Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Urotensin Ⅱinduces pulmonary arterial smooth muscle cell proliferation and up-regulates Egr-1 expression through ERK1/2 pathway in rats

AIM: To investigate the effect of urotensinⅡ (UⅡ) on the proliferation of cultured rat pulmonary arterial smooth muscle cells (PASMCs), and to explore whether mitogen-activated protein kinase (MAPK) signaling pathways and early growth response factor-1 (Egr-1) involved in the regulation of the PASMCs proliferation stimulated by UⅡ. METHODS: The rat PASMCs were isolated and cultured in vitrowith explant culture technique. The proliferation of cultured PASMCs stimulated by different doses of UⅡwas detected by BrdU incorporation. The mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase (SAPK), p38 MAPK and Egr-1 in cultured PASMCs treated with UⅡ, UⅡ-specific antagonist urantide, and ERK1/2 inhibitor PD98059 was detected by real-time PCR. The protein levels of phosphorylated ERK1/2 (p-ERK1/2), p-SAPK, p-p38 and Egr-1 in cultured PASMCs were determined by Western blotting. RESULTS: UⅡ at concentrations of 1 μmol/L, 0.1 μmol/L and 0.01 μmol/L increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.01 or P<0.05), with the maximal effect at a concentration of 1 μmol/L. However, urantide inhibited the promotion effect of UⅡ on PASMC proliferation (P<0.05). UⅡ up-regulated the mRNA expression of ERK1/2, SAPK and Egr-1 (P<0.01 or P<0.05), but not the p38 MAPK. However, the up-regulatory effect of UⅡ on ERK1/2 and Egr-1 expression was inhibited by PD98059 and/or urantide (P<0.01 or P<0.05). UⅡ also increased the protein levels of p-ERK1/2, p-SAPK and Egr-1 (P<0.01 or P<0.05), but the promotion effect was also inhibited by PD98059 and/or urantide (P<0.01 or P<0.05).CONCLUSION: UⅡ increases the proliferation of PASMCs, and U Ⅱand Egr-1 participates in UⅡ-mediated proliferation of cultured PASMCs through activation of ERK1/2 signal pathway.     

Effect of IL-1β and IL-6 on expression of nerve growth factor in human nucleus pulposus cells

AIM: To determine the effect of interleukin-1β(IL-1β)and IL-6 on the expression of nerve growth factor (NGF) by human nucleus pulposus (NP) cells. METHODS: The NP cells from the normal disc of operative patients with scoliosis were isolated, cultured and identified. After 7 days preculture, the NP cells were treated with IL-1β (10 μg/L, 50 μg/L) or IL-6 (10 μg/L, 50 μg/L) for 48 h in the experimental groups and 0.3% PBS was used in the control groups. The expression of NGF was detected by the methods of immunohistochemistry, semi-quantitative RT-PCR and Western blotting.RESULTS: The NP cells were chondrocyte-like cellular morphology with positive staining of toluidine blue, safranine O and anti-collagen II antibody. The NP cells cultured in monolayer showed immunoreactivity to NGF either in control condition or in experimental group. IL-1β and IL-6 up-regulated the mRNA expression of NGF and the protein production of NGF. The effect of this up-regulation was higher by treating with IL-6 than by treating with IL-1β in the same concentration.CONCLUSION: Our results demonstrate that the proinflammatory cytokines IL-1β and IL-6 stimulate the production of NGF in NP cells. The effect of IL-6 is more significant than that of IL-1β.     

TCRVβ7.1 transfection activates signal protein ERK and insreases IFN-γ expression in PBMCs

AIM: To investigate the effect of tumor-specific T cell receptor (TCR) gene transfection on production of cytokine and signaling activation in T cells.METHODS: TCRVβ7.1 gene was transferred into peripheral blood mononuclear cells (PBMCs) obtained from healthy adults, and the expression of Vβ7.1 was detected by flow cytometry before and after transfection. The total quantities of protein and phosphorylation of ERK1/2 were detected by Western blotting. The expressions of IL-4 and IFN-γ were detected by ELISA.RESULTS: The results of flow cytometry showed that TCRVβ7.1 protein was efficiently expressed after transfection. The phosphorylation level of ERK increased significantly in TCRVβ7.1-modified PBMCs, and was related with the activation of T cells. The expression of IFN-γ was significantly higher in TCR-transfected cells than that in non-transfected cells. The expression of IL-4, however, has no distinct difference between groups.CONCLUSION: The transfection of TCRVβ7.1 induces phosphorylation of ERK1/2 and production of IFN-γ, and activates T lymphocytes.     

