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Objective:To investigate the role of integrin-linked kinase (ILK)-small interfering RNA (siRNA)-adeno-associated virus (AAV) in scar formation after glaucoma filtering surgery in rat eyes.Methods:Forty-eight Sprague Dawley rats of SPF grade, aged 8 to 9 weeks old, were selected and divided into blank control group, ILK-siRNA-AAV group, NC-siRNA-AAV group and mitomycin C (MMC) group by random number table method, with 12 rats in each group.Left eyes of the rats were taken as experimental eyes, and no intervention was administered to fellow eyes.The bulbar conjunctival filtering bleb after glaucoma filtration surgery in rats was established by anterior chamber drainage tube implantation.One day after operation, phosphate buffer solution, ILK-siRNA-AAV, and NC-siRNA-AAV were injected into the filtering bleb of blank control group, ILK-siRNA-AAV group and NC-siRNA-AAV group, 5 μl each group, respectively.Cotton tablets containing 0.4 mg/ml MMC were placed under conjunctival flap for 5 minutes during operation in MMC group.Intraocular pressure (IOP) was measured with a handheld tonometer before surgery and at 1, 2, 3, 7, 14, 21, 28 days after surgery.Formation of filtering blebs in rats was observed with a surgical microscope at 1, 2, 3, 7, 14, 21, 28 days after operation, and the bleb survival time was calculated by Kaplan-Meier survival analysis.The mRNA and protein expression levels of ILK in conjunctival and subconjunctival tissues at the surgical sites were detected by reverse transcription PCR and Western blot, respectively, on the 28th day after operation.Silencing of ILK gene was identified.Effect of ILK gene silencing on the morphology of drainage pathway was observed by hematoxylin-eosin staining.Effect of ILK gene silencing on collagen fiber deposition in the bulbar conjunctiva at filtration area was examined by Masson staining, and the percentage of positive area of collagen fiber staining in the total tissue visual field was calculated.The use and care of the animals complied with the ARVO Statement.This research protocol was approved by an Ethics Committee of Xi'an Jiaotong University (No.2013-772). Results:There were statistically significant differences in IOP at different time points between before and after surgery among four groups ( Fgroup=76.84, P<0.001; Ftime=114.49, P<0.001). The IOP of ILK-siRNA-AAV group on the 1st, 7th, 14th and 28th day after operation and the IOP of MMC group on the 2nd, 3rd, 7th, 14th and 28th day after operation were lower than those of blank control group, and the differences were statistically significant (all at P<0.05). The IOP of ILK-siRNA-AAV group was lower on 7th, 14th, 21st and 28th day after operation than those of NC-siRNA-AAV group, with statistically significant differences (all at P<0.05). The bleb survival time of blank control group, NC-siRNA-AAV group, ILK-siRNA-AAV group and MMC group was (3.50±1.51), (5.00±3.41), (31.50±3.15) and (31.33±2.46) days, respectively, with a significant difference among them ( F=395.83, P<0.05). The bleb survival time of ILK-siRNA-AAV group and MMC group was higher than that of blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of ILK mRNA and protein among four groups ( F=222.32, 752.69; both at P<0.05), and the relative expression levels of ILK mRNA and protein were significantly lower in ILK-siRNA-AAV group than blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Proliferative fibrous connective tissue and a large number of cells at surgical sites were found in blank control group and NC-siRNA-AAV group, and the fibroblasts were of a high density and grew in clumps.In ILK-siRNA-AAV group, the bulbar conjunctiva was thin, and the arrangement of fibrous connective tissue was loose, and a few proliferative fibroblasts were found.In MMC group, the conjunctival fibrous layer was loose and thin, forming cavities, and scarce cells were found.There was statistically significant difference in the percentage of collagen fiber positive staining area among four groups ( F=741.66, P<0.05). The positive staining percentage of ILK-siRNA-AAV group and MMC group was significantly lower than that of blank control group, among which there was lower positive staining percentage in ILK-siRNA-AAV group than NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Conclusions:Silencing of ILK can inhibit the scar formation after glaucoma filtering surgery and maintain low IOP in rats.
