Angiotensin II-induced matrix metalloproteinase-9 expression mediated by NF-κB pathway in human THP-1 cells

AIM: The present study was undertaken to investigate the effect of angiotensin II (AngⅡ) on expression of MMP-9 in THP-1 macrophages. METHODS: Macrophages converted from THP-1 monocytes by incubating with PMA (0.1 μmol/L) for 48 h were divided into PMA group; PMA+AngⅡ group (10-7mol/L, 1 h); PMA+AngⅡ+PDTC group (10 μmol/L, 30 min) and PDTC group. Western blotting was used to detect the MMP-9 and phosphorylation of NF-κB p65, and the expression of MMP-9 mRNA in THP-1 macrophages was measured by RT-PCR.RESULTS: Compared to control group, the expression of MMP-9 (1.06±0.11, P<0.05) and phosphorylation of NF-κB p65 (1.02±0.10, P<0.05) in THP-1 macrophages were expressed when treated with AngⅡ (10-7mol/L); and the expression of MMP-9 mRNA were upregulated (1.22±0.08, P<0.05). However, NF-κB inhibitor PDTC reduced the NF-κB p65 (0.99±0.12, P<0.01) and MMP-9 (1.04±0.14, P<0.01) expressions and decreased the expression of MMP-9 mRNA (0.90±0.06,P<0.01). CONCLUSION: NF-κB signaling pathway contributes to the expression of MMP-9 in THP-1 macrophage induced by AngⅡ.     

he effects of constant magnetic field on apoptosis,adhesion molecule expression in endothelial cells and their adhesion rates with monocytes

AIM: To investigate the effects of constant magnetic field on apoptosis, secretion and expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC), and their adhesion rates with THP-1 monocytes induced by angiotensin Ⅱ (AngⅡ).METHODS: The third passage of cultured HUVEC was used.There were six groups: control group, Ang Ⅱ (10^-6 mol/L) group, Ang Ⅱ with 1, 5, 10 or 20 gausses of constant magnetic field group.Samples were collected 24 h after incubation with or without magnetic field.Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidinm iodide staining with flow cytometry.Secretion and expression of ICAM-1 and VCAM-1 were detected by ELISA and immunocytochemistry, respectively.Adhesion rate between HUVEC and THP-1 was measured by counting method.RESULTS: Ang Ⅱ at concentration of 10^-6mol/L induced apoptosis in HUVECs (P<0.05 vs control), whereas in 1, 5, 10 and 20 gausses group, apoptosis of HUVECs was significantly lower than that in Ang Ⅱ group (P<0.05).Ang Ⅱ at concentration of 10-6 mol/L significantly increased secretion and expression of ICAM-1 and VCAM-1 (P<0.05 vs control), whereas secretion and expression of ICAM-1 and VCAM-1 in 1, 5, 10 and 20 gausses group significantly decreased, compared with Ang Ⅱ group (P<0.05).The adhesion rates between HUVEC and THP-1 significantly increased 24 h after incubation of HUVEC with Ang Ⅱ (P<0.05 vs control), in contrast, the adhesion rates between HUVEC and THP-1 in 1, 5, 10 and 20 gausses group significantly decreased, compaed with Ang Ⅱ group (P<0.05).CONCLUSIONS: One gauss to 20 gausses of constant magnetic field antagonizes the effects of Ang Ⅱ on HUVEC, decreases apoptosis and expression of ICAM-1 and VCAM-1 in HUVEC, and also decreases the adhesion rates between HUVEC and monocytes induced by Ang Ⅱ.     

Inhibition of HMGB1 expression and release by nicotine in RAW264.7 cells

AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α7 subunit-containing nicotinic receptor (α7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α7nAch receptor expression.     

Role of cAMP and cGMP in the regulation of bFGF gene expression of rat cardiomyocytes

AIM: To investigate the role of cAMP and cGMP in the regulation of bFGF gene expression of rat cardiomyocytes. METHODS: 8-Bromo-cAMP and sodium nitroprusside were added into the media to raise the concentrations of cAMP and cGMP of cultured cardiomyocytes, in situ hybridization and immunohistochemistry methods were used to identify the mRNA and protein expression of bFGF. The amount of bFGF mRNA and protein expression were detected by computor imaging analysis system MIAS exe. RESULTS: At 24 h after adding 8-Bromo-cAMP(0 1 mmol/L), bFGF mRNA and protein expression elevated significantly , at 48 h and 72 h it still expressed higher than that of control group( P< 0.05). But after adding sodium nitroprusside (1 mmol/L),bFGF expression decreased significantly,and especially at 48 h. CONCLUSION: The regulation of bFGF expression of cardiomyocytes is related to cAMP signal pathway.     

