Notch2 regulates matrix metallopeptidase 9 via PI3K/AKT signaling in human gastric carcinoma cell MKN-45

AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway

Salvianolic acid B promotes the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 via the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway

OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.

Effect of exercise training on left ventricular remodeling in patients with myocardial infarction and possible mechanisms

BACKGROUND A growing amount of evidence provides support for the hypothesis that acute myocardial infarction(AMI)patients should go through cardiopulmonary exercise testing(CPET)about 3-5 d after AMI is diagnosed,make reasonable exercising prescription,and conduct exercise training under guidance.AIM To investigate the effect of exercise training(ET)on left ventricular systolic function and left ventricular remodeling(LVRM)and to study the possible mechanisms of LVRM by the changes of matrix metallopeptidase 9(MMP-9)and tissue inhibitor of metalloproteinases 1(TIMP-1)in patients with acute STsegment elevation myocardial infarction(STEMI).METHODS Sixty patients with first STEMI undergoing direct percutaneous coronary intervention from February 2008 to October 2008 were randomly assigned to an exercise group(n=30)and a control group(n=30).The levels of MMP-9 and TIMP-1 were measured in all patients at 1 d,10-14 d,30 d,and 6 mo after admission.Two-dimensional echocardiography and cardiopulmonary exercise testing were done in patients at 10-14 d and 6 mo after admission.RESULTS There was no significant difference in CPET at baseline between the exercise group and the control group.At 6 mo,the time of exercise,peak and anaerobic threshold values of O2 uptake,and metabolic equivalents increased in both groups,but markedly increased in the exercise group.At baseline,there were no significant differences in left ventricular ejection fraction(LVEF)between the two groups.At 6 mo,LVEF increased in the exercise group,but not in the control group.At 6 mo,the percentage of patients with positive result of LVRM was 26.6%in the exercise group and 52.6%in the control group(P<0.05).The levels of plasma MMP-9 and TIMP-1 and the ratio of MMP-9 to TIMP-1 in both groups had no significant difference at 1 d and 10-14 d after AMI,but at 30 d and 6 mo,the levels of plasma MMP-9 and TIMP-1 in the exercise group were significantly lower than those in the control group;the ratio of MMP-9 to TIMP-1 in the exercise group was significantly higher than that in the control group.CONCLUSION ET under supervision based on home condition in early and recovery stage of AMI can improve exercise cardiopulmonary function and prevent the LVRM.Therefore,it may reduce unfavorable remodeling response by decreasing the levels of plasma MMP-9 and TIMP-1 and adjusting the ratio of MMP-9 to TIMP-1 hereafter.

Protective Effect of Simvastatin on Impaired Intestine Tight Junction Protein ZO-1 in a Mouse Model of Parkinson's Disease

Summary： Recently, several studies showed that gastrointestinal tract may be associated with patho- physiology of Parkinson＇s disease （PD）. Intestine tight junction protein zonula occluden-1 （ZO-1） is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 （MMP-9）. In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine （MPTP）-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-I expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvas- tatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that＇pass across the intestinal barrier.