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OBJECTIVE: To observe the effect of herbal cake-partitioned moxibustion on renal function and expression of connective tissue growth factor (CTGF), integrin-linked kinase (ILK) and bone morphogenetic protein-7 (BMP-7) in chronic renal failure (CRF) rabbits, so as to reveal its mechanisms underlying improvement of CRF. METHODS: Twenty-four male New Zealand rabbits were randomly divided into control, model, medication and herbal cake-partitioned moxibustion (moxibustion) groups (n=6 rabbits in each group). The CRF model was established by gavage of suspension of Adenine (150 mg·kg-1·d-1) for 21 days. Herbal cake-partitioned moxibustion was applied to "Mingmen"(GV4) and bilateral "Shenshu"(BL23), "Pishu"(BL20) and for 5 moxa-cones every time. Rabbits of the medication group was treated by gavage of Losartan Potassium (2.33 mg·kg-1·d-1). All the treatments were conducted once daily,12 times a course for consecutive 3 courses with a two-day rest after each course of treatment. Serum creatinine (Scr), blood urea nitrogen (BUN) and 24 h urine protein contents were detected by using an automatic biochemical analyzer. The expression levels of CTGF, ILK and BMP-7 proteins and mRNA in the kidney tissue were determined by Western blot and quantitative real time-PCR, separately. RESULTS: Following modeling, Scr and BUN and 24 h urine protein contents were significantly increased in the model group in comparison with the control group (P0.05). CONCLUSION: Herbal cake-partitioned moxibustion can improve renal function in CRF rabbits, which may be related to its effects in suppressing the expression of ILK and CTGF, and in up-regulating the expression of BMP-7 in the kidney tissue.
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Objective To investigate the expression of integrin-linked kinase (ILK) and Toll-like receptor (TLR-2) in ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma and the effects on patients' outcomes.Methods Tissue specimens from 46 patients with MALT lymphoma were collected in this study.The expression of ILK and TLR-2 protein was detected by immunohistochemical methods,and the correlation of ILK and TLR-2 protein expression with clinicopathological features and patients' outcomes was analyzed.Results The positive rates of ILK and TLR-2 protein expression in tumor tissues were 67.4％ (31/46) and 71.7％ (33/46),respectively,which was related to the clinical stage of AnnArbor (P ＜ 0.05),rather than to sex,age and lesion location (all P ＞0.05).The survival of patients with ILK positive expression was less than that of ones with ILK negative expression [(21.5 ± 2.7)months vs.(29.2 ± 2.1) months] (P ＜ 0.05);meanwhile,the survival of patients with TLR-2 positive expression was less than that of ones with TLR-2 negative expression [(20.4 ±1.7) months v.(27.6 ± 2.3) months] (P ＜ 0.05).Conclusion ILK and TLR-2 are closely related to biological behavior of ocular adnexal MALT lymphoma,and combination detection of ILK and TLR-2 has a certain guiding value for diagnosis and prognosis.
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Objective To investigate the molecular mechanism of endothelial-mesenchymal transition (EndMT) induced by uremic toxin,parathyroid hormone (PTH),in vascular endothelial cells.Methods PTH (1 × 10-8 mol/L) was used to induce EndMT in human aortic endothelial cells (HAECs).TGF-β signaling inhibitor,including SB431542 and pirfenidone (PFD),and integrin-linked kinase (ILK) inhibitor Cpd22 were used to investigate the potential mechanism of EndMT induced by PTH in HAECs.Then the vascular endothelial cell markers VE-cadherin and CD31,and the mesenchymal marker α-SMA were detected by western blot.Results PTH reduced the expression levels of vascular endothelial cell marker CD31 and VE-cadherin (P＜0.05),while significantly increased the expression level of fiber cell marker α-SMA(P＜0.05).Furthermore,the TGF-βsignaling inhibitors (SB431542 and PFD) and ILK inhibitor (Cpd22) were able to partially reverse the EndMT induced by PTH in HAECs,which reversed the effect of PTH on reducing vascular endothelial cell marker expression and increasing fiber cell marker expression.Conclusion PTH could induce EndMT in HAECs via TGF-β/ILK pathway.
