Effect of Portulacae Herba free granuleson integrin-β1 of rats’ lens with diabetic cataract / 国际中医中药杂志

Objective:To investigate the effect of Portulacae Herba free granules on the degree of lens opacity and integrin β1 of lens in diabetic cataract rats. Methods:50 rats were randomly divided into control group, model group and the low, medium and high dose group of Portulacae Herba, 10 rats in each group. Except the control group, the diabetic cataract rat model was established by intraperitoneal injection of 1% STZ 60 mg/kg. The low, medium and high dose groups of Portulacae Herba were gavaged with Portulacae Herba 125, 250, 500 mg/kg, once a day for 12 weeks. The degree of lens opacity was observed with slit lamp. After 12 weeks, the lens was stained with HE. The expression of integrin β1 was detected by Western blot.Results:Compared with the model group, the opacification degree of lens in the low, medium and high dose groups of Portulacae Herba was significantly decreased ( P<0.05), the expression of integrin β1 (1.58 ± 0.14, 1.46 ± 0.24, 1.12 ± 0.19 vs. 1.76 ± 0.23) in rat lens was decreased ( P<0.05). Conclusion:For diabetic cataract rats, Portulacae Herba free granules can influence the expression of integrin β1 and inhibit the occurance of lens opacity.

Differential Expression of Integrin β1 in Two Brain Injury Models of Rats / 法医学杂志

Objective To study the characteristics of positive expression of integrin β1 in the rat brain tissue of two kinds of traumatic brain injury models and to explore the feasibility of inferring the mode of traumatic brain injury using the positive expression of integrin β1. Methods The occipital region of rats was hit by hydraulic impact method and pendulum striking method to produce two closed brain injury models of linear and rotation acceleration respectively, then 120 SD rats were randomly divided into linear acceleration injury group, rotation acceleration injury group, sham operation group and normal control group. Immunohistochemistry staining and Western blotting method were used to detect the positive expression of integrin β1 in different parts of the brain tissue at 30 min, 3 h, 6 h, 12 h, 3 d and 7 d after rat injury. The data was processed statistically by SPSS 18.0 software. Results The positive expression of integrin β1 was detected 30 min after brain injury and reached the peak 6 h after brain injury. With the extension of injury time, the expression tended to enhance. At the same time points after injury, the differences in the positive expression of integrin β1 between the linear acceleration injury group and the rotation acceleration injury group in the occipital strike point and thalamus had no statistical significance （ P>0.05）, but the differences in the expression of integrin β1 in the frontal lobe and brain stem had statistical significance （P<0.05）. Conclusion The characteristics of positive expression of integrin β1 in brain tissue can be used to infer the strike point and the manner of injury and has application value for the reconstruction of craniocerebral injury process.

Effects of hyperbaric oxygen on the expression of osteoprotegerin and integrin β1 in rabbit's mandibular distraction gap / 中华医学美学美容杂志

Objective To investigate the effect of hyperbaric-oxygen therapy on osteoprotegerin (OPG) and integrin β1 (Itgβ1 ) expression during mandibular distraction osteogenesis .Methods Forty New-Zealand rabbits were used .The animals were randomly divided into two groups (experimental group and control group) with 20 animals each ;after accomplished osteotomy and implant distraction devices on mandible bilaterally for 3 days of latency period ,the device was activated at the rate of 0 .8 mm per day for 10 days .All animals in the experimental group were subjected to hyperbaric oxy -gen for 90 minutes once a day since the beginning of distraction ,and lasted for four weeks .In control group ,all the distractors were activated following the same distraction protocol as the experimental group ,but without hyperbaric oxygen therapy .Five animals of each group were sacrificed at 10th day after distraction ,7th ,14th and 28th day of consolidation , respectively .The lengthened mandibles were harvested and processed for immunohistochemical examinations to detect OPG and Itg β1 expres-sion in the distraction gap .Semi-quantitative analysis was carried out by image analysis software . Results OPG staining was mainly located in the membrane and cytoplasm of osteoprogenitor cells and osteoblasts in the distraction zones .The expression of OPG increased after distraction accomplished and reached to the peak at 7th day of consolidation ,and then decreased gradually .At every time point , the level of expression of OPG in the experimental group was remarkably higher than those in control group .There were significant differences between the experimental group and control group ( P <0 .01) .Itgβ1 mainly located in actively proliferating osteoblasts ,fibroblasts and mesenchymal cells . The expression of Itgβ1 decreased significantly after reached to the peak at the 10th day distraction .At 10th day distraction ,7th and 14th day of consolidation ,Itgβ1 expressed more strongly than that in the experimental group ,which was remarkably higher than those in control group .There were significant differences between experimental group and control group (P < 0 .05) .At 28th day of consolidation , Itgβ1 expressed weakly ; there was no significant difference between the two groups ( P > 0 .05 ) . Conclusions Hyperbaric oxygen therapy can up-regulate the expression of OPG and Itgβ1 in the dis-traction gap ,which may promote osteoblast differentiation ,proliferation ,enhance osteoblast func-tion ,and new bone formation in distraction gap .