Regulation of estrogen on the expression of KLK6 mRNA and protein in human breast cancer cell line MCF-7

AIM:To investigate the effects of estrogen and tamoxifen on the expression of KLK6 mRNA and protein (hK6) in human breast cancer cell line MCF-7. METHODS:MCF-7 cells were incubated with 17-βE2 and tamoxifen at different concentrations for 72 hours, respectively. The expression levels of kallikrein 6 (KLK6) mRNA and protein were evaluated by fluorescence quantitative RT-PCR and flow cytometry, respectively. RESULTS:Compared with ethanol control, KLK6 mRNA expression levels were significantly decreased when 17-βE2 was added at concentrations of 10^-10 and 10^-8 mol/L (P<0.01). No statistical change was observed when 17-βE2 was at 10^-12 mol/L (P>0.05). Flow cytometry showed the same results. The average fluorescence intensity (AFI) that represents the level of hK6 was decreased after incubated with 17-βE2 (P<0.01). After incubation with tamoxifen, the levels of KLK6 mRNA and hK6 were increased (P<0.01). CONCLUSION:Estrogen down-regulates the expression levels of KLK6 mRNA and protein (hK6), while tamoxifen has an opposite effect.     

Effects of dehydroepiandrosterone on expression of ICAM-1 in rabbit aorta and HUVECs induced by high lipid levels

AIM: To investigate the effects of dehydroepiandrosterone (DHEA) on the expression of intercellular adhesion molecule-1 (ICAM-1) induced by high lipid levels in rabbit aorta and human umbilical venous endothelial cells (HUVECs), and the effects of all-trans retinoic acid (ATRA) in this process.METHODS: For in vitro experiments, the cultured HUVEC were divided into control group, oxidized low-density lipoprotein (ox-LDL) group, ox-LDL+DHEA group, ox-LDL+DHEA+ATRA group and DHEA group. The HUVECs in all groups were treated with the corresponding reagents for 24 h. The expression of ICAM-1 at mRNA and protein levels in all groups were determined by RT-PCR and ELISA, respectively. For in vivo experiments, the rabbits were divided into control group, high lipid group, high lipid+DHEA group, high lipid+DHEA+ATRA group and DHEA group. The rabbits in all groups were fed with the corresponding diets for 10 weeks. The expression of ICAM-1 in the rabbit aorta at mRNA and protein levels was determined by RT-PCR and immunohistochemistry. RESULTS: The expression of ICAM-1 in the HUVECs in ox-LDL group was significantly increased compared with control group (P<0.05). Compared with ox-LDL group, the expression of ICAM-1 in ox-LDL+DHEA group was obviously decreased (P<0.05). The expression of ICAM-1 was similar in both control group and DHEA group (P>0.05). The expression of ICAM-1 was similar in both ox-LDL+DHEA group and ox-LDL+DHEA+ATRA group (P>0.05). The expression of ICAM-1 in the rabbit aorta in high lipid group was significantly increased compared with control group (P<0.05). Compared with high lipid group, the expression of ICAM-1 in high lipid+DHEA group was obviously decreased (P<0.05). No remarkable difference in the expression of ICAM-1 between control group and DHEA group was observed (P>0.05), so did between high lipid+DHEA group and high lipid+DHEA+ATRA group (P>0.05). CONCLUSION: DHEA inhibits high lipid-induced ICAM-1 expression in rabbit aorta and HUVECs. That may be one of the mechanisms of antiatherosclerotic effect of DHEA. ATRA seems no positive effect on DHEA function.     