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Objective To investigate the effect of complement C3a on mouse podocytes phenotype transformation.Methods Purified C3a recombinant protein was used to stimulate mature mouse podocytes.The expression of the mature podocyte markers synaptopodin,podocin,nephrin,CD2-associated protein (CD2AP) and the mesenchymal cell markers fibroblast specific protein 1 (FSP-1),α-smooth muscle actin (α-SMA) were detected by RT-PCR,Western blotting,immunochemistry and immunofluorescence,respectively.Some podocytes were transfected with integrin-linked kinase (ILK) siRNA before the administration of C3a,the expression of nephrin and α-SMA were accessed by Western blotting,and the expression of Snail and α-actinin 4 were accessed by Western blotting and immunochemical method.The migration ability of podocytes was observed by scratch test.Results Immunocytochemistry and immunofluorescence analysis showed that synaptopodin,podocin,nephrin,CD2AP were highly expressed by mature mouse podocytes.The expression of these podocyte markers could be markedly inhibited after 24 h of C3a (0.1 μmol/L) treatment,and accompanied by the induction of mesenchymal markers FSP-1 and α-SMA.Compared with control group,the mRNA levels of synaptopodin,podocin,CD2AP and nephrin were significantly repressed by the administration of C3a in a dose-dependent manner,whereas the transcription of FSP-1 and α-SMA were remarkably up-regulated by C3a treatment (P ＜ 0.05,respectively).Western blotting analysis also confirmed the decrease of synaptopodin,podocin,nephrin and CD2AP protein and the increase of FSP-1 and α-SMA protein were closely depend on the C3a concentration (P ＜ 0.05,respectively).To further assess the downstream of C3a,some podocytes were transfected with ILK siRNA before the administration of C3a.Compared with C3a group,the protein levels of nephrin and α-SMA were significantly changed by the administration of ILK siRNA (P ＜ 0.05,respectively).The expression of α-actinin 4 and Snail induced by C3a were inhibited by ILK knockdown (P ＜ 0.05,respectively),accompanied by a decline of cell migration potency.Conclusion Complement fragment C3a can induce transformation of mouse podocytes to mesenehymal cells,and ILK signaling pathway is involved in this cell type transformation.
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Objective:To investigate the effect of specific inhibition of transforming growth factor-β1 (TGF-β1) with SB-431542 on the Smad2/3 and integrin-linked kinase (ILK) signaling molecules in tubule interstitial fibrosis(TIF)-induced cyclosporine A(CsA) in mouse.Methods:50 BALB/c mice were randomly divided into 5 groups (10 mice per group):the CsA model group (CMG),the interventional model group (IMG),the solvent control group (SCG),the low-salt control group (LCG),and the normal control group (NCG).The model mouse was established with low-sodium diet and intragastric administration of cyclosporine A,which was dissolved in olive oil at a dose of 60 mg/(kg·d).After 4 weeks,a specific inhibitor of TGF-β1 (SB-431542)was administered intraperitoneally with 10 mg/(kg·2 d) for 10 days (every other days).Mice were sacrificed at day 38.Serum creatinine (Scr) was measured,hydroxyp roline (Hyp)level and morphological changes of renal tissue were analyzed,expression levels of TGF-β1,P-Smad 2/3 and ILK were respectively detected by immunohistochemistry or Western blot,mRNA levels of TGF-β1,Smad 2/3 and ILK were respectively detected real-time polymerase chain reaction (RT-PCR).Results:Compared with three control groups (NCG,LCG and SCG),mice weight was decreased significantly,Scr level was increased significantly in two modeling groups (CMG and IMG) (P<0.01),and these changes in CMG were more obvious than those of IMG (P<0.05).Different levels of tubulointerstitial injury,interstitial infiltration of inflammatory cells and blue collagen staining in two modeling groups were observed,and particularly evident in CMG.TGF-β1,P-Smad2/3 and ILK immunostaining were mainly expressed in tubulointerstitium.The TGF-β1,P-Smad2/3 and ILK mRNA and immunostaining levels in two modeling groups were significantly increased as compared with three control groups (P<0.01),but their levels in IMG were significantly lower than those of CMG (P<0.05).The level of Hyp in renal tissue was positively correlated with Scr,TGF-β1,Smad2/3 and ILK (r=0.860,0.711,0.776,0.676,P<0.01).Conclusion:The activation of the TGF-β1/Smads signaling pathway plays an important role in the development of chronic CsA-induced TIF.The activation of ILK is closely correlated with the development of TIF,and may be used as a downstream factor of TGF-β1/Smads signaling pathway in regulating CsA-induced TIF.