Role of TGF-β1/Smads and ERK expression in vascular remodeling induced by high-salt diet in Wistar rats

AIM: To investigate the role of transforming growth factor β₁ (TGF-β₁)/Smads and extracellular signal-regulated kinase(ERK) expression in vascular remodeling induced by high-salt diet in Wistar rats. METHODS: Wistar rats were randomly divided into 3 groups: normal control group (n=13), high salt (8%) model group and high salt+telmisartan group (n=13). Tail-cuff arterial pressure was determined every 2 weeks. After 24 weeks, the rats in high salt model group were divided into model animals with hypertension group (MH, n=12) and model animals without hypertension group (MN, n=12). The remodeling of aorta and mesenteric artery was observed by HE and Masson staining. In addition, the techniques of immunohistochemistry and real-time PCR were applied to detect the expression of proliferating cell nuclear antigen (PCNA), TGF-β₁, p-Smad2/3, p-ERK1/2 and Smad7 at both protein and mRNA levels. RESULTS: Compared with normal control group, blood pressure in MH group was much higher, and media thickness (MT) and collagen volume fraction (CVF) of arteries in MH and MN groups were higher.The mRNA expression of TGF-β₁, Smad2 and Smad7 in the aorta was significantly increased, and the protein levels of PCNA, p-ERK1/2, TGF-β₁ and p-Smad2/3 in the aorta and mesenteric artery media were elevated, but Smad7 decreased. After telmisartan treatment, MT and CVF were much lower,and the protein levels of PCNA, TGF-β₁, p-Smad2/3 and p-ERK1/2 were significantly reduced, whereas Smad7 was increased. CONCLUSION: The abnormal expression of TGF-β₁/Smads and ERK may be involved in the mechanism of remodeling of aorta and mesenteric artery induced by high-salt diet. Telmisartan prevents the vascular remodeling via regulating TGF-β₁/Smads and ERK signal pathways mediated by angiotensinⅡ type 1 (AT₁) receptor, at least in part.     

Influence of losartan potassium on renal expression of TGF-β1, CD68 and MCP-1 in type 2 diabetic nephropathy rats

AIM: To observe the effects of losartan potassium on renal expression of transforming growth factor beta 1(TGF-β₁), CD68 and monocyte chemoattractant protein-1 (MCP-1) in type 2 diabetic nephropathy rats for exploring the protective mechanism of losartan potassium on type 2 diabetic rat kidney. METHODS: Thirty Sprague-Dawley rats were randomized into 3 groups: normal control group, model group and treatment group. The morphology of kidney tissues, the renal function, and the change of 24 h urinary protein quantitative index were measured after 15 weeks of treatment, while TGF-β₁, CD68 and MCP-1 expression in kidney cortex was observed by immunohistochemistry. RESULTS: Compared with the normal control rats, the body weight of the rats was lower in other groups, but the levels of blood glucose, triglyceride and cholesterol were higher.The expression of CD68, MCP-1 and TGF-β₁, 24 h urinary protein quantitative index and serum creatinine were higher in model group than those in normal control rats. However, compared with model group, serum creatinine, 24 h urinary protein quantitative index and the expression of CD68, MCP-1 and TGF-β₁ were decreased in treatment group. CONCLUSION: Losartan potassium protects the kidney of diabetic nephropathy rats through inhibiting the expression of TGF-β₁ and MCP-1 in the kidney and restraining macrophage infiltration.     

Effects of IOX1 on proliferation,apoptosis and extracellular matrix-related protein expression in LX2 cells induced by TGF-β