Effects of insulin on osteoblast and its post-receptor mechanism

AIM:To study the effects of insulin on the proliferation and function of osteoblasts and the relationship between insulin post-receptor change in osteoblasts and osteoblastic cell growth.METHODS:The effects of different levels of insulin on osteoblasts were assessed by MTT colorimetry.Osteocalcin in medium was measured by RIM.IGF-1 mRNA expression levels were determined by RT-PCR.The concentrations of free IGF-1 protein in serum-free medium were measured by ELISA.In addition,the protein level and phosphorylated protein of P44/42MAPK were determined by Western blotting analysis.RESULTS:Insulin enhanced the proliferation of osteoblasts,depending on its dose and exposure time.Insulin at concentration of 10-7 mol/L showed the strongest effect,and the action attained the plateau phase beyond 96 h.The best concentration that stimulated synthesis of osteocalcin by insulin was 10-7 mol/L.When the insulin concentration beyond 10-7 mol/L,the osteocalcin concentration was decreased.Exposure time had no effect on insulin-stimulated synthesis of osteocalcin of osteoblastic cells.When the concentration of insulin reaches 10-6 mol/L,the IGF-1 mRNA expression stimulated by insulin was also decreased.The concentrations of free IGF-1 protein in insulin-stimulated groups were all higher than that in control group (P<0.05),but there was no statistically significant difference among insulin-stimulated groups (P>0.05).Insulin acute stimulation rapidly induced the activity of tyrosine phosphorylation of P44/42MAPK.The degree of tyrosine phosphorylation of P44/42MAPK was increased step by step along with the increasing doses of insulin from 0 to 10-7 mol/L (P<0.05,between groups).After insulin chronicity treatment,there was a marked reduction in the tyrosine phosphorylation of P44/42 MAPK (P<0.05,between groups).There was no significant change in protein level of P44/42MAPK.CONCLUSIONS:Insulin enhances the proliferation of osteoblasts as a growth factor at a suitable concentration,but this effect disappears at chronic high insulin stimulation.The MAPK may be involved in the proliferating effect of insulin on osteoblasts.Transient stimulation of insulin activates the P44/42MAPK,however chronic high insulin stimulation results in down-regulation of P44/42MAPK signal activity.     

Contributions of cardiotrophin-1 to cardiac fibroblast proliferation

AIM: To investigated the role of CT-1 in the hypertensive ventricular remodeling. METHODS: The cardiac fibroblasts in 3-4 passages were cultured in vitro, and divided into eight groups according to the different intervention factors: group C (control); group DM: dimethyl sulphoxide (DMSO); group P: cultured under high hydrostatic pressure (160 mmHg); group ASODN: interfered with CT-1 antisense oligodeoxynucleotides (10 μmol/L); group SODN: incubated with CT-1 sense oligodeoxynucleotides (10 μmol/L); group AG: interfered with AG490 (25 μmol/L); group PD: interfered with PD98059 (20 μmol/L); group LY: interfered with LY 294002 (10 μmol/L). Western blotting was employed to assess the expression of STAT3, ERK1/2 and PI3-K respectively at protein level. Cell proliferation was quantified by MTT. RESULTS: High hydrostatic pressure stimulated the proliferation of cardiac fibroblasts, upregulated the expression of CT-1. CT-1ASODN inhibited the proliferation of cardiac fibroblasts (0.132±0.013 vs 0.154±0.011, P<0.05). ASODN extensively inhibited the expression of STAT3, ERK1/2 and PI3-k respectively at protein level (2.09±0.25 vs 2.47±0.28, P<0.05), (1.13±0.19 vs 1.61±0.22, P<0.05), (1.25±0.23 vs1.71±0.25, P<0.05). AG490, a JAK-STAT3 inhibitor, reversed the increase in the proliferation of cardiac fibroblasts stimulated by high hydrostatic pressure and the expression of ERK1 phosphorylation (0.118±0.018 vs 0.155±0.010, P<0.05). PD98059, a MAPK-ERK1/2 inhibitor, increased the proliferation of cardiac fibroblasts stimulated by high hydrostatic pressure (0.185±0.011 vs 0.155±0.010, P<0.05) and the expression of STAT3 phosphorylation (1.83±0.23 vs 1.58±0.22, P<0.05). LY294002, a PI3-K inhibitor, had no effect on the proliferation of cardiac fibroblasts stimulated by high hydrostatic pressure (0.157±0.015 vs 0.155±0.010, P>0.05). No difference of the expression of the above factors was observed in SOND (0.151±0.010 vs 0.154±0.011, P>0.05) and DMSO (0.141±0.017 vs 0.155±0.010, P>0.05) groups as compared with the control group. CONCLUSION: Under high hydrostatic pressure, the proliferation of cardiac fibroblasts is essentially mediated by STAT3, independent of PI3-K and the action is negatively regulated by ERK1/2 via inhibiting STAT3.The interaction between STAT3 and ERK1/2 may assist CT-1 in developing adequate cardiac fibroblast proliferation.     