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Previous studies have demonstrated that integrin-linked kinases (ILKs) are abundantly expressed in extracellular matrix (ECM) riche dermis, hair follicles, and basal cells of epidermis. ILKs are not only essential for the maintenance of skin structure, but also play important roles in wound healing. ILKs can promote the formation of granulation tissue by stimulating the proliferation of fibroblasts and secretion of ECM, accelerate wound contraction by inducing the differentiation of fibroblasts to myofibroblasts, and boost reepithelization by promoting proliferation, migration, and differentiation of keratinocytes and follicle epidermal stem cells.
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Objective@#To observe the expression of integrin-linked kinase on pulmonary fibrosis of paraquat (PQ) poisoning rats, and to discuss the relationship between ILK with pulmonary fibrosis induced by paraquat.@*Methods@#Forty male Sprague-Dawley (SD) rats were randomly divided into control group and paraquat group, 20 in each group; the PQ group rats were intraperitoneally injected PQ liquid (18 mg/kg) , and the control group rats were injected the same volume of saline; 5 rats of these two groups were respectively sacrificed after 7, 14, 28, 56 days of PQ injection; according to the results of lung biopsy HE staining and Masson staining to observe the lung pathologic changes, measure the value of lung hydroxyproline and the expression of ILK.@*Results@#HE and Masson staining of lung pathological biopsy showed, the 7th day after PQ exposure lung tissue mostly had congestion, edema, inflammatory cells infiltration; the 14th inflammation reduced, fibrosis change appeared gradually; the 28th and 56th showed the lung tissue structure disorder and occurred apparent hydroproline with blue dye in pulmonary interstitium. Compared with control group in the same experiment period, the value of lung hydroxyproline in each experiment period of PQ group increased (P<0.05) , and the value of lung hydroxyproline of PQ group rose with the increasing of the time of PQ poisoning. The expression of ILK mRNA and protein in each experiment period of PQ group was higher than the control group in the same experiment period (P<0.05) ; ILK mRNA and protein of PQ group began to increase on 7 day phase, reached the highest on 28 day phase, and decreased on 56 day phase.@*Conclusion@#The expression of ILK mRNA and protein increased with the lung fibrosis progression of PQ poisoning rats, so ILK could be the key molecule target which induced pulmonary fibrosis of PQ poisoning.
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Integrin-linked kinase (ILK) has been found for twenty years, and its biological characteristics have been extensively studied by multi-discipline. At present, studies of ILK are mainly focused on its roles in angiogenesis, tumor formation, and tissue fibrosis, etc. In recent years, the regulation effect of ILK in angiogenesis attracts attention of researchers. The studies showed that ILK can stimulate the secretion of angiogenic factor, promote the proliferation and migration of endothelial cells and inhibit their apoptosis, and therefore play an important role in the regulation of angiogenesis. Further research on molecular mechanism about the role of ILK playing in angiogenesis may provide an effective method for the treatment of some diseases.
