched and used to run time lapse experiments by using time lapse video microscopy (TLVM) to quantify reparation rate while considering a dose-response effect. Contemporarily, the molecular mechanisms that are involved in tissue repair were studied. In fact, key biomarkers that are involved in remodeling, such as MMP-2 and MMP-9, and in matrix structure assembly, such as collagen I, elastin, integrin αV and aquaporin 3, were evaluated with gene expression analyses (RT-PCR) and protein quantification in western blotting. RESULTS: All TVE doses tested on the HaCat-supported cell proliferation. TVE also prompted cell migration in respect to the control, correctly modulating the timing of metalloproteases expression toward a consistent and well-assessed matrix remodeling. Furthermore, TVE treatments upregulated and positively modulated the expression of the analyzed biomarkers, thus resulting in a better remodeling of dermal tissue during healing. CONCLUSIONS: The in vitro results on the beneficial effects of TVE on tissue elasticity and regeneration may support a better understanding of the action mechanism of TVE as active principles in pharmaceutical preparation in wound treatment.

Discovery of an Orally Bioavailable Pan αv Integrin Inhibitor for Idiopathic Pulmonary Fibrosis.

The heterodimeric transmembrane αv integrin receptors have recently emerged as potential targets for the treatment of idiopathic pulmonary fibrosis. Herein, we describe how subtle modifications of the central aromatic ring of a series of phenylbutyrate-based antagonists of the vitronectin receptors αvß3 and αvß5 significantly change the biological activities against αvß6 and αvß8. This resulted in the discovery of a pan αv antagonist (compound 39, 4-40 nM for the integrin receptors named above) possessing excellent oral pharmacokinetic properties in rats (with a clearance of 7.6 mL/(min kg) and a bioavailability of 97%).

Ganoderma immunomodulatory protein and chidamide down-regulate integrin-related signaling pathway result in migration inhibition and apoptosis induction.

BACKGROUND: In terms of melanoma, recent advances have been made in target therapies and immune checkpoint inhibitors, but durable remission is rare. Ganoderma immunomodulatory proteins (GMI) induce a cytotoxic effect in cancer cells via autophagy. However, the role of GMI in melanoma is not clear. PURPOSE: The aims of this study are to investigate the inhibiting effects of GMI combined with chidamide on survival and metastases of melanoma cells via integrin-related signaling pathway and to propose strategies for combining GMI and chidamide using animal model. METHODS: Cell viability was measured by cell CCK-8. The activities of apoptosis- and migration-related proteins were detected on Western blot. Flow cytometry was used to analyze cell cycle distribution and sub-G1 fraction in treated melanoma cells. To evaluate the activity of combination GMI and chidamide treatment, an in vivo anti-tumor metastasis study was performed. RESULTS: GMI combined with chidamide additively induced apoptosis. GMI inhibited the expressions of Integrin α5, αV, ß1, and ß3. The level of p-FAK was inhibited by GMI. Combination treatment of GMI and chidamide decreased survivin and increased cleaved caspase-7 and LC3 II/I. Integrin-αV overexpression activated p-FAK pathways in A375.S2 cells. GMI significantly inhibited cell growth and migration of A375.S2 cells on wound healing assay. In vivo, GMI combined with chidamide suppressed distal tumor metastasis. CONCLUSION: GMI inhibits the migration and growth of melanoma cells via integrin-related signaling pathway. GMI and chidamide induces apoptosis. In vivo, GMI and chidamide additively reduce distant metastases. GMI and chidamide are potential immunotherapeutic adjuvant for metastatic melanoma.

Targeted delivery of p-coumaric acid encapsulated mannosylated liposomes to the synovial macrophages inhibits osteoclast formation and bone resorption in the rheumatoid arthritis animal model.

The current study aimed to target the delivery of p-coumaric acid (CA), a dietary polyphenol to the synovial macrophages of AIA rats via mannose incorporated liposomal delivery system (ML) with reference to osteoclastogenesis and bone resorption. In vivo imaging and in vitro drug release study indicated the efficiency of mannosylated liposomes to localize at the site of inflammation and increased sustain drug release respectively. Morphological assessment of isolated synovial macrophages with respect to CD86 (synovial macrophages) and CD51 (pre-/osteoclast) indicated that p-coumaric acid encapsulated mannosylated liposomes (ML-CA) inhibited the osteoclasts differentiation. ML-CA treatment inhibited the TRAP staining, downregulated the expression of MMP-9 and NFATc1 and inflammatory cytokines. The ex-vivo study specified the ability of CA to induce the OPG production in bone marrow stromal cell triggered macrophage-osteoclasts differentiation and to preserve the calcium content. Taken together, our results demonstrated that ML-CA could intervene in the osteoclast formation.