Investigation of protective mechanisms of Atorvastatin against high glucose environment-induced injuries of myocardial microvascular endothelial cells / 中华老年医学杂志

Objective To investigate the protective mechanisms of Atorvastatin against high glucose environment-induced injuries of myocardial microvascular endothelial cells. Methods Myocardial microvascular endothelial cells(MMECs)in SD rat were cultured and divided into groups of control group ,hyperglycemia group ,atorvastatin group ,and atorvastatin + high glucose group. The level of reactive oxygen species (ROS)was assayed using Superoxide Assay Kit. Apoptosis of cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL) . The expression levels of Akt1 and β1-Integrin were assayed by short-interfering RNA (siRNA ) technique ,and the levels of small GTP-binding protein dissociation stimulator (SmgGDS) expression were measured using Western blot. Results (1)The level of ROS was higher in the high glucose group than in the control group(t=4.154 ,P <0.01) ,and lower in both Atorvastatin group and the Atorvastatin + high glucose group than in the high glucose group (t= 4.233 and 2.893 ,both P <0.05). (2)The proportion of apoptotic cells was higher in the high glucose group than in the control group(t= 4.058 ,P < 0.01) ,and lower in both Atorvastatin group and the Atorvastatin + high glucose group than in the high glucose group(t=4.157 and 2.601 ,both P<0.05).(3)The expression level of Akt1 was lower in the high glucose group and the high glucose + Atorvastatin group than in the mock control group after transfection of Akt1-siRNA(t=4.058 and 4.167 ,both P<0.01).The expression level of β1-integrin was lower in the high glucose group and the high glucose + atorvastatin group than in the mock control group after transfection of β1-integrin-siRNA (t=4.073 and 4.215 , both P<0.01). (4)Western blot analysis showed the following results. First ,the relative expression levels of SmgGDS in both the low dose(1 μmol/L)and high dose(10 μmol/L)of atorvastatin group were higher than in the control group (t= 2.671 and 2.832 ,both P < 0.05).Second ,the relative expression level of SmgGDS in the high dose group were higher than in the low dose group (t=2.612 , P< 0.05 ). Third ,after transfection of Akt1-siRNA ,the expression level of SmgGDS in the high glucose + Atorvastatin group and the high glucose group was decreased ;and the level was higher in the high glucose + atorvastatin + mock group than in the high glucose + mock group(t=4.051 ,P<0.01).Fourth ,after transfection of β1-integrin-siRNA ,the expression level of SmgGDS was lower in high glucose + Atorvastatin group and the high glucose group than in the high glucose +Atorvastatin + mock group ;the level was higher in the high glucose + Atorvastatin + mock group than in the high glucose + mock group(t= 4.068 ,P < 0.01).Fifth ,the expression level of Akt phosphorylation in the high glucose group and the high glucose + Atorvastatin group was higher at 10 minutes than at five minutes(t=2.608 ,P<0.05) ,and higher at 15 minutes than at 10 minutes(t=3.127 ,P <0.05). After transfection of β1-integrin-siRNA ,the expression level of p-Akt /t-Akt was lower in the high glucose group than in the high glucose + mock group(t= 3.371 ,P < 0.05). Conclusions Atorvastatin treatment protects myocardial microvascular endothelial cells possibly by up-regulating SmgGDS through β1-integrin/Akt1 against high glucose environment-induced oxidative stress and apoptosis injuries.

CD98 activation increases surface expression and clusteringof beta 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates beta 1 integrin- mediated cell adhesion. However, the molecular mechanisms underlying CD98-mediated activation of beta 1 integrin are presently unclear. In this study, the effects of CD98 signaling on the expression and clustering of beta 1 integrin were investigated. Activation of CD98 augmented surface expression of beta 1 integrin on MCF-7 cells. Cross-linking CD98 induced clustering of beta 1 integrins. Inhibition of phosphorylation of focal adhesion kimase (FAK) by PP2, an inhibitor of Src family kinase, reduced cell-extracellular matrix adhesion, but not surface expression and clustering of beta1 integrin on MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, phalloidin or cytochalasin D inhibited CD98-mediated induction of cell-ECM adhesion, but not surface expression and clustering of b1 integrins. The inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated cell adhesion were diminished by pretreatment of cells with Mn2+, which is shown to induce conformational change of integrins. These results provide the first evidence that CD98 activation increases not only beta1 integrin affinity but also its surface expression and clustering and the latter is independent of FAK/Src and cytoskeleton.