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Objective@#To investigate the effect of cardiac stem cells (CSC) overexpressing integrin-linked kinase (ILK-CSC) transplantation on cardiac function after myocardial infarction (MI) and related mechanism.@*Methods@#CSCs were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and ILK-CSC were established by transfecting recombinant adenoviral vector harboring human wild-type ILK cDNA. Forty 8-week-old rats were randomly divided into 4 groups (n=10 each group): sham group, MI plus saline injection group(saline group), MI plus CSC transfected with null vector injection group (Ad-null-CSC group), and MI plus ILK-CSC injection group(ILK-CSC group). MI was induced through coronary artery ligation, and after 15 minutes, 30 μl saline, Ad-null-CSC (1×105 cells/30 μl) or ILK-CSC (1×105 cells/30 μl) were injected into the hearts of MI rats at 3 different points in infracted zone and infarct border zone. After 4 weeks, left ventricular (LV) function was examined by echocardiography, LV fibrosis was detected by HE and Masson staining, and myocardial protein expression of Ki-67 and p-H3 was evaluated by immuohistochemistry and mRNA expression of cyclinD1 and PCNA was detected by real-time RT-PCR.@*Results@#(1) Thirty-seven rats (sham group=10, saline group=8, Ad-null-CSC group=9 and ILK-CSC group=10) survived at 4 weeks after operation. Left ventricular end-systolic dimension (LVESD, P=0.009) and left ventricular end-diastolic dimension (LVEDD, P=0.002) were significantly increased, and left ventricular ejection fraction(LVEF, P=0.006) was decreased in saline group compared with those of sham group.In Ad-null-CSC group LVESD (P=0.005) and LVEDD (P=0.003) were decreased, but LVEF remained unchanged (P=0.113) compared with those of saline group. LVESD (P=0.004) and LVEDD (P=0.000 1) of ILK-CSC group were significantly decreased, and LVEF (P=0.004) was significantly increased compared with those of Ad-null-CSC group. (2) LV histology and myocardial fibrosis: there were marked myocyte loss and significant increase of myocardial fibrosis in the saline group((70.6±5.1) %, P=0.002) and Ad-null-CSC group((57.7±3.4) %, P=0.001) compared with sham group ((8.2±2.2) %), while the fibrosis was significantly attenuated post injection of ILK-CSC ((30.6±7.0)%, P=0.005 vs. Ad-null-CSC). (3) Protein levels of mitotic genes: the results of immuohistochemistry showed that the brown positive particles were presented in the nuclei of cardiac myocytes in saline, Ad-null-CSC and ILK-CSC groups, but they were negative in sham group. The protein expression of Ki-67 (P=0.007) and phosphohistone-H3 (p-H3) (P=0.003) in ILK-CSC group was significantly higher than in Ad-null-CSC group. (4) mRNA levels of mitotic genes: the results of real-time RT-PCR showed that the mRNA levels of cyclinD1 and proliferating cell nuclear antigen (PCNA) in saline and Ad-null-CSC groups were similar as in sham group (all P>0.05). However, in ILK-CSC group they were significantly increased compared with those in Ad-null-CSC group (cyclinD1, P=0.003; PCNA, P=0.004).@*Conclusion@#Our results suggest that myocardial transplantation of CSC overexpressing ILK improves cardiac function in the post-MI rats, and this beneficial effect may be related to the enhanced proliferation of cardiac myocytes and attenuated myocardial fibrosis.
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AIM:To investigate the effects of integrin-linked kinase (ILK) overexpression on survival and proliferation of cardiac c-Kit + cells,and the role of ILK-overexpressing c-Kit + cell transplantation in cardiac function in a rat myocardial infarction (MI) model.METHODS:Cardiac c-Kit + cells were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and cultured to prepare the ILK-c-Kit+ cells by infected with recombinant adenoviral vector harboring human wild-type ILK cDNA.The survival and proliferation of cardiac c-Kit + cells were detected by cell counting and CCK-8 assay at 48 h after infection,respectively.The protein levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) in the cardiac c-Kit+ cells were examined by Western blot.MI was induced by coronary artery ligation in 40 adult rats.After 15 min,ILK-c-Kit + cells were transplanted into the hearts by myocardial injection at 3 different sites in the infracted zone and border zone.All rats were randomly divided into 4 groups:sham group,MI plus saline injection group (MI group),MI plus null vector-infected cardiac c-Kit+ cell injection group (Ad-null-c-Kit+ cell group),and MI plus ILK-overexpressing cardiac c-Kit + cells injection group (ILK-c-Kit+ cell group),with 10 rats in each group.At 2 weeks after MI,the protein levels of c-Kit in MI hearts were investigated by immunohistochemical assay.At 4 weeks,left ventricular function was examined by hemodynamic measurement.RESULTS:The survival and proliferation of cardiac c-Kit + cells and the protein levels of cyclin D1 and PCNA were enhanced by ILK overexpression compared with Ad-null group.In MI rat model,the number of c-Kit + cells was increased by ILK-c-Kit + cell injection compared with Ad-null-c-Kit + cell group at 2 weeks after MI.Cardiac function was significantly improved in ILK-c-Kit + cell-transplanted rats.CONCLUSION:ILK overexpression improves survival and proliferation of cardiac c-Kit + cells by increasing the protein levels of cyclin D1 and PCNA.ILK-c-Kit + cell transplantation enhances the therapeutic efficiency of cardiac c-Kit + cells in the postMI hearts of rats.