Molecular ultrasound imaging using contrast agents targeting endoglin, vascular endothelial growth factor receptor 2 and integrin.

Expression levels of endoglin, αv integrin and vascular endothelial growth factor receptor 2 (VEGFR2) were investigated using targeted, contrast-enhanced ultrasonography in murine melanoma tumor models. Microvasculature and expression levels of biomarkers were investigated using specific contrast agents conjugated with biotinylated monoclonal antibodies. Ultrasound signal intensity from bound contrast agents was evaluated in two groups of mice: control mice and mice treated with sorafenib. Expression levels were analyzed by immunohistochemistry. Endoglin biomarkers were more highly expressed than αv integrin and VEGFR2. Endoglin decreased in the sorafenib group, whereas it tended to increase with time in the control group. Targeted ultrasound contrast agents may be used for non-invasive longitudinal evaluation of tumor angiogenesis during tumor growth or therapeutic treatment in preclinical studies. Endoglin protein, which plays an important role in angiogenesis, seems to be a target of interest for detection of cancer and for prediction of therapeutic efficacy.

Tanshinone II a protects against lipopolysaccharides-induced endothelial cell injury via Rho/Rho kinase pathway.

OBJECTIVE: To test whether tanshinone II A (Tan II A), a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin (lipopolysaccharides, LPS)-induced endothelial injury. METHODS: Endothelial cell injury was induced by treating human umbilical vein endothelial cells (HUVECs) with 0.2 µg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone II A on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase (ROCK) pathway-associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray. RESULTS: Tan II A improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan II A, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 (ITG A5), Ras homolog gene family member A (RhoA), myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K, or PIP2 in Western blotting), focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan II A, Y27632 and valsartan. CONCLUSION: Tan II A exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of ß3, ß5 or ß6 integrins or conditional loss of ß8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Quantifying amyloid beta (Aß)-mediated changes in neuronal morphology in primary cultures: implications for phenotypic screening.

Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aß) in the brain. Herein, the authors quantify Aß-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aß caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aß-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aß-mediated cytotoxicity. Finally, we showed that Aß challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aß-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

Metal ring on 4th or 5th finger markedly increases both cardiac troponin I at left ventricle and cancer-related parameters such as oncogen C-fosAb2 & integrin α5ß1[corrected] by 4-12 times. Thus these metal rings appear to promote both heart problems & cancer.

We examined patients wearing a metal ring on the left 4th finger with abnormally increased Cardiac Troponin I (which is known to increase in the presence of myocardial injury or left ventricular hypertrophy) of 5-14ng BDORT units (depending on the ring and individual) at left ventricle compared with normal value of 1ng BDORT units or less. Although shape of the ECG does not change significantly regardless of whether metal rings are on or not, when rings are on, the Bi-Digital O-Ring Test evaluation of trace of ECG revealed "Vulnerable Period of Rising Part of T-wave" of ECG waves (which correspond to the left ventricle and AV node) become abnormal with increased Cardiac Troponin I. DHEA in various parts of the body reduced significantly and maximum decrease in DHEA was found when metal ring was on the left 4th and 5th fingers. Telomere reduced with each of the 5 fingers, but the 2nd, 4th, and 5th fingers produced the maximum reduction of telomere. When metal ring was inserted onto the left 1st finger and left 2nd finger, Cardiac Troponin I did not change significantly. Additional abnormality was found when patients with cancer wore metal ring(s); namely both Cardiac Troponin I and cancer parameters, such as Integrin α5ß1[corrected] and Oncogen C-fos Ab2, increase anywhere between 4-12 times. However, when the ring was cut, creating a 1mm or longer empty space, no increase in cancer markers and Cardiac Troponin I were observed. Similar findings were found with metal bracelets.

[Effects of bushen zhuyun recipe on protein expressions of estrogen receptor, progesterone receptor and integrin alpha5 and beta3 in endometrium of rats at the implantation stage].