In vivo characterization of the integrin beta3 as a receptor for Hantaan virus cellular entry

Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the alpha v beta3 integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether beta3 is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of beta3 integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 +/- 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either beta3 or av prolonged survival significantly to a maximal average survival of 19.7 +/- 1.5 days (P<0.01) and 18.4 +/- 0.9 days (P<0.01), respectively. XT-199, a chemical blocker of the alpha v beta3 receptor also prolonged survival to 19.5 +/- 1.3 days (P<0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine beta1 antibody comparatively prolonged survival (19.0 +/- 1.2 days), suggesting that HTNV infection is partly mediated through integrin beta1 receptors as well as through beta3 receptors in vivo. Our data demonstrate that the beta3 receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond beta3 for cellular entry within an organism.

The Control of Tumor Cell Invasiveness in Chondrosarcoma Cell Lines by Modifying Focal Adhesion Kinase Expression / 대한정형외과학회잡지

PURPOSE: We propose that cell attachment and invasion can be regulated by the modulation of FAK expression in chondrosarcoma cell lines. MATERIALS AND METHODS: The C-terminal domain of FAK (FAK-CD) was transfected by recombinant adenovirus infection in chondrosarcoma cell lines, JJ012 and 105KC. The expression of FAK, FAK-CD and tyrosine phosphorylation were checked. Chondrocytes and chondrosarcoma cells were used in cell attachment tests by blocking or not blocking integrin-beta 1 antibodies and synthetic peptides on type II collagen. To evaluate the effect of cell invasiveness, a wound healing assay and a Boyden chamber assay were done after FAKCD transfection. RESULTS: We observed higher FAK expression in the chondrosarcoma cells than in chondrocytes. The level of attachment to type II collagen was significantly inhibited by blocking with the antibody of integrin-beta 1 and synthetic RGD peptides. Also, the adenovirus mediated transfection of FAK-CD resulted in the inhibition of the phosphorylation of FAK and significant inhibition of cell attachment in only JJ012, without changing FAK expression. Moreover, migration after transfection with FAK-CD was reduced by up to 79.9% for JJ012 and 75.5% for 105KC. CONCLUSION: Attachment of chondrosarcoma cells could be mediated through integrin-beta 1. We conclude that modified FAK expression contributes to the suppression of tumor cell attachment and invasion.

The control of chondroid cell's adhesiveness by modulation of focal adhesion kinase(FAK) expression / 대한정형외과연구학회지

PURPOSE: We propose that cell attachment can be regulated by the modulation of FAK expression using an adenovirus vector. MATERIALS AND METHODS: Chondrocytes and chondroid cells were used in cell attachment test by blocking or non-blocking of antibodies and synthetic peptides on type II collagen precoated 96-well immunoplates. The C-terminal domain of FAK(FAK-CD) was transfected through infection of the recombinant adenovirus. Also tyrosine phosphorylation of FAK was checked by immunoprecipitation of FAK followed by western blot analysis with anti-phosphotyrosine antibody. For evaluating the change of integrin expression, semi-quantitative reverse-transcription polymerase chain(RT-PCR) reactions were done after transfection of FAK-CD. RESULTS: We observed more increased expression of FAK in the chondroid cells than that in chondrocytes using western blotting. The level of attachment to type II collagen was significantly inhibited by blocking with the monoclonal antibody of integrin-beta1 and synthetic RGD peptides. Also adenovirus mediated transfection of FAK-CD resulted in inhibition of phosphorylation of FAK and significantly inhibited cell attachment in only JJ102. Integrin-beta1 antibody blocking after transfection with FAK-CD showed inhibition of cell attachment in more than 95% of all cells. The mRNA expression of both Integrin a2 and integrin a5 was increased but was not significant. Protein expression of integrin a2 and integrin a5 showed no changes. CONCLUSION: We found that the attachment of FAK-overexpressing cells could be mediated through integrin-beta1 receptor. We concluded that the modification of FAK expression will contribute to increase the cell attachment to biomaterials and regeneration of cartilage defects.