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Liver is the only organ in the human body that can regenerate to its original size after partial resection.Most studies focus on the mechanisms of the initiation and development of liver regeneration.Recently studies about termination of liver regeneration have been gradually investigating.Exploring the termination of liver regeneration can help us understand the mechanism of "hepatostat",and it can also help us discover the relationship between the destruction of liver regeneration termination process and tumorigenesis.This review will summarize the currently known signaling pathways in termination of liver regeneration.
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AIM:To investigate the effects of integrin-linked kinase (ILK) overexpression on survival and proliferation of cardiac c-Kit + cells,and the role of ILK-overexpressing c-Kit + cell transplantation in cardiac function in a rat myocardial infarction (MI) model.METHODS:Cardiac c-Kit + cells were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and cultured to prepare the ILK-c-Kit+ cells by infected with recombinant adenoviral vector harboring human wild-type ILK cDNA.The survival and proliferation of cardiac c-Kit + cells were detected by cell counting and CCK-8 assay at 48 h after infection,respectively.The protein levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) in the cardiac c-Kit+ cells were examined by Western blot.MI was induced by coronary artery ligation in 40 adult rats.After 15 min,ILK-c-Kit + cells were transplanted into the hearts by myocardial injection at 3 different sites in the infracted zone and border zone.All rats were randomly divided into 4 groups:sham group,MI plus saline injection group (MI group),MI plus null vector-infected cardiac c-Kit+ cell injection group (Ad-null-c-Kit+ cell group),and MI plus ILK-overexpressing cardiac c-Kit + cells injection group (ILK-c-Kit+ cell group),with 10 rats in each group.At 2 weeks after MI,the protein levels of c-Kit in MI hearts were investigated by immunohistochemical assay.At 4 weeks,left ventricular function was examined by hemodynamic measurement.RESULTS:The survival and proliferation of cardiac c-Kit + cells and the protein levels of cyclin D1 and PCNA were enhanced by ILK overexpression compared with Ad-null group.In MI rat model,the number of c-Kit + cells was increased by ILK-c-Kit + cell injection compared with Ad-null-c-Kit + cell group at 2 weeks after MI.Cardiac function was significantly improved in ILK-c-Kit + cell-transplanted rats.CONCLUSION:ILK overexpression improves survival and proliferation of cardiac c-Kit + cells by increasing the protein levels of cyclin D1 and PCNA.ILK-c-Kit + cell transplantation enhances the therapeutic efficiency of cardiac c-Kit + cells in the postMI hearts of rats.
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Liver is the only organ in the human body that can regenerate to its original size after partial resection.Most studies focus on the mechanisms of the initiation and development of liver regeneration.Recently studies about termination of liver regeneration have been gradually investigating.Exploring the termination of liver regeneration can help us understand the mechanism of "hepatostat",and it can also help us discover the relationship between the destruction of liver regeneration termination process and tumorigenesis.This review will summarize the currently known signaling pathways in termination of liver regeneration.
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Integrin is a cell surface receptor that is widely expressed in mammals.Integrin-linked kinase (ILK) is a key kinase of the integrin signaling pathway which combines with integrins to communicate cell and extracellular matrix.Recent studies have shown that ILK can activate phosphatidylinositol-3-kinase/serine protein kinase (PI3K/AKT) and transforming growth factor beta/Smad (TGF-β/Smad) signaling pathways,which can promote cell proliferation,adhesion and migration of lens epithelial cells.It also can activate glycogen synthase kinase 3β/β-catenin (GSK3β/β-catenin) and other signaling pathways mediate aquaporins to regulate the water transport process.Eventually these changes can affect osmotic pressure of lens and lead to the formation of cataract.Cataract is a leading cause of visual impairment worldwide.It is a multi-factorial optic disorder associated with various risk factors such as aging,genetic,metabolic abnormalities,trauma,ultraviolet light exposure,poisoning and malnutrition.But the pathogenesis of cataract is not fully understood.ILK can mediate the migration,adhesion,proliferation and apoptosis of human lens epithelial cells through a variety of signaling pathways.Therefore,it is very important to study the role of ILK in the pathogenesis of cataract in the prevention and treatment of cataract.In this article,we reviewed the role of ILK in the pathogenesis of cataract from recent years.