OBJECTIVE: To observe the effects of Bushen Zhuyun Recipe (BZR) on protein expressions of estrogen receptor (ER), progesterone receptor (PR) and integrin alpha5 and beta3 in endometrium of rats at the implantation stage, for exploring the possible mechanism of the recipe in treating luteal phase defect (LPD) infertility. METHODS: Female SD rats were randomly divided into 6 groups, the blank group, the model group, the WM group treated by Western medicine, and the three BZR groups treated by low-, middle- and high-dose BZR respectively. Rats were made to pregnancy and sacrificed at the implantation stage, their middle segment of uterus, about 1 cm in length was gotten for detecting the protein expressions by Western blot. Results The protein expressions of endometrial ER and PR were significantly higher, while those of integrin alpha5 and beta3 were significantly lower than those of the control group (P < 0.05). The protein expressions of endometrial ER and PR were significantly lower, but those of integrin alpha5 and integrin beta3 were higher in rats treated by middle- and high- dose BZR than those in model rats (P < 0.05). CONCLUSION: BZR can raise the receptivity of rats' endometrium through down-regulating the expressions of ER, PR and increasing the protein expression of integrin alpha5 and beta3 in endometrium and thus to enhance the pregnant rate.

Novel near-infrared fluorescent integrin-targeted DFO analogue.

Desferrioxamine (DFO), a siderophore initially isolated from Streptomyces pilosus, possesses extraordinary metal binding properties with wide biomedical applications that include chelation therapy, nuclear imaging, and antiproliferation. In this work, we prepared a novel multifunctional agent consisting of (i) a near-infrared (NIR) fluorescent probe-cypate; (ii) an integrin alpha vbeta3 receptor (ABIR)-avid cyclic RGD peptide, and (iii) a DFO moiety, DFO-cypate-cyclo[RGDfK(approximately)] (1, with approximately representing the cypate conjugation site at the side chain of lysine; f is d-phenylalanine). Compound 1 and two control compounds, cypate-cyclo[RGDfK(approximately)] ( 2) and cypate-DFO ( 3), were synthesized by modular assembly of the corresponding protected RGD peptide cyclo[R(Pbf)GD(OBut)fK] and DFO on the dicarboxylic acid-containing cypate scaffold in solution. The three compounds exhibited similar UV-vis and emission spectral properties. Metal binding analysis shows that DFO as well as 1 and 3 exhibited relatively high binding affinity with Fe(III), Al(III), and Ga(III). In contrast to Ga(III), the binding of Fe to 1 and 3 quenched the fluorescence emission of cypate significantly, suggesting an efficient metal-mediated approach to perturb the spectral properties of NIR fluorescent carbocyanine probes. In vitro, 1 showed a high ABIR binding affinity (10 (-7) M) comparable to that of 2 and the reference peptide cyclo(RGDfV), indicating that both DFO and cypate motifs did not interfere significantly with the molecular recognition of the cyclic RGD motif with ABIR. Fluorescence microscopy showed that internalization of 1 and 2 in ABIR-positive A549 cells at 1 h postincubation was higher than 3 and cypate alone, demonstrating that incorporating ABIR-targeting RGD motif could improve cellular internalization of DFO analogues. The ensemble of these findings demonstrate the use of multifunctional NIR fluorescent ABIR-targeting DFO analogues to modulate the spectral properties of the NIR fluorescent probe by the chelating properties of DFO and visualize intracellular delivery of DFO by receptor-specific peptides. These features provide a strategy to explore the potential of 1 in tumor imaging and treatment as well as some molecular recognition processes mediated by metal ions.

Expression of alpha V-associated integrin beta subunits in epithelial ovarian cancer and its relation to prognosis in patients treated with platinum-based regimens.

The aim of the study was to investigate the relationships between the expression of alphav, beta1, beta3, beta5, and beta6, integrin subunits and clinical parameters in ovarian cancers. Ovarian surface epithelium (OSE) from five donors and tumour samples from 39 patients with an epithelial ovarian cancer (39 primary tumours and 21 associated peritoneal metastases) were analysed using immunohistochemistry on paraffin-embedded or frozen tissue sections. The alphav and beta5 integrin subunits were always present in normal OSE and in tumours. beta1 and beta3 subunit expression was significantly less frequent in grade 3 than in grade 1-2 tumours. The proportion of stage IV tumours expressing beta3 was significantly lower as compared to other stages. The beta6 subunit was undetectable in OSE but was expressed in about 40% of primary tumours. For all integrin, there was a strong relationship between the expression in primary tumours and in associated peritoneal metastases. Survival analyses restricted to patients receiving platinum-based chemotherapy did not reveal any relationship between integrin subunit expression and 3-year survival rate, in this limited series of patients. In conclusion, the expression of the various beta integrin subunits was differentially altered in ovarian carcinoma, evocative of complementary roles of alphav integrins during tumour development.