Rapamycin inhibits HMGB1 expression and releases in RAW264.7 cells induced by lipopolysaccharides in vitro

AIM: To observe the mechanism that rapamycin (RPM) affects HMGB1 expression and release in RAW264.7 cells induced by lipopolysaccharides (LPS).METHODS: RAW264.7 cells were cultured in six wells plate and divided into five groups: control group, 250 μg/L LPS treatment group, 100 μg/L RPM treatment group, 50 μg/L rTNF-α treatment group and 100 μg/L TNF-α antibody treatment group. After 4 h treatment, the TNF-α level in the culture media was evaluated by ELISA assay. After 24 h, the expression of HMGB1 mRNA was measured by RT-PCR, and HMGB1 protein level in the culture media was determined by Western blotting analysis.RESULTS: TNF-α level in the culture media of RAW264.7 cells has no significant difference between RPM treatment group and control group (P>0.05). Both HMGB1 mRNA expression and HMGB1 protein level were remarkably higher in LPS treatment group than that in control group (P<0.05). RPM attenuated LPS-induced HMGB1 mRNA and HMGB1 accumulation. Compared with that in RPM treatment group, HMGB1 accumulation was increased in rTNF-α treatment group, and had no significant difference in TNF-α antibody treatment group (P>0.05).CONCLUSION: RPM inhibits HMGB1 expression not only by directly suppressing STAT3 activation, but also by indirectly reducing TNF-α level.     

Effect of bFGF on human kidney fibroblasts

AIM: To study the effect of bFGF on cell proliferation, secretion of type I collagen and expression of integrin β₁ in human kidney fibroblasts(KFB). METHODS: The KFB was cultured and stimulated by bFGF in vitro. The proliferation and collagen I secreting of KFB, the expression of integrin β₁ were measured by MTT, ELISA and flow cytometer, respectively. RESULTS: bFGF(25-50 μg/L) could obviously stimulate the cell proliferation(P< 0.05), promote the secretion of collagen I(P< 0.05) and enhance the expression of integrin β₁(P< 0.05) in human kidney fibroblast. CONCLUSION: bFGF could induce renal interstitial fibrosis by promoting cell proliferation, secretion of collagen I and integrin β₁ expression of KFB.     

Effect of AG490 on expression of VEGF and HIF-1α in HEL cells

AIM: To investigate the effect of AG490 on the expression of VEGF and HIF-1α, and the capacity of invasion in human erythroleukemia (HEL) cells. METHODS: The HEL cells were treated with AG490 at different concentrations. The cell viability was detected by CCK-8 assay. The apoptosis was detected by Hoechst staining. The apoptosis and the cell cycle were analyzed by flow cytometry. The capacity of migration was evaluated by Transwell assay. The mRNA expression level of JAK2 was measured by RT-PCR. The protein levels of p-JAK2, VEGF and HIF-1α were determined by Western blot. RESULTS: The HEL cell viabilities were 88%, 75%, 48%, 10% and 0.12% after treated with AG490 at 20, 40, 60, 80 and 100 μmol/L for 48 h, respectively. The results of Hoechst staining showed that brilliant blue cells in 80 μmol/L AG490 group was significantly increased compared with control group for 48 h. The apoptosis rate of 80 μmol/L AG490 group was significantly increased compared with control group at 48 h after AG490 treatment. The number of membrane-permeating HEL cells in 20 μmol/L AG490 group at 24 h after AG490 treatment was significantly lower than that in control group (P<0.05). The mRNA level of JAK2 decreased in a concentration-dependent manner after the HEL cells were treated with different concentrations of AG490 for 48 h. The protein levels of p-JAK2, VEGF and HIF-1α were lower in AG490 treatment groups than those in control group (P<0.05). CONCLUSION: AG490 inhibits the expression of VEGF and HIF-1α in HEL cells by inhibiting JAK2 pathway.     