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Objective To investigate the biological behavior and expression of intergrin-linked kinase (ILK) after exposure to transforming growth factor beta 2 (TGF-β32) in human Tenon fibroblasts (HTFs).Methods The primary ceils of HTFs were cultured by tissue attached culture method and identified by immunofluorescence analysis with Vimentin and keratin.The proliferation levels of HTFs induced by different concentrations of TGF-β2 were analyzed by MTT.The α-smooth muscle actin (α-SMA) and ILK mRNA expression were analyzed by quantitative Real-time PCR.The protein expression of α-SMA,ILK and E-cadherin were analyzed by Western Blot,and the protein expression of α-SMA and E-cadherin were also analyzed by immunofluorescence staining.Results MTT analysis showed that the optical density levels of 5.0 μg · L-1 and 10.0 μg · L-1 TGF-β2 were significantly higher than those of 0.1 μg · L 1,1.0 μg · L-1 and the control after exposure for 48 hours and 72 hours,and all these optical density levels were significantly higher than that for 24 hours (all P ＜0.05).The expression of α-SMA and ILK mRNA increased significantly when cells were treated with 5.0 μg · L-1 TGF-β2 for 48 hours in comparison with the control group (all P ＜ 0.05).The protein of α-SMA,ILK and E-cadherin were expressed both in TGF-β2 treated groups and control group,and TGF-β2 up-regulated the expression of them.There were significant differences when compared with the control group (all P ＜ 0.05).Immunofluorescent staining showed that α-SMA and E-cadherin were detected in TGF-β2 treated groups.α-SMA expressed in cytoplasm,while E-cadherin both in cytoplasm and nucleus.Conclusion TGF-β2 can induce the proliferation,transdifferentiation and adhesion of HTFs,and up-regulate the expression of ILK in vitro,suggests that ILK may play a role in the process of scar formation after glaucoma filtration surgery.
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@#The aim of this study was to investigate the effects and mechanisms of a nature product, amygdalin, in non-small cell lung cancer NCI-H1299 cell line invasion and migration in vitro. NCI-H1299(H1299)cells were treated with amygdalin. MTS assay was employed to determine cell proliferation, transwell chamber and wound-healing assay were employed to determine invasion and migration in vitro, western blotting and RT-PCR assay were employed to determine the expression of integrin β1, integrin β4, integrin-linked kinase(ILK), focal adhesion kinase(FAK), p-FAK and β-catenin, and the phosphorylation of Akt and RICTOR. Results showed that in vitro proliferation of H1299 cells were inhibited significantly after treated with 0. 5 and 1 mg/mL amygdalin for 48 h. The invasion and migration potential was decreased. The protein and mRNA expression of MMP-2/9, integrin β1/4, ILK, FAK, β-catenin, the activity of MMP-2/9, the phosphorylation of FAK, Akt and RICTOR were all decreased significantly(all of P< 0. 05).
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Objective To observe the effect of TGF‐β1 in the endothelial‐mesenchymal transition EndM T of glomeruli ,and to explore the effect of TGF‐β/Smad signaling pathway mediated by integrin linked kinase(ILK ) in the progress of renal fibrosis . Methods Human glomerular endothelial cells (HGEnC) incubated in vitro were divided into blank control group ,TGF‐β1 (12 .5 , 25 .0 ,50 .0 ng/mL) group .TGF‐β1 50 .0 ng/mL receptor type one antagonist (LY364749)5 .0 μmol/L group ,ILK (QLT‐0267)5 .0μmol/L antagonist group .The mRNA level of P‐Smad 2/3 and ILK was determined by RT‐PCR ,and the protein level of P‐Smad 2/3 ,ILK ,E‐cadherin ,CD31 ,α‐SMA and FSP‐1 was determined by Western blot after 48 h and 72 h after incubation in each group un‐der the above‐mentioned condition .Results (1)TGF‐β1 could significantly increased the mRNA level of P‐Smad2/3 and ILK (P0 .05) .Conclusion TGF‐β1 as the effector molecule in downstream can promote endothelial‐mesenchymal transition of HGEnC ,and TGF‐β/Smad signaling pathway mediated integrin linked kinase participate in this process ,which probably play important role in the progress of renal fibrosis .