AIM To investigate the effects of histone demethylase inhibitor IOX1 (5-carboxy-8-hydroxyquinoline) on the proliferation, apoptosis and extracellular matrix (ECM)-related protein expression in transforming growth factor-β (TGF-β)-induced human hepatic stellate LX2 cells. METHODS The proliferation and apoptosis of the LX2 cells were determined by real-time cell analysis and flow cytometry, respectively. The level of histone H3 lysine 9 dimethylation (H3K9me2) and the protein expression of ECM-related molecules [α-smooth muscle actin (α-SMA), collagen type I (Col I), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1)] in the LX2 cells were detected by Western blot. RESULTS Treatment with IOX1 at 50~300 μmol/L significantly inhibited LX2 cell proliferation, and 300 μmol/L IOX1 significantly promoted the apoptosis of the LX2 cells. In addition, different concentrations of IOX1 increased the levels of H3K9me2 and MMP-1, and down-regulated the expression of α-SMA, Col I and TIMP-1 in TGF-β-induced LX2 cells (P<0.05). CONCLUSION Treatment with IOX1 inhibits the proliferation of LX2 cells induced by TGF-β, promotes the cell apoptosis, and regulates the synthesis and metabolism of ECM by elevating H3K9me2 level, thus attenuating hepatic fibrosis.     

Effects of aldosterone on expression of angiotensin Ⅱ receptors in cultured rat mesangial cells

AIM: To investigate the effect of aldosterone (ALD) on the mRNA expression of angiotensin Ⅱ (Ang II) type 1 (AT-1a R and AT-1bR) and 2 (AT-2R) receptors in cultured rat mesangial cells (RMCs) treated with high glucose. METHODS: Rat mesangial cells were cultured in high glucose medium containing different concentrations of ALD (10-8-10-6 mol/L). The antagonists of ALD and Ang II receptors including pironolactone (10-7 mol/L, aldosterone receptor antagonist, SPI), losartan (10-7 mol/L, Ang II type 1 receptor blocker, Los) or PD123319 (10-9 mol/L, Ang II type 2 receptor antagonist, PD) were added in the cell culture for 12 h. The control cells were only treated with high (30 mmol/L) or normal (5.6 mmol/L) glucose medium. The viability and proliferation of the RMCs were evaluated by MTT assay. The mRNA expression of AT-1aR, AT-1b R and AT-2R was detected by semi-quantitative RT- PCR. The expression of MCP-1 in cultured RMCs was detected by ELISA. RESULTS: The mRNA expression of AT-1aR, AT-1b R and AT-2R was increased significantly by treatment with ALD in a dose-dependent manner (1.62-1.77, 9.61-9.89 and 7.26-7.35 folds of high glucose control, respectively, P<0.01). SPI significantly reduced the mRNA expression of AT-1aR and AT-1b R (P<0.01) but not affected the mRNA expression of AT-2R. The ratio of AT-1aR/AT-1b R in cultured RMCs treated with high glucose decreased significantly after stimulated with ALD (P<0.01). However, the effect of ALD was inhibited by SPI (P<0.01). Aldosterone treatment induced a significant upregulation of MCP-1 expression in a dose-dependent manner, and previous treatment with spironolactone, losartan or PD123319 abolished this aldosterone-induced MCP-1 expression. CONCLUSION: The results suggest that aldosterone is involved in the inflammatory response by up-regulating the expression of AT-1aR, AT-1bR and AT-2R, changing the proportion of AT-1R subtype, and inducing MCP-1 overproduction in cultured RMCs treated with high glucose.     

AngⅡ/AT1R pathway activates PP2A to down-regulate p-eNOS (Ser1177) in human umbilical vein endothelial cells

AIM: To investigate whether angiotensinⅡ (AngⅡ)/angiotensin Ⅱ type 1 receptor (AT₁R) pathway down-regulates endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation level in human umbilical vein endothelial cells by activating protein phosphatase 2A (PP2A).METHODS: Human umbilical vein endothelial cells were randomly divided into normal control (control) group, Ang Ⅱ group, candesartan (CAN; specific AT₁R blocker) group and CAN pretreatment+AngⅡ group. The protein levels of total eNOS, p-eNOS (Ser1177), PP2Ac, I₂^PP2A and p-PP2Ac (Tyr307) were determined by Western blot. The content of NO in the cell culture medium was detected by chemical colorimetry.RESULTS: Compared with control group, the level of p-eNOS (Ser1177) and the content of NO decreased (P<0.05). Compared with the same concentration of AngⅡ group, CAN pretreatment increased the level of p-eNOS (Ser1177) and the content of NO (P<0.05), but the protein expression of eNOS showed no significant difference. Compared with control group, the levels of p-PP2Ac (Tyr307) and I₂^PP2A decreased (P<0.05). Compared with the same concentration of AngⅡ group, CAN pretreatment increased the levels of p-PP2Ac (Tyr307) and I₂^PP2A (P<0.05), but the protein expression of PP2Ac showed no significant difference.CONCLUSION: AngⅡ down-regulates the level of p-eNOS (Ser1177), and decreases the production of NO in human umbilical vein endothelial cells via AT₁R pathway. This effect may be related to the reduction of p-PP2Ac (Tyr307) and protein expression of I₂^PP2A, which results in the enhancement of PP2A activity. Pretreatment with AT₁R blocker CAN increases p-PP2Ac (Tyr307) level and I₂^PP2A protein expression, thus reducing the PP2A activity, and ultimately restoring eNOS Ser1177 phosphorylation level and eNOS activity.     