Metoprolol inhibits norepinephrine-induced rat cardiomyocyte apoptosis and connexin 43 phosphorylation

AIM: To study the effects of metoprolol (Meto) on the apoptosis of neonatal rat cardiomyocytes and the phosphorylation of connexin 43 (Cx43) induced by norepinephrine (NE). METHODS: Neonatal SD rat cardiomyocytes were divided into the following five groups (n=6 in each group): (1) control (Con) group: no treatment; (2) NE group: treatment with NE at 0.1 μmol/L for 24 h; (3) NE+Meto group: simultaneous treatment with NE and Meto both at 01 μmol/L for 24 h; (4) NE+Meto+PD98059 group: pretreatment with extracellular signal-regulated kinase (ERK) phosphorylation inhibitor PD98059 at 10 μmol/L for 30 min and then treatment with NE and Meto both at 01 μmol/L for 24 h; (5) NE+PD98059 group: pretreatment with PD98059 at 10 μmol/L for 30 min and then treatment with NE at 01 μmol/L for 24 h. The beating rates of cardiomyocytes in various groups were calculated, and the viability of cardiomyocytes was assayed by MTT method. The Cx43 mRNA expression was detected by RT-PCR, and the protein expression of phosphorylated Cx43 (p-Cx43), phosphorylated ERK1/2 (p-ERK1/2) and cleaved caspase-3 was detected by Western blotting. RESULTS: (1) Separate NE treatment could significantly increased the beating rate of cardiomyocytes and reduced cell viability, while Meto showed the opposite effects. PD98059 treatment had no significant effect on cardiomyocyte beating rate, but suppressed Meto to improve cell viability to some extent. (2) Compared with Con group, separate NE treatment significantly increased the Cx43 mRNA expression (P<001). Compared with NE group, Meto or PD98059 intervention could significantly inhibited Cx43 mRNA expression (both P<001), and simultaneous treatment with Meto and PD98059 could further suppress Cx43 mRNA expression up-regulated by NE (P<001). (3) Compared with NE group, Meto significantly inhibited the increased p-Cx43, p-ERK1/2 and cleaved caspase-3 expression induced by NE (P<001), and simultaneous treatment with Meto and PD98059 could further enhance the inhibition of p-Cx43, p-ERK1/2 and cleaved caspase-3 expression by Meto (P<001). PD98059 treatment had no significant effect on the increased p-Cx43 and cleaved caspase-3 expression induced by NE (P>005). CONCLUSION: The inhibitory effect of Meto on NE-induced cardiomyocyte apoptosis is related to the inhibition of Cx43 phosphorylation, which may be partly mediated via ERK1/2 pathway.     

Effects of simvastatin on cigarette smoke extract-induced sEPCR and EPCR expression in human umbilical vein endothelial cells

AIM: To investigate the effects of simvastatin on cigarette smoke extract (CSE)-induced expression levels of soluble endothelial cell protein C receptor (sEPCR) and membrane-associated endothelial cell protein C receptor (mEPCR ) in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs at passage 4 to 6 were randomly divided into control group, 5% CSE group, simvastatin groups and simvastatin+CSE groups. In simvastatin groups, HUVECs were incubated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 24 h. In simvastatin+CSE groups, the cells were treated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 2 h, and then exposed to CSE for 24 h. The protein level of sEPCR in the culture supernatants was measured by ELISA. The cells were collected for determining the mRNA expression of mEPCR by real-time PCR. RESULTS: Compared with control group, the protein level of sEPCR was significantly increased, and the mRNA expression of mEPCR was significantly decreased in 5% CSE group (both P<0.05). The protein levels of sEPCR were significantly increased, and the mRNA expression of mEPCR was significantly decreased in 100 μmol/L and 200 μmol/L simvastatin groups. However, the protein levels of sEPCR were lower, and the mRNA expression of mEPCR was significantly higher in 100 μmol/L and 200 μmol/L simvastatin groups than those in 5% CSE group. Compared with 5% CSE group, the protein levels of sEPCR in simvastatin+CSE groups were significantly decreased, but higher than those in control group and simvastatin group with corresponding concentration. On the contrary, the mRNA expression of mEPCR in simvastatin+CSE groups was significantly increased, but lower than that in control group and simvastatin group with corresponding concentration (all P<0.05). CONCLUSION: Simvastatin obviously increases the mRNA expression of mEPCR, decreases the protein level of sEPCR, and attenuates the CSE-induced endothelial injury in vitro .     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.