Résumé
ObjectiveTo investigate the effect and mechanism of pachymic acid （PA） in Poria on the invasion and metastasis of renal carcinoma cells. MethodThe effect of PA （0， 20， 40， 80， 160 μmol·L-1） on cell viability was detected by cell counting kit-8（CCK-8）， and the dose of PA was selected for subsequent experiments. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell proliferation was evaluated by colony formation assay. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell adhesion ability was observed by cell adhesion assay. The effect of PA （0， 20， 40， and 80 μmol·L-1） on cell invasion and metastasis was investigated by Wound healing assay and Transwell invasion assay. The inhibitory effect of PA （0， 20， 40， 80 μmol·L-1） on cell motility was further observed and verified by high-content imaging technology. The effects of PA （0， 20， 40， 80 μmol·L-1） on the expression of matrix metalloproteinase （MMP）/tissue inhibitor of metalloproteinasas （TIMP） related to invasion and metastasis and Smads were detected by Western blot. ResultCCK-8 results showed that compared with the blank group， the PA groups showed decreased cell viability（P<0.01）， with the half-maximal inhibitory concentration （IC50） of ACHN cells of 70.42 μmol·L-1 at 24 h. Colony formation assay showed that the number of cell clonal groups in the PA groups was reduced compared with that in the blank group（P<0.01）. Cell adhesion assay showed that compared with the blank group， the PA groups displayed reduced cell adhesion（P<0.01）. Wound healing assay showed that the wound healing rate of cells in the PA groups was lower than that in the blank group （P<0.05，P<0.01）. Transwell invasion assay showed that compared with the blank group， the number of transmembrane cells in PA groups was reduced（P<0.01）. High-content imaging showed that the cumulative migration distance of cells in the PA groups was shorter than that in the blank group（P<0.01）. The results of Western blot showed that the protein expression of MMP-2 and MMP-9 in the PA groups decreased （P<0.01）， and TIMP-1 protein expression increased （P<0.01） compared with those in the blank group. In addition， compared with the blank group， the PA groups showed decreased protein expression of Smad2 and Smad3 （P<0.01）. ConclusionPA can inhibit the invasion and metastasis of renal carcinoma cells presumably through regulating the homeostasis of MMP/TIMP by Smad2/3.
Résumé
Objective To determine the expression of matrix metalloproteinase-1 (MMP-1) and its inhibitor in the development of exercise-induced atrial fibrosis.Methods Totally 48 eight-week-old male adult sprague-dawley rats were randomly divided into a control(C) group and a highly intensive exercise(H) group,each of 24.Group C was fed normally,while group H took one hour treadmill running with the gradient of 10°and speed of 28 m/min every weekday,lasting 5 weeks.The mRNA and protein expression of MMP-1 and matrix metalloproteinase tissue inhibitor-1 (TIMP-1) were detected using the real-time PCR and Western blotting.Results The MMP-1 expression of group H increased significantly after 8 weeks' training,compared to the control group.However,there was no significant difference in MMP-1 expression between group C and H after 12 or 16 weeks of training.The MMP-1 mRNA expression decreased with the extending of exercise,and that of group H after 16 weeks' training was significantly lower than 8 weeks' (P＜0.05).The TIMP-1 expression had an increasing trend without significance after 8-week exercise.After 12 and 16-week exercise,the mRNA and protein expression of TIMP-1 increased significantly(P＜0.01 and P＜0.05).The TIMP-1 mRNA and protein expression increased gradually with the extension of exercise,and the TIMP-1 mRNA expression of group H after 16 weeks of training was significantly higher than that after 8 weeks(P＜0.01).The ratio of MMP-1/TIMP-1 mRNA and protein increased at first and decreased afterwards.The ratio of MMP-1/TIMP-1 of group H after 16 weeks of exercise was significantly lower than group C at the same time point,and group H after 8 weeks' Conclusion After a long-term high-intensity exercise,the MMP-1 expression of atrial first increases and then decreases,while the TIMP-1 expression increases gradually.Moreover,such exercise can induce disbalance between MMP-1 and TIMP-1,maybe due to the molecular pathological mechanism of exercise-induced atrial damage and fibrosis.
Résumé
Small hairpin RNA (shRNA) was used to silence the HIF1α gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2 to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1α mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1α was interfered in RPE cultured under hypoxia (induced by 150 μmol/L CoCl2 ). RT-PCR was employed to detect the expression of HIF1α and TIMP1. The expression levels of HIF1α and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1α mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1α gene silencing in RPE was 83. 4 %. Western blotting revealed that the expression levels of HIF1α protein was dramatically dropped. In addition, RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9 %, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1α mRNA could effectively silence the HIF1α gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.