Sphingosine 1-phosphate inhibits TGF-β-induced endothelial-to-mesenchymal transition in human umbilical vein endothelial cells

AIMTo investigate the effect of sphingosine 1-phosphate (S1P) on endothelial-to-mesenchymal transition (End-MT) in human umbilical vein endothelial cells (HUVECs) induced by transforming growth factor-β (TGF-β) in vitro. METHODSThe HUVECs in different groups were treated with TGF-β, S1P or sphingosine 1-phosphate receptor 1(S1PR1) inhibitor VPC23019. Western blot was used to detect the protein levels of endothelial cell markers (CD31 and VE-cadherin), mesenchymal cell markers (α-smooth muscle actin and fibroblast-specific protein 1), S1PR1 and p-Smad3. Immunofluorescence staining was used to analyze the nuclear translocation of Smad3. RESULTSCompared with TGF-β group, the process of End-MT was significantly inhibited, and the phosphorylation and nuclear translocation of Smad3 were significantly reduced in TGF-β+S1P group (P<0.05). However, the above effects of SP1 were reversed after the addition of S1PR1 inhibitor (P<0.05). CONCLUSION S1P inhibits TGF-β-induced End-MT via S1PR1 in HUVECs. This effect may be associated with decreases in Smad3 phosphorylation and nuclear translocation.     

Effect of Buyanghuanwu decoction on the expression of ERK2 and CaMKⅡβ in hippocampus tissue and on ability of learning and memory in vascular dementia rats

AIM: To investigate the effect of Buyanghuanwu decoction, a Chinese medicine, on the ability of learning and memory in the rats with vascular dementia (VD) and on the protein expression of extracellular signal-regulated kinase 2(ERK2) and calcium/calmodulin-dependent protein kinase Ⅱβ(CaMKⅡβ) in hippocampus CA1 area.METHODS: The rats were divided into 4 groups: sham group, VD group, VD+Buyanghuanwu decoction group and VD+nimodipine group. The VD rat model was prepared by Pulsinelli's four-vessel occlusion. At 7th day, 14th day or 28th day after operation, the behaviors of the rats were tested by Morris water maze. The morphological changes of the neurons in hippocampus CA1 area were observed by HE staining 30 d after operation. Western blotting was used to observe the protein expression of ERK2 and CaMKⅡβ in the brain tissues of hippocampal CA1 area of the VD rats. RESULTS: Compared with sham group, the pathological changes such as irregular arrangement, coagulation necrosis and obvious deletion in the neurons of hippocampus CA1 area in VD group appeared significantly. The obstacle of learning and memory ability was observed and the protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area was significantly decreased (P<0.05). Compared with VD group, the neurons in hippocampal CA1 area of VD+Buyanghuanwu decoction group and VD+nimodipine group were in eumorphism, lined up in order, and the structure was close to that in sham group. The ability of learning and memory also significantly improved (P<0.05). The protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area significantly increased (P<0.05). CONCLUSION: Buyanghuanwu decoction promotes the protein expression of ERK2 and CaMKⅡβ in hippocampus CA1 area to protect the neurons from injury, builds up the synapses and promotes the ability of learning and memory in VD rats.     

Ovarian carcinoma cells inhibit ζ chain expression and the secretion of Tc1/Tc2 type cytokines in CD8+ T cells

AIM: To investigate the effect of ovarian carcinoma cells on ζ chain expression and the secretion of Tc1/Tc2 type cytokine in CD8+ T cells, and its role in the ovarian carcinoma induced immunosuppression.METHODS: The supernatants of human ovarian carcinoma cell lines of OVCAR3, CAOV3 and SKOV3 and RPMI-1640 were added into CD8+ T cells (groups I, II, III, and control), which were isolated from the peripheral venous blood of healthy persons. The expression of ζ chain was analyzed by Western blotting. Thiazolyl blue(MTT) method was used to detect the effects of those cell line supernatants on the growth of CD8+ T cells. The secretion of the Tc1 type cytokine interferon (IFN)-γ mRNA and the Tc2 type cytokine interferon (IL)-10 mRNA were detected by RT-PCR. RESULTS: The expression of ζ chain was significantly lower in groups I, II, and III in comparison with that in control group. The absorbance at the wavelength 570 nm of CD8+ T cells culture in the group I, II, and III was all significantly lower than that in the control group. The IFN-γ expression was significantly lower in groups I, II, and III in comparison with that in control group, while the expression of IL-10 was significantly higher. CONCLUSION: Ovarian carcinoma may suppress CD8+ T cell proliferation and secretion of the Tc1/Tc2 type cytokine through inhibition of ζ chain, which may play an important role in the ovarian carcinoma induced immunosuppression.     

Proapoptotic mechanism and changes of endogenous TGF-β1 in NB4 cells induced by exogenous TGF-β1

AIM: To study the effects of transforming growth factor-β1 (TGF-β1) on cell apoptosis, cell cycle, production of endogenous TGF-β1, expressions of P27Kip1, cyclin E and bcl-2 mRNA levels in NB4 cells. METHODS: Apoptotic morphological changes were observed by Wright-Giemsa staining. Cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine the mRNA levels of endogenous TGF-β1, P27Kip1, cyclin E and bcl-2. RESULTS: TGF-β1 significantly restrained the growth and promoted the apoptosis of NB4 cells. The blockage of NB4 cells treated by TGF-β1 at concentration of 5 μg/L was in G1 phase. Endogenous TGF-β1 mRNA expression in NB4 cells was up-regulated when the concentration of exogenous TGF-β1 was <5 μg/L. Meanwhile, the expression of endogenous TGF-β1 mRNA was down-regulated when the concentration of exogenous TGF-β1 was 10 μg/L. After treated with TGF-β1 at concentration of 5 μg/L, P27Kip1 mRNA expression in NB4 cells was up-regulated, cyclin E and bcl-2 were reduced. CONCLUSION: TGF-β1 is able to induce apoptosis and cell cycle distribution abnormally in NB4 cells by (1) Up-regulation of endogenous TGF-β1, so that NB4 cells was induced into apoptosis through consequently high expression of P27Kip1. (2) TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by inhibiting the activity of cyclin E through the increased expression of P27Kip1. (3) Down-regulation of bcl-2 induces apoptosis of NB4 cells.     

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.     

Interleukin-1β inhibits AKT to down-regulate eNOS Ser1177 phosphorylation in human umbilical vein endothelial cells

AIM To investigate whether interleukin-1β （IL-1β） regulates endothelial nitric oxide synthase （eNOS） phosphorylation at Ser1177 site in human umbilical vein endothelial cells （HUVECs）, and to explore its possible mechanism. METHODS The HUVECs were randomly divided into normal control group, tumor necrosis factor-α （TNF-α） group, IL-1β group, IL-6 group, SC79 ［protein kinase B （PKB/AKT） specific agonist］ group and SC79+IL-1β group. Western blot was used to determine the protein levels of eNOS, p-eNOS-Ser1177, AKT and p-AKT-Ser473 in the HUVECs. Chemical colorimetry was used to detect the nitric oxide （NO） content in the culture medium of HUVECs. RESULTS No statistically significant difference of p-eNOS-Ser1177 level in HUVECs treated with TNF-α and IL-6 was observed as compared with normal control group （P>0.05）, while the protein level of p-eNOS-Ser1177 in the HUVECs and the content of NO in the culture medium of HUVECs decreased significantly in IL-1β group （P<0.05）, and the protein level of p-AKT-Ser473 in the HUVECs was decreased as compared with normal control group （P<0.05）. The AKT agonist SC79 blocked the down-regulation effect of IL-1β on p-eNOS-Ser1177 level in the HUVECs and NO content in the culture medium of HUVECs （P<0.05）. CONCLUSION IL-1β down-regulates the protein level of p-eNOS-Ser1177 in HUVECs and affects the activity of eNOS, which may be involved in AKT/eNOS signaling pathway.