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Objective To investigate the role of integrin-linked kinase (ILK) on migration and invasion of lung cancer cell by upregulating matrix metalloproteinase-9 through nuclear factor-κB (NF-κB) pathway.Methods A549 cell line were overexpressed ILK and matrix metalloproteinase-9 (MMP-9) confirmed by cell transfection,siRNA interference,cell scratch test,real-time quantitative PCR and Western Blot.Results Over-expression of ILK stimulated MMP-9 expression in lung cancer cells(P ＜ 0.01).The addition of MMP-9 inhibitor doxycycline and anti-MMP-9 neutralizing antibody significantly impaired the wound healing capacity of ILK-transfected cells(P ＜ 0.01),as well as by in vitro matrigel invasion assay (P ＜ 0.01).In addition,overexpression ILK induced phosphorylation and nuclear translocation of nuclear factor-κB subunit p65.Upregulation of MMP-9 was severely abolished by either BAY 11-7028,a specific NF-κB inhibitor,or siRNA targeted to NF-κB p65 in ILK over-expression cells.Conclusion The finding indicate that over-expression of ILK can promote the migration and invasion of lung cancer cell,and upregulate MMP-9 through the NF-κB pathway.
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Objective To investigate the possible function of integrin-linked kinase (ILK)/protein kinase B (PKB/Akt) signaling in repair of neonatal rat hypoxia-ischemia brain damage (HIBD). Methods Postnatal day 10 SD rats were randomly divided into hypoxia ischemia (HI) group and sham control group. Rat brains were collected at 0 h, 4 h, 6 h, 12 h, 24 h, 48 h and 72 h after hypoxia ischemia damage. Immunolfuorescence staining was used to observe the distribution and expression of ILK. Western blot was used to detect the expression of ILK, Akt, phosphorylated Akt (p-Akt) and vascular endothelial growth factor (VEGF). Lentiviral vectors expressing ILK shRNA were constructed to inhibit the expression of ILK in neonatal rats. After intracerebroventricular injections of LV-ILK shRNA lentivirus and LV-control respectively, HIBD model was established. Rat brains were collected at 4 h and 24 h after HIBD. Western blot was used to detect the expression of ILK, p-Akt, and VEGF. TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to detect cell apoptosis. Results Immunolfuorescence staining showed that ILK was widely distributed in cortex and hippocampus both in HI group and sham control group. ILK located at cell membrane and cytoplasm. Western blot results demonstrated that ILK protein increased after HI, with a peak at 24 h, and maintained higher level than those in sham control group. The p-Akt protein signiifcantly increased at 4 h after HI, and signiifcantly decreased in the following 24 h, and then increased again, with a peak at 48 h, but the level of p-Akt protein was higher than that of sham control group. The VEGF protein increased at 4 h after HI, with a peak at 12 h, higher than that of sham control group. The expression of Akt protein showed no signiifcant difference between HI group and sham control group. Lentiviral vectors containing RNAi targeting ILK was applied successfully in vivo. At 4 h and 24 h after HIBD model, the expression of ILK, p-Akt, and VEGF proteins in right side brain received LV-ILK shRNA signiifcantly decreased compared with those of right side brain received LV-control at the same time point. And cell apoptosis signiifcantly increased in LV-ILK shRNA group. Conclusions The expression of ILK, p-Akt, VEGF proteins increased after HI. By inhibiting the expression of ILK, the expression of p-Akt and VEGF proteins can be reduced, and cell apoptosis could increase in newborn rats after HIBD. The results suggest that ILK may induce the expression of VEGF through activating the PI3K/Akt signaling pathway, and promote cell survival and angiogenesis after